Physiol Rev 93: 189 –268, 2013
doi:10.1152/physrev.00015.2012
GASTRIC ACID, CALCIUM ABSORPTION, AND
THEIR IMPACT ON BONE HEALTH
Sascha Kopic and John P. Geibel
Departments of Surgery and Cellular and Molecular Physiology, Yale School of Medicine,
New Haven, Connecticut
Kopic S, Geibel JP. Gastric Acid, Calcium Absorption, and Their Impact on Bone
Health. Physiol Rev 93:189 –268, 2013; doi:10.1152/physrev.00015.2012.—Calcium balance is essential for a multitude of physiological processes, ranging from cell
signaling to maintenance of bone health. Adequate intestinal absorption of calcium is a
major factor for maintaining systemic calcium homeostasis. Recent observations indicate that a reduction of gastric acidity may impair effective calcium uptake through the intestine.
This article reviews the physiology of gastric acid secretion, intestinal calcium absorption, and their
respective neuroendocrine regulation and explores the physiological basis of a potential link between these individual systems.
L
I.
II.
III.
IV.
V.
VI.
INTRODUCTION
GASTRIC ACID SECRETION
INTESTINAL CALCIUM ABSORPTION
REGULATION OF CALCIUM HOMEOSTASIS
THE STOMACH AND CALCIUM
CONCLUSIONS
189
189
203
212
231
238
I. INTRODUCTION
The average adult human body contains ⬃1.6% calcium,
which relates to ⬃1,120 g in a 70-kg individual (743). Ninetynine percent of the calcium is stored in bone and teeth and
is therefore inaccessible to most physiological processes
(743). Although the amount of the immediately accessible
11 g (1%) of calcium may seem miniscule, this fraction
represents a pivotal constituent of our body. It serves a
broad diversity of roles, which range from intracellular signaling and maintenance of membrane integrity to muscle
contraction and neuronal transmission.
To allow for these calcium-dependent processes to function, our body undertakes extensive measures to keep the
intracellular and extracellular calcium concentrations and
the gradient between these two compartments stable. The
extracellular calcium concentration is typically clamped at
⬃1.1 mM, whereas the intracellular environment is kept at
a 10,000 times lower concentration. In consequence, relatively small disturbances in calcium homeostasis can lead to
severe symptoms, such as cardiac arrhythmias or cognitive
dysfunctions. To maintain eucalcemia, our body is therefore tightly regulating the balance between calcium absorption by the intestine and calcium excretion by the kidney. In
addition, calcium is deposited in or extracted from bone,
which serves as a dynamic calcium reservoir. These three
organ systems, i.e., the intestine, the kidney, and bone, are
precisely controlled by a complex endocrine network,
which primarily consists of the calcitropic hormones: 1,25dihydroxyvitamin D [1,25(OH)2-vitamin D], parathyroid
hormone (PTH), and calcitonin.
This review mainly focuses on the question as to how calcium
enters the body through the intestine and how this mechanism
is regulated via the endocrine system. Furthermore, the process
of gastric acid secretion as related to calcium homeostasis will
be reviewed in detail. This may seem surprising, as gastric acid
secretion and intestinal calcium absorption are two distinct
physiological processes, which on first examination may not
seem to be interdependent. However, recent clinical studies
suggest that there may be a relationship between reduced gastric acid secretion and increased risk for sustaining bone fractures, which asks the question whether we need gastric acid to
absorb calcium efficiently through the intestine, or whether the
stomach exerts endocrine functions that impact bone health.
Indeed, it has been put forward several decades ago that gastric acid solubilizes calcium that is then complexed with other
dietary constituents, thereby allowing for a more efficient absorption in the intestine (18, 520, 699, 797). Furthermore, it is
long known that a partial or complete resection of the stomach
results in decreased bone density, also leading to fractures (58,
305, 732, 876). The stomach, the intestine, and bone are therefore functionally more intertwined than one may initially assume. This review will independently analyze the processes of
gastric acid secretion, intestinal calcium absorption, and their
respective neuroendocrine control and will conclude with a
critical attempt at illustrating where these two seemingly independent organ systems intersect in terms of calcium homeostasis and bone health.
II. GASTRIC ACID SECRETION
The stomach is a unique organ that fulfills multiple roles.
The main function of the gastric mucosa is to secrete con-
0031-9333/13 Copyright © 2013 the American Physiological Society
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SASCHA KOPIC AND JOHN P. GEIBEL
centrated hydrochloric acid, which provides a chemical barrier against ingested pathogens and aids in the digestion of
foodstuffs. To achieve these functions, the gastric gland
contains specialized cells that pump protons into the gastric
lumen in an effort to acidify the contents of the stomach.
These cells are known as parietal cells, or oxyntic cells.
Since concentrated acid is a noxious substance, the gastric
mucosa has to undertake extensive measures to protect itself from tissue injury. The protection is accomplished by
secreting mucus from mucus neck cells, but also by tightly
regulating the secretion of acid (see sect. IIB). A variety of
specialized endocrine cells in the gastric mucosa are involved in the regulation of gastric acid secretion. A perturbation of either protective mechanism can lead to severe
tissue damage, resulting in gastric ulcers. This section discusses the process of how gastric acid is secreted by reviewing the molecular mechanism underlying acid secretion in
the parietal cell and its neuroendocrine regulation.
A. Apical Ion Transport in the Parietal Cell
The gastric parietal cell is responsible for acidifying the
stomach by secreting concentrated acid. Gastric acid secre-
tion depends on the apical extrusion of three ions. Protons
are pumped into the gastric lumen by a proton pump, the
gastric H⫹-K⫹-ATPase, to acidify the gastric content to a
pH of as low as 1. Chloride is secreted via apical chloride
channels to ensure formation of HCl and to provide the
counter-ion conductance to protons. Lastly, potassium
leaves the parietal cell apically in a recycling mechanism,
thereby fueling reciprocal proton transport by the H⫹-K⫹ATPase (FIGURE 1). It has been demonstrated in numerous
investigations that disruption of one of these ion transport
mechanism renders the parietal cell incapable of secreting
gastric acid (705, 820, 1013, 1029).
1. H⫹-K⫹-ATPase
The gastric H⫹-K⫹-ATPase belongs to the
family of P2-type ATPases, which also includes the ubiquitous Na⫹-K⫹-ATPase and the sarcoplasmic reticulum
Ca2⫹-ATPase (SERCA). As the name implies, it exchanges
one intracellular hydrogen ion for one extracellular potassium ion at the expense of ATP. ATP is provided to the
pump by a large network of mitochondria, which occupy
up to 40% of the cell volume, making the parietal cell one of
the most mitochondria-rich cells in the body (292). In the
A) STRUCTURE.
Apical
Basolateral
PPIs
APAs
H+
SSTR
SST
H2
Hist
CCK2
Gast
M3
ACh
K+
cAMP
K+
KCNQ1
Kir
Ca2+
Cl–
CFTR
CIC-2?
SLC26A9
Parietal cell
FIGURE 1. Parietal cell model. The gastric parietal cell is equipped with apical ion transport mechanisms that
allow for the secretion of concentrated hydrochloric acid. Activation of basolateral secretagogue receptors
mainly leads to an increase in either cAMP (histamine) or calcium (acetylcholine, gastrin), causing apical
insertion and activation of the H⫹-K⫹-ATPase. Somatostatin reduces intracellular cAMP levels. ACh, acetylcholine; APAs, acid pump antagonists; Gast, gastrin; Hist, histamine; PPIs, proton pump inhibitors; SST,
somatostatin.
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GASTRIC ACID, CALCIUM ABSORPTION, AND BONE HEALTH
process of proton extrusion, the H⫹-K⫹-ATPase can overcome a massive acid gradient of 6 pH units, which is necessary to achieve sufficient gastric acidification. The pump
itself is a heterodimer, consisting of a ␣ subunit and a ␤
subunit, while the individual pumps assemble as (␣␤)4 tetramers on the parietal cell surface (1). The ␣ subunit consists of 10 transmembrane domains and contains the catalytic site, which mediates ion exchange. The ␤ subunit stabilizes the ␣ subunit and is heavily glycosylated (41, 1105).
Mutational analysis of the glycosylated asparagine residues
suggests that these sites are critical for adequate membrane
delivery of the entire pump (41, 1105). Furthermore, the ␤
subunit prevents a reversal of ion transport by a “ratchet”like mechanism, which allows H⫹-K⫹-ATPase to pump
against the imposed high proton gradient (4, 294). Both
subunits share a significant degree of homology to Na⫹-K⫹ATPase (697, 1012). This close relationship to other P2type ATPase has historically been exploited for homology
modeling of H⫹-K⫹-ATPase based on the crystal structure
of SERCA, which had been acquired in several conformational states (762, 815, 1092, 1093). Recently, however,
direct structural information on H⫹-K⫹-ATPase has been
obtained by electron crystallography, also in the presence of
the acid pump antagonist SCH28080 (2– 4).
In the resting parietal cell, H⫹-K⫹-ATPase is
stored in tubulovesicles throughout the cell (292). Following neuronal or hormonal stimulation (see sect. IIB), these
vesicles are postulated to fuse with the apical pole, which is
characterized by multiple microvilli-lined membrane invaginations, the so-called secretory canaliculi (292). This
distinct apical morphology of the parietal cells maximizes
cell surface and thereby allows for insertion of a high number of proton pumps per cell following stimulation. The
changes in membrane morphology and insertion of H⫹-K⫹ATPase are extremely dynamic to ensure fine regulation of
gastric acid secretion (973). H⫹-K⫹-ATPase containing
tubulovesicle fusion relies on SNARE complex formation. In particular, the SNARE proteins syntaxin 3/7/12/
13, VAMP2/8, and SNAP-25 were implicated to be candidates mediating this process (548 –550, 624). The functional significance of these proteins was, for example,
demonstrated in primary rabbit parietal cell cultures expressing a SNAP-25 mutation, which was shown to reduce
their capacity to secrete gastric acid (548).
B) TRAFFICKING.
Apart from SNARE proteins, the small GTPases of the rab
family (rab2/11a/25/27b) are involved in the regulation of
H⫹-K⫹-ATPase vesicle trafficking (147, 293, 386, 387,
1049, 1070). Functional data especially substantiate the
importance of rab11a and rab27b. In parallel to SNAP-25
defective cells, parietal cells transfected with a rab11a and
rab27b mutant secrete acid less effectively (293, 1049).
After stimulation, in the off-phase of gastric acid secretion,
H⫹-K⫹-ATPase has to be retrieved from the plasma mem-
brane for recycling (336). It is plausible that the initial step
of this process relies on the formation of clathrin-coated pits
and subsequent vesicle budding. Indeed, clathrin was identified fairly early on H⫹-K⫹-ATPase containing tubulovesicles, although a functional role was not demonstrated
(813). One of the multiple clathrin binding proteins is Huntingtin interacting protein 1 related (Hip1r) which aids in
vesicle formation and membrane trafficking (309). It is
strongly expressed in parietal cells, especially in the vicinity
of secretory canaliculi (522). Functionally, Hip1r-deficient
animals present with a decreased number of parietal cells,
loss of tubulovesicles, and decreased acid output (522,
561).
2. Chloride secretion
Apical chloride secretion provides the second component
for the formation of concentrated HCl and maintains overall electroneutrality during acid secretion. The importance
of chloride efflux for the process of gastric acid secretion
has been established in the 1980s. Patch-clamp measurements demonstrated the presence of chloride conductance
on the apical pole of the parietal cell in Necturus, the human
parietal cell line HGT-1, and rabbit parietal cells (259, 935,
940). All reports demonstrated a sensitivity of the chloride
current to cAMP or histamine, which is a common second
messenger promoting acid secretion or a direct acid secretagogue, respectively (259, 935, 940). Simple flux measurements in isolated parietal cell vesicles had indicated the
presence of a chloride conductance pathway even earlier
(232, 895, 1169). In these early experiments, inhibition of
chloride flux with chloride channel blockers also abolished
proton transport which underlines the necessity of intact
chloride secretion for acid secretion to take place (232, 895,
1169). However, the molecular identity of the chloride
pathway remained elusive. Today, at least three candidates
have been put forward as potential mediators of apical chloride secretion in the parietal cell: the cystic fibrosis conductance regulator (CFTR), chloride channel protein 2 (ClC-2),
and solute carrier 26 A 9 (SLC26A9) (FIGURE 1).
A) CFTR. CFTR represents a common apical chloride conduc-
tance pathway in a broad variety of epithelia, such as the
airways, intestine, and pancreas. Its mutation is responsible
for the most widespread inherited disease, namely, cystic
fibrosis (CF), which results in increased mortality due to
secretory defects and concomitant infections. The presence
of CFTR has been confirmed in gastric mucosa by in situ
hybridization, albeit at low quantities (1044). Nevertheless,
functional measurements in isolated gastric glands demonstrated a decreased acid secretory capacity in animals carrying the most common mutation responsible for CF
(⌬F508) (1013). Furthermore, acid secretion was reduced
in wild-type animals when a specific CFTR inhibitor was
applied (1013). Although these observations may suggest a
direct involvement of CFTR in the process of chloride secretion, it is plausible that CFTR rather has a regulatory
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SASCHA KOPIC AND JOHN P. GEIBEL
effect on H⫹-K⫹-ATPase (1013). In other tissues, CFTR
can interact with a variety of ion transport proteins, such as
NHE, forming regulatory complexes, making an interaction with H⫹-K⫹-ATPase plausible (1013).
B) CLC-2. ClC-2 has been proposed as an alternative chloride
secretion pathway to CFTR in other epithelia, such as the
lung and intestine (207, 404, 675, 766). ClC-2 has been
cloned from rabbit gastric mucosa, which led to the hypothesis that the channel may also be involved in acid secretion
(706). However, follow-up investigations revealed that the
role of ClC-2 is much less clear. The studies revealed controversial results regarding the channel’s expression in the
gastric mucosa (488, 706, 1001). While the initial observations reported mRNA and cDNA expression in rabbit gastric mucosa, no protein could be detected in human and rat
gastric glands (488, 706, 1001). The importance of ClC-2 in
the stomach has further been severely challenged by the
creation of a ClC-2 (⫺/⫺) animal model. Although ClC-2deficient animals present with a distinct phenotype characterized by testicular and retinal abnormalities, no defect in
acid secretion was observed (118).
C) SLC26A9. Lastly, evidence suggests that chloride may leave
the apical pole via SLC26A9, a chloride-bicarbonate antiporter. Both SLC26A9 and an antiporter from the same
anion exchanger family (SLC26A6) have been detected in
the tubulovesicles of parietal cells (845, 1179, 1180). Concerning the functional involvement, the authors speculate
about two potential roles SLC26A9 may play in parietal cell
physiology. Being a chloride-bicarbonate exchanger, its activation would entail alkalinization of the gastric lumen
by bicarbonate efflux and simultaneous chloride uptake
(1180). Since this would neutralize H ⫹ -K ⫹ -ATPasemediated proton extrusion, it has been suggested that
SLC26A9 activates in the off-phase of acid secretion to
neutralize tubulovesicular pH during vesicle retrieval
(1180). Alternatively, SLC26A9 may function as a chloride
secretion pathway that contributes to acid secretion. This
hypothesis is based on the observation that SLC26A9 can
also exhibit the behavior of a bona fide chloride channel,
rather than an anion antiporter (88, 281). Undoubtedly,
further functional investigations are needed to delineate its
exact role in the parietal cell. Its genetic disruption, however, leads to a severely altered parietal cell morphology
that is characterized by dilation of gastric glands, loss of
tubulovesicles, and decreased acid output (1180). Although
these results do not answer whether SLC26A9 serves as an
apical chloride efflux pathway, they indicate that it may be
necessary for normal parietal cell function.
3. Potassium recycling
Even before the identification of H⫹-K⫹-ATPase, it has
been observed that potassium is necessary for acid secretion
to take place (335). To prevent the luminal depletion of
potassium, which would impair proton pumping by H⫹-
192
K⫹-ATPase, potassium has to leak through potassium
channels or transporters into the gland lumen to ensure
adequate supply to H⫹-K⫹-ATPase (FIGURE 1). This process is referred to as potassium recycling. Early flux measurements in isolated H⫹-K⫹-ATPase containing parietal
cell vesicles had already indicated the presence of a large
potassium conductance during H⫹-K⫹-ATPase activity
(1169). The exact molecular identity of the potassium efflux
pathway is, however, under debate. The list of candidates
that have been put forward to be responsible for potassium
recycling during acid secretion is long and includes KCNQ1
(Kv7.1), KCNJ10 (Kir4.1), KCNJ15 (Kir4.2), KCNJ2
(Kir2.1.), and KCC4.
A) KCNQ1. KCNQ1 is a typical “shaker”-like six transmembrane spanning domain voltage-gated potassium channel
(1144). It was initially identified in the heart, where its
mutation can be responsible for cardiac arrhythmias
(1144). Yet, studies in KCNQ1 (⫺/⫺) animals revealed no
electrocardiographical abnormalities (641). Rather than
suffering from cardiac abnormalities, these animals surprisingly exhibited a distinct gastric phenotype with gastric hyperplasia, dilated gastric glands, vacuolated parietal cells,
hypochlorhydria, and hypergastrinemia (641). This observation led to the speculation that KCNQ1 may be the channel responsible for potassium recycling. Subsequently, immunohistochemical studies confirmed a colocalization of
the channel with H⫹-K⫹-ATPase, and acid secretion was
shown to be inhibited by pharmacological blockade (253,
391). Direct measurement of acid secretion in KCNQ1
(⫺/⫺) mice with modified Ussing chambers (pH stat) later
confirmed the initially observed hypochlorhydria (1029).
Interestingly, luminal substitution of potassium could rescue the acid secretory deficit, indicating that hypochlorhydria ensued from a true lack of apical potassium secretion
rather than a general morphological defect of the KCNQ1
(⫺/⫺) parietal cell (1029).
KCNQ1 is a peculiar channel in that it has a low conductance in acidic environments. In the context of the extreme
acidic milieu surrounding the parietal cell, this would impede its function as a potassium recycling pathway. To
circumvent this limitation, KCNQ1 attaches to a regulatory
subunit (KCNE2), which modulates the channel’s gating
properties and current amplitude (253, 391, 1087). Coassembly with KCNE2 activates KCNQ1 at acidic pH values
and thus facilitates the process of potassium secretion into
the gland lumen (391, 436). The importance of KCNE2 for
proper channel function is underlined by the observation
that KNCE2 (⫺/⫺) animals display a phenotype similar to
KCNQ1 (⫺/⫺) mice, i.e., hypochlorhydria, altered parietal
cell morphology, and hypergastrinemia (917).
B) KIR CHANNELS.
Apart from KCNQ1, several members of
the inward-rectifier potassium channel (Kir) family have
been proposed to be involved in gastric acid secretion, albeit
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GASTRIC ACID, CALCIUM ABSORPTION, AND BONE HEALTH
the amount of functional evidence supporting a role of these
channels is smaller and the field is divided about the relative
contribution of each channel. Kir 2.1, 4.1, 4.2, and 7.1 were
all confirmed on an mRNA level in gastric mucosa (353,
431, 707). On a protein level, immunohistochemistry demonstrated colocalization of Kir 2.1, 4.1, and 4.2 with H⫹K⫹-ATPase (353, 431, 556, 707). Cell fractionation experiments further indicated trafficking of Kir 4.1 and 4.2 to the
cell surface, following parietal cell stimulation (431, 556).
A most recent observation monitored acid secretion in Kir
4.1 (⫺/⫺) mice (1028). Surprisingly, loss of Kir 4.1 results in
augmented rather than impaired acid secretion, accompanied by upregulated H⫹-K⫹-ATPase expression (1028).
This makes a contribution of Kir 4.1 to potassium recycling
highly unlikely. Instead, it has been proposed that the channel may balance excessive potassium loss through KCNQ1
and may be involved in membrane recycling (1028). In summary, more investigations will be necessary to clarify the
roles of the individual Kir channels.
C) KCC4.
Apart from being secreted through channels, potassium and chloride may exit the parietal cell through transporters. This alternative hypothesis is corroborated by a
recent observation of Fuji et al. (352). The group reported
that the K-2Cl cotransporter KCC4 coimmunoprecipitates
with H⫹-K⫹-ATPase in apical membrane fractions of parietal cells (352). Furthermore, flux measurements in H⫹-K⫹ATPase containing vesicles showed decreased chloride and
proton transport under pharmacological blockade of
KCC4, suggesting a functional coupling of KCC4 to H⫹K⫹-ATPase (352). Although the hypothesis that both potassium and chloride leave the cell via a transporter is intriguing, the observation is, as of now, solitary and needs
further experimental validation.
B. Control of Acid Secretion
Gastric acid secretion is subjected to precise regulation. The
complex regulatory machinery that orchestrates the secretion
of gastric acid consists of hormonal (gastrin, somatostatin),
paracrine (histamine, somatostatin), and neuronal components (FIGURE 2). The need for this tight regulation is highlighted by conditions that lead to a hypersecretion of gastric
acid, such as Zollinger-Ellisson syndrome (ZES; gastrinoma).
Gastric hypersecretion can overcome the measures our body
undertakes to protect itself from the acid and thereby lead to
peptic ulcers. A fine on-demand regulation of acid secretion is
thus pivotal to ensure the balance between an adequately low
intragastric pH and tissue protection.
According to the well-established model of acid secretion,
the parietal cell is activated by neuronal input from the
vagus nerve, endocrine input from gastrin-producing G
cells, and paracrine input from histamine-producing enterochromaffin-like (ECL) cells (FIGURES 1 AND 2). The
distinct substances released by these cells, i.e., acetylcho-
line, histamine, and gastrin, directly or indirectly stimulate
the parietal cell by inducing insertion of H⫹-K⫹-ATPase at
the apical membrane and are thus commonly referred to as
acid secretagogues. The main inhibitor of parietal cell acid
secretion is somatostatin, which is secreted by the D-cells
of the gastric mucosa (FIGURES 1 AND 2). Because of the
complexity of the network that controls the release of
acid into the stomach, it has been historically challenging
to dissect the relative role of each individual regulatory
component. Without a doubt, knockout models have
greatly aided us in the last years to gain a more profound
understanding of this process, despite their limitations of
chronic compensation. The subsequent chapter aims to
summarize the key players in our canonical model of acid
regulation.
1. Cholinergic stimulation/vagus nerve
Since the seminal experiments conducted by Pavlov on
dogs, we know that the mere prospect of food ingestion or
sham-feeding is sufficient to trigger the secretion of gastric
acid (833). This first of three phases of acid secretion is
called the cephalic phase and is mostly mediated through
the vagus nerve (595, 725, 910). Hence, before the advent
of pharmacological inhibitors, vagotomy has been an effective surgical procedure to control acid-related disorders
(301).
The parietal cell receives neuronal input from the vagus nerve
that is relayed via cholinergic postganglionic enteric fibers in
the enteric nervous system (ENS) (FIGURES 1 AND 2). In addition, the vagus nerve activates G-cells to release gastrin,
resulting in an indirect stimulation of the parietal cell. Direct cholinergic activation occurs mostly via muscarinic M3
receptors, which have been identified on the surface of the
parietal cell (507, 541, 846). The M3 receptor is a classic
seven-transmembrane domain GPCR. Predictably, knockout of M3 receptors leads to an impairment of gastric acid
secretion and compensatory hypergastrinemia due to negative feedback (9). Following acetylcholine binding, M3 receptor activation mostly causes an increase in intracellular
calcium concentrations (44, 1163). Calcium rises in response to PLC-mediated IP3 generation and subsequent mobilization from intracellular stores (190). The primary kinases activated by the M3 receptor are protein kinase C
(PKC) and calcium/calmodulin-dependent protein kinase II
(CaMKII) (136, 196, 314 –316, 773, 774, 1095). While
activation of CaMKII has a clear stimulatory effect on acid
secretion, PKC has been reported to have dual effects, although reports of an inhibitory role predominate numerically (23, 73, 136, 196, 313, 314, 316, 597, 755, 773,
1095). It has been postulated that the expression of different PKC isoforms may account for this dichotomy (313,
314). Current evidence suggests that the PKC-␣ isoform has
a suppressing effect by trans-inhibiting CaMKII activity,
whereas PKC-⑀ increases the baseline levels of intracellular
calcium, thereby sensitizing the parietal cell to subsequent
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SASCHA KOPIC AND JOHN P. GEIBEL
Oxyntic mucosa
Lumenal
Antrum
Basolateral
D-cell
Lumenal
Somatostatin
Basolateral
D-cell
?
VPAC
PACAP
VIP
CCK1
CCK
ENS
Somatostatin
Low
lumenal
pH
ECL-cell
SSTR
SST
G-cell
PAC1
PACAP
CCK2
Gast
SSTR
ENS
CaSR
CaSR
ENS
Histamine
Calcium
Amino acids
Polyamines
PPIs
APAs
SST
Gastrin
ACh
GRP
CaSR
H+
H,K-ATPase
H2
Hist
Circulation
K+
CCK2
Gast
M3
ACh
ENS
SSTR
SST
Parietal cell
Somatostatin
D-cell
FIGURE 2. Neuroendocrine regulation of gastric acid secretion. In addition to direct neuronal regulation, the
parietal cell receives paracrine signals from neighboring ECL- and D-cells. Gastrin is produced in the antral
mucosa of the stomach and reaches the oxyntic mucosa via the circulation (endocrine regulation). Gastrinmediated histamine release represents one of the major stimulatory pathways leading to the secretion of
gastric acid (gastrin-histamine axis). The secretion of gastrin is closely tied to intragastric pH (via somatostatin), thereby creating a negative-feedback loop. ACh, acetylcholine; APAs, acid pump antagonists; ENS, enteric
nervous system; Gast, gastrin; Hist, histamine; PPIs, proton pump inhibitors; SST, somatostatin.
stimulation (313, 314). Apart from PKC and CaMKII activation, cholinergic signaling activates parietal cell MAPKs,
which is partially a downstream effect of PKC activation
(771, 1039, 1062, 1063). MAPK activation seems to have a
biphasic effect on acid secretion (acute inhibition and
chronic augmentation) and also serves as a mediator of
trophic responses in the parietal cell. For example, pro-
194
longed MAPK activation (72h) has been shown to serve as
a maturation and differentiation signal leading to a transformation of parietal cell morphology in vitro (1039). The
change in morphology is accompanied by a downregulation
of H⫹-K⫹-ATPase gene expression (1039). As of now, it is
challenging to put these findings into a physiological perspective.
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GASTRIC ACID, CALCIUM ABSORPTION, AND BONE HEALTH
In addition to M3 receptors, M1 receptors have also been
implicated to play a role in the process of acid secretion.
This hypothesis was derived from the observation that the
M1 receptor is expressed in gastric mucosa and that its
blocker pirenzepine can inhibit gastric acid secretion (29,
466). Most evidence pointed to an expression of M1 on
ECL-cells, where it was speculated to regulate the release of
histamine (437, 507). More recent findings somewhat surprisingly report that pirenzepine also suppresses acid secretion in M1-deficient animals. Furthermore, these animals
show a normal phenotype in terms of acid output (10).
These observations question both the involvement of M1
receptors in acid secretion and the specificity of pirenzepine.
Lastly, knockout studies point towards a contribution of the
M5 receptor to the regulation of acid secretion, as its deletion
correlates with decreased acid output (10). Yet, M5 receptor
mRNA could only be detected in whole stomach homogenates, but not in gastric mucosa per se, making its localization
to the submucosal enteric plexus more likely (10).
2. Gastrin/G-cell
Gastrin has been discovered in 1906 by John S. Edkins, who
injected gastric extracts of pig and cat stomachs into the
jugular vein of cats and observed a subsequent increase in
acid secretion (298). Gastrin is a peptide hormone that is
produced in specialized G-cells, located in the antral section
of the stomach (FIGURE 2) and endocrine cells in the duodenum, small intestine, colon, pancreas, testis, and pituitary. It is the main mediator of the so-called gastric phase of
acid secretion, which initiates when the ingested food enters
the stomach. The gastric phase accounts for the majority of
the acid secretory response of the stomach.
A) SYNTHESIS.
The gastrin cDNA encodes a 101-amino acid
pre-pro-hormone that undergoes extensive posttranslational processing (113, 519, 551, 552, 1161). In brief, the
pre-pro-hormone is first cleaved NH2-terminally to create
progastrin and then truncated to two main core proteins,
G17 and G34, which can exist in glycine extended (G17Gly; G34-Gly) or terminally amidated (G17-NH2, G34NH2) forms. Furthermore, a fraction of progastrin (⬃47%
in humans) is sulfated at Tyr66 in the course of its passage
through the Golgi apparatus, thereby giving rise to sulfated
and nonsulfated isoforms of gastrin (22). Sulfation has no
influence on the acid secretory response, as the affinity to
the gastrin receptor remains unchanged (399, 596). G17NH2 is the main circulating form that mediates the secretory effects of gastrin. Although the glycine-extended forms
have a low affinity towards the gastrin receptor (CCK2) and
thus play no role in gastric acid secretion (they are four to
five orders of magnitude less potent in inducing acid secretion), it is still important to acknowledge their existence
(178, 722). First, they serve as substrates for the synthesis of
amidated gastrin and are cosecreted with gastrin by the
G-cells (1040, 1051). Second, they potentiate the acid se-
cretory response to amidated gastrin, although they have no
intrinsic ability to induce acid secretion (178). Third, progastrin and glycine extended gastrins were shown to act as
a proliferative signal, especially in the colon (20, 482, 994,
1145). This is also of pathophysiological relevance as both
forms can promote cancer growth by presumably inhibiting
apoptosis and inducing angiogenesis (71, 87, 900). For example, it was shown that overexpression of progastrin in
mice is a predisposing factor for the development of colorectal or bronchoalveolar cancers (587, 1017).
B) REGULATION OF RELEASE. Gastrin is released by the G-cell in
response to a variety of stimuli of different origin. Direct
neuronal stimulation of the G-cell occurs via ACh and gastrin releasing peptide (GRP), which are released by postganglionic neurons of the enteric nervous system. The postganglionic fibers themselves receive input from the efferent
fraction of the vagus nerve (86, 485). On the other hand,
food-related signals, such as calcium, amino acids, and
amines, can also directly trigger gastrin secretion (FIGURE 2)
(257). The secretory stimuli culminate in an increase in
intracellular calcium concentrations, leading to vesicle
fusion and gastrin secretion. The main inhibitory signal
for gastrin secretion is somatostatin, which reaches the
G-cells in a paracrine fashion from neighboring D-cells
(632, 975).
With regard to the neuronal control of gastrin secretion, it is
generally thought that vagal stimulation increases the release of gastrin, although some conflicting evidence exists
(310, 694). Latest experiments that assessed local gastrin
concentrations utilizing microdialysis, however, clearly
show an increase in gastrin levels following acute electrical
vagal stimulation (310). The vagus nerve then synapses on
neurons of the ENS, which are thought to release either the
neurotransmitter ACh or GRP on a G-cell, leading to secretion of gastrin (295, 635, 694, 960, 978). It should be noted
that a recent investigation failed to observe increased gastrin levels, following exogenous GRP administration in humans (456). Yet, GRP itself serves as a clear acid secretagogue, although potentially not via gastrin (456). Whether
these conflicting observations are attributable to species differences (most earlier observations utilized rodent models)
remains to be elucidated. The ENS is also thought to mediate parietal and G-cell activation in response to mechanical
distension of the stomach (455, 962, 977). The neurohormonal response to gastric stretch is an integral part of the
gastric phase of acid secretion. Closer examination, however, reveals that the reports are very conflicting in that it is
not clear whether a pure mechanical distension stimulates
or inhibits gastrin release (455, 664, 803, 962, 977). A
biphasic model characterized by initial inhibition of gastrin
secretion under low volumes followed by stimulation under
high volumes has been suggested, but awaits further confirmation (977).
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Dietary components, such as amino acids and calcium, can
directly promote the secretion of gastrin and can thus sustain acid secretion in the gastric and intestinal phase as
digestion progresses (652, 1074). A rise in serum calcium
concentrations evokes a similar effect. The correlation between calcium and gastrin is discussed in a separate section
(see sect. VD2). It has been unclear for a long time as to how
these dietary components activate the G-cell. An involvement of the ENS has been proposed as the most likely explanation in the past. More recent observations, however,
strongly indicate that the calcium-sensing receptor (CaSR)
represents the molecular link between luminal dietary constituents and G-cell activation (325). The CaSR and its role
in the stomach are discussed separately and shall only be
summarized at this point (FIGURE 8) (see sect. IVD). First,
the same dietary components, i.e., amino acids, amines, and
calcium, which have all been shown to trigger gastrin release also function as activators of CaSR (652, 1074). Second, CaSR is expressed on the apical and basolateral side of
the G-cell, which allows it to act as nutrient sensor both in
the gastric lumen and the circulation (142, 182, 886).
Third, direct activation of the CaSR is known to stimulate
acid secretion (145, 291, 373). Finally, and most importantly, CaSR (⫺/⫺) animals lack the gastrin secretory response to intraluminal instillation of peptone, calcium, and
phenylalanine (325). In light of this evidence, it is highly
likely that CaSR is the long elusive luminal nutrient sensor
that regulates the secretion of gastrin from the G-cell.
The plasma levels of gastrin are closely tied to the intragastric pH. Low intraluminal pH is a potent inhibitor of gastrin
release, which serves as a negative-feedback mechanism to
impede an overproduction of acid. Conversely, a more alkali intragastric pH induces the secretion of gastrin, which
accounts for the commonly observed hypergastrinemia in
states of acid suppression, such as during proton pump
inhibitor (PPI) therapy. The pH dependency of serum gastrin levels is mainly relayed via somatostatin, as acid directly stimulates somatostatin release (see sect. IIB4). Somatostatin, released by neighboring antral D-cells, in turn acts
as the main inhibitor of gastrin secretion (FIGURE 2). The
physical proximity to G-cells allows for a fine paracrine
regulation of gastrin release. Although it is generally accepted that intragastric pH mostly modulates local somatostatin levels, the G-cell may also directly sense intragastric
pH via CaSR. CaSR is acid sensitive, and it has been shown
that isolated rat G-cells secrete less gastrin when the extracellular pH is dropped from 7.4 to 5.5 (569). However,
more investigations are needed to substantiate this evidence. Furthermore, gastrin release is also inhibited by neuronal regulation by the ENS. The neurotransmitter galanin
has been demonstrated to exert a direct inhibitory effect on
isolated G-cells (695, 961).
C) CELLULAR EFFECTS. Following secretion, gastrin enters the
bloodstream and acts on its target cells in an endocrine
196
fashion. Its half-life is determined by its rate of elimination
from the plasma which mainly occurs by metabolism in the
kidney, gut, and brain (419, 420). The importance of renal
elimination is corroborated by the observation that patients
with renal failure present with higher plasma gastrin levels
(818, 1075).
The two primary target cells of gastrin are the histaminesecreting ECL cell and the parietal cell. Gastrin exerts its
functions via binding to the cholecystokinin receptor type 2
(CCK2), a seven transmembrane domain G protein-coupled
receptor, which is expressed on mature parietal and ECL
cells, but also on gastric stem cells (560, 596, 608, 769, 772,
904). On the ECL cell, gastrin binding causes the release of
histamine, which in turn stimulates the parietal cell in a
paracrine fashion (FIGURE 2) (see sect. IIB3) (412). This
activation cascade is commonly referred to as the gastrinhistamine axis. Evidence for a direct, i.e., nonhistaminerelayed, activation of H⫹-K⫹-ATPase in the parietal cell by
gastrin exists, but is far less substantiated (459, 1024,
1025). Gastrin may sensitize the parietal cell to subsequent
secretagogue stimulation, rather than acting as a bona fide
secretagogue itself. Canonically it is widely accepted that
gastrin exerts its physiological effects mostly via activation
of ECL cells (24, 1131). Knock-out of gastrin leads to a
severe impairment of basal and stimulated acid secretion
(179, 347). Apart from stimulating acid secretion, gastrin
serves as a pivotal proliferative signal for the gastric mucosa
in general (60, 410, 534, 631, 816). It is commonly observed that elevated plasma gastrin levels lead to substantial
mucosal proliferation (60, 410, 534, 631, 816). This phenomenon has been extensively described in various knockout animals suffering from hypochlorhydria and concomitant hypergastrinemia, but also in patients with ZES (39,
515, 584, 1066). The source of mucosal cell proliferation is
progenitor cells located in the isthmus region of the gastric
gland (545). Expression of the CCK2 has been confirmed on
several gastric progenitor cells (560, 769). Furthermore,
gastrin has been shown to stimulate cell migration from the
progenitor region along the gastric gland axis (578). Mucosal hyperplasia thus ensues most likely via a direct activation of precursor cells by gastrin. Although hypergastrinemia causes a generalized mucosal hyperplasia, ECL cells
seem to be particularly regulated by gastrin, as their relative
fraction compared with other mucosal cells increases under
prolonged gastrin exposure (60, 410, 631). Conversely, the
absence of the CCK2 almost entirely eliminates mature
ECL-cells from the gastric mucosa (177, 622). [Somewhat
surprisingly this does not occur when gastrin itself is
knocked out (179, 347).] It should be noted that the M3
receptor seems to be necessary as a cofactor mediating the
trophic effects of gastrin, as its absence is associated with a
normal mucosal phenotype despite elevated serum gastrin
levels (see above) (9). The mechanism underlying this interdependency between gastrin and the M3 receptor is as of
now elusive. The cholinergic and gastrin systems also seem
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to be intertwined with regard to acid secretion. In the absence of the CCK2 receptor, the parietal cell’s acid secretory
response to the secretagogue carbachol (ACh analog) is
abolished, while the response to histamine remains intact
(543). Again, one can only speculate about the molecular
basis of this interaction.
In conclusion, gastrin is the most important activator of
acid secretion in the stomach. The role of gastrin, and especially its glycine extended forms, has evolved beyond being
a mere acid secretagogue to being an important global regulator of cell growth and differentiation. Furthermore, the
regulation of gastrin by the levels of plasma calcium provokes the question as to whether gastrin itself in turn has an
impact on global calcium homeostasis. A subsequent section makes an attempt at addressing this question (see sect.
VD2).
3. Histamine/ECL cell
Histamine has been discovered as early as 1910 by Dale,
Barger and Laidlow in extracts of ergot fungi (63, 237). In
1920, Popielski for the first time described its effect on the
secretion of gastric acid (866). He observed that subcutaneous administration of histamine resulted in increased acid
secretion (866). Furthermore, he concluded that this effect
was independent of the vagus nerve, as secretion still took
place after vagotomy and administration of atropine. This
led Popielski to postulate that histamine exerts its effects
directly on the level of the gastric gland (866). The hypothesis that histamine acts in a paracrine fashion on parietal
cells and that its release is regulated by the levels of gastrin
has been put forward for the first time by Emmelin and
Kahlson in 1944 (306). At this point, the cellular source of
histamine was still obscure. It was only in the late 1960s
that histamine had been histochemically localized to the
ECL cells of the gastric gland (413, 1084).
A) SYNTHESIS AND REGULATION OF RELEASE.
Histamine is the
effector of the gastrin-histamine axis and directly stimulates
the parietal cell to secrete hydrochloric acid (FIGURE 2).
Histamine is derived from the amino acid histidine, which is
enzymatically converted to histamine by L-histidine decarboxylase (HDC) (957). The effects of genetic HDC deletion
are predictably severe: animals lacking HDC have a low
basal acid output that does not respond to exogenous administration of gastrin (1066).
Histamine is stored in secretory granules of the ECL cell and
is released into the surrounding milieu in response to stimulation by gastrin and neuronal signals. Stimulation by gastrin occurs via activation of its GPCR CCK2 (772, 904).
Gastrin affects the ECL cell in multiple ways. First, gastrin
exposure increases the levels of HDC expression by enhancing its transcription and inhibiting its degradation, to allow
for increased synthesis of histamine (268, 331). The molecular mechanism underlying increased HDC transcription is
fairly well understood. Following CCK2 activation, increased transcription of HDC is mediated via a PKC- and
ERK-dependent pathway (470, 472). The HDC gene promoter is then activated by at least three distinct nuclear
factors which bind to gastrin response elements, resulting in
gene transcription (889, 890). Apart from augmenting gene
transcription, gastrin regulates the degradation of HDC,
which further increases intracellular enzyme levels (331,
1214). Second, gastrin enhances the transcription of the vesicular monoamine transporter type 2 (VMAT2; SLC18A2),
which is responsible for accumulating histamine in the secretory vesicles (376). Similarly to HDC, this effect depends
on PKC and ERK activation and binding of a nuclear factor
to a gastrin response element in the VMAT2 promoter region (164, 1154). It should be mentioned that gastrin regulates the transcription of a plethora of other genes which
serve a diverse array of roles, ranging from growth to metabolism (346). Amongst many others these include chromogranin A, which is essential for granule packaging and is
a precursor of pancreastatin (see sect.VD3) (231). Third,
gastrin induces the fusion of secretory granules and the
release of histamine into the gland environment. Secretion
follows a biphasic elevation of intracellular calcium concentrations after activation of CCK2 (1201). The biphasic increase has been proposed to result from initial IP3-mediated
release from intracellular stores, which is followed by subsequent influx of calcium via L-type calcium channels from
the extracellular space (1201). The importance of intracellular store mobilization has been contested by a different
group, which proposed that solely influx trough L-type, and
to a lesser extent N-type, calcium channels triggers the secretory response (673). Lastly, gastrin has a trophic effect
on the ECL cell (see sect. IIB2).
Apart from gastrin, ECL cells are stimulated by pituitary
adenylate cyclase activating polypeptide (PACAP), which is
a neuropeptide expressed in the ENS of the gastric mucosa
(737, 1054). PACAP has homology to vasoactive intestinal
polypeptide (VIP) and binds to a distinct receptor (PAC-1)
on the ECL cell (1207, 1208). Binding of PACAP to PAC-1
induces release of histamine (672, 798, 944, 1207). Similar
results have been obtained with VIP, which is attributable
to partial agonism at PAC-1 (798, 941). Historically, investigations yielded controversial results with regard to the
effects of exogenously administrated PACAP on acid secretion. Both an inhibition and stimulation of acid secretion
following PACAP injection are reported (760, 862, 944,
1207). This discrepancy is most likely attributable to the
fact that PACAP can also act as an agonist of the VIP receptor (VPAC) on the somatostatin-secreting D-cell, leading to a concomitant suppression of acid secretion by somatostatin release (1207). Indeed, if an anti-somatostatin antibody is injected into rats simultaneously with PACAP,
acid secretion is elevated threefold from baseline (compared
with 1.5-fold in the absence of an anti somatostatin antibody) (1207). Evidence points to the fact that the PACAP-
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stimulated release of somatostatin is of particular importance in the mouse, as most studies showing a suppression
of acid secretion after PACAP administration were conducted in murine models.
PACAP has very similar effects on the ECL cell as gastrin.
Similarly to gastrin, PACAP causes histamine release by
increasing intracellular calcium concentrations via calcium
influx through L-type, but also ligand-gated calcium channels (673). In further analogy to gastrin, PACAP upregulates the expression of HDC and exerts trophic effects on
the ECL cell (590, 729, 810). Contradictory results with
regard to the effects of acetylcholine on histamine release
exist. It has been reported that acetylcholine can either stimulate or has no effect on the secretion of histamine in in vitro
experiments on isolated ECL cells (481, 672, 674, 941,
946). In vivo application of muscarinic agonists, followed
by measurement of histamine concentrations using microdialysis, also yielded no evidence for cholinergic stimulation
(798). Conversely, it is well accepted that adrenergic stimulation leads to an increase in histamine release; however,
the physiological relevance of adrenergic activation of ECL
cells is not entirely clear (636, 672, 674, 798, 871, 941).
The ECL cell is inhibited by a variety of substances, the
most prominent of which is somatostatin (204, 590, 798,
941). Somatostatin is produced in D-cells of the oxyntic
mucosa and reaches the ECL cell in a paracrine fashion
where it binds to the somatostatin receptor (SST2 and potentially SST5) (FIGURE 2) (570, 873). Receptor binding
leads to inhibition of histamine exocytosis via blockade of
mostly L-type calcium channels (105). This impedes the
elevation of intracellular calcium concentrations caused by
ECL activators, such as gastrin (see above) (873). In addition, somatostatin also inhibits the proliferation of ECL
cells (570). Somatostatin can thus be seen as the global
hormonal antagonist to gastrin with regard to ECL cell
function and proliferation. The neuronal inhibition of ECL
cells is mainly carried out by the neuropeptide galanin (105,
672, 798, 1209). Galanin is localized to neurons of the ENS
and demonstrated an inhibitory effect on histamine secretion in in vitro and in vivo models (105, 302, 672, 731, 798,
1209). Similarly to somatostatin, the molecular mechanism
underlying its inhibitory effect is an interference with calcium signaling via closure of L-type calcium channels (105).
Lastly, prostaglandin E and nitric oxide also act as inhibitors of histamine release (105, 554, 798, 1002). Although
neuropeptide YY (PYY) and calcitonin gene-related peptide
(CGRP) have also been implicated in playing a role in ECL
cell regulation, a detailed discussion is omitted in light of
contradictory results which range from stimulation to inhibition of secretion (672, 674, 798, 1210).
B) CELLULAR EFFECTS. As mentioned earlier, stimulation of the
ECL cell is translated into an elevation in intracellular calcium concentrations, leading to exocytosis of preformed
198
histamine-containing secretory vesicles. The molecular
mechanism of vesicle fusion with the apical membrane relies on the formation of the core SNARE complex, consisting of syntaxin, synaptobrevin, and SNAP-25. Synaptotagmin presumably acts as a calcium sensor relaying the
intracellular calcium signal to the vesicle fusion protein
apparatus. The expression of all SNARE complex proteins has been confirmed in the ECL cell (471, 477,
1215). In accordance with these findings, introduction of
the neurotoxins tetanus toxin light chain and botulinum
toxin, which cleave constituents of the SNARE complex
apparatus and thereby render it nonfunctional, result in
inhibition of histamine secretion (477).
Very small amounts of histamine are sufficient to induce
acid secretion. Histamine acts via the H2 receptor on the
parietal cell, which has been discovered by Sir J. W. Black in
1972 (106). For this seminal discovery, he was later
awarded the Nobel Prize in Physiology and Medicine. The
H2 receptor belongs to the family of seven-transmembrane
domain GPCRs. Its activation predominantly leads to increases in the intracellular levels of cAMP, but also of calcium, which serve as stimulatory signals for H⫹-K⫹-ATPase
trafficking (67, 189, 738, 840, 1026, 1143). In analogy,
pharmacological agents that elevate cAMP, such as IBMX
or forskolin, induce acid secretion (1026, 1191). The increase in cAMP is due to activation of adenylate cyclase via
Gs. The role of calcium in the process of histamine secretion
remains a controversial matter. First, the mechanism leading to histamine-induced increases in intracellular calcium
has been subject of discussion. Evidence exists that histamine can, apart from adenylate cyclase, also activate PLC,
leading to calcium release from intracellular stores (607,
1142, 1143). Conversely, it has been suggested that the
observed increases in intracellular calcium are a byproduct
of cAMP-mediated PKA activation, which in turn can regulate the opening of calcium channels (144, 189, 840). Second, it is questionable to what degree the calcium signal is
an integral and necessary part of the acid secretory response
to histamine (738, 840). Chelation of the transitory calcium
increases with BAPTA abolishes only the secretory response
of isolated gastric glands to histamine by ⬃40%, while it
completely eliminates the response to cholinergic stimulation (738). Also, live fluorescence imaging in isolated glands
showed no spatiotemporal correlation between the histamine-induced increases in calcium and the onset of acid
secretion, thereby questioning an involvement of calcium in
the secretory response (840).
H2 receptor knockout animals effectively illustrate the significance of the histamine-gastrin axis in gastric physiology.
Lack of the H2 receptor leads to a complete failure of gastrin or histamine to induce acid secretion (584). The secretory response to carbachol, however, remains intact (584).
Hypergastrinemia develops as a feedback mechanism with
the aim of reestablishing acid secretion, leading to mucosal
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hypertrophy (584). In light of the central role of the H2
receptor in parietal cell physiology, it has been successfully
used as a pharmacological target with the aim of suppressing gastric acid output (see sect. IIC2).
4. Somatostatin/D-cell
Somatostatin was isolated for the first time in 1973 from
ovine hypothalamus and characterized as an inhibitor of
growth hormone release from the pituitary gland (124). A
few years later somatostatin was identified in endocrine
cells of the stomach, which we now know as D-cells (638).
A) SYNTHESIS AND REGULATION OF RELEASE. Somatostatin is a
peptide hormone that exists in two primary forms that differ in their respective peptide length. The most abundant
form in the gastric mucosa is somatostatin-14 (consisting of
14 amino acids), whereas somatostatin-28 only constitutes
a minute fraction of the total gastric somatostatin content
(198, 1125). The two forms of somatostatin are cleavage
products of a larger 116-amino acid pre-prohormone (preprosomatostatin), which in turn is processed to the 92amino acid-long prosomatostatin (1000). It should be mentioned that other cleavage products, such as antrin or somatostatin-28(1–12) exist and are secreted together with
somatostatin (77, 885). Their physiological significance is,
however, less well understood.
Somatostatin is the global antagonist of the acid secretagogues. It is produced by intestinal and gastric D-cells, the
latter of which exist in two populations in the stomach (61):
an antral population locally inhibits the release of gastrin
from G-cells, whereas a population localized to the acidproducing oxyntic mucosa directly regulates the parietal
cell and inhibits histamine release from the ECL cell (FIGURE 2) (19). The morphology of the D-cell is characteristic
in that it possesses long cytoplasmic processes, which allow
it to communicate with and regulate neighboring cells in a
paracrine fashion (620, 632). It is worthwhile to distinguish
the two populations of gastric D-cells, as each population
possesses unique physiological properties (1202).
The antral D-cell is mostly regulated by the local concentrations of gastrin, cholecystokinin, and intraluminal pH.
Gastrin induces somatostatin secretion from D-cells, which
causes reciprocal inhibition of gastrin release from neighboring G-cells, thereby creating a local negative-feedback
loop (976, 1011, 1202). The molecular mechanism underlying this loop is, however, less clear. CCK2 receptor is, if at
all, only expressed at very low levels in the antral mucosa
(749, 905, 967). It has been proposed that gastrin stimulates somatostatin release in the antrum in a receptor-independent mechanism (1202). This may be accomplished via
direct cell-cell contacts between the G- and the D-cell,
which have been demonstrated with electron microscopy
(620). Conversely, evidence for cholecystokinin and its
stimulatory role for somatostatin release via CCK1 is more
substantiated (749, 905, 967, 1202). Cholecystokinin is
structurally closely related to gastrin (both share an identical 5-amino acid COOH terminus) and also exists in various peptide lengths (767). It is secreted by I-cells of the small
intestine following protein and fat-rich chyme entering the
duodenum, and thus represents a classical mediator of the
intestinal phase of acid secretion (594). As its name implies,
cholecystokinin has originally been described as a stimulator of gallbladder contraction; however, its inhibitory influence on gastric acid secretion is now well accepted and
extensively described (593, 1203). Cholecystokinin can
bind to both the CCK1 and CCK2 receptor with almost
equal affinity, whereas the actions of gastrin are almost
exclusively mediated by the CCK2 receptor. The dual affinity of cholecystokinin would imply a possible stimulatory
effect on acid secretion via activation of CCK2 on ECL cells;
however, in vivo the inhibitory effect mediated by activation of CCK1 and CCK2 on D-cells prevails (593, 966,
1202, 1203).
One of the most important stimulators of D-cell secretion is
the intragastric pH. A seminal observation demonstrating a
correlation between gastric acidity and the amount of secreted somatostatin was made in dogs in the 1970s. It has
been shown that the amount of somatostatin directly increases in antral venous blood following gastric HCl infusion, while somatostatin levels were unaffected in venous
blood from the oxyntic mucosa (982). Similar observations
were later made in isolated mouse stomach, however without topographic discrimination (975). Two main hypotheses as to how somatostatin is regulated by intragastric pH
exist. The first states that the D-cell can directly act as a pH
sensor, and the second postulates that the pH sensing is
mediated by neurons, which in turn act on D-cells. To accomplish putative direct pH sensing, several antral D-cells
are equipped with a distinct morphological feature. They
possess apical projections that are in contact with the glandular lumen, potentially allowing them to constantly monitor the intraluminal milieu (620). These D-cells have been
termed open type. Conversely, the D-cells of the oxyntic
mucosa are mostly of the closed type, meaning that they are
embedded in the mucosa without luminal contact. The molecular identity of the putative apical pH sensor remains
elusive. However, the presence of CaSR, which has pH
sensing properties, was recently confirmed in preliminary
studies on the D-cell and may represent a possible candidate
for this mechanism (770). Apart from directly acting on
D-cells, the effect of pH on somatostatin secretion may be
mediated via afferent spinal neurons. Over 80% of the spinal afferent neurons contain the neuropeptide CGRP (397,
758, 1047, 1116). Perfusion models of antral sleeves have
shown that the acid-induced rise in somatostatin is accompanied by a concomitant increase in the concentrations of
the neuropeptide CGRP (708). Furthermore, application of
a CGRP receptor blocker inhibited the release of somatostatin following acid exposure (708). As D-cells are known to
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express the CGRP receptor, an involvement of CGRP in
acid sensing is plausible (558). Again, this provokes the
question of how CGRP-containing neurons may sense acidity on a molecular basis. The acid-sensitive channels transient receptor potential vanilloid channel (TRPV1) and the
acid-sensing ion channel 3 (ASIC3) had been proposed as
molecular acid sensors; however, latest experiments have
shown that the increase in CGRP still occurs in the genetic
absence of the channels (47, 96, 163).
Neuropeptides of the gastric ENS that stimulate the secretion of somatostatin include PACAP and VIP, which both
bind to the VPAC receptor expressed on D-cells (199, 657,
1207). The presence of VIP and PACAP containing neurons, which integrate signals from the vagus nerve, has been
demonstrated in the gastric mucosa (302, 737). Furthermore, cholinergic signals can act on the antral D-cell via the
M3 receptor to promote secretion of somatostatin (140).
This is in sharp contrast to D-cells from the oxyntic mucosa
that are inhibited by cholinergic signals (197, 200, 1182).
As mentioned earlier, the D-cells in the oxyntic mucosa also
differ in their morphology. D-cells in the oxyntic mucosa
are of the closed type and have thus not been implicated to
participate in acid sensing. They exert their acid-suppressive effects by the paracrine regulation of ECL and parietal
cells. Further functional divergence between antrum and the
oxyntic mucosa has been demonstrated in the regulation of the
somatostatin mRNA. For example, suppression of acid secretion with omeprazole in fasted animals markedly decreased
somatostatin mRNA levels in the antrum, whereas the levels in
the oxyntic mucosa were affected to a much lesser extent,
which further corroborates the hypothesis that the antral cells
are involved in luminal chemosensation (945).
B) CELLULAR EFFECTS.
The effects of somatostatin on its target
cells are mediated by the SST2 receptor. Knockout of the receptor causes a 10-fold increase in basal acid output, which
exemplifies the pivotal role somatostatin plays as a global suppressant of acid secretion (715). Somatostatin acts on all main
cell types that are involved in the process of acid secretion, i.e.,
parietal cells, ECL cells, and G-cells (FIGURE 2). The inhibition
of the G- and ECL cells has been discussed in the respective
sections. In the parietal cell, somatostatin has a clear direct
inhibitory effect on secretagogue-induced acid secretion (827,
1177). This effect is partially attributable to activation of Gi,
leading to inhibition of adenylate cyclase and a subsequent
decrease in intracellular cAMP levels (827).
interest of conciseness, their physiological effects will only
be discussed briefly at this point.
A) SECRETIN. Secretin is a 27-amino acid peptide hormone
that is synthesized in duodenal S-cells and secreted into the
circulation in response to a low duodenal pH or passage of
digestive products, such as fat (195, 955, 1153). A subpopulation of secretin-producing cells is also present in the
gastric mucosa, where it may influence acid secretion in a
paracrine manner (191–193). Given its secretory stimulus,
it is regarded as a classic effector of the intestinal phase of
acid secretion. When it was first discovered in 1902 by
Bayliss and Starling (interestingly secretin was the first hormone ever to be discovered), it was noted that secretin induces pancreatic bicarbonate secretion, which leads to a
buffering of the gastric acid entering the duodenum (68). In
the stomach, secretin acts as an inhibitor of gastric motility
and acid secretion (141, 194, 269, 374, 532, 564, 656, 677,
1107). The exact mechanism as to how secretin attenuates
the secretion of acid is not exactly known, and several hypotheses have been put forward. For example, it has been
shown that secretin induces the secretion of somatostatin
from isolated D-cells (141). Increases in somatostatin levels
were also observed in isolated perfused stomach models (205,
374). Others have proposed that secretin activates vagal primary afferent neurons, which in turn leads to neuronal modulation of acid secretion (656, 659). In opposition to this theory, it has also been demonstrated that the inhibitory effects of
secretin are independent of vagotomy (677).
B) OXYNTOMODULIN. Oxyntomodulin is a peptide hormone
produced in the mammalian intestine. It is closely related to
glucagon and contains its entire amino acid sequence, extended by a COOH-terminal octapeptide (66). In isolated
parietal cells, oxyntomodulin acts as an activator of acid
secretion (959). The integrated response to oxyntomodulin
is, however, opposite. Systemic injection decreases gastric
acid secretion in rat, cat, and human test subjects (53, 157,
285, 524, 525, 965). The inhibitory effect is most likely
mediated via somatostatin release (53).
In conclusion, somatostatin acts as the global brake on acid
secretion. By acting on G-cells, ECL cells, and parietal cells,
it exerts its inhibitory action on every link in the regulatory
chain leading to the secretion of gastric acid.
It was recognized in the early 1950s that serotonin was present in the antral mucosa of dog stomachs
(323). Serotonin is stored in granules of enterochromaffin
cells of the antrum (1099). It is released into the circulation
and the gastric lumen in response to vagal stimulation (107,
649). Intraluminal acidification serves as another stimulus
for serotonin release (1196). Serotonin has an inhibitory
effect on the secretion of gastric acid (107, 153, 521, 650,
720, 903). It is still poorly understood where serotonin
interferes with acid secretion.
5. Other substances
D) NEUROTENSIN.
A variety of other substances have been shown to have
either direct or indirect effects on acid secretion. In the
200
C) SEROTONIN.
Neurotensin is a 13-amino acid neuropeptide that was originally isolated from calf hypothalamus
(161). In the periphery, it is also produced and secreted
postprandially by specialized endocrine cells (N-cells) of the
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small intestine (863). Various investigations have demonstrated that neurotensin suppresses the secretion of gastric
acid and delays gastric emptying (25, 108, 486, 985). This
has been shown by direct systemic injection, but also by
immunoneutralization of endogenous neurotensin in a reverse approach (25, 108, 486, 985). In disagreement with
these findings, other investigators could only inhibit acid
secretion at unphysiologically high serum concentrations of
⬃750 pmol (747). Of note, physiological postprandial neurotensin levels were measured to be ⬃15 pmol by the same
investigators, questioning the role of neurotensin as a physiological endocrine inhibitor of acid secretion (747). Neurotensin is also located to nerve fibers of the enteric nervous
system in the stomach, indicating that it may act as a local
neuronal rather than an endocrine regulator. It has been
proposed that neurotensin may induce the secretion of somatostatin and thereby exert its inhibitory action on acid
secretion (53, 414). Most recently, however, the low-affinity neurotenstin type 2 receptor (NTS2) has been identified
on the parietal cell, suggesting a direct influence.
E) GHRELIN. Ghrelin is a recently discovered 28-amino acid
peptide hormone that is synthesized in P/D1 cells of the
fundus (242). Since its discovery, multiple functions have
been ascribed to it, ranging from being a regulator of appetite to being a modulator of bone remodeling. Its effects on
bone are described in a separate section of this review (see
sect. VD1). Apart from these functions, ghrelin also has
been implicated to affect gastric acid secretion, although it
remains a matter of discussion in which direction, as peripherally administered ghrelin has been reported to stimulate,
inhibit, or not affect acid secretion (278, 355, 654, 719).
The reason for these dichotomic results is largely unclear.
The fact that ghrelin circulates in acylated and desacylated
forms adds further complexity to the subject (491). Indeed,
acylated ghrelin has been shown to stimulate acid secretion
following peripheral injection, whereas desacylated ghrelin
remained without effect (938). Further investigations are
needed to clarify the controversy surrounding ghrelin and
its influence on gastric acid secretion.
F) NITRIC OXIDE.
Nitric oxide (NO) is an important signaling
molecule that plays a role in multiple physiological processes, such as vasodilation or the immune response. Indeed, NO has been shown to mediate the hyperemic response of the gastric mucosa that occurs during acid secretion (553). However, NO also directly influences the
production of acid. The effect of NO on acid secretion is
most likely inhibitory (81, 82, 555, 1002; opposed by Ref.
426). NO is produced by various forms of NO synthases,
one of which has been localized at high concentrations in
cells in the vicinity of parietal cells, allowing for a putative
paracrine regulation (80). NO has been proposed to exert
its inhibitory action by either directly inhibiting the parietal
cell or by suppressing the release of histamine from ECL
cells (81, 82, 555, 1002). Intracellular increases in cGMP
concentrations have been observed in both cell types after
NO exposure, suggesting that guanylate cyclase is an intracellular target for NO (82, 1002).
G) INTERLEUKINS. Interleukins (IL) are cytokines that mainly
coordinate immune responses. In particular, IL-1␤ has been
shown to impact gastric acid secretion. IL-1␤ is a general
proinflammatory cytokine that plays an important role in
the stomach in the context of Helicobacter pylori infection.
H. pylori infection triggers an elevation of IL-1␤ levels as
part of the host’s immune response (65). Peripheral injection of IL-1␤ can profoundly suppress gastric acid secretion
(912, 947, 1059, 1101, 1132). Multiple explanations for
this observation have been put forward. It has been suggested the IL-1␤ acts in the CNS, as intrathecal injection
also has an acid-suppressive effect (948, 949). Others have
suggested that IL-1␤ promotes formation of prostaglandins
or NO, which in turn inhibit acid secretion (312, 947,
1101). Yet, a direct effect on parietal cells and ECL cells is
the most likely explanation, as both cell types express the
IL-1 receptor and have been shown to be inhibited in their
function in isolated cell models (69, 70, 872, 958).
C. The Pharmacological Suppression
of Acid Secretion
Decreasing gastric acidity is indicated in many pathological
contexts, including gastric reflux disease or peptic ulcer disease. This target can be achieved by two main pharmacological approaches: 1) the inhibition of gastric acid secretion or 2) the intraluminal neutralization of already secreted
gastric acid (antacids). Gastric acid secretion can be attenuated by either directly blocking its final molecular effector,
namely, H⫹-K⫹-ATPase (PPIs and acid pump antagonists),
or by interfering with the neurohormonal signaling pathway leading to its secretion (H2 antagonists). The following
section attempts to discuss the four most common substance classes employed to increase intragastric pH.
1. Direct pharmacological inhibition of H⫹-K⫹-ATPase
The inhibition of H⫹-K⫹-ATPase-mediated proton transport represents the main contemporary pharmacological
strategy for reducing gastric acidity. An increase of gastric
pH is the main factor ameliorating acid-related disorders
and has been show to directly correlate with healing rates of,
for example, GERD (74). Two main substance groups exert
their acid-reducing effect via inhibiting H⫹-K⫹-ATPase function: PPIs and acid pump antagonists (APAs). Both substances
achieve this aim by distinct mechanisms.
Omeprazole was the first clinically available PPI (324). The
first patent on omeprazole was filed in 1979 by the Swedish
company Astra AB (today AstraZeneca). The introduction
as a prescription PPI followed in 1989. Today, the omeprazole enantiomer esomeprazole (S-omeprazole) generates the
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SASCHA KOPIC AND JOHN P. GEIBEL
second highest revenue of all pharmaceuticals in the United
States and is only surpassed by the statin atorvastatin (512).
Furthermore, in the United States, PPIs are available as
over-the-counter formulations, making them accessible for
the broad public. This is partially made possible by the high
safety profile of PPIs with a low incidence of unspecific
adverse effects. Recently, however, concerns about the
long-term effects of chronic acid suppression have emerged
with regard to its impact on bone health (see sect. VA).
PPIs are delivered as pro-drugs through the bloodstream to
the parietal cell. They are weak bases (pKa ⬃4), which can
easily pass the cell membrane and accumulate in acidic compartments, such as the secretory canaliculus of the parietal
cell. The pro-drug is then converted to the pharmacologically active cyclic sulphenamide by the acidic pH in the
secretory canaliculus (670, 1007, 1135). Their specific accumulation in acidic milieus and their pH-catalyzed conversion to active substances confers specificity and thus a high
safety profile to PPIs. Once activated, PPIs bind covalently
via disulfide bonds to H⫹-K⫹-ATPase, thereby inhibiting its
capacity to pump protons (89, 90, 1005, 1006, 1008). The
pattern of the cysteine residues, which are involved in PPI
binding, differ among the respective members of the PPI
family: cysteine-813 reacts with all PPIs. In addition,
omeprazole reacts with cysteine-892, lansoprazole with
cysteine-321, and pantoprazole and tenatoprazole, respectively, with cysteine-822 (89, 90, 1005, 1006, 1008). Since
the binding is covalent and irreversible, the inhibitory effect
of PPIs lasts long beyond their plasma half-life, which usually ranges between 0.5 and 2 h depending on the specific
PPI (581, 1036). PPIs are generally metabolized by the hepatic cytochrome P-450 system, in particular CYP2C19
and CYP3A4. This is of particular clinical importance, as
CYP2C19 polymorphisms are known to exist. These polymorphisms can impact the pharmacokinetics of PPIs by
affecting the metabolic rate of CYP2C19, which may have
consequences for the optimal therapeutic regimen (582).
Esomeprazole and rabeprazole seem to be less dependent on
CYP2C19 metabolism (516, 983). Apart from the pattern
of cysteine reactivity, half-life and metabolism, PPIs also
vary in oral bioavailability (581).
Suppression of acid secretion can never be complete, as
H⫹-K⫹-ATPase is subjected to a constant turnover (half-life
⬃50 h) and needs to be stimulated for the conversion of the
PPI to take place (936). Nevertheless, PPIs are highly effective in reducing gastric acidity. Depending on the PPI and
the regimen, overall intragastric pH can be elevated by several pH units, up to a pH of 6 (compared with 1–2 at
baseline) (137, 425, 1036). For an excellent summary of PPI
efficacy, please refer to Reference 1036.
APAs represent the second class of H⫹-K⫹-ATPase inhibitors. Unlike PPIs, they do not undergo irreversible binding,
but rather act as potassium competitive antagonists. The
202
duration of inhibition is thus directly dependent on the
plasma concentration of the inhibitor. As predicted by homology modeling, mutational analysis, but also recent
structural data, APAs bind in the luminal cavity of H⫹-K⫹ATPase in the vicinity of the potassium entry site where they
exert their inhibitory action (2, 42, 761, 1104, 1106). Although the inhibition of acid secretion has been shown to be
very effective, these substances are generally not in clinical use
(577). For example, clinical trials of the APA AZD08650 have
shown no additional therapeutic effect compared with the PPI
gold-standard, which resulted in abandonment of the drug in a
clinical setting (262, 539).
2. H2 antagonists
The development of H2 blockers is inseparably intertwined
with Sir Black’s discovery of the H2 receptor on the gastric
parietal cell at the Smith Kline and French Laboratories
(now GlaxoSmithKline) (106). In his original publication,
Sir Black also describes burimamide as a competitive H2
antagonist that can effectively inhibit pentagastrin-stimulated gastric acid output in human volunteers (106). Further
development of the antagonist led to the synthesis of cimetidine, which was first commercially introduced in 1976 in
the United Kingdom, followed by the United States in 1977.
Other commonly used members of H2-antagonist family
now include ranitidine, famotidine, and nizatidine.
H2 antagonists prevent histamine-mediated stimulation of
the parietal cell by competitively interfering with its receptor. Although this effectively terminates the gastrin-histamine axis, the parietal cell is still susceptible to cholinergic
stimulation via the M3 receptor. This partial inhibition
mainly accounts for the lower clinical efficacy of H2 antagonists compared with PPIs, which directly target H⫹-K⫹ATPase as the final target of all parietal cell stimuli (384).
For example, a meta-analysis concluded that patients
treated for bleeding peptic ulcers are about twice as likely to
suffer from persistent or recurrent bleeding if treated with
H2 antagonists compared with PPIs (384). Another metaanalysis also demonstrated a higher efficacy of PPIs in treating esophagitis (83% healing rate with PPIs compared with
52% with H2 antagonists)(567). Today, H2 antagonists are
largely superseded by PPIs due to their higher clinical efficacy. Furthermore, with the exception of famotidine, H2
antagonists are extensively metabolized in the liver by the
CYP-450 system, leading to substantial drug-drug interaction profile (for review, see Ref. 506).
3. Antacids
Antacids directly neutralize gastric acid allowing immediate
short-term control of heartburn. They exist in various salt
formulations, the most common of which are carbonate
salts, such as CaCO3, MgCO3, or NaHCO3. The use of
calcium carbonate as a dietary calcium supplement is dis-
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cussed in a separate section (see sect. VC). Although each
formulation possesses its own spectrum of side effects, a
notable condition in the context of this review is the milkalkali syndrome, which is the result of a concomitant overingestion of calcium and alkali, such as CaCO3. Although
the syndrome became less prevalent with the introduction
of modern ulcer therapies, it still poses a significant risk for
patients that may ingest CaCO3 as a calcium supplement
for the prevention of osteoporosis or as an antacid on a
regular basis. Milk-alkali syndrome presents with the triad
of metabolic alkalosis (carbonate), hpercalcemia (calcium),
and renal insufficiency. The above average ingestion of calcium leads to increased plasma calcium levels due to excess
absorption and impaired renal secretion. PTH levels are low
due to negative feedback (6). Hypercalcemia further causes
renal vasoconstriction, which decreases the glomerular filtration rate, and renal fluid loss because of activation of the
CaSR, which in turn has loop-diuretic-like effects (see sect.
IVD3). The activation of the CaSR is further potentiated by
the metabolic alkalosis, which increases its sensitivity to
calcium. All abnormalities are usually reversible after withdrawal of the offending agent and adequate treatment.
III. INTESTINAL CALCIUM ABSORPTION
The intestine is responsible for the absorption of dietary
calcium into our systemic circulation. Although the kidney
also plays a pivotal role in calcium homeostasis by retaining
and balancing systemic calcium via regulating its excretion
into the urine, renal calcium handling will not be the subject
of this review.
Current recommendations suggest that an average 40-yrold adult should ingest ⬃1,000 mg of calcium on a daily
basis (218). In the United States, this requirement is mostly
met (57). Up to 72% of the dietary calcium intake is attributable to dairy products (218). Typically, the intestine absorbs between 25 and 35% of the ingested calcium (1193).
This occurs via two distinct pathways: 1) a paracellular
pathway and 2) a transcellular pathway.
Since a concentration gradient is not a prerequisite for this
process, transcellular transport allows us to absorb calcium
even when the calcium concentration in the chyme is fairly
low. The relative importance of each respective absorption
pathway thus alternates with the amount of ingested calcium (46, 824, 1224). The rate of paracellular calcium uptake is canonically thought to remain constant, while transcellular transport can be upregulated under conditions of
dietary calcium restriction (46, 824, 1224). This regulation
occurs via the active metabolite of vitamin D [1,25(OH)2vitamin D], which serves as a stimulator for transcellular
calcium uptake to prevent systemic calcium depletion.
A. Transcellular Calcium Absorption
1. Calcium entry
Evidence for active transport of calcium across the intestinal
epithelium was established very early. With the use of a calcium radioisotope, various groups demonstrated 1,25(OH)2vitamin D-dependent calcium transport against an imposed
concentration gradient in the small intestine of the rat (952,
954, 1150). It has also been observed that active transport
can be induced by a low-calcium diet, which we now know
to stimulate the production of 1,25(OH)2-vitamin D (1133,
1134). The degree of transport was highest in the duodenum and decreased in the more distal segments (952). The
duodenum is still considered the primary site where the bulk
of transcellular transport occurs. To conduct active transcellular calcium absorption, the enterocyte has to be equipped
with an apical calcium entry pathway, a mechanism for cytosolic calcium shuttling, and a basolateral calcium exit pathway
(FIGURE 3). As the cytosolic concentration of free calcium
is kept at a constant low level with typical concentrations
of ⬃100 nM, apical calcium influx follows its electrochemical gradient into the cell. In contrast, the extrusion
of calcium on the basolateral membrane against an uphill
gradient either directly requires ATP or energy stored in
the sodium gradient.
A) THE SEARCH FOR THE APICAL CALCIUM ENTRY PATHWAY.
Calcium absorption via the paracellular route is tied to a
downhill concentration gradient between the luminal and
the extracellular compartment and occurs throughout the
entire intestine (although solvent drag induced paracellular
flux may also play a role at low luminal calcium concentrations; Refs. 266, 1071, 1098). Conversely, transcellular absorption can also take place against an uphill gradient, but
requires molecular machinery in the form of distinct calcium transport proteins which are expressed on the apical
and basolateral membranes of the enterocyte. This process
directly requires energy in the form of hydrolyzable ATP
and is alternatively termed “active” transport (versus “passive” paracellular transport). The proximal small intestine,
i.e., the duodenum and the jejunum, is the main site for
transcellular calcium absorption (825).
The molecular identity of the apical calcium entry pathway was unclear for a long time. Early experiments in isolated duodenal
brush-border vesicles revealed that calcium uptake was passive, saturable, sensitive to ruthenium red, 1,25(OH)2-vitamin
D dependent, and functionally optimal at a pH of 7.5 (740).
This black box characterization suggested that a “specific
carrier” was responsible for calcium absorption and in retrospect already provided us with accurate key characteristics of the transient receptor potential vanilloid channel
type 6 (TRPV6), which was later established as the primary
apical calcium uptake channel (740). In subsequent attempts to further unravel the nature of the calcium uptake
mechanism, various voltage-gated L-type calcium channel
blockers were used (474, 717, 838). Although isolated duodenal cells accumulated less calcium following application
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SASCHA KOPIC AND JOHN P. GEIBEL
Enterocyte
D
D
VDR
D
RXR VDR
Transcription
Ca2+
PMCA
Transcription
Ca2+
Ca2+
Na+
Ca2+
Calbindin-D 9k
NCX
TRPV6
Ca2+
D
FIGURE 3. Transcellular and paracellular calcium absorption in the intestine. The transcellular intestinal
absorption of calcium relies on apical calcium entry through TRPV6, intracellular calcium transport by calbindin-D9k, and basolateral calcium extrusion via either NCX or PMCA. 1,25(OH)2-vitamin D regulates most of
these ion transport proteins on a transcriptional level. 1,25(OH)2-vitamin D passes the plasma membrane of
the enterocyte and binds to its receptor (VDR), which then heterodimerizes with RXR to initiate transcription.
Evidence also suggests that 1,25(OH)2-vitamin D regulates the permeability of tight junctions, which gate the
paracellular absorption of calcium. D, 1,25(OH)2-vitamin D.
of the inhibitors and 1,25(OH)2-vitamin D stimulation
(717), in vivo calcium entry proved to be fairly insensitive to
their effects (with the exception of verapamil, which demonstrated some degree of inhibition if applied at very high
concentration in the millimolar range) (474, 838). The conflicting reports may be attributable to the different experimental models that were used, as isolated single cells do not
allow discrimination between apical and basolateral transport mechanisms. Furthermore, it has been argued that Ltype calcium channels may play a role in the stimulatory
pathway of vitamin D, rather than in calcium uptake per se
(717). In conclusion, an involvement of L-type calcium
channels seemed rather inconclusive and the identity of the
calcium entry channel remained elusive.
B) TRPV6. The cloning of the calcium transport protein subtype 1 (CaT1) in 1999 finally marked a turning point in the
search for the elusive intestinal calcium entry channels
(838). The work was pioneered by Hoenderop and colleagues who had identified the main calcium entry protein
in the kidney (epithelial calcium channel type 1, ECaC) via
an expression cloning strategy a few months earlier (474).
204
CaT1 was identified by a similar approach. A rat duodenal
cDNA library was functionally screened using a calcium
uptake assay in a Xenopus oocyte expression system (838).
This screening process yielded the 727-amino acid protein
CaT1, which showed a 75% sequence homology to rabbit
ECaC. Homology analysis also demonstrated a relationship
to the vanilloid reptor type 1 (VR1), a nonspecific cation
channel that is activated by capsaicin, the pungent ingredient in chili peppers, and mostly mediates pain signaling
through afferent sensory neurons (838). The nomenclature
changed over time as new channel proteins were identified,
and today we consider CaT1, ECaC, and VR1 to be members of the same transient potential receptor vanilloid
(TRPV) ion channel family. Literature now refers to ECaC
as TRPV5, to CaT1 as TRPV6, and to VR1 as TRPV1.
The structure of TRPV6 was predicted to have six transmembrane domains and four ankyrin repeat domains,
which serve as cytoskeletal linking sites (838). Channel conductance was not dependent on other ions and was inhibitable by a low extracellular pH (838). Calcium uptake was
reduced by as much as ⬃70% at a pH of 5.5, which con-
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firmed the early black box data observations made in intestinal brush-border vesicles (838). This behavior seemed
counterintuitive to the investigators, as TRPV6 expression
was highest in the duodenum, which is exposed to an acid
load from the stomach (838). Duodenal pH has been reported to be as low as ⬃6.1– 6.6 but is even lower acutely
after gastric emptying (⬃5.4), which would entail significant inhibition of TRPV6-mediated calcium uptake (284,
290, 838). Conductance was also modestly sensitive (10 –
15% inhibition) to L-type calcium channel blockers at high
concentrations, which partially clarified the preceding ambiguous observations made by other groups with these inhibitors (318, 337, 717, 838).
Subsequently, the human analog of rat TRPV6 was cloned
(it had 97% sequence homology and was rather confusingly
named ECaC2), and its expression was confirmed in human
duodenum (64). The generation of a TRPV6 antibody allowed for first localization studies, which confirmed an apical localization of TRPV6 and thus reaffirmed its role as the
primary apical calcium entry pathway in the intestine
(1220). Tissue expression of TRPV6 varies among species
(461). In humans, TRPV6 was identified in the duodenum,
jejunum, stomach, esophagus, kidney, placenta, mammary
gland, pancreas, prostate, testis, and salivary gland, but was
also found to be upregulated in a series of malignancies,
including prostate, breast, colon, and ovarian cancer (461,
792, 836, 839, 1168, 1220). The role of TRPV6 in these
tissues mostly remains to be elucidated.
TRPV6 displays some unique biophysical properties. As
mentioned earlier, TRPV6 consists of six transmembrane
segments, like many voltage-gated cation channels. Unlike
these channels, TRPV6 lacks the voltage sensor domain in
the fourth ␣-helix and is in a constitutively open state at
resting membrane potential. Analysis of the quarterny
structure shows that it assembles in tetramers (476). The
ankyrin domains were implicated to be responsible for tetramer formation; however, more recent structural data obtained by crystallography question this hypothesis (311,
848). It is thought that heterotetramers can also be formed
with subunits of closely related TRPV5 (476). In contrast to
most other members of the TRP channel family, TRPV6 is a
very selective channel for calcium (475, 1058, 1197). The
relative permeability of TRPV6 for calcium over sodium
(PCa/PNa) is ⬎100, whereas TRPV1, for example, has a
selectivity of PCa/PNa ⬃10 (although this has recently been
shown to be variable) (206, 475, 1197). This high selectivity is crucial for the maintenance of a constant membrane
potential in the enterocyte during calcium absorption.
TRPV6 is strongly inward rectifying, which has been attributed to magnesium ions plugging the channel pore for outward ion movement during states of depolarization (1126).
Magnesium also exerts a voltage-independent inhibitory effect on TRPV6 current; however, the mechanism underlying this observation is unclear (475, 1126). Furthermore,
TRPV6 gating is sensitive to intracellular calcium. Increases
in calcium were shown to inhibit the channel, resembling a
negative-feedback mechanism (475). Although global calcium concentrations in the cell largely remain constant, the
channel microenvironment is exposed to fluctuations in local calcium. This feedback loop may be important for finely
tuning the amount of calcium influx and preventing calcium
overload of the enterocyte. The putative mechanism of this
regulation will be discussed later in this section.
In 2001 it was demonstrated that ATP modulates TRPV6
activity by preventing channel rundown (475). Recent work
by Al-Ansary et al. (15) suggests that ATP directly binds to
the channel, thereby inhibiting inactivation by locking the
channel in the open conformation. Binding of ATP may be
antagonized by channel phosphorylation through PKC
(15). In conclusion, TRPV6 is precisely regulated by its own
microenvironment. Calcium, magnesium, and protons have
an inhibitory effect on the channel, whereas ATP prevents
channel inactivation.
It is important to remember that apart from being a nutrient, calcium is a crucial intracellular messaging molecule.
Therefore, the enterocyte has to tightly control its intracellular concentration and adapt uptake to energy status and
basolateral extrusion. To ensure this delicate intracellular
homeostasis, TRPV6 is associated with and regulated by a
variety of auxiliary proteins. The first protein that has been
identified to interact with TRPV6 was the S100A10-annexin2 complex (1112). The S100A10-annexin2 complex
has been implicated to play a role in protein trafficking and
membrane anchoring. Its association with TRPV6 is essential for channel trafficking to occur, as a disruption of the
interaction leads to cytosolic scattering of the channel
(1112). Formation of the S100A10-annexin2 complex and
its association with TRPV6 involves activation of PKA and
calcineurinA (CnA) (117). Another protein that is required
for TRPV6 membrane insertion is rab11a (1111). In analogy to S100A10-annexin2, perturbation of rab11a binding
results in decreased surface expression of TRPV6 and channel retention in the cytosol (1111). Furthermore, the protein
kinases SGK1 and WNK3 are known to promote membrane insertion of TRPV6; however, their exact site of action is still unclear (114, 1031). Once trafficked to the membrane, physical channel stability is maintained by a variety
of auxiliary proteins. The COOH-terminal tail of TRPV6
contains a PDZ protein binding motif. PDZ proteins serve
as membrane anchors and protein scaffolds and mediate the
assembly of multiprotein complexes, thereby regulating
channel activity. The PDZ protein sodium hydrogen exchanger regulating factor (NHERF4) (aka PDZK2) has recently been identified to interact with TRPV6 (574, 1113).
It has been speculated that NHERF4 serves as a scaffold for
TRPV6 at the apical pole, which is underlined by the observation that knockdown of NHERF4 by RNAi leads to decreased TRPV6 current in HEK293 cells (574, 1113). Apart
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SASCHA KOPIC AND JOHN P. GEIBEL
from trafficking to the apical membrane, channel number
can be regulated by internalization and degradation. One
possibility of decreasing functional channels at the cell surface is protein ubiquitination. The process of ubiquitination
involves enzymatic tagging of target proteins, thus directing
them to the proteasome or lysosome for rapid degradation.
The degradation tag is transferred to the target protein by
E3 ubiquitin protein ligases, such as Nedd4 –2. It is already
well understood how Nedd4 –2-mediated ubiquitination
can decrease the number, but also directly modulate the
activity, of the epithelial sodium channel (ENaC) in the
kidney (825). A recent study demonstrated that a similar
mechanism applies to TRPV6 (1212). Coexpression of
Nedd4 –2 and TRPV6 in oocytes resulted in decreased calcium flux and channel numbers (1212). Nedd4 –2-mediated
TRPV6 ubiquitination could thus serve as a mechanism of
rapidly regulating channel retrieval from the plasma membrane (1212).
Several other proteins have been identified to associate with
TRPV6 that do not affect channel numbers, but rather directly regulate channel activity. The nisnap1 gene was identified in the late 1990s; however, its function remained elusive (992). Recently, TRPV6 was shown to interact with
nisnap1 (971). It is expressed in the intestine, but not the
kidney, which is unusual as TRPV6 and TRPV5 are generally regulated by similar auxiliary proteins. Electrophysiological measurements showed that nisnap1 inhibits TRPV6
without affecting its surface expression (971). Very similar
functional properties were attributed to RGS2, a protein
that is mainly known to alter the GTPase activity of G
proteins. In analogy to nisnap1, RGS2 can inhibit TRPV6
at the plasma membrane without affecting its trafficking
dynamics (970).
As previously discussed, the activity of TRPV6 is finely
regulated by intracellular calcium concentrations. Elevations in intracellular calcium levels exert an autoinhibitory effect on channel opening (475). Shortly after the
identification of TRPV6, calmodulin (CaM) was shown
to bind to the channel in a calcium-dependent manner,
and it was speculated that it mediates the calcium feedback response (461, 791). Subsequent investigations demonstrated that CaM may indeed represent the molecular
calcium sensor (263, 619). Elegant FRET studies reported
that CaM dynamically associates with TRPV6 in the presence of calcium and that this association is terminated when
intracellular calcium is depleted (263). The association between TRPV6 and CaM also correlates with decreased current flux through the channel (263). Although the ankyrin
repeat domains of the channel were thought to possibly
serve as binding sites for CaM, more recent structural insights provide rebutting evidence for this hypothesis (848).
Instead, the COOH-terminal tail of TRPV6 has been suggested as the site where CaM binding occurs (263, 619,
791). Hence, CaM can tune ion flux through TRPV6 and
206
most likely mediates the channel’s sensitivity to intracellular calcium.
Phosphorylation by kinases and dephosphorylation by
phosphatases represent a common cellular strategy for rapidly modulating channel gating properties. The non-receptor tyrosine kinase Src has emerged as a candidate for the
direct phosphorylation of TRPV6, thereby increasing channel conductance (1042). Src was previously shown to modulate TRPV4 activity (1178). The effects of Src are antagonized by the phosphatase PTP1B. Both enzymes act on the
tyrosine residues Y161/162, which are located in the NH2terminal tail of the channel (1041).
An interesting interaction without direct relevance for the
intestine has been reported between renal TRPV5 and
klotho (170). Klotho is a ␤-glucuronidase with a transmembrane anchor that can be cleaved, resulting in shedding of
the enzymatically active domain of the protein into the
urine (170, 612). Klotho can then increase TRPV5-mediated calcium uptake by hydrolyzing N-linked oligosaccharides on extracellular channel domains, thereby preventing
channel retrieval from the plasma membrane (170). Although a recent report indicates that klotho can also affect
TRPV6 activity in vitro, it is not expressed in the intestine
and thus may only interact with renal TRPV6 (612, 683).
Although this finding has no implications for intestinal calcium uptake, various bacteria and neutrophils produce
␤-glucuronidase, which may affect TRPV6 activity in the
intestine (359, 1004).
Recently, Stumpf et al. (1046) described an association between cyclophilin B (CyB) and TRPV6 in the placenta. Coexpression of both proteins in oocytes increased calcium
uptake (1046). CyB was also detected by Western blot analysis in the small intestine and colon; however, colocalization and functional studies in intestinal tissue remain to be
performed (1046).
C) THE TRPV6 KNOCKOUT CONUNDRUM.
As will be discussed in
more detail later, 1,25(OH)2-vitamin D is one of the key
hormonal regulators of systemic calcium homeostasis (see
sect. IVA). Increased levels of 1,25(OH)2-vitamin D lead to
enhanced intestinal calcium absorption. Shortly after cloning of TRPV6, it was recognized that the channel is positively regulated at the mRNA level by 1,25(OH)2-vitamin D
(614, 1030, 1109, 1110). The TRPV6 promoter has multiple binding sites for the 1,25(OH)2-vitamin D receptor
(VDR) and is thus directly sensitive to increases in
1,25(OH)2-vitamin D levels (736). Consequently, VDR-deficient animals display a marked decrease in TRPV6 mRNA
levels, whereas administration of 1,25(OH)2-vitamin D in
wild-type animals results in an increase in mRNA transcription (1030, 1109, 1110). In conjunction with the rapidly expanding characterization of the channel itself, these observations supported the dogma that TRPV6 is the essential player
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in transcellular calcium uptake and that this pathway is
strongly regulated by 1,25(OH)2-vitamin D.
With the introduction of novel genetic techniques, a TRPV6
(⫺/⫺) mouse was created in 2007 by Bianco et al. (94). The
animals presented with decreased intestinal calcium uptake,
as measured by serum concentrations of a calcium radioisotope following gavage, decreased femoral bone density
(9.3%) and increased 1,25(OH)2-vitamin D levels, as a result of feedback regulation (94). These findings were in
accordance with the postulated role of TRPV6 as the primary 1,25(OH)2-vitamin D-sensitive calcium uptake mechanism in the intestine. However, the generation of another
TRPV6 (⫺/⫺) animal line by Benn et al. (76) a year later
spawned a controversy in the field. In these animals, baseline calcium uptake was identical between wild-type and
(⫺/⫺) groups. Surprisingly, calcium uptake could be increased in the (⫺/⫺) group by a low calcium diet and, more
importantly, by 1,25(OH)2-vitamin D (an observation that
was also confirmed by another group; Ref. 615) (76). These
findings profoundly questioned the role of TRPV6 in calcium absorption and suggested that a different molecular
target of 1,25(OH)2-vitamin D mediates the increase in calcium uptake after exposure. All these observations were in
sharp contrast to the initial report by Bianco et al. (94).
However, Benn et al. (76) also reported that compared with
wild-type animals calcium uptake was reduced in TRPV6deficient animals that were fed a low-calcium diet. This
suggests that the animals may not be able to adequately
increase absorption, when confronted with a low dietary
availability of calcium, which in turn suggests a role of
TRPV6 during states of dietary calcium insufficiency.
These controversial findings allow several conclusions and
speculations. 1) As with any model of targeted gene disruption, a compensatory mechanism may be in place that
masks and distorts the physiological importance of TRPV6.
The animals may upregulate other, yet unidentified, calcium transport mechanisms to compensate for the loss of
TRPV6 function. 2) Rather than being a constitutively
active pathway, transcellular calcium absorption through
TRPV6 only occurs in states of low dietary calcium intake.
This hypothesis has been put forward very early and has been
reaffirmed by a recent study that observed an increase in bone
turnover in TRPV6 (⫺/⫺) animals on a low-calcium diet
(46, 666, 824, 1224). Mobilization of bone calcium may be
necessary in these animals to maintain systemic calcium
concentrations, which are double challenged by the lack of
TRPV6 and the insufficient calcium intake (666). Furthermore, it has been extensively described that a low-calcium
diet induces gene expression of TRPV6 [presumably
through an increase in 1,25(OH)2-vitamin D levels], suggesting a role of the channel during states of insufficient
dietary calcium supply (76, 132, 639). 3) 1,25(OH)2-vitamin D may have many more targets in the intestinal mucosa
than previously anticipated and may also regulate calcium
uptake through the paracellular pathway (76, 615). In conclusion, the generation of TRPV6 (⫺/⫺) animals has sustainably challenged our model of transcellular calcium absorption by questioning the relative importance of TRPV6
and by introducing new potential targets of 1,25(OH)2vitamin D regulation.
D) GENETIC POLYMORPHISMS OF TRPV6.
Single nucleotide polymorphisms (SNPs) are variations in the genomic sequence
that occur in one single base. If the frequency of a SNP or a
specific set of SNPs (haplotype) increases in a population
over time compared with other SNPs, it can be concluded
that this set of SNPs is associated with an evolutionary
advantage, meaning that this gene locus was under selection. Recent reports from various groups indicate that
TRPV6 was subjected to strong selection (12, 502, 1023,
1050). Traces for selection can be found in any non-African
population (12, 502, 1023). The selective event in Europeans has presumably occurred ⬃7,000 years ago and
coincides with the development of agriculture (502). This
correlation also holds true for other populations (502). It
has been speculated that a change in diet or resistance to
emerging disease led to selection and fixation of the now
conserved haplotype (502). Interestingly, selection took
place in parallel in many populations, pointing towards a
strong selective stress that developed independently in
each region (502).
The electrophysiological properties of ancestral and derived
TRPV6 were investigated by two groups (502, 1050).
Hughes et al. (502) observed no statistically significant divergence in channel behavior. The derived channel only
displayed a tendency towards decreased sensitivity to the
autoinhibition by calcium, albeit not significant (P ⫽
⫺0.094) (502). The authors speculated that differences in
protein-protein interactions may have led to selection
(502). Conversely, Sudo et al. (1050) reported increased
calcium influx through the derived channel, which may constitute an evolutionary advantage by facilitating dietary calcium uptake. Regardless of the controversial functional
data, these insights indirectly underline the importance of
TRPV6 and provide a counterweight to the conclusions
drawn from the TRPV6 (⫺/⫺) animal model. It is unquestionable that TRPV6 was under parallel independent selection in many regions across the planet. This provokes the
question of how strong selection can occur for a derived
channel whose physiological difference to the ancestral
form seems to be very subtle, yet complete disruption of the
channel in the mouse model only causes a very mild phenotype. This inconsistency adequately exemplifies the caveat
that underlies the conclusions drawn from (⫺/⫺) animals
and reminds us of the fact that we are far from understanding the exact physiology of transcellular calcium uptake.
E) CAV1.3. TRPV6 may not be the only channel mediating
apical calcium absorption in the intestine. A recent investi-
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SASCHA KOPIC AND JOHN P. GEIBEL
gation provides another explanation for the initially observed partial sensitivity of transcellular calcium uptake to
L-type calcium channel blockers. The voltage-gated calcium channel Cav1.3, a member of the L-type calcium channel family, was recently identified in the apical membranes
of the distal jejunum and proximal ileum (751). Previous
observations were emulated, as the investigators again demonstrated a decrease in calcium absorption following application of L-type calcium channel inhibitors in the corresponding segments (751). The authors argued that uptake
through Cav1.3 may have previously been misinterpreted as
paracellular calcium uptake (751). However, it should be
noted that the calcium uptake assay used in this report did
not discriminate between transcellular and paracellular calcium movement. Subsequently, it has been observed that
L-type inhibitor-sensitive calcium flux is linked to stimulation of glucose uptake through the glucose transporter type
2 (GLUT2) (692, 750). Cav1.3 may serve as an alternative
calcium entry pathway that is active in states of luminal
calcium abundance and that coregulates glucose absorption
(692, 750). Further investigations will be needed to determine the contribution of Cav1.3 to dietary calcium absorption.
2. Calbindin-D 9k
The following section will address the question of what
happens to dietary calcium after it enters the enterocyte. It
should be considered that transcellular calcium uptake and
cytosolic calcium homeostasis are two contradicting requirements for the enterocyte. A prominent transcellular
flux of free calcium will inevitably interfere with housekeeping functions of the cell, such as intracellular signaling.
Furthermore, it has been calculated from in vivo data that if
calcium were to diffuse freely (simple diffusion) through the
cytosol, uptake rates would only be 1/70th of the actually
measured values (129). Simple diffusion would constitute a
bottleneck in the process of transcellular calcium absorption if cytosolic calcium concentrations should be kept low.
A partial solution to this problem was found as early
as 1966, when Wasserman et al. (1151) identified a
1,25(OH)2-vitamin D-inducible calcium binding protein in
the chick intestine. The authors observed that calcium radioisotopes traveled faster across a cellophane membrane if
suspended in intestinal homogenates from rachitic, i.e.,
1,25(OH)2-vitamin D deficient, chicks than if suspended in
the homogenates from rachitic animals treated with
1,25(OH)2-vitamin D (1151). This indicated that calcium
was bound to a protein in the enterocyte and that expression of this protein was controlled by 1,25(OH)2-vitamin D
(1151, 1152). Subsequently, the calcium binding protein
was further characterized (1073, 1149, 1152). Expression
levels were shown to be highest in the duodenum and to
gradually decrease in more distal segments, which correlated with the degree of calcium uptake that has been attributed to each intestinal segment respectively in prior
functional investigations (1073). The mammalian isoform,
208
which had a lower molecular mass of ⬃9 kDa (hence the
name calbindin-D 9k), compared with the 28 kDa of the
avian isoform, was later identified (138, 280, 283, 358).
Concerning the functional role of calbindin-D 9k, it had
already been speculated very early after its discovery that it
may serve as a calcium shuttling protein (951). Indeed, initial calculations and later very basic experimental data confirmed that calbindin-D 9k may mediate facilitated diffusion of calcium between the two poles of the enterocyte, in
analogy to the transport of oxygen by myoglobin in the
muscle (321, 602). In a very fundamental investigation,
calcium flux was measured between chambers that were
separated by dialysis membranes. A 51% increase in transchamber calcium flux occurred in the presence of calbindin-D 9k (321). However, it is hard to relate this number to
physiological values, given the simplification underlying the
experimental model. It has subsequently been calculated
that calbindin-D 9k may facilitate diffusion of calcium by a
factor of up to ⬃60 (129). Rather than envisioning calbindin-D 9k as a protein that individually shuttles calcium ions
across the cell, one should imagine calbindin-D 9k as a
calcium gradient amplifier (129, 602). As the intracellular
concentration of free calcium is in the nanomolar range, the
intracellular gradient of free calcium between the apical and
basolateral pole can also only be in the nanomolar range.
Diffusional flux, however, directly correlates with the concentration gradient and can in consequence only be very
small. Calbindin-D 9k is expressed in the enterocyte in the
micromolar range and dynamically binds and releases calcium (714). Since the association between calbindin-D 9k
and calcium is dynamic, the local concentration of calbindin-D 9k-bound calcium will directly correlate with the
concentration of free calcium. It is thus the concentration
gradient of calbindin-D 9k-bound calcium throughout the
cell that determines the flux rate of calcium. As this gradient
can be in the micromolar range, calbindin-D 9k serves as a
calcium gradient amplifier (129, 602). It should be noted
that the real experimental data on calbindin-D 9k are fairly
scarce and that the bulk of scientific effort has gone into
mathematical modeling of facilitated diffusion and calcium
binding kinetics (129, 321, 322, 602, 1021).
Functionally, several correlations between calbindin-D 9k
and calcium uptake were identified. The calbindin-D 9k
content of each intestinal segment correlates linearly with
its ability for calcium uptake (129, 824, 1021). Although
this relation holds true on the experimental and the mathematical modeling level, it does not prove causality (129,
824, 1021). Furthermore, 1,25(OH)2-vitamin D and a lowcalcium diet induce calbindin-D 9k on both the mRNA and
protein level, suggesting that it is involved in the process of
regulated calcium absorption (289, 604). In accordance
with these findings, VDR-deficient animals have a decreased calbindin-D 9k mRNA content that cannot be rescued by exogenous 1,25(OH)2-vitamin D administration
(663, 1194). Although a 1,25(OH)2-vitamin D responsive
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element (VDRE) had been identified in the 5=-flanking region of the calbindin-D 9k gene, later mutational analysis
demonstrated that this site was not essential for transcriptional regulation of calbindin-D 9k by 1,25(OH)2-vitamin
D (217, 240). Hence, it is not clear how 1,25(OH)2-vitamin
D exactly regulates calbindin-D 9k on a molecular level.
Interestingly, intestinal calbindin-D 9k mRNA levels can be
rescued in VDR (⫺/⫺) animals by a high-calcium/phosphorus/lactose diet, while extraintestinal calbindin-D 9k
mRNA levels are unaffected (663). This suggests that, apart
from endocrine regulation through 1,25(OH)2-vitamin D,
intestinal calbindin-D 9k is also regulated by local factors
(663). A direct short-term stimulatory effect of oral calcium
intake on calbindin-D 9k levels had been observed before in
a wild-type background (647). The mechanisms underlying
the effect of diet on calbindin-D 9k levels are, however, still
obscure.
Despite the correlation between calbindin-D 9k levels and
1,25(OH)2-vitamin D and the mathematical and experimental models, which support its role as a calcium shuttling
protein, no clear evidence for the involvement of calbindin-D 9k in transcellular calcium absorption existed. This
had sparked controversy among investigators. In fact, a
very early study concluded that although 1,25(OH)2-vitamin D induced both calbindin-D 9k and calcium absorption, a discrepancy existed in the time course of both effects
(424). Later, synthetic 1,25(OH)2-vitamin D derivatives,
designed for a maximized effect on cell differentiation and
suppressed calciotropic activity, failed to increase serum
calcium levels while causing a sevenfold increase in calbindin-D 9k mRNA [native 1,25(OH)2-vitamin D increases
both serum calcium and calbindin-D 9k mRNA](604). In
2006, a calbindin-D 9k (⫺/⫺) mouse was created by Kutuzova et al. (613). The animals presented with no apparent
phenotype abnormalities and had normal serum calcium
concentrations (613). Subsequent analysis of these animals
showed that they responded equally well to 1,25(OH)2vitamin D with regard to calcium absorption as wild-type
animals (13). Of note, calcium absorption was assessed by
measuring serum calcium following gavage of a calcium
radioisotope and did not discriminate between transcellular
and paracellular absorption (13). A different group replicated the findings made in the calbindin-D 9k (⫺/⫺) mouse
and also did not find any appreciable variation in phenotype or serum calcium concentrations (639). However, the
investigators observed that during preweaning, when both
calcium demand and absorption are at their peak, gene
expression of TRPV6 and the basolateral calcium extruder,
plasma membrane calcium ATPase isoform 1b (PMCA1b),
were highly induced in the calbindin-D 9k (⫺/⫺) animals
(128, 639). This has been interpreted as a compensatory
upregulation of calcium transport proteins to ensure sufficient uptake in a state of high metabolic demand for calcium (639). Subsequently, a TRPV6 and calbindin-D 9k
double (⫺/⫺) animal was created. The mature animals
show no disturbances in calcium homeostasis, although,
similarly to TRPV 6 (⫺/⫺) animals, they cannot increase
calcium uptake in response to a dietary calcium challenge to
the same extent as wild-type animals (76).
In light of the insights gained through knockout animals,
the role of calbindin-D 9k remains somewhat unclear.
Again, it is difficult to dissect the physiological function of
calbindin-D 9k in these animal models, given the assumption that the organism will pursue compensation for the loss
of a gene product. In conclusion, it is undisputed that calbindin-D 9k is regulated by 1,25(OH)2-vitamin D, that it
can bind calcium and that theoretical modeling and very
fundamental experimental models verify that it can facilitate diffusion of calcium across the enterocyte. The (⫺/⫺)
models suggest that animals can compensate for the loss of
calbindin-D 9k and maintain normal calcium homeostasis
(13, 76, 613, 639). Furthermore, TRPV6, calbindin-D 9k
and double (⫺/⫺) animals can still increase their calcium
uptake in response to 1,25(OH)2-vitamin D or a low-calcium diet, respectively, albeit not to the same extent as
wild-type animals (13, 76, 615). One may conclude that
neither of these proteins is necessary for transcellular uptake; however, it seems more likely that 1,25(OH)2-vitamin
D has more targets than previously postulated and that
compensatory mechanisms are in place.
3. Basolateral calcium extrusion
Once calcium reaches the basolateral membrane, it is extruded into the extracellular space, which completes the
process of intestinal absorption. As extracellular calcium
concentrations are higher than cytosolic concentrations,
this process requires energy. Two proteins are responsible
for this task. 1) The PMCA extrudes calcium at the direct
expense of ATP, whereas 2) the sodium-calcium exchanger
(NCX) utilizes the energy stored in the sodium gradient to
transport calcium out of the cell (FIGURE 3). It is this basolateral exit process that requires energy during transcellular
calcium uptake and that allows us to absorb calcium against
an uphill gradient when dietary concentrations are low.
A) THE PLASMA MEMBRANE CALCIUM ATPASE. The plasma membrane calcium ATPase (PMCA) is a virtually ubiquitous
protein that is responsible for intracellular calcium homeostasis by pumping calcium into the extracellular milieu,
thereby keeping intracellular concentrations low. The
pump belongs to the family of P-type primary ion transport
ATPases, which among others also includes gastric H⫹-K⫹ATPase (see sect. IIA1). Four isoforms of the protein exist;
however, variety is increased by splicing (562, 1043). The
PMCA1b splice variant is most predominant in the small
intestine and the duodenum in particular, which suggest an
involvement of this isoform in the process of transcellular
calcium absorption (341, 492). Given the ubiquitous expression of PMCA, a detailed review of its structure and
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SASCHA KOPIC AND JOHN P. GEIBEL
function will be omitted. For a current and detailed review,
please refer to Reference 1043.
PMCA was first characterized in the 1960s in the membrane
of erythrocytes (956). Very early experiments in basolateral
membranes isolated from enterocytes identified a calciumdependent enzyme with phosphatase activity, which served
as first evidence for PMCA in the intestine (100, 742). Subsequent investigations in basolateral vesicles from rat intestine concluded that two distinct calcium transport mechanisms existed in their membranes: an ATP-dependent
(PMCA) and a sodium-dependent (NCX) mechanism (382,
457, 573). Furthermore, it was shown that inhibition of
calmodulin halved the amount of ATP-dependent calcium
transport (573). Today, we know that PMCA is highly regulated by CaM, which can increase both the affinity of the
pump to calcium and its turnover speed by a factor of up to
10 (526, 627, 628, 756).
Interestingly, it has also been suggested that calbindin-D 9k
can directly stimulate calcium transport via PMCA (1136,
1137). In addition to intracellular proteins, PMCA is also
quantitatively regulated by endocrine factors. 1,25(OH)2vitamin D can increase both the PMCA mRNA and protein
content in enterocytes (35, 36, 62, 146, 382, 489, 614, 823,
1109; contested by Ref. 1138). The data on the effects of a
low-calcium diet on PMCA transcription are more controversial, as one study suggests induction whereas two other studies
observed reduced mRNA levels (146, 583, 639). Of note,
these observations were made in different species (chick vs.
mouse). The sensitivity of the tissue to 1,25(OH)2-vitamin D is
also reported to decrease with age, which may represent
another confounding factor underlying these observations
(35, 36). In conclusion, the transcriptional regulation of
PMCA1b by 1,25(OH)2-vitamin D and its decreasing expression along the length of the small intestine correlate
well with the canonical model of transcellular calcium absorption. It is challenging to investigate the functional
contribution of PMCA1b in the process of calcium absorption, given its role as a housekeeping protein.
PMCA1 is crucial during development, which results in
embryonic death if knocked out (814, 1198). Conversely,
heterozygote (⫹/⫺) animals present with no apparent
phenotype, albeit parameters linked to calcium homeostasis were not assessed (814).
B) THE NCX.
Long before the molecular identities of PMCA
and NCX were known, it had been observed that calcium
uptake in the intestine was dependent on extracellular sodium (713). The authors concluded that “a sodium, calciumexchange diffusion carrier may exist at the basal membrane
of the cell,” which proved to be a remarkably accurate
prediction (713). As mentioned before, later investigations
delineated between an ATP and a sodium-dependent transport of calcium on the basolateral enterocyte membrane
(382, 457, 573).
210
For a recent review on the structure and function of NCX,
please refer to Reference 687. In brief, NCX exists in three
isoforms (NCX1–3), which are expressed in a broad variety
of cell types (687). The function of NCX has mainly been
investigated in excitatory tissues, given its role as a highcapacity calcium extrusion mechanism following excitation. To transport calcium against its strong electrochemical gradient, NCX has to utilize three sodium ions to accomplish extrusion of one calcium ion (85, 460). NCX1 is
the predominant isoform in the small intestine, where it has
been detected on the mRNA and the protein levels (275,
688). However, NCX had been postulated to play a more
important role during calcium absorption in the kidney
than in the enterocyte, hence not much data on intestinal
NCX are available to us (473). Furthermore, genetic disruption of NCX1 is embryonically lethal, which imposes
some limitations on our experimental methodology (600).
A more recent study provides some functional evidence for
NCX in the intestine. By measuring intracellular calcium
concentrations with a fluorescent indicator dye, Dong et al.
(275) demonstrated that calcium uptake in a sodium-free
environment (to run NCX in its reversed configuration) was
significantly decreased when NCX was pharmacologically
inhibited. Despite our knowledge that NCX exists in enterocytes and that it can extrude calcium under experimental conditions, it is difficult to assess its relative contribution
to transcellular calcium absorption.
In addition, NCX is not subjected to regulation by 1,25(OH)2vitamin D, which further contributes to the ambiguity concerning its importance in the process of calcium absorption.
This has been observed very early, when exogenous administration of 1,25(OH)2-vitamin D to vitamin D-deficient
animals did not increase sodium-dependent calcium transport, while doubling transport through PMCA (382). Furthermore, it was recently shown that a calcium-depleted
diet decreases duodenal NCX1 mRNA levels (583).
Of note, two members of the potassium-dependent sodium
calcium exchanger (NCKX) family, namely, NCKX 3 and
4, were also identified in the small intestine (658, 687, 688).
However, further functional investigations will be needed
to clarify their role.
B. Paracellular Calcium Absorption
If we plot the amount of duodenal calcium absorption as a
function of the luminal calcium concentration, we can observe that the absorption curve is comprised of two distinct
kinetic components: a saturable/exponential component
and a nonsaturable/linear component (824, 825, 1134).
The saturable component represents transcellular calcium
uptake through the enterocyte, as both the number of calcium transport proteins and their turnover rate is limited.
The nonsaturable component reflects calcium uptake
through the paracellular pathway. The saturable compo-
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nent is less pronounced in the jejunum and disappears completely in the ileum, indicating that transcellular calcium
absorption is restricted to the proximal segments of the
intestine, as discussed previously (825) [this has been contested more recently, when transcellular flux was also noted
in the ileum (46)]. Compared with the transcellular pathway, the paracellular route has not received much scientific
attention. It has been put forward that the rate of paracellular calcium absorption is constant across the length of the
intestine and is neither sensitive to 1,25(OH)2-vitamin D
nor a low-calcium diet (824, 825, 1224). However, provided that enough dietary calcium is available to saturate
the transcellular pathway, observations indicate that net
calcium absorption is highest in the ileum, which has been
attributed to the sojourn time, rather than alterations in
paracellular permeability (290, 710). In the rat, the chyme
spends some 74% of its transit time in the ileum, which
allows for a long exchange period between lumen and
plasma (290). The diffusion rate itself is fairly low and only
amounts to 2% of the rate if calcium were to diffuse freely
between intestinal lumen and plasma (290). This effect is a
consequence of the diffusion barrier that tight junctions,
which act as functional gating molecules of the paracellular
pathway, impose on calcium flux.
Epithelial tight junctions are adhesion points between two
neighboring cells that seal their intercellular space against a
lumen, thereby restricting ion and water movement between the two compartments. As water cannot be directly
transported, its movement is tied to ion fluxes, which in
turn are determined by their respective concentration gradients across the epithelium. Each epithelial cell expresses
an annulus of tight junction proteins at the apical end of the
lateral membrane. Tight junctions are protein complexes
that consist of a variety of intra- and transcellular proteins.
A detailed review of their structure would exceed the scope
of this article (for a recent review, please refer to Ref. 999).
Briefly, the composition of the involved proteins determines
the pore size, ion selectivity, and thereby the relative leakiness of each tight junction and the whole epithelium in
general. Furthermore, tight junctions create a lateral diffusion barrier for membrane proteins and help to maintain the
functional polarity of the epithelial cell. It is important to
recognize that tight junctions are not static complexes, but
can be rather dynamically regulated (999). This allows the
epithelium to change its ion and water permeability in response to various stimuli (999).
As discussed previously, the rate of the nonsaturable
component of calcium absorption is documented to be
fairly constant and insensitive to 1,25(OH)2-vitamin D,
which indicated that 1,25(OH)2-vitamin D may have no
effect on paracellular transport (824, 825, 1224). However, in the late 1990s, Chirayath et al. (201) postulated
that 1,25(OH)2-vitamin D can increase paracellular calcium flux in confluent Caco-2 cell cultures, which form
an epithelia-like monolayer with tight junctions. This
conclusion is based on the observations that 1,25(OH)2vitamin D decreased the transepithelial electric resistance, which is often used as a measure of tight junction
permeability, and induced bidirectional, i.e., also “serosal”
to “mucosal,” calcium flux in the cultured monolayers
(201). A more recent investigation further substantiated
this hypothesis. It has been shown that 1,25(OH)2-vitamin
D regulates the mRNA levels of some tight junction proteins
in rat duodenum, which may alter their gating characteristics (354, 614). For example, the mRNA levels of claudin-3,
a protein that directly determines tight junction permeability, were decreased 2.2-fold following 1,25(OH)2-vitamin
D administration (614). Conversely, claudin-2 and -12
mRNA and protein levels were increased (354). Functional
observations in Caco-2 monolayers further demonstrate
that overexpression of claudin-2 and -12 increases electrical
conductivity and facilitates paracellular calcium flux (354).
All of these events are tied to a 1,25(OH)2-vitamin D-mediated promotion of transcriptional events. However, it is
known that 1,25(OH)2-vitamin D can also exert short-term
nongenomic effects (1122) (see sect. IVA6B). A recent investigation presents evidence for augmented solvent-drag
paracellular calcium flux after acute administration of
1,25(OH)2-vitamin D (1098). Solvent-drag mediated flux
may occur at low calcium concentrations in the absence of
a calcium gradient. In this model, calcium is dragged
through the paracellular space by water flux that is fueled
by the hyperosmolar milieu in the paracellular space (266,
626). 1,25(OH)2-vitamin D was shown to induce this calcium flux in the presence of initially equimolar calcium
concentrations between the mucosal and serosal compartments (1098).
Over the last years, evidence for the regulation of the paracellular pathway by 1,25(OH)2-vitamin D has slowly accumulated. It is apparent that the canonical dogma which
postulates that 1,25(OH)2-vitamin D only targets the transcellular pathway has to be revisited. Still, more experimental investigations will be needed to ultimately clarify the
effects of 1,25(OH)2-vitamin D on paracellular calcium
transport. Should tight junctions indeed be subjected to regulation by 1,25(OH)2-vitamin D, this mechanism may partially
explain the sensitivity of calcium uptake in TRPV6 and calbindin-D 9k (⫺/⫺) animals to 1,25(OH)2-vitamin D.
C. Alternative Models of Transcellular
Calcium Absorption
Two alternative models of transcellular calcium absorption
have been put forward. One suggests that calcium is transported through the enterocyte by vesicles (vesicular transport model) rather than facilitated diffusion, and the other
postulates that 1,25(OH)2-vitamin D can induce intestinal calcium absorption in a rapid, nongenomic fashion via a putative
1,25(OH)2-vitamin D surface receptor (transcaltachia).
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The assumption that calcium is transported through the cell
in vesicles is corroborated by a handful of observations. It
has been demonstrated that 1,25(OH)2-vitamin D treatment increased the number of supranuclear lysosomes in
rachitic chicks, compared with nontreated animals (247). It
was later shown that the calcium content of lysosomes can
more than double following treatment with 1,25(OH)2-vitamin D, suggesting that they may play a role in the process
of transcellular calcium movement (780). Compared with
the cumulative evidence that is in accordance with our canonical model of calcium absorption, the role of vesicular
transport of calcium is still fairly obscure.
The model of transcaltachia mainly relies on the observation that 1,25(OH)2-vitamin D can exert effects that are too
acute to be attributable to transcriptional events. For example, intestinal calcium absorption was increased in chicks
within 14 min of 1,25(OH)2-vitamin D exposure, an onset
which is too rapid as to be of a genomic nature (785). It
should be noted that rapid effects of 1,25(OH)2-vitamin D
have also been suggested to influence the paracellular pathway (see above). Undoubtedly a careful evaluation of the
route of calcium flux, i.e., transcellular versus paracellular,
is necessary. At least, isolated intestinal cells respond with
increased uptake of radiolabeled calcium to acute 1,25(OH)2vitamin D exposure (568). It has been postulated that apart
from the VDR, a membrane-bound 1,25(OH)2-vitamin D exposure receptor, the 1,25(OH)2-vitamin D-MARRS (membrane-associated, rapid response, steroid binding) protein, mediates the acute effects of 1,25(OH)2-vitamin D (568, 779,
785).
IV. REGULATION OF
CALCIUM HOMEOSTASIS
Eucalcemia is maintained by the concerted effort of vitamin
D, PTH, and, to a lesser extent, calcitonin. All three hormones can influence serum calcium concentrations by acting on the intestine, the kidney, or bone. 1,25(OH)2-vitamin D, the active vitamin D metabolite, primarily modulates the intestinal absorption of calcium and will therefore
be discussed in most detail (FIGURE 4). Apart from hormonal regulators that influence the absorption, excretion,
and deposition of calcium, our body needs a mechanism
that allows it to sense the current levels of plasma calcium.
This task is fulfilled by the CaSR. It oversees the precise
regulation of the calcitropic hormones.
A. Vitamin D
As discussed previously, vitamin D is one of the key regulators of calcium homeostasis. Our body has two sources
for vitamin D, namely, a dietary source (vitamin D2⫹3) and
an endogenous source that relies on ultraviolet (UV) light
catalyzed synthesis in the skin (vitamin D3) (FIGURE 5).
212
Nomenclature in this case is fairly misleading, as vitamins are
per definition substances that cannot be generated by our body
and have to be ingested from an external source. The fact that
we can synthesize vitamin D3 in our skin classifies the substance as a prohormone rather than a vitamin. As we will see,
our current nomenclature is a byproduct of the historic events
leading to the discovery of vitamin D.
1. Historical perspective
From a historical perspective, the identification of vitamin
D is closely intertwined with attempts to understand the
pathophysiology of rickets. Rickets is characterized by
childhood skeletal deformities resulting from inadequate
osteoid mineralization and calcification of cartilage due to
decreased serum calcium levels during development. The
adult form equivalent of rickets is termed osteomalacia.
With the onset of industrialization, rickets became a prevalent problem in the 18th, 19th, and the beginning of the
20th century. In fact, the disease was so widespread at the
beginning of the 20th century that an investigation conducted by the German pathologist Schmorl on 386 children,
who had died before the age of 4 years, concluded that 90%
of them had had rickets (968). Even in present times nutritional rickets still remains a major public health concern in
developing countries (303, 757, 809). The seminal observations that led to the identification of vitamin D were provided by Mellanby in 1919 (733). He observed that dog
pups who were fed a severely restricted diet consisting of
porridge or bread were consistently developing rickets
(733). Development of rickets could be averted if their diet
was supplemented with cod liver oil, which we now know
to contain a high concentration of vitamin D (733). Mellanby (733) concluded that “rickets is a deficiency disease
which develops in consequence of the absence of some accessory food factor or factors.” Vitamin A had been discovered shortly before, and it was subsequently speculated that
it may represent the factor promoting bone formation
(733). This hypothesis was later rebutted by American biochemist McCollum who concluded that “a substance which
is distinct from fat-soluble [vitamin] A” must be responsible
for preventing rickets (727). Furthermore, he stated that his
experiments “demonstrate the existence of a fourth Vitamin [vitamin D] whose specific property [. . .] is to regulate
the metabolism of bones” (727). In parallel to the unraveling of the dietary component of rickets, scientists were independently discovering the importance of sunlight for disease prevention. The Polish pediatrician Raczynski was most
likely the first to demonstrate evidence for this hypothesis experimentally (881). He kept one dog pup in the shade while a
littermate was kept in the sunlight. Both dogs were breastfed
by their mother. After 6 wk, the bones of the dog that was kept
in the shade contained 36% less calcium (881). These observations were followed up by the German pediatrician Huldschinsky, who healed rachitic children after exposing them
intermittently for 2 months to the UV rays generated by a
mercury vapor quartz lamp (504). Hess and Unger (447) rep-
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GASTRIC ACID, CALCIUM ABSORPTION, AND BONE HEALTH
Hypocalcemia
Stimulates PTH secretion
Parathyroid glands
Increases bone resorption
Bone
PTH
Increases renal calcium absorption
Stimulates 1,2
1,25(OH)2-D synthesis
Kidney
Intestine
eases renal
Increases
calcium absorption
Increases intestinal
calcium absorption
1,25(OH)2-D
FIGURE 4. Endocrine regulation of serum calcium levels. Calcium homeostasis is mainly regulated by PTH and
1,25(OH)2-vitamin D. Both hormones act at their respective target organs to increase serum calcium levels.
licated these findings by using sunlight treatment. Hess (446)
later summed up the impact of these revolutionary observations, which now seem intuitive to us: “We have known that a
growing plant cannot thrive in the dark, but have failed to
realize that the same laws apply to growing animals.”
It was later recognized that it was sufficient to irradiate the
food administered to animals rather than the whole animal
to prevent development of or heal rickets. Thus initially
“inert” dietary substances with no antirachitic properties
could be activated by UV light (385, 448 – 450, 505, 1037).
It was concluded that irradiation caused the conversion of a
biological precursor to an active form and that the same
mechanism was physiologically taking place in the skin.
Initially, it was speculated that cholesterol may serve as this
pro-vitamin. It was Windaus and Hess (in collaboration
with Rosenheim) who were the first to uncover its exact
molecular identity. They stated: “We conclude from our
experiments with complete certainty that ergosterol [. . .]
represents the anti-rachitic provitamin (1166). In 1928,
Windaus received the Nobel Prize in Chemistry for “the
services rendered through his research into the constitution
of the sterols and their connection with the vitamins.” The
irradiation product of ergosterol was later purified and
named vitamin D2 (ergocalciferol) (43, 896, 1164, 1167).
Although these findings solved the question as to how UV
irradiation generates Vitamin D2 from ergosterol, which has
antirachitic properties if ingested, the molecular mechanisms
underlying the antirachitic effects of cutaneous sunlight exposure still remained obscure. Since ergosterol is an exclusive
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SASCHA KOPIC AND JOHN P. GEIBEL
Keratinocyte
Dietary vitamin D
7-dehydrocholesterol
UV-B (290-315nm)
Intestinal uptake
Previtamin D
Isomerization
Vitamin D
Vitamin D
Vitamin D
DBP
DBP
Hepatocyte
1,25(OH)2-vitamin D
Vitamin D
Mitochondria:
CYP27A1
ER:
CYP2R1
25(OH)-vitamin D
25(OH)-vitamin D
DBP
Proximal tubule
Glomerular
filtration
25(OH)-vitamin D
PTH
25(OH)-vitamin D
DBP
Mitochondria:
CYP27B1
Megalin
Calcium
1,25(OH)2-vitamin D
Target organs
FIGURE 5. Vitamin D metabolism. Vitamin D can either be synthesized in the skin or absorbed from our diet.
It is then transported to the liver where it undergoes 25-hydroxylation by one of two hepatic enzymes (CYP27A1
or CYP2R1). During transport through the circulation, vitamin D is bound to a carrier protein (DBP). The
25(OH)-vitamin-D-DBP complex passes the glomerular filter and is scavenged from the primary urine by the
apical megalin receptor of the proximal tubule. Here, 25(OH)-vitamin D is converted to the active vitamin D
metabolite 1,25(OH)2-vitamin D. DBP, vitamin D binding protein.
component of yeast and fungal membranes, a different precursor substance had to exist in animal skin. Again, it was
Windaus and colleagues who identified 7-dehydrocholesterol
as the provitamin in porcine skin, which is converted to vitamin D3 (cholecalciferol) under irradiation (1165). After these
discoveries, industrially produced vitamin D has rapidly been
used in medical applications and as a food fortification. To-
214
day, the main portion of dietary vitamin D ingestion in the
United States stems from fortified dairy products (150).
2. Intestinal vitamin D absorption
To exert its antirachitic effects, dietary vitamin D has to be
absorbed into our circulation. Early everted gut sack exper-
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iments demonstrated that this process has linear, nonsaturable, and passive kinetics, suggesting that no specific carrier mechanism for vitamin D is in place (484). These observations were later replicated in in vivo models (483).
Absorption is highest in the proximal and mid small intestine (484). Since vitamin D is fat soluble, its absorption
mechanism is similar to that of dietary lipids. In an aqueous
media, vitamin D aggregates in micelle-like structures
(735). Its absorption is aided by the secretion of bile acids,
which is underscored by the observation that patients suffering from cholestasis can present with vitamin D deficiency and develop bone disease, such as osteomalacia or
osteoporosis (245, 445, 563, 721, 894, 953, 1018, 1080).
Apart from bile salts, formation of mixed micelles containing monoglycerides and free fatty acids represents another
factor that aids in vitamin D absorption (884, 1082; contested by Ref. 483). These substances increase micelle size,
which promotes the solubilization of vitamin D, thereby
increasing uptake (884). Clinically, pancreatic insufficiency, causing an impairment of triglyceride breakdown
through insufficient lipase secretion, leads to decreased vitamin D absorption (1080, 1127). This is a particular problem in cystic fibrosis patients, who often develop pancreatic
and concomitant vitamin D insufficiencies (33, 230, 625,
678, 924).
Following uptake into the enterocyte, vitamin D is packed
into chylomicrons and secreted into the lymph (288, 953). It
has been demonstrated that after intestinal administration
of radioactive labeled vitamin D3, up to 90% of the recovered radioactive tracer was associated with the chylomicron
fraction of the collected intestinal lymph (288). Furthermore, patients suffering from the autosomal recessive chylomicron retention disease (Anderson disease; OMIM
246700), which causes impairment of chylomicron processing and secretion in the enterocyte, can present with insufficient levels of fat-soluble vitamins, such as vitamins D and
E (535). The chylomicron remnants, which include vitamin
D, are then scavenged by the liver after the lymph has entered the circulation through the thoracic duct (287, 407).
However, it has been demonstrated in vitro and in vivo in
hepatectomized and normal rats that vitamin D can directly
transfer from chylomicrons to a vitamin D binding protein
(DBP; see sect. IVA5) in the blood plasma (286, 288). It has
therefore been suggested that at least a fraction of hepatic
vitamin D uptake is mediated through DBP rather than
chylomicrons (286; contested by Ref. 407). In the liver,
vitamin D is then hydroxylated at its 25 position to
25(OH)-vitamin D. The metabolism of vitamin D will be
the subject of a later section.
The intestinal absorption of 25(OH)-vitamin D has been
investigated by many groups, with the aim of optimizing
vitamin D administration in a therapeutic context by bypassing the first metabolic step in the liver (219, 245, 287,
445, 645, 721, 1018, 1019, 1127). In general, enteric up-
take of 25(OH)-vitamin D is more effective than that of
vitamin D, which is partially attributable to a comparably
lower dependency on bile acid secretion (219, 245, 287,
645, 1018, 1019). The observation that patients with
cholestasis still absorb 25(OH)-vitamin D effectively,
whereas vitamin D absorption is impaired, corroborates
this hypothesis (1018). There is some controversy with regard to the transport of 25(OH)-vitamin D following its
absorption (287, 701, 1019). It has been argued that it may
be transported predominantly in the protein fraction of the
lymph (287), i.e., not in chylomicrons, or that it is directly
absorbed into the portal blood (701, 1019).
3. Cutaneous vitamin D synthesis
Cutaneous synthesis is our second source of vitamin D.
Cutaneous production depends on exposure to UVB (290 –
315 nm) light (FIGURE 5). The UVB photons convert 7-dehydrocholesterol, which is located in the plasma membrane
of keratinocytes, to previtamin D3 (1117–1120). This dependency on sunlight causes a seasonal variation in vitamin
D3 production, with synthesis being low during the winter
months when the radiation angle of the sun flattens (187,
478, 538, 1035, 1156). In consequence, changes in latitude
result in similar variability in production. Further factors
that decrease production include pigmentation of the skin
and application of sunscreen (209, 723), whereas an increase in altitude promotes production (478). Following
conversion from 7-dehydrocholesterol, previtamin D3
isomerizes to vitamin D3 (479). The isomerization process
is temperature dependent and fairly slow (479, 1117). It has
been calculated that the half-life for the formation of vitamin D3 is ⬃2.5 h (1085). Interestingly, in vitro experiments
conducted in isotropic medium demonstrated that the
isomerization rate was 10 times slower than in in vivo experiments (1085). This was later attributed to the fact that
amphiphatic interactions with phospholipids of the cell
membrane stabilize the previtamin D3 conformer which
then isomerizes to vitamin D3 (1086). The cellular microenvironment of the reaction thus greatly optimizes the isomerization to vitamin D3. After its synthesis, vitamin D3 is
bound to DBP and carried though the bloodstream to its
target organs. Observations made in patients indicate that
the vitamin D3 plasma levels peak ⬃2 days after sunlight
exposure, which is due to the slow isomerization rate in the
skin (7).
4. Vitamin D metabolism and its regulation
Irrespective of the source (endogenous or exogenous), vitamin D is metabolized in the liver to 25(OH)-vitamin D
(FIGURE 5). Evidence for the existence of biologically active
vitamin D metabolites emerged in the 1960s (686, 799).
25(OH)-vitamin D was identified by means of injecting rats
with radiolabeled vitamin D and subsequent silicic acid column chromatography of lipid extracts from serum and var-
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SASCHA KOPIC AND JOHN P. GEIBEL
ious tissues (686). Chromatography revealed the presence
of a vitamin D metabolite in serum, liver, bone, and feces in
rats and in human serum (256, 686). Furthermore, it was
shown that the vitamin D metabolite was biologically active
by reverting rickets in diseased animals and by increasing
intestinal calcium transport and bone metabolism (686,
752). The metabolite was characterized as 25(OH)-vitamin
D and subsequently successfully synthesized (111, 112). It
was soon recognized that hepatectomized rats were not effectively converting vitamin D to 25(OH)-vitamin D, suggesting that the liver was the main organ responsible for
25-hydroxylation (864, 865). The ability of the liver to
metabolize vitamin D was also confirmed in perfused livers
and tissue homogenates (490). Historically, a long controversy existed with regard to the subcellular localization of
the enzyme that hydroxylates vitamin D (25-hydroxylase)
in the liver, as enzyme activity was observed in both mitochondrial and microsomal fractions of liver homogenates
(93, 102, 103, 258, 696, 1053). Extensive investigations
indicated that both the microsomal and mitochondrial 25hydroxylase are members of the cytochrome P-450 family
(102–104, 696, 835). Both fractions demonstrated distinct
enzymatic kinetics. The mitochondrial enzyme was characterized as a low-affinity high-capacity enzyme, whereas the
microsomal fraction displayed high-affinity low-capacity
characteristics (103, 104, 356, 696).
The mitochondrial 25-hydroxylase (CYP27A1) was first
purified to homogeneity from rabbit liver mitochondria
(235). It was demonstrated that this cytochrome P-450 was
not specific to vitamin D and could also hydroxylate other
substrates, most notably cholesterol (27-hydroxylation),
which represents an important step in the formation of bile
acids (235). In retrospect, CYP27A1 had been purified 4
years earlier; however, only the 27-hydroxylation of cholesterol had been investigated and its effects on vitamin D
had remained obscure (1162). Subsequently, the enzyme’s
cDNA was cloned from rabbit, rat, and human, and its dual
role in vitamin D and steroid conversion was confirmed (26,
149, 1048, 1103). The full gene structure was identified a
few years later (646). Although no crystal structure of the
enzyme is available to us, a homology model based on other
CYP family members has been proposed (874). In the liver,
CYP27A1 is expressed on the mRNA level in hepatocytes,
endothelial, stellate, and Kupffer cells (1078). Furthermore,
CYP27A1 expression was confirmed in a variety of other
tissues, including duodenum, adrenal glands, kidney, lung,
vascular endothelium, brain, retina, skin, muscle, and osteoblasts; however, their potential contribution to vitamin
D metabolism remains unclear (26, 51, 135, 184, 370, 383,
508, 640, 898, 979). Interestingly, an extrahepatic conversion of vitamin D had been suggested previously to occur in
the kidney and the intestine (1097). Marked differences in
CYP27A1 activity can be observed between males and females. For example, CYP27A1 enzyme activity and mRNA
expression were demonstrated to be increased in female rats
216
(933, 1078). Higher expression in females was also confirmed in biopsy samples from human subjects (370). A
regulation via sex hormones may underlie this phenomenon, as injection of estradiol was shown to induce
CYP27A1 activity (933). Interestingly, seasonal variations
in expression were also observed, which may represent a
confounding factor for decreased 25(OH)-vitamin D levels
during the winter months (370).
It should be noted that CYP27A1 can also hydroxylate
vitamin D3 at other positions (402, 950). These include
positions 27 and 26; however, the ratio for 25-:27-:26hydroxylation has been estimated to be only 100:15:3,
which demonstrates that 25-hydroxylation of vitamin D3 is
the most essential reaction catalyzed by the enzyme (950).
More importantly, CYP27A1 can also use its own product
25(OH)-vitamin D as a substrate to further act as a 1␣hydroxylase and produce the hormonally active form of
vitamin D, namely, 1,25(OH)2-vitamin D (50, 51, 950). As
will be discussed later, this reaction is normally catalyzed in
the kidney by another CYP family member (CYP27B1). At
the moment, it is not clear what the physiological significance of 1␣-hydroxylation by CYP27A1 is.
Mutations in CYP27A1 cause the autosomal recessive disorder cerebrotendinous xanthomatosis (CTX; OMIM
213700). The disease was first described in 1937 and is
characterized by cholestanol deposits that are most prominent in tendons, especially the Achilles tendon, the brain,
and the lung. Patients present with progressive neurologic
defects, atherosclerosis, and cataracts and commonly suffer
from diarrhea. The inadequate bile acid synthesis was first
noted in 1974 by Setoguchi et al. (993). Shortly after the
cDNA of CYP27A1 was cloned, it was demonstrated that
mutations of this enzyme were responsible for CTX (148).
In agreement with the dual role of CYP27A1, CTX patients
also suffer from osteoporosis, low 25(OH)-vitamin D levels, and impaired intestinal calcium absorption (83, 320).
Three CYP27A1 mutations that are known to cause CTX
and still lead to protein expression were recreated in vitro,
and enzymatic activity was assayed. Depending on the expression system, these mutants showed lower or higher 25hydroxylation activity than the wt enzyme, which led the
authors to questions the enzyme’s role in vitamin D metabolism (403). It should be considered that 1) many more
(⬃38) mutations underlying CTX exist, 2) by far not all
patients exhibit disturbances in bone or vitamin D homeostasis, and 3) the investigated mutant enzymes may only
cause disturbances in cholesterol, rather than vitamin D
metabolism (83, 320, 403). In the light of these limitations,
it should be questioned whether CTX represents an apt
model system to evaluate CYP27A1 in the context of vitamin D metabolism.
With the introduction of novel genetic tools, a cyp27a1
(⫺/⫺) mouse was created in 1998 (918). However, the phe-
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notype of CTX could not be reproduced, albeit fecal bile
acid content was markedly decreased (918). Rather surprisingly, serum 25(OH)-vitamin D levels were increased in
(⫺/⫺) animals, which either pointed to compensatory upregulation of other, maybe microsomal, enzymes or a noninvolvement of cyp27a1 in vitamin D metabolism in the
mouse (918). The (⫺/⫺) mouse model was later characterized in more detail, but no new conclusions with regard to
vitamin D were drawn (902).
Another caveat that needs to be considered when assessing
the physiological importance of CYP27A1 for vitamin D
metabolism is that the enzyme cannot 25-hydroxylate dietary vitamin D2 (402). This observation underlines the
necessity for another 25-hydroxylase which physiologically
metabolizes vitamin D2. The microsomal 25-hydroxylase
was a clear candidate for this process; however, it has only
recently received more extensive scientific attention.
The molecular identity of the microsomal 25-hydroxylase
was unclear, until it was unmasked by Cheng et al. (184) in
2003 as CYP2R1. CYP2R1 is expressed on the mRNA level
in a plethora of tissues, but most prominently in the liver
and testes (184). As illustrated by a recent investigation, the
testes may play a role in calcium homeostasis. Patients with
testiculopathy (and concomitant lower CYP2R1 expression) were shown to have decreased 25(OH)-vitamin D levels and osteoporosis (333). The protein’s crystal structure
has recently been resolved in complex with vitamin D3
(1045). Unlike CYP27A1, CYP2R1 has been shown to 25hydroxylate both vitamin D2 and vitamin D3, which may be
a solution to the enigma of vitamin D2 metabolism (184).
The physiological relevance of CYP2R1 was underscored
by the characterization of a patient who presented with low
25(OH)-vitamin D levels and was shown to have a transition mutation in the CYP2R1 gene (183). Furthermore, a
remarkable large-scale study has recently tried to establish a
correlation between 25(OH)-vitamin D status and the genotype of 33,996 individuals. It was found that lower
25(OH)-vitamin D levels correlated with variants in the
CYP2R1 gene (1146) (a smaller study came to similar conclusions, Ref. 139). This represents an outstanding finding,
as the influence of CYP27A1 mutations on 25(OH)-vitamin
D production is far less conclusive. Given the disproportionate quantity of scientific data, it is at this moment difficult to evaluate the relative contribution of each 25-hydroxylase to vitamin D metabolism, but more and more
evidence for the importance of CYP2R1 is accumulating.
The regulation of 25-hydroxylation has been the subject of
controversy in the past, which is partially due to the fact
that multiple enzymes may metabolize vitamin D, and that
it was challenging to experimentally discriminate between
these enzyme entities. Thus evidence which supports (91,
92, 741, 1078) and questions (258, 1097) the existence of
25-hydroxylase regulation can be found. A detailed analysis
of these observations is beyond the scope of this review, but
the most recent investigation should be considered in more
detail, given the advances in our experimental repertoire: it
has been demonstrated in the rat that 1,25(OH)2-vitamin D
can downregulate hepatic CYP27A1 transcription with a
concomitant reduction in enzyme activity (1078). These
observations strongly corroborate the hypothesis that the
25-hydroxylation step is subjected to negative-feedback
regulation. The exact mechanism of this regulatory mechanism remains elusive, especially since the CYP27A1 gene is
not under control of a VDRE (369, 988, 1078).
Following its synthesis, 25(OH)-vitamin D binds to DBP
and is transported to the kidney, where it undergoes further
conversion to 1,25(OH)2-vitamin D, the hormonally active
form of vitamin D (FIGURE 5). Historically, 1,25(OH)2vitamin D was first identified in the nuclei of intestinal cells
as an uncharacterized vitamin D metabolite (429, 634). The
biological activity of the metabolite was determined to be
much higher than that of vitamin D, and it was eventually
isolated and identified as 1,25(OH)2-vitamin D (480, 586,
633, 800). 1,25(OH)2-vitamin D is up to 10 times more
potent than vitamin D. The importance of the kidney for the
synthesis of 1,25(OH)2-vitamin D was soon discovered, as
nephrectomized rats were neither able to convert 25(OH)vitamin D, nor absorb calcium effectively (339, 458).
Briefly thereafter, patients with chronic renal insufficiency
were shown to lack the capability to metabolize 25(OH)vitamin D, which further established the role of the kidney
as the major conversion site (724). The impairment of
1,25(OH)2-vitamin D synthesis in the course of chronic
renal deficiency is a cofactor in the development of renal
osteodystrophy, a bone mineralization deficiency due to
deranged mineral balance. Of note, the kidney is not the
exclusive site of CYP27B1 expression. 1␣-Hydroxylase has
been detected in a variety of other tissues, including the
placenta, decidua, skin, brain, vascular endothelium, pancreas, colon, but also in monocytes and dendritic cells (451–
453, 603, 1204 –1206). The role of extrarenal 1,25(OH)2vitamin D synthesis is not entirely clear. Beyond modulating
calcium homeostasis, vitamin D has been shown to have immunomodulatory and antiproliferative effects (see sect. IVA6).
Given these observations, it has been speculated that extrarenal local 1,25(OH)2-vitamin D production and paracrine secretion may represent important factors for the maintenance
of the “barrier function” in these tissues (451, 453).
In the kidney, 1␣-hydroxylation of 25(OH)-vitamin D
takes place in the inner mitochondrial membrane of the
epithelial cells of the proximal tubule (393).
The cellular uptake mechanism of 25(OH)-vitamin D by the
tubule cells will be subject of a later section. The 1␣-hydroxylase responsible for the conversion is also a member of
the cytochrome P-450 family (CYP27B1) and was first
cloned in 1997 by St-Arnaud et al. from rat cDNA (1034).
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The human homolog was cloned shortly thereafter (349,
748). Interestingly, mapping of the CYP27B1 gene revealed
that the locus was identical to the gene locus of the autosomal recessive disorder pseudo vitamin D deficiency rickets, type 1A (PDDR1A, OMIM 264700) (1034). PDDR is
characterized by low serum calcium, secondary hyperparathyroidism, and low 1,25(OH)2-vitamin D levels (869) (of
note, the original article wrongly suggests an autosomal
dominant inheritance pattern). Patients can exhibit rickets
or osteomalacia due to increased mobilization of calcium.
The gene locus of PDDR1A had been mapped by linkage
analysis; however, before the cloning of CYP27B1, it was
not clear whether a defect in the enzyme itself or disturbances in its regulation were responsible for the disease
(617, 618, 1223). The clear-cut phenotype of PPDR1A effectively illustrates the pivotal role of CYP27B1 and the
lack of redundancy at this essential step of vitamin D metabolism. The characteristic features of PDDR1A can essentially be emulated if cyp27b1 is knocked out in a mouse
model (238, 822). The serum calcium levels and the secondary hyperparathyroidism can be normalized if these animals
are fed a high-calcium, phosphorus, lactose diet, albeit bone
growth remains impaired (239).
The activity of CYP27B1 is subjected to tight hormonal
regulation. The key regulator of 1,25(OH)2-vitamin D synthesis is PTH, which is secreted by the parathyroid glands in
response to low serum calcium concentrations in an effort
to increase calcium uptake and release bone calcium into
the circulation. The regulation of calcium homeostasis by
the parathyroid glands and the CaSR will be covered in
subsequent sections (see sect. IV, B and D). Apart from this
stimulatory input, CYP27B1 is under negative control by its
own product. 1,25(OH)2-vitamin D represses CYP27B1 on
a transcriptional level (116, 125, 443, 591, 764). Although
studies in (⫺/⫺) animals suggest that the VDR is essential
for autoinhibition to take place, the promoter region of
CYP27B1 does not include a canonical VDRE (125, 591,
764). It is thus most likely that the transcriptional regulation through 1,25(OH)2-vitamin D is indirect (125,
591). Alternatively, so-called E-box-type elements were
recently proposed to act as negative VDREs (575). Moreover, CYP27B1 can be directly regulated by the local
calcium concentrations. High extracellular calcium inhibits 1,25(OH)2-vitamin D synthesis, whereas low calcium
concentrations induce its production (109). It has been proposed that changes in calcium modulate VDR expression
and thereby the sensitivity of cell to the local negative feedback by 1,25(OH)2-vitamin D (702). Some evidence suggests that the CaSR mediates the regulatory effects of calcium on CYP27B1 activity (FIGURE 8) (702). Other factors
that regulate CYP27B1 activity include fibroblast growth
factor 23 (FGF23), calcitonin, prolactin, sex steroids (at
least in avian species), and phosphate (716, 913, 1067,
1068, 1217).
218
Apart from regulating the synthesis of the vitamin D
metabolites, our body also tightly controls their degradation. The first step of vitamin D catabolism is 24-hydroxylation, which is carried out by the mitochondrial
enzyme CYP24A1. The primary site for vitamin D catabolism is the kidney, but CYP24A1 is also strongly expressed
in other extrarenal tissues, such as the intestine, osteoblasts,
keratinocytes, prostate, placenta, brain, and heart (11, 181,
796). CYP24A1 can hydroxylate both 25(OH)-vitamin D
and 1,25(OH)2-vitamin D, thereby creating 24,25(OH)2vitamin D and 1,24,25(OH)3-vitamin D, respectively (14).
24-Hydroxylation is followed by a series of oxidation/reduction reactions which finally yield the excretable product
calcitroic acid (703, 893). At least in the proximal tubule of
the kidney, the regulation of CYP24A1 is reciprocal to that
of CYP27B1. 1,25(OH)2-vitamin D upregulates CYP24A1,
thereby stimulating its own breakdown, whereas PTH inhibits
CYP24A1 (37, 180, 812, 1069, 1222). While 1,25(OH)2-vitamin D increases the transcription of CYP24A1 (the gene has
two upstream VDREs), PTH exerts its inhibitory effects by
decreasing CYP24A1 mRNA stability (1222). In osteoblastic
and distal convoluted tubule cell lines, PTH has synergistic
effects with 1,25(OH)2-vitamin D in inducing CYP24A1 (37,
1189). In analogy to CYP27B1, CYP24A1 is further regulated
by FGF23, calcitonin, and phosphate (361, 513, 1175).
5. Vitamin D transport and cellular uptake
The hypothesis that vitamin D may be bound to a carrier
substance in serum was first expressed in the late 1950s,
when it was demonstrated that the alpha fraction of human
serum had high anti-rachitic properties (1079). It was later
shown that DBP is identical to the group specific component
(Gc) protein, which had been characterized independently
by Hirschfeld and colleagues around the same time (236,
464, 465). DBP (⬃58 kDa; 458 amino acids) is synthesized
in the liver and is closely related to albumin and ␣-fetoprotein, which are all derived from the same ancestral gene
(221, 875, 1158, 1187, 1188). The crystal structure of DBP
has been resolved at a resolution of 2.3 Å in complex with
25(OH)-vitamin D (1121). DBP can bind vitamin D and all
of its metabolites (408). There is, however, a difference in
the relative affinity for the vitamin D steroids, with the
affinity for 25(OH)-vitamin D being highest (Kd ⬃10⫺8 M),
followed by 1,25(OH)2-vitamin D and vitamin D (Kd
⬃10⫺7 M) (408). In humans, vitamin D2 and D3 metabolites are bound with equal affinity to DBP (406). Given its
long plasma half-life, 25(OH)-vitamin D is also measured
as the primary clinical parameter to assess the vitamin D
status of patients. Although the plasma concentration of
DBP is ⬃4 – 8 ␮M, only ⬍5% of the binding sites are occupied by vitamin D sterols (408, 409). DBP has a very fast
turnover rate. It has been estimated that up to 28% of DBP
are replaced every day (557). This turnover entails a high
demand for synthesis output by the liver. In consequence,
patients with liver disease demonstrate lower DBP and total
vitamin D levels than healthy subjects (97, 409). This rela-
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GASTRIC ACID, CALCIUM ABSORPTION, AND BONE HEALTH
tionship is reversed during pregnancy, when the levels of
DBP and vitamin D sterols are increased (97, 409, 609, 685,
892). Of note, DBP is not the exclusive carrier substance for
vitamin D sterols. Albumin and lipoproteins were shown to
transport a fraction (⬃15%) of vitamin D (1015).
Given its multitude of functions, DBP has been regarded as an
essential protein. Indeed, an analysis of over 80,000 human
serum samples showed that DBP was present in all of them,
which led to the hypothesis that deleterious mutations of
DBP were lethal (213). Rather surprisingly, DBP (⫺/⫺)
mice thrive well with growth curves identical to their
littermates, although their 25(OH)-vitamin D and
1,25(OH)2-vitamin D levels (total) are severely decreased
(937). If challenged with a low vitamin D diet, the DBP
(⫺/⫺) animals develop secondary hyperparathyroidism,
leading to defects in bone mineralization (937). It is unclear, however, to what extent albumin and lipoproteins
compensate for the loss of the primary vitamin D carrier
substance.
Not much is known about the cellular uptake of vitamin D.
As all other steroids, free vitamin D can passively diffuse
through the plasma membrane because of its lipohilic nature. However, due to the high concentration of DBP and its
affinity towards vitamin D ligands, only a fraction of vitamin D circulates in the free form. For example, 0.003% of
25(OH)-vitamin D is transported as an unbound sterol in
serum, which raises the question whether passive diffusion
represents a sufficient uptake pathway (98). At least
25(OH)-vitamin D has been proposed to be delivered to
the proximal kidney tubule for further conversion to
1,25(OH)2-vitamin D by a different and remarkable mechanism. Rather than diffusing passively, it has been proposed
that the 25(OH)-vitamin D-DBP complex passes the glomerular filter and is endocytosed by the epithelial cell of the
proximal tubule (FIGURE 5) (805). The endocytotic process
is mediated by megalin (aka gp330), which is a multifunctional clearance receptor on the luminal membrane. Mice
lacking functional megalin were shown to lose DBP and
vitamin D in the urine and develop vitamin D deficiency
(805). A kidney specific megalin (⫺/⫺) animal was recently
created, and the observations made in the global (⫺/⫺)
animal, which had a very low perinatal survival rate of 2%,
could essentially be replicated (643). Furthermore, defects
in cubulin, a membrane protein that colocalizes with megalin, cause a similar phenotype (806). The experimental data
are further supported by clinical observations made in patients suffering from Fanconi syndrome. Fanconi syndrome is a global reabsorption deficiency of the proximal
tubule, which can develop as a result of heavy metal
poisoning and/or drugs or may have inherited causes.
These patients were shown to lose DBP in their urine,
which may reflect the inability of the tubule cell to endocytose DBP (805, 1076, 1100). A similar endocytotic
uptake mechanism has been proposed for mammary
cells, which can also convert 25(OH)-vitamin D to
1.25(OH)2-vitamin D(926).
6. Cellular effects of vitamin D
The cellular effects of 1,25(OH)2-vitamin D can be categorized into two major pathways, which are defined by their
respective speed of onset: 1) slow genomic responses and
2) rapid nongenomic responses. Both pathways require
binding of 1,25(OH)2-vitamin D to its intracellular receptor, the VDR (503, 787). The VDR (NR1I1, nuclear receptor subfamily 1, group I, member 1) belongs to the superfamily of nuclear receptors, which amongst others also
includes the estrogen, testosterone, or glucocorticoid receptors. VDR was first identified in the late 1960s in the chromatin fraction of chick intestinal mucosa, where
1,25(OH)2-vitamin D increases the rate of intestinal calcium uptake (430). The 427-amino acid protein (molecular
mass 48.3 kDa) was cloned in 1988 by Baker et al. (59). The
crystal structure of the VDR is available to us with
1,25(OH)2-vitamin D bound to the receptor’s ligand binding domain (914).
A) GENOMIC EFFECTS. Following ligand binding, nuclear receptors typically act as transcription factors and induce or repress the transcription of certain target genes. In the case of
VDR, 1,25(OH)2-vitamin D binds to the receptor, which
subsequently heterodimerizes with the retinoid X receptor
(RXR) (FIGURE 3) (1027, 1083). The VDR-RXR complex
then interacts with a VDRE in the 5= promoter region of the
regulated gene resulting in transactivation. Alternatively, it
has been proposed that VDR can bind to the VDRE before
ligand binding occurs (920).
I) Intestine. The intestine is one of the primary target sites of
1,25(OH)2-vitamin D. 1,25(OH)2-vitamin D upregulates
the expression of intestinal TRPV6, calbindin-D 9k, and
PMCA, which are canonically regarded to mediate the process of transcellular calcium absorption (FIGURE 3) (see sect.
IIIA). Furthermore, 1,25(OH)2-vitamin D may modulate
calcium uptake through the paracellular route (see sect.
IIIB). By increasing the amount of absorbed calcium,
1,25(OH)2-vitamin D directly elevates serum calcium levels. This represents one of the final links in the regulatory
chain of calcium homeostasis which starts with sensing of
low calcium levels by the parathyroid gland and ends with
increased synthesis of 1,25(OH)2-vitamin D by the kidney.
The significance of 1,25(OH)2-vitamin D for intestinal calcium absorption is illustrated by 1,25(OH)2-vitamin D-deficient patients, which absorb up to 80% less calcium from
their meal compared with healthy individuals (996). Although similar results were obtained in animal models,
probably one of the more illustrative observations has been
made in VDR-deficient animals (825, 1110). Deletion of
VDR correlates with a massively impaired capacity to absorb intestinal calcium resulting in low plasma calcium levels and hyperparathyroidism (1110). This phenotype is re-
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219
SASCHA KOPIC AND JOHN P. GEIBEL
versible by intestine-specific expression of VDR on a VDR
(⫺/⫺) background (712, 1181). Thus animals that lack the
VDR, except in the intestine, present with normal serum
calcium and PTH levels (1181). In conclusion, it is unquestionable that 1,25(OH)2-vitamin D and its receptor are the
key regulators of intestinal calcium absorption.
II) Bone. Some evidence exists that 1,25(OH)2-vitamin D
may have direct influences on the formation of bone (FIGURE 4). The VDR is expressed in osteoblasts, osteoclasts,
and chondrocytes (115, 533, 623, 730). Most of the direct
effects of 1,25(OH)2-vitamin D are thought to be mediated
by osteoblasts. 1,25(OH)2-vitamin D has been shown to
promote osteoblast differentiation from mesenchymal stem
cells and to regulate the synthesis of various osteoblast proteins, such as osteocalcin, alkaline phosphatase, collagen I,
osteopontin, and RANKL (45, 78, 79, 665, 709, 804, 870,
925). In general, however, the effects of 1,25(OH)2-vitamin
D on osteoblast maturation and protein synthesis are very
pleiotropic and depended on the duration of the exposure
and the differentiation stage at which the osteoblast is exposed (817). Effects of 1,25(OH)2-vitamin D on the mineralization of bone have also been reported. For example, an
increase in the mineralization of extracellular matrix was
observed following concomitant 1,25(OH)2-vitamin D and
vitamin K exposure (599, 745). Yet, the overall direct influence of 1,25(OH)2-vitamin D on bone metabolism is somewhat obscure. This is illustrated by VDR-deficient animals.
Naturally, these animals develop rickets and osteomalacia
due to impaired intestinal calcium absorption. If the animals are, however, maintained normocalcemic, the skeletal
phenotype is completely rescued (21, 661). Although, these
observations question the importance of 1,25(OH)2-vitamin D as a direct regulator of bone metabolism, a subsequent investigation by Panda et al. (821) came to a different
conclusion. The authors reported that osteoblast numbers,
mineral apposition rates, and overall bone volume were
reduced in normocalcemic (rescue diet) cyp27b1/VDR
(double ⫺/⫺) animals, suggesting that the 1,25(OH)2-vitamin D system is necessary for intact bone formation (821).
The reason for these discrepant findings is generally unclear; however, differences in the length of exposure to the
rescue diet have been put forward as a possible cause (821).
For a more detailed review of the effects of vitamin D on
bone, please refer to References 1033, 1114.
III) Kidney. The kidney is not only the major site of
1,25(OH)2-vitamin D synthesis (see above), but also represents a vitamin D target organ. The kidney acts as key
regulator of calcium homeostasis by changing the amount
of calcium that is reabsorbed from the primary urine. The
majority of the calcium that is filtered through the glomerulus is reabsorbed in the proximal tubule through the paracellular space, with the amount of absorbed calcium gradually decreasing along the nephron. Fine regulation of calcium absorption occurs in the distal tubule and collecting
220
duct. The mechanism by which the distal tubule conducts
transcellular calcium absorption is highly analogous to the
proximal intestine. The epithelial cells of the distal tubule
express TRPV5 (the “sister” channel of TRPV6) as the apical calcium entry channel, calbindin-D 28k and NCX1 and
PMCA1b as basolateral calcium extruders (203, 474, 610,
837, 922). In analogy to the intestine, 1,25(OH)2-vitamin D
upregulates the majority of these proteins in an effort to
increase renal calcium reabsorption (203, 474, 610, 837,
922).
B) NONGENOMIC EFFECTS.
In contrast to the genomic effects of
1,25(OH)2-vitamin D, which have been known for decades,
the rapid cellular responses have only recently received
more scientific attention. While the transcriptional events of
1,25(OH)2-vitamin D take place on a time scale of a few
hours to days, the rapid nongenomic responses occur within
minutes of exposure. One of the first evidences, which in
retrospect can be attributed to a nongenomic response, was
the observation made in 1941 by Selye that intraperitoneal
injection of steroids had an anesthetic effect (990). Interestingly, the rapid responses to 1,25(OH)2-vitamin D also require the presence of the VDR, as these responses cannot be
elicited in VDR deficient animals (503, 787). It should be
noted that other proteins such as 1,25(OH)2-vitamin-DMARRS have also been suggested as candidates for a membrane associated 1,25(OH)2-vitamin D receptor (782). Attempts at identifying the subcellular localization of the
VDR in the rapid response context have yielded that VDR is
also present in plasma membrane invaginations, the socalled caveolae (503, 802). These VDR-containing membrane microdomains have been identified in multiple tissues, including the intestine, kidney, and lung, and are identified by coexpression of caveolin-1, which is used as a
marker protein for caveolae (503, 802). Functionally, not
many rapid response effects of 1,25(OH)2-vitamin D have
been characterized. For example, it has been demonstrated
that 1,25(OH)2-vitamin D can influence ion channel gating
in osteoblasts, modulate the contraction of cardiomyocytes,
lead to insulin secretion in pancreatic ␤-cells via elevating
intracellular calcium, and cause photoprotection in keratinocytes (273, 540, 1088, 1200, 1216). In the intestine, the
phenomenon of transcaltachia (see sect. IIIC) has been attributed to rapid actions of 1,25(OH)2-vitamin D (801).
In addition to 1,25(OH)2-vitamin D, the VDR also binds
the secondary bile acid lithocholic acid (704). Secondary
bile acids are bile acids that have been metabolized by the
intestinal gut flora. Lithocholic acid is toxic and has been
implicated to play a role in intestinal carcinogenesis (601).
It has been suggested that the VDR may serve as a secondary bile acid sensor and induce lithocholic acid breakdown
through CYP3A activation (537). The noncanonical VDR
stimulation by lithocholic acid may thus serve as an autoprotective mechanism (704). Apart from inducing CYP3A,
lithocholic acid has been demonstrated to increase expres-
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GASTRIC ACID, CALCIUM ABSORPTION, AND BONE HEALTH
sion of TRPV6 in intestinal cell lines, which corroborates its
role as a physiological VDR agonist (517).
We are far from understanding the full spectrum of the
effects of 1,25(OH)2-vitamin D, and unfortunately, the
scope of this review does not allow a detailed analysis of
these processes. In general, our knowledge concerning the
physiological role of 1,25(OH)2-vitamin D is massively expanding beyond the horizon of calcium homeostasis. Evidence is accumulating that 1,25(OH)2-vitamin D can influence the renin-angiotensin-aldosterone system and may
thereby act as a regulator of blood pressure (660, 662,
1219). Furthermore, low vitamin D status is associated with
an increased incidence of colorectal, ovarian, and breast
cancer(363, 364, 642, 984). Vitamin D also acts as a potent
modulator of the immune response and cell proliferation.
B. PTH
Experiments dating back to the beginning of the 1900s have
demonstrated that surgical removal of the parathyroid
gland results in tetany. It was recognized very early that
administration of calcium could ameliorate or prevent the
manifestation of tetany, and it was subsequently concluded
that the parathyroid glands play an important role in calcium metabolism (690, 826, 939). In 1925, extracts from
the parathyroids were for the first time shown to control
tetany in dogs (216). This finding marked the discovery of
PTH, which is one of the three prime hormones regulating
calcium homeostasis.
1. Production and secretion
PTH is a 84-amino acid peptide hormone produced in the
parathyroid glands. Its amino acid sequence was first established in 1970 in bovine (126, 788). Cloning of the human
cDNA followed a decade later (440). The PTH gene encodes a 115-amino acid precursor hormone (preproPTH),
which is enzymatically cleaved in a two step process to its
mature 84-amino acid secreted form (565). The NH2-terminal prepro-signaling sequence is necessary for correct
hormone processing and trafficking (340, 546). This
knowledge is mainly founded on truncation studies and
observations made in patients with mutations in the PTH
gene, which can result in familial isolated hypothyroidism,
a disorder characterized by hypocalcemia and low PTH
levels (38, 340, 828, 1055). For example, a well-characterized T-to-C point mutation in the prepro signaling sequence
that causes FIH leads to accumulation of the precursor hormone in the ER (243, 546). The impaired processing triggers ER stress, ultimately apoptosis and PTH insufficiency
(243).
Following its cleavage to mature PTH, the hormone is
stored in secretory vesicles and released into the circulation
in response to low plasma calcium. The regulation of PTH
secretion is extremely tight, given the body’s need to maintain the calcium concentration within a narrow window
(1.1–1.3 mM). Small alterations in calcium homeostasis can
have deleterious effects, for example, on the excitability of
neurons and muscles. The low plasma half-life of PTH of ⬍5
min allows for a precise regulation of this balance (95). To
achieve controlled and rapid on-demand secretion of PTH, the
parathyroid is equipped with an ultrasensitive extracellular
CaSR, which constantly monitors the plasma calcium levels
and triggers intracellular signaling events and PTH release
upon imminent drops in calcium levels (FIGURE 6) (see sect.
IVD). PTH is further regulated on a transcriptional level by
1,25(OH)2-vitamin D, creating a negative-feedback loop
(154, 930, 931).
2. PTH1R
PTH exerts its physiological effects via activation of a membrane-bound GPCR, the parathyroid hormone receptor
type 1 (PTH1R) (PTH2R is mostly expressed in the CNS
and tissues that are not involved in calcium handling and
will thus not be reviewed). Of note, PTH1R is not exclusively located at the plasma membrane but can also localize
to the cell nucleus. The physiological significance of nuclear
PTH1R is currently unclear but may represent a novel signaling paradigm for the actions of PTH (830, 856, 857,
1155).
Full-length PTH is not required to activate PTH1R. The
NH2-terminal domain of PTH mediates most of the physiological effects of the hormone and is responsible for binding in the ␣-␤-␤␣ binding fold of PTH1R (861). This is why
clinically PTH(1–34) is used as a PTH analog with identical
biological activity (753, 867, 919, 1094). Conversely, NH2terminal truncation of PTH(1–34) to PTH(2–34) changes
the characteristics to a partial receptor agonist, whereas
further truncation to PTH(3–34) results in loss of biological
activity (1094).
PTH1R was first cloned from opossum in 1991, which was
followed by identification of the highly homologous human
cDNA shortly thereafter (536, 964). In the nonactivated
state the receptor is expressed as a homodimer at the cell
surface, which dissociates upon PTH binding (860).
PTH1R is a member of the class B (class 2, secretin family)
GPCRs. As many other GPCRs, it undergoes N-linked glycosylation at four asparagine residues (1218). Mutational
analysis revealed that site-specific mutation of all four sites
decreases cell surface expression, whereas impairment of
fewer glycosylation sites does not seem to have significant
effects on trafficking or ligand binding (1218). Furthermore, the extracellular domain of the receptor includes a
characteristic disulfide bond pattern involving six cysteine
residues (392). These residues are thought to be essential for
stabilizing the hydrophobic ␣-␤-␤␣ binding pocket for
PTH, which is conserved among all members of class B
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SASCHA KOPIC AND JOHN P. GEIBEL
Ca
Ca
Ca
Calcium
Ca
PLC
Gq
PIP2
DAG
Kinase phosphorylation
Inhibition of
cell growth
IP3
Calcium
1,25(OH)2-D D
PLA2
ER
D
VDR
D
RXR VDR
AA
Increases VDR expression
Nucleus
Inhibits PTH
secretion
Reduces PTH
synthesis
Parathyroid gland
PTH
FIGURE 6. CaSR signaling in the parathyroid gland. Increased serum calcium levels lead to an inhibition of
PTH secretion. Serum calcium levels are measured by the CaSR receptor. Activation of CaSR causes
generation of arachidonic acid (AA) metabolites, which inhibit the release of PTH and increase the expression
of VDR, thereby increasing the cell’s sensitivity to the negative feedback exerted by 1,25(OH)2-vitamin D.
1,25(OH)2-vitamin D suppresses the synthesis of PTH. Furthermore, CaSR activation inhibits parathyroid gland
growth.
GPCRs and has recently been crystallized in the presence of
PTH (861).
Binding of PTH causes activation of at least two distinct G
proteins. G␣q/11 mediates intracellular calcium release via
phospholipase C (PLC) activation and increases in inositol
trisphosphate (IP3), whereas G␣s activates adenylyl cyclase
leading to rises in cAMP (5, 188, 808, 859).
PTH1R can be regulated on a variety of levels, ranging from
trafficking and internalization to direct protein interactions
at the cell surface. Desensitization of PTH1R is mediated by
GRK2 binding/phosphorylation and ␤-arrestin binding,
which uncouples the receptor from its associated G proteins
and triggers its internalization (267, 326, 330, 1123). For
222
example, knockout of ␤-arrestin causes increased and sustained levels of the second messenger cAMP in primary
osteoblast cultures upon PTH stimulation (327). PTH1R
also associates with the scaffolding protein NHERF1,
which stabilizes the receptor at the cell membrane and prevents its endocytosis and desensitization (1022, 1139). This
effect of NHERF1 is partially attributable to a prevention of
an interaction between ␤-arrestin and PTH1R (1140). Colocalization of NHERF1, ␤-arrestin, and PTH1R has been
demonstrated and suggests that NHERF1 is constitutively
bound, whereas ␤-arrestin association is more dependent
on receptor activation (580). Interestingly, it has recently
become apparent that ␤-arrestin not only plays a role in
receptor desensitization, but also mediates activation of
downstream signaling cascades, such as MAPKs, in a G
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protein-independent manner (380). Current scientific effort
thus focuses on the development of so-called “biased”
PTH1R agonists, which can selectively trigger G protein- or
␤-arrestin-dependent signaling events, allowing for a more
selective therapeutic repertoire (381, 1160). Furthermore,
NHERF may also modulate the cell’s response to PTH binding. In the presence of NHERF2, the cell’s calcium response
via PLC is augmented, whereas the cAMP response is dampened, presumably via recruitment of G␣i (698). Both
NHERF and ␤-arrestin provide effective examples of how
receptor-associated proteins can modulate canonical signaling events or even, as is the case with ␤-arrestin, initiate
signaling events in their own right.
PTH1R can also be regulated by its external environment.
The extracellular receptor domain can be cleaved by metalloproteases, resulting in receptor degradation (579). PTH
binding prevents this proteolytic cleavage, thus stabilizing
PTH1R at the cell surface. The physiological importance of
this mechanism is not yet fully understood. However,
MMPs are involved in bone remodeling and may in consequence locally regulate the sensitivity of osteoblasts to PTH
(579).
3. Cellular effects
PTH exerts its effects in two primary target tissues: bone
and the kidney.
In the kidney, PTH causes phostphaturia, increases calcium
absorption, and induces the synthesis of 1,25(OH)2-vitamin D. In-detail analysis of renal phosphate handling is
beyond the scope of this review and has been summarized
previously (334, 765). In brief, phosphaturia mainly results
from downregulation of the Na-Pi transporter type IIa
(NaPi-IIa) at the apical membrane of the proximal tubule,
thereby reducing the amount of reabsorbed phosphate from
the primary urine. Rather than directly modulating the
transporter’s activity, PTH exposure mainly affects the
number of active cotransporters on the plasma membrane.
Activation of basolateral PTH1R causes retrieval of NaPiIIa and targets it for lysosomal degradation, resulting in
diminished Pi reuptake (566, 682, 847). Apart from NaPiIIa, at least two other apical phosphate transporters are
present in the proximal tubule: NaPi-IIc and PiT-2 (986,
1124). Currently, there is some evidence that PTH can also
regulate NaPi-IIc and PiT-2, but additional studies remain
to be conducted (855, 987). Although a contribution of
these transporters to renal phosphate reabsorption is highly
likely, knockout studies suggest that NaPi-IIa is responsible
for ⬃80% of total phosphate transport, thus representing
the major uptake mechanism (72, 468).
It has been recognized for over 30 years that PTH can
stimulate the synthesis of the active vitamin D metabolite
1,25(OH)2-vitamin D in the kidney (116, 338) (see sect.
IVA4). 1,25(OH)2-vitamin D in consequence enhances the
intestinal and renal uptake of calcium in an effort to counteract hypocalcemia, which initially led to secretion of PTH.
The PTH-stimulated increase in 1,25(OH)2-vitamin D levels is achieved on a transcriptional level. PTH upregulates
the transcription of CYP27B1, the mitochondrial enzyme
which is responsible for the conversion from 25(OH)-vitamin D to 1,25(OH)2-vitamin D. Transcriptional upregulation occurs via PTH binding to PTH1R, leading to increases
in the second messenger cAMP and activation of PKA (125,
442, 763, 764, 921).
Apart from inducing the synthesis of 1,25(OH)2-vitamin D,
PTH can directly upregulate the renal reabsorption of calcium. The regulation of calcium absorption by PTH occurs
in distal segments of the nephron, mostly in the distal convoluted tubule and the connecting tubule (99, 379, 1003,
1172). In close analogy to the duodenum, these segments
express calcium transport proteins, which are responsible
for mediating the process of active transcellular calcium
absorption in the kidney, namely, TRPV5, calbindin-D
28k, and NCX1. PTH can regulate all of these protein levels
on a transcriptional level (1108). Interestingly, this seems to
be accomplished independently of 1,25(OH)2-vitamin D,
which also positively regulates most of these transporters
(see sect. IVA6) (1108). It is therefore difficult to dissect the
relative contribution of each of the two hormones in the
physiological regulation of the calcium transport proteins.
In addition to transcriptional activation, PTH was shown to
cause direct phosphorylation of TRPV5, thereby increasing
its opening probability (249). The channel is phosphorylated at threonine-709 in a PKA-dependent fashion (249).
Elevation of intracellular cAMP levels and concomitant
PKA activation are classical downstream effects of PTH1R
activation in the kidney (248, 1172).
In bone, PTH exerts a dichotomous effect depending on the
pattern of exposure. It is well documented that pulsatile
PTH exposure has anabolic effects on bone mass, whereas
continuous release increases plasma calcium by bone catabolism (FIGURE 7) (27, 348, 401, 469, 1065). The observation that intermittent PTH administration increases bone
mass has led to the use of PTH as a treatment strategy for
osteoporosis (621, 671, 897). The enhanced bone formation mainly results from an increase in osteoblast numbers.
This phenomenon has been partially attributed to a PTHmediated induction of osteoblast differentiation and an inhibition of their apoptosis (75, 274, 518, 530, 531, 684,
969, 1032). Multiple mechanisms underlying the anti-apoptotic effects of intermittent PTH on osteoblasts have been
suggested. Among others, these include runt-related transcription factor 2 (Runx2)-mediated transcription of survival genes and increased DNA repair (75, 969). It has
further been shown that fibroblast growth factor 2 (FGF2)
is partially needed as an endogenous cofactor for the anabolic effects of PTH to take place (934).
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SASCHA KOPIC AND JOHN P. GEIBEL
Intermittent
Chronic
PTH
PTH1R
OPG
RANKL
Osteoblast
proliferation
RANK
Osteoclast
differentiation
H+
Osteoblast
Osteoclast
V-ATPase
FIGURE 7. The effects of PTH on bone. PTH has a dual effect on bone. Intermittent PTH exposure causes
osteoblast proliferation, leading to an increase in bone mass. Continuous PTH exposure results in RANKL
upregulation and concomitant OPG suppression (OPG serves as a decoy receptor for RANKL and prevents its
interaction with osteoclast RANK). The stimulated RANKL-RANK interaction leads to osteoclast proliferation
and increased bone turnover.
Continuous PTH exposure, on the other hand, mainly affects osteoclast numbers and activation, thereby increasing
bone turnover. Since osteoclasts are canonically thought to
not express PTH1R (although this view has recently been
challenged, Ref. 260), the catabolic effects of PTH are relayed through osteoblast signaling. The PTH-induced
crosstalk between osteoblasts and osteoclast is mainly mediated by receptor activator of nuclear factor ␬B (RANK),
osteoprotegrin (OPG), and RANK ligand (RANKL). Both
RANKL and OPG are expressed by osteoblasts and exert
opposing actions on osteoclasts. RANKL promotes osteoclastogenesis by binding to RANK on osteoclasts. Conversely, OPG serves as a soluble decoy receptor for RANKL
and thus inhibits its interaction with RANK, thereby suppressing osteoclastogenesis. In accordance with this model,
RANKL-deficient animals develop osteopetrosis because of
insufficient osteoclasts activation (592). Sustained PTH exposure affects RANK-RANKL signaling by downregulating antiresorptive OPG, while simultaneously stimulating production of RANKL by osteoblasts (FIGURE 7)
(350, 497, 679, 689). The enhanced RANK-RANKL signaling induces formation of osteoclasts, which in turn
leads to enhanced bone resorption and elevates serum
calcium levels.
In the intestine, several observations suggest that PTH may
have a direct, i.e., non-1,25(OH)2-vitamin-D mediated, effect on intestinal calcium absorption. Both isolated enterocytes and intestinal loops demonstrated an increase in calcium uptake following acute PTH exposure (781, 783,
784). However, more investigations are needed to clearly
establish a direct regulatory role of PTH in the context of
intestinal calcium uptake.
224
4. PTH fragments
It should be noted that full-length PTH(1– 84) is not the
only circulating form of the hormone in the body. Various
PTH fragments can be found in the circulation, which partially originate directly from the parathyroid gland and partially represent products of peripheral cleavage. The parathyroid itself releases COOH- and NH2-terminal hormone
fragments, which are generated by cysteine proteases (cathepsin B and H) in distinct secretory vesicles of the gland
(418, 427, 693). Interestingly, the fraction of secreted hormone fragments changes with extracellular calcium conditions. It has been reported that more fragments are released
under conditions of hypercalcemia, when secretion of fulllength PTH is suppressed (417, 418, 605, 726). Peripheral
proteolysis represents the second source of PTH fragments.
This process occurs predominantly in liver and the kidney
(127, 234, 989). The group of fragments that have received
the most amount of scientific attention is the large NH2terminally truncated non-PTH(1– 84) fragments. PTH(7–
84) is the quantitatively major member of this group, which
is secreted by the parathyroids (233). The group of nonPTH(1– 84) fragments can represent up to 20% of circulating PTH, but can increase in patients with renal failure
dramatically to up to 50% because of impaired renal clearance (130, 131, 444, 648). This is of particular interest, as it
has become recently apparent that the non-PTH(1– 84)
fragments exert biological activity. In general, non-PTH(1–
84) fragments antagonize the effects of PTH in its primary
target tissues, bone and the kidney but also the parathyroid
gland directly. It has been shown that PTH(7– 84) can inhibit PTH release from the parathyroid, presumably in an
autocrine fashion, despite low serum calcium concentra-
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GASTRIC ACID, CALCIUM ABSORPTION, AND BONE HEALTH
tions (493). In bone, PTH(7– 84) blocks the effects of PTH
on calcium mobilization in thyroparathyroidectomized rats
(786). More detailed investigations revealed that PTH(7–
84) reduces calcium release from bone in vivo and inhibits
the formation of osteoclast-like cells in murine primary
marrow cultures (272). In the kidney, PTH(7– 84) can inhibit the formation of 1,25(OH)2-vitamin D, presumably
via a posttranscriptional mechanism (768, 1102).
Since activation of PTH1R requires an intact NH2-terminal
domain of PTH, it has been speculated that non-PTH(1–
84) fragments exert their function through a pathway distinct of PTH1R. The existence of a COOH-terminal PTH
receptor has thus been postulated (270 –272, 786). The molecular identity of this receptor is, however, not yet resolved. Another mechanism through which non-PTH(1–
84) fragments may exert their biological activity is downregulation of PTH1R. It has been demonstrated that
PTH(7– 84) causes internalization of PTH1R, thus offsetting the effects of PTH by decreasing the number of available receptors at the cell surface (1022).
In conclusion, non-PTH(1– 84) fragments act as PTH antagonists and are secreted by the parathyroid in response to
hypercalcemia.
2. The calcitonin receptor
Calcitonin exerts its physiological functions via activation
of the calcitonin receptor. The calcitonin receptor is a seventransmembrane domain GPCR. In particular, it is a member
of the family B subfamily of GPCRs (669). It shares significant homology with other receptors of the family, which
include the PTH, GHRH, PACAP, VIP, secretin, glucagon,
and glucagon-like peptide receptors.
The calcitonin receptor is expressed in the two most extensively described calcitonin target tissues, i.e., osteoclasts
and the kidney, but also in other adult tissues, such as the
prostate, CNS, skeletal muscle, and placenta (17, 329, 428,
790, 878, 1174). Multiple isoforms of the calcitonin receptor occur in the body, which results from different RNA
splicing directed by tissue-specific promoters (17, 30, 389,
606). Activation of the calcitonin receptor mostly translates
into a rise of intracellular cAMP levels via Gs-dependent
activation of adenylate cyclase (167, 332, 435, 669). Although the cAMP/PKA pathway appears to be dominant,
activation of both PLC and PLD have also been reported
(167, 332, 776).
3. Cellular effects
A) OSTEOCLASTS.
1. Production and secretion
Shortly after the discovery of calcitonin and
its hypocalcemic effects, investigators set out to identify its
physiological site of action. First evidence for an effect of
calcitonin on bone metabolism came from experiments on
rat embryonic bone in tissue culture. It was observed that
calcitonin caused a decreased basal release of calcium from
these preparations (343). These observations served as the
first evidence of how calcitonin lowers serum calcium levels.
Furthermore, calcitonin blocked the resorptive actions of
PTH on bone, albeit only temporarily (342, 343). After 4 – 6
days of combined treatment (calcitonin ⫹ PTH), calcium
release rose again (342). This desensitization to the effects
of calcitonin has been coined as the calcitonin “escape”
phenomenon (342). Today we know that the transient effect of calcitonin on osteoclasts is attributable to a downregulation of surface calcitonin receptors and their synthesis (882, 1061, 1129, 1130).
Calcitonin is encoded by the CALCA gene and is initially
synthesized as a 141-amino acid precursor (preprocalcitonin), which is later processed to the mature 32-amino acid
hormone (637). The same gene also encodes the neuropeptide CGRP. Production of either peptide is dependent on
tissue-specific RNA splicing. Although encoded by a different gene, amylin also belongs to the calcitonin peptide family. All three peptides, i.e., calcitonin, CGRP, and amylin,
share some overlapping functions with regard to osteoclast
suppression (16, 229, 1199). Calcitonin is released from the
C-cells in response to rising concentrations of plasma calcium. The CaSR is responsible for the molecular process of
calcium sensing on the parafollicular cells (351, 367, 728).
As alluded to before, calcitonin directly inhibits the action
of osteoclasts, causing the balance between bone absorption and formation to shift towards anabolism. Calcitonin
exerts its inhibitory effects on osteoclasts via activation of
its receptor, which is expressed in abundance on their surface (428, 790, 878). Exposure to calcitonin triggers distinct morphological changes in the osteoclast. Osteoclasts
are highly motile cells that resorb bone via formation of
so-called resorptive pits, which are membrane invaginations that are luminally acidified by active proton secretion.
Calcitonin has been shown to inhibit the formation of these
resorptive bays in vitro (487, 1057). Furthermore, osteoclast motility is markedly decreased, causing the cell to
C. Calcitonin
Calcitonin is a peptide hormone that has been discovered by
Copp et al. in 1962 as a factor that reduces serum calcium
concentrations (160, 227). Calcitonin production was initially falsely ascribed to the parathyroid glands, and it was
only later that the thyroid gland had been established as the
source of calcitonin (463). The primary sites of calcitonin
production are the parafollicular cells (C-cells) of the thyroid gland. Calcitonin exerts its hypocalcemic effects primarily by inhibition of osteoclast activity. It should be
noted that the importance of calcitonin in day-to-day calcium homeostasis in humans is rather negligible (see sect.
IVC4). For this reason, it will only be reviewed concisely.
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SASCHA KOPIC AND JOHN P. GEIBEL
enter a state of functional quiescence (169). Apart from
directly altering osteoclast function, calcitonin also affects
osteoclast differentiation. Calcitonin inhibits the formation
of multinucleated mature osteoclasts by arresting their differentiation in more immature stages (1060).
Renal calcium handling is the second organ
function that is influenced by calcitonin. However, it is
not entirely clear whether calcitonin causes calciuria or
enhances calcium reabsorption from the urine. The conflicting results may be to some extent attributable to species differences. In humans, calcitonin likely increases the
excretion calcium through the urine and thereby acts in
concert with its inhibitory action on osteoclasts to lower
serum calcium levels (31, 32, 143, 215, 819, 1016). Conversely, most studies demonstrating an increase in the
renal reabsorption of calcium and magnesium were conducted in rats, rabbits, or mice (84, 158, 159, 265, 304,
877, 883, 1225). A single investigation established a calcium-conserving effect of calcitonin in human (160). The
primary site of action for calcitonin in the rat is the thick
ascending limb (TAL) of the loop of Henle, where calcitonin has been demonstrated to bind its receptor, increase local adenylate cyclase activity, and promote calcium reabsorption (166, 168). Apart from enhancing the
reabsorption of calcium, calcitonin also promotes the
vectorial transport of NaCl in the rat TAL, thereby amplifying the corticomedullary concentration gradient,
which is a prerequisite for the subsequent concentration
of urine in the collecting duct (265, 304). In the rabbit,
the calcium-conserving effects of calcitonin seem to be
mediated by the distal tubule (1225).
duced in HEK-293 cells following calcitonin exposure,
suggesting a regulatory role of calcitonin in vitamin D
catabolism (361). In addition to its putative direct effect
on calcium reabsorption, calcitonin may thus affect normal calcium metabolism indirectly by modulating the
levels of circulating 1,25(OH)2-vitamin D.
B) KIDNEY.
It has also been speculated that calcitonin may act directly on the collecting duct in a similar fashion to antidiuretic hormone (ADH or vasopressin), i.e., to concentrate the urine by increasing the reabsorption of water
from the primary urine (251). Indeed, calcitonin was
shown to increase the apical expression of aquaporin 2
(AQP2) in principal cells of the collecting duct (119).
Apical insertion of AQP2 and subsequent transepithelial
water movement is the primary mechanism by which
ADH causes concentration of the urine to lower plasma
osmolarity.
In conclusion, the direct impact of calcitonin on renal
calcium handling is quite vague and may be of minor
importance. However, calcitonin also has another, indirect effect on calcium homeostasis. Calcitonin was
shown to be an important regulator of the expression of
CYP27B1, the renal enzyme responsible for the conversion of 25(OH)-vitamin D to 1,25(OH)2-vitamin D
(1009, 1217). In normocalcemic rats, CYP27B1 mRNA
levels were inducible by calcitonin administration, leading to an increase in the production of 1,25(OH)2-vitamin D (1009, 1217). Furthermore, CYP24A1 was in-
226
4. The relevance of calcitonin for
calcium homeostasis
The relevance of calcitonin for day-to-day calcium balance
is highly debatable. This is corroborated by fundamental
observations during conditions of decreased or increased
calcitonin levels, neither of which result in an appreciable
phenotype in terms of calcium balance. For example, patients with medullary carcinomas of the thyroid (MTC), a
tumor of the thyroid C-cells resulting in the hypersecretion
of calcitonin, were shown to have normal bone mineral
densities (1176). Animal studies further substantiate this
conundrum. A reduction in serum calcitonin levels by thyroidectomy in rats did not impact serum calcium levels
(223, 1064). In light of this evidence, the question arises as
to what the physiological role of calcitonin is.
It has been suggested that calcitonin is an evolutionary remnant (462). This is substantiated by the fact that calcitonin
from other species is more potent than human calcitonin.
Teleost calcitonin has the highest biological activity in humans, which may be a result of their higher dependence on
the hormone. For example, salmon calcitonin has an approximately sixfold higher affinity to calcitonin receptor
than human calcitonin (28, 328). Furthermore, it is less
effectively eliminated by the kidney, resulting in a longer
plasma half-life (405). The differences in the biological activity between calcitonin forms led to the introduction of
salmon calcitonin as a treatment for skeletal disorders, such
as osteoporosis or Paget’s disease (1077).
Given its ambiguous role in regular calcium homeostasis, it
has been postulated that calcitonin may be of importance
during states of high calcium demand, such as during lactation (1170). It has been demonstrated that calcitonin and
CGRP (⫺/⫺) mice show greater loss of skeletal mass during
lactation than wt animals (1170). Since calcitonin and
CGRP are encoded by the same gene, animals were controlled by CGRP substitution, which was without effect
(1170). Another mechanism by which calcitonin may be
osteoprotective during lactation or pregnancy is by inducing the renal synthesis of 1,25(OH)2-vitamin D (1217).
D. The CaSR
The CaSR is a G protein-coupled membrane-bound receptor that is the primary sensor for calcium and is the first link
in the regulatory chain of calcium homeostasis. By regulating the release of PTH from the parathyroid to modulate
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GASTRIC ACID, CALCIUM ABSORPTION, AND BONE HEALTH
current serum calcium levels, it presides over the subsequent
hierarchical cascade of vitamin D synthesis and calcium
handling in the vitamin D target organs, such as the intestine, kidney, and bone. More recent investigations have
demonstrated that the CaSR is not exclusively expressed in
the parathyroid gland, but is also present locally in the
vitamin D target organs. This diverse expression suggests
that CaSR can modulate organ function locally and outside
of the strict PTH-vitamin D-organ axis. Thus a short and
local feedback loop is created, which allows the organs to
respond rapidly to the local calcium environment. A detailed description of the receptor’s role in each tissue is
beyond the scope of this article, but an attempt will be made
to identify the key features of CaSR in these local sites in a
subsequent section (FIGURE 8). For excellent in-depth reviews, please refer to Refs. 372, 711, 906.
The importance of CaSR as a regulator of calcium balance
is underlined by clinical pathologies that are caused by its
mutation. Loss-of-function mutations cause familial hypocalciuric hypercalcaemia (FHH; OMIM 145980) and
neonatal severe hyperparathyroidism (NSHPT; OMIM
239200), whereas activating mutations cause autosomal
dominant hypocalcemia (OMIM 601198). These mutations mostly change the threshold of receptor activation in
either direction. Currently, ⬃300 different mutations are
reported, two-thirds of which represent inactivating mutations (241). Given its broad expression pattern, the deranged sensing of blood calcium levels in these disorders not
only affects the secretion of PTH from the parathyroid
glands, but also causes local dysfunction in other organs,
such as the kidney where calcium absorption is perturbed.
1. Structure and signaling
The CaSR was first cloned by Brown et al. (134) in 1993
from bovine parathyroid using a Xenopus oocyte expression cloning system. Cloning of the human CaSR followed
2 years later (366). The CaSR is a 1,028-amino acid protein
that belongs to the superfamily of classic 7-transmembrane
domain G protein-coupled receptors (134, 366). It is mainly
expressed as a homodimer on the cell surface (55). The
dimerization process has been shown to take place in the ER
and is mediated by the formation of disulfide bonds between cysteine residues (C129, C131) and noncovalent interactions of leucine residues (L112, L156) in the extracellular domain of the receptor (54, 529, 858, 888, 1213).
Following assembly in the ER, the CaSR undergoes
N-linked glycosylation in the Golgi apparatus, some of
which is pivotal for cell surface expression (887). The trafficking between ER and Golgi apparatus is regulated by the
small GTP-binding protein Rab1 (1221). Knockdown and
mutations of Rab1 in HEK cells results in decreased numbers of CaSR at the cell surface (1221). Conversely, the
internalization of CaSR is thought to be mediated by ubiquitination by the E3 ligase dorfin (498).
On the cell surface, the CaSR resides in caveolin-1-rich
plasma membrane domains, which also contain associated
signaling proteins (571). These signaling complexes are
formed with the help of scaffolding proteins. The COOHterminal tail of CaSR binds to filamin A, an actin binding
protein (48, 467). Silencing of filamin A with siRNAs results in the attenuation of MAPK signaling by the receptor
(496) (see below).
In lack of a crystal structure of CaSR, the exact binding sites
of calcium on the extracellular domain remain subject of
speculation. So far, applicable structural data has only been
obtained from the metabotropic glutamate receptor type I
(mGluR1), which belongs to the same family of type C
GPCRs. In this model, glutamate binds to key residues
which are located in a cavity, embedded in between two
lobular domains (LB1 and LB2) of the extracellular tail
(611). This structural hallmark has been aptly coined the
receptor’s Venus fly trap module. This motif is conserved
among other GPCRs of the same family. Multiple attempts
to identify the calcium binding sites have been undertaken
using computational homology modeling (499, 500, 1014).
These models have postulated between one and five calcium
binding sites (499, 500, 1014). With the employment of
mutational analysis, it has been possible to validate functionality of some of these putative binding sites (500). Monitoring of the intracellular calcium response to increasing
extracellular calcium levels in CaSR transfected HEK cells
had indicated previously that the Hill coefficient for this
response was ⬃3.1, suggesting that multiple calcium binding sites may exist (834).
It should be noted that the CaSR can also be stimulated by
other polyvalent cations (Mg2⫹, Pb2⫹, Cd2⫹, Fe2⫹, Ba2⫹,
Ni2⫹, Co2⫹, or Gd3⫹) and larger polycationic molecules,
such as spermine, spermidine, putricine, protamine, and
neomycin (134, 416, 880). Furthermore, several substances
can allosterically modify the receptor and potentiate its sensitivity to its direct agonists. These include pharmacological
small molecule substances (calcimimetics) that are in clinical use for the treatment of conditions, such as secondary
hyperparathyroidism, and L-type amino acids, which enable the CaSR to act as a nutrient sensor (220). Truncation
studies have demonstrated that the Venus fly trap motif is
necessary for allosteric modification by L-type amino acids
(759). The affinity of calcium to the CaSR can also be modulated by changes in extracellular pH (279, 879). An acidic
extracellular milieu has been shown to decrease the sensitivity of CaSR to its agonists, whereas an increased extracellular pH has converse effects (879).
The intracellular domain of CaSR contains five PKC phosphorylation sites (366). Mutational analysis demonstrated
that PKC-mediated phosphorylation of the CaSR at Thr888 blunts its response to extracellular calcium, as evidenced by inhibited calcium release from intracellular
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SASCHA KOPIC AND JOHN P. GEIBEL
Calcium-sensing receptor
Kidney
Proximal tubule
Collecting duct
Distal convoluted tubule
CaSR
CaSR
CaSR
HPO42–
CaSR
CaSR
NaPi-IIa
Na+
H+
Ca2+
V-ATPase
TRPV5
25(OH)-vitamin D
CaSR
CaSR
Mitochondria:
CYP27B1
H2O
1,25(OH)2-vitamin D
AQP2
Intestine
Thick ascending limb of
the loop of Henle
Enterocyte
ROMK
K+
1
CaSR
CaSR
NKCC2
2
CaSR
Na+
CFTR
2Cl–
Cl–
K+
3
1
2
Ca2+
H2O
Stomach
Bone
G-cell
PPIs
APAs
CaSR
CaSR
H+
H,K-ATPase
CaSR
CaSR
K+
?
Gastrin
CaSR
ECL-cell +
parietal cell
activation
Differentiation
and function
?
RANKL
RANK
?
Osteoblast
Circulation
H+
V-ATPase
Parietal cell
FIGURE 8. CaSR in the gastrointestinal tract, kidney, and bone. Kidney: the effects of CaSR activation on ion
transport in various nephron segments are shown. In the proximal tubule, CaSR stimulates phosphate
absorption and 1,25(OH)2-vitamin D synthesis. In the thick ascending limb of the loop of Henle, CaSR inhibits
apical potassium channels (ROMK), thereby inhibiting NKCC2 (potassium recycling). The resulting changes in
the lumen-positive potential inhibit paracellular calcium uptake. In the distal convoluted tubule, CaSR presumably stimulates apical calcium entry through TRPV5. In the collecting duct, CaSR stimulates proton extrusion
through the V-type ATPase and inhibits urine concentration through AQP2. Stomach: in the parietal cell, CaSR
induces acid secretion by activating H⫹-K⫹-ATPase. In the G-cell, CaSR activation results in gastrin secretion.
Of note, CaSR serves as a luminal nutrient and calcium sensor on the G-cell. Bone: CaSR on osteoblasts
presumably regulates their differentiation and RANKL expression. Intestine: in the intestine, CaSR activation
reduces water secretion by inhibiting chloride secretion through CFTR.
228
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Osteoclast
GASTRIC ACID, CALCIUM ABSORPTION, AND BONE HEALTH
stores (56, 246). The phosphorylation occurs in response to
receptor activation and thus represents an autoinhibitory
feedback mechanism (246). ␤-Arrestins are most likely involved in the process of PKC-associated desensitization
(681, 853). Conversely, dephosphorylation of the Thr-888
residue is carried out by a calyculin-sensitive phosphatase,
thereby restoring the receptor’s initial sensitivity (246). Another mechanism of desensitization is mediated by a G protein receptor kinase (GRK), most likely by interfering with
G␣q regulated pathways (see below) (681, 853).
Once stimulated, the CaSR activates a variety of intracellular signaling cascades. Being a GPCR, most of these processes are mediated by G proteins. Specifically, G␣q/11, G␣i,
and G␣12/13 have been shown to be coupled to the CaSR
(40, 175, 495, 849). The expression of all subunits was
confirmed in bovine parathyroid (1115). G␣i mediates the
suppression of cAMP levels by inhibiting adenylyl cyclase
and activates the ERK/MAPK pathway (175, 250, 375,
572). Activation of G␣q/11 results in increased intracellular
calcium concentrations via activation of PLC and IP3 triggered calcium release (133, 1010). As demonstrated in HEK
cells, this cascade can also activate further downstream
phospholipase A2 leading to production of arachidonic acid
metabolites (415). G␣12/13 is thought to regulate phospholipase D and phosphatidylinositol 4-kinase (PI 4-K); however, this interaction has only been demonstrated in heterologous cell culture system (494, 495).
2. CaSR in the parathyroid
CaSR regulates parathyroid function at three levels: 1) the
release of PTH from secretory granules, 2) de novo synthesis of PTH, and 3) parathyroid cell growth.
Activation of CaSR by increasing plasma calcium results in
an inhibition of PTH release, thereby lowering calcium levels. It is thought that this response is mediated by the generation of arachidonic acid metabolites via G␣q and PLA2
activation (FIGURE 6) (121, 152). Cultured porcine parathyroid cells demonstrated an increase in arachidonic acid production after CaSR stimulation while PTH release was inhibited (121). Furthermore, exogenous administration of
arachidonic acid suppressed PTH release from the parathyroid cells (121). Similar effects were demonstrated for the
arachidonic acid metabolites 12- and 15-hydroxyeicosatetranoic acid, suggesting that they represent the downstream
effectors of arachidonic acid production (120).
Apart from directly controlling PTH release, CaSR also
modulates PTH synthesis. PTH gene transcription is mainly
regulated by 1,25(OH)2-vitamin D. Binding of 1,25(OH)2vitamin D to the VDR causes a decrease in pre-pro-PTH
mRNA levels creating a negative-feedback loop (154, 930,
931). However, it was recognized before the identification
of the CaSR that serum calcium can modulate the actions of
1,25(OH)2-vitamin D on PTH gene transcription (930). It
was shown that increases in calcium can potentiate the inhibitory effects of 1,25(OH)2-vitamin D (930). This effect is
most likely mediated by CaSR, whose activation can decrease
PTH transcription by augmenting the inhibitory effects of
1,25(OH)2-vitamin D. Molecularly this is achieved by upregulating the expression of the VDR (151, 162, 362, 653, 916).
The current working model states that activation of CaSR
causes an increase of arachidonic acid metabolites and activation of the MAPK pathway, which in turn results in increased
VDR mRNA levels (FIGURE 6) (151). This allows the parathyroid to adjust its 1,25(OH)2-vitamin D sensitivity to the current plasma calcium levels.
The molecular mechanisms underlying the trophic effects of
CaSR activation are less clear. Earlier observations had already suggested that hypocalcemia is associated with parathyroid cell proliferation (778). Currently, the CaSR specific calcimimetics provide the most useful insight into the
regulation of parathyroid growth by CaSR. Calcimimetics
administered in the context of both animal models and
clinical studies of hyperparathyroidism demonstrate that
activation of CaSR leads to a reduction in gland size
(510, 589, 746, 1128). Conversely, inactivating mutations of CaSR result in parathyroid enlargement. Parathyroid-selective genetic disruption of G␣q was furthermore shown to cause moderate hyperparathyroidism
with increased plasma PTH levels and gland hyperplasia,
suggesting a role of G␣q in the regulation of parathyroid
cell growth (849). Similar findings were reported in
G␣q/11 double KO animals (1159).
3. CaSR in the kidney
The CaSR acts as an important regulator of ion and water
homeostasis in the kidney. It should be noted that it can
exert its effects on calcium transport independently of other
hormonal regulators, such as PTH and 1,25(OH)2-vitamin
D. The CaSR is expressed along most of the nephron, albeit
in varying subcellular localizations (FIGURE 8) (907, 908).
In the proximal tubule, CaSR is localized apically at the
base of the brush border, where it has been implicated to
play a role in phosphate transport (52, 907, 909). The primary regulator of phosphate transport in the proximal tubule of the kidney is PTH. In brief, increased PTH levels
inhibit phosphate reabsorption from the lumen. Activation
of the apical CaSR can partially reverse the effects of PTH
and restore phosphate absorption (52). Conversely, PTH
and high phosphate levels reduce CaSR expression (909).
Furthermore, it is likely that the CaSR mediates the inhibitory effects of calcium on 1,25(OH)2-vitamin D synthesis in
the proximal tubule (109, 702).
In the thick ascending limb of the loop of Henle, CaSR is
located on the basolateral membrane (907). In this nephron
segment, the receptor acts as a major modulator of monovalent and polyvalent ion absorption. Activation of CaSR
leads to an inhibition of the apical renal outer medullary
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SASCHA KOPIC AND JOHN P. GEIBEL
potassium (ROMK; Kir1.1) channel, mainly through arachidonic acid metabolites created by PLA2 (1147, 1148).
Apical ROMK releases potassium ions into the lumen,
which in turn are needed to fuel apical ion uptake through
the Na⫹-K⫹-2Cl⫺ (NKCC2) cotransporter. By decreasing
apical potassium efflux, CaSR inhibits sodium and chloride
uptake through NKCC2 (906). This correlation is reflected
in much earlier observations, which report that calcium
infusions can decrease tubular sodium clearance (300, 718,
1052). In addition, impairment of NKCC2 has also implications for calcium absorption. Reduced NKCC2 activity
decreases the lumen-positive potential and negatively affects countercurrent multiplication, and in consequence the
nephron’s ability to concentrate urine (434). Both mechanisms will lead to impaired calcium absorption (434). Calcium absorption in the medullary portion of the thick ascending limb is thought to occur predominantly as passive
uptake through the paracellular route (995). Similar observations have been made when blocking NKCC2 pharmacologically with the loop diuretic furosemide (299). It has thus
been proposed that activation of basolateral CaSR has
“loop diuretic-like” effects, reducing NaCl but also calcium
absorption in the kidney (434).
In contrast to the medullary section, the cortical portion of
the thick ascending limb has been proposed to have predominantly active calcium uptake properties, which are under the hormonal regulation of PTH and calcitonin (344,
509). PTH increases calcium absorption in this segment,
and it has been shown that, similarly to phosphate absorption in the proximal tubule, activation of CaSR can suppress the effects of PTH (264, 754). The absorption of NaCl
does not seem to be affected by CaSR (264, 754).
The distal convoluted tubule and the connecting tubule are
responsible for the fine-tuning of calcium reabsorption in
the kidney. To achieve this goal, they are equipped with
molecular machinery, similar to that in the duodenum
(TRPV5, calbindin-D 28k, NCX1, and PMCA1b) to absorb calcium against its electrochemical gradient through
the transcellular pathway (680). In analogy to the proximal
small intestine, these transporters are predominantly regulated through 1,25(OH)2-vitamin D, but also PTH. CaSR
colocalizes with TRPV5 in this segment (1090). Its activation causes increase calcium influx through TRPV5 and
may thereby locally and rapidly adapt active absorption to
the urine calcium concentration (FIGURE 8) (1090).
Apart from regulating calcium and phosphate absorption in
the kidney, CaSR modulates proton and water movement in
the collecting duct. In the intercalated cells of the collecting
duct, apical V-ATPase acidifies the urine in an effort to
maintain systemic acid-base homeostasis. It has been shown
that luminal calcium and neomycin can induce V-ATPase
activity via activation of CaSR, thereby causing proton secretion into the urine (FIGURE 8) (901). Since the formation
230
of calcium kidney stones is dependent on luminal pH, it has
been speculated that this may represent an autoprotective
mechanism that prevents nephrolithiasis (901). Furthermore, stimulation of apical CaSR in the principal cells of the
collecting duct leads to decreased ADH (vasopressin)-stimulated water reabsorption through AQP2 (FIGURE 8) (942,
943). Taken together, activation of CaSR has diuretic effects via inhibiting NKCC2 in the thick ascending limb of
the loop of Henle and by inhibiting AQP2-mediated water
reabsorption the collecting duct.
4. CaSR in the gastrointestinal tract
The CaSR is distributed along most of the gastrointestinal
tract, ranging from the stomach to the large intestine (186,
360, 744, 932, 998). We are now only slowly beginning to
unravel its function in this diverse array of tissues. In the
stomach, CaSR localizes to the basolateral membrane of
the acid-secreting parietal cells and to all membranes of the
gastrin-secreting G-cells (FIGURE 8) (142, 182, 886). Primary cultures of G-cells were shown to release gastrin after
stimulation of the CaSR with calcium (142, 886). The release is mediated via calcium influx into the cytosol through
nonselective cation channels opening after CaSR stimulation (142). These findings provide the molecular basis for
the observation that rises in serum calcium can increase
serum gastrin levels (see sect. IIB2). The apical expression of
CaSR in G-cells theoretically enables it to act as a luminal
nutrient sensor modulating gastric acid secretion and other
parameters. In recent studies there is direct evidence showing that gastrin levels increase in mice after calcium and
L-type amino acid ingestion (325). This effect was abolished in CaSR (⫺/⫺) animals (325). In healthy human test
subjects, pharmacological stimulation of CaSR leads to a
concomitant increase in gastrin levels and gastric acid output (165). CaSR on G-cells was thus postulated to play an
important role in the gastric phase of acid secretion by
maintaining acid output by maintaining gastrin secretion
(325).
Apart from being expressed on G-cells, CaSR is also localized on the basolateral membrane of the acid-secreting parietal cell, where it exerts effects that are independent of
gastrin and other secretagogues. Activation of parietal cell
CaSR has been reported to increase H⫹-K⫹-ATPase-mediated proton secretion, thereby acidifying the gastric lumen
(FIGURE 8) (145, 291, 373). This stimulatory effect was
demonstrated for direct activators, such as calcium or
Gd3⫹, but also allosteric modifiers, such as L-type amino
acids (145, 291, 373). In parallel to other tissues, the intracellular activation signal for H⫹-K⫹-ATPase is mediated by
rises in intracellular calcium, PLC, MAPK, and PKC (899).
In conclusion, both rises in luminal and plasma calcium
concentrations can induce gastric acid secretion either indirectly through gastrin release or directly through parietal
cell activation. The physiological significance of this observation remains the subject of speculation, but may be linked
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to facilitating calcium uptake by increasing acid output. As
described in a subsequent section, it has been speculated
that gastric acid increases the bioavailability of ingested
calcium (see sect. V). Alternatively, CaSR may primarily
function as a nutrient sensor in the stomach (amino acid
sensing), which maintains constant acid output in the gastric (apical G-cell sensing) and postprandial (basolateral
parietal cell) phase of digestion, when circulating levels of
amino acids are high (325).
In the intestine, functional investigations on CaSR have
mainly been conducted in colonic epithelia, where CaSR
localizes to both the basolateral and apical membranes of
the colonic crypt (173, 186, 360). Expression patterns vary
slightly in the small intestine, with general basolateral expression and additional weak apical expression in the villus
(173, 360). Furthermore, CaSR is expressed in both the
Meissner’s and Auberach’s plexuses. Early experiments on
single perfused colonic crypts demonstrated that intracellular calcium concentrations could be increased when exposing the crypts to classic CaSR agonists and that forskolinstimulated fluid secretion could be inhibited (186). This and
subsequent investigations indicate that CaSR plays an important role as a modulator of colonic fluid secretion (185,
186). Subsequently, attempts have been made to take advantage of the “constipatory” effects of CaSR activation in
pathophysiological settings. Activations of CaSR in the
course of diarrheagenic enterotoxin exposure was shown to
decrease fluid secretion via increased breakdown of cyclic
nucleotides (371). Although the potential clinical applications of ameliorating the symptoms of secretory diarrhea
are promising, more efforts will have to be made to fully
unravel the physiological role of CaSR in intestinal ion and
fluid transport. So far it is not clear whether intestinal CaSR
can modulate calcium absorption, as is the case in the kidney.
5. CaSR in bone
It is well established that CaSR is expressed in osteoblasts,
osteoclasts, and their respective precursors (172, 174, 542,
1183–1185, 1192). The functional role of CaSR in these
cells is, however, less clear. Undoubtedly, both cell lines are
exposed to local fluctuations in calcium concentrations
making an adaptive response to the calcium environment
plausible. Indeed, changes in extracellular calcium concentration have been shown to regulate various cell functions,
mostly in in vitro models. Extracellular calcium can stimulate the proliferation, migration, and differentiation of osteoblasts (174, 297, 1183, 1184, 1186). Similarly, calcium
was proposed as a differentiation signal for osteoclasts
(542, 544, 734). Significant doubt about the in vivo importance of CaSR in bone has emerged with the generation of
the CaSR (⫺/⫺) mice. Although CaSR knockout results in
rickets, these animals suffer from severe hyperparathyroidism, which did not allow a discrimination between the effects of high PTH and CaSR on bone turnover (365). Con-
comitant genetic ablation of the parathyroid gland or PTH
secretion, however, revealed that the skeletal phenotype of
CaSR single mutation (⫺/⫺) could mostly be rescued, suggesting that the skeletal abnormalities were due to high
circulating PTH levels rather than CaSR inactivation (598,
1096). Furthermore, CaSR does not seem to be the exclusive calcium-sensing mechanism in osteoblasts, as changes
in extracellular calcium can still elicit functional responses
in CaSR (⫺/⫺) osteoblasts (852). This observation has been
attributed to another GPCR with calcium-sensing capabilities, namely, GPRC6A (850, 851). Although GPRC6A has
a higher activation threshold for calcium, it also responds to
the CaSR allosteric activator R568 (851). GPRC6A activation may thus represent a confounding factor in most in
vitro studies on osteoblasts and their modulation by CaSR.
Also, GPRC6A knockout leads to osteopenia, further underlining the possibility of an alternate calcium-sensing
pathway in bone (850). Osteoblasts extracted from these
GPRC6A-deficient animals show decreased sensitivity to
extracellular calcium and in vitro mineralization defects
(854).
Although these observations have profoundly questioned
the physiological significance of CaSR in bone, closer examination still favors a role of CaSR in bone turnover. With
the recent advances in genetic methods, an osteoblast-specific CaSR (⫺/⫺) model has been created (171). These animals have severely stunted growth and skeletal development, clearly suggesting an involvement of CaSR in normal
osteoblast function (171). The previous conflicting evidence
gained from global CaSR (⫺/⫺) models with survival rescue
by elimination of PTH synthesis have been attributed to the
possible expression of alternate CaSR splice variants, which
may compensate for the deletion of full-length CaSR in
these animals (171, 915). In an attempt to further elucidate
the function of CaSR in osteoblasts, the reverse approach
has been executed by specifically upregulating CaSR in osteoblasts with use of a constitutively active receptor mutant
(296). Upregulation of CaSR results in bone loss, as evidenced
by a decrease in bone volume and density, specifically of trabecular bone (296). These findings are accompanied by an
increased number of osteoclasts, whereas osteoblast parameters were essentially unchanged (296). Activation of CaSR has
been speculated to promote RANKL production by osteoblasts, which serves as an osteoclastogenic signal (296). Osteoblasts may thus recruit osteoclasts and induce their maturation
via CaSR signaling and increased RANKL expression, which
would explain the observed increase in bone turnover and
osteoclast numbers in the setting of constitutive CaSR activation (241).
V. THE STOMACH AND CALCIUM
Preceding parts of this review have independently summarized the physiology of acid secretion, intestinal calcium
absorption, and their respective regulation. The following
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SASCHA KOPIC AND JOHN P. GEIBEL
section will attempt to illustrate the functional intersections
between these seemingly unrelated fields. In particular, the
question of whether acid is needed to absorb calcium effectively from the gut or whether the stomach contributes to
the regulation of calcium homeostasis by secretion of an
endocrine substance will be investigated.
A. Proton Pump Inhibitors and the Risk
of Fracture
In May 2010, the Food and Drug Administration (FDA)
released the following safety announcement: “Healthcare
professionals and users of proton pump inhibitors should
be aware of the possible increased risk of fractures of the
hip, wrist, and spine with the use of proton pump inhibitors, and weigh the known benefits against the potential
risks when deciding to use them” (319).
Proton pump inhibitors (PPIs, see sect. IIC1) are in widespread use for the treatment of acid-related disorders, such
as gastroesophageal reflux disease (GERD) or gastric ulcer
disease. They exert their curative effects by inhibiting the
acid output of the stomach. Over the recent years, mostly
epidemiological evidence has accumulated which links the
intake of PPIs to an increased risk of sustaining fractures,
especially in the elderly population. Yang et al. (1190) published one of the earliest and largest studies investigating
this potential correlation in 2006. Examining a population
of over 13,000 hip fracture cases and over 135,000 controls
over the age of 50, the authors concluded that long-term
(over 1 year) PPI use was associated with an increase in hip
fractures (AOR ⫽ 1.44) (1190). Although the likelihood of
sustaining a fracture following PPI intake may seem fairly
low, the implications for public health are substantial. This
has multiple reasons: PPIs represent the third most commonly prescribed medication in the United States and are
also available as over-the-counter formulations. Furthermore, there is an ongoing debate whether PPIs are overprescribed, putting certain populations at unnecessary risk of
side effects. In combination with the high incidence of osteoporotic fractures, the mean incidence of hip fractures
alone between 1986 and 2005 was 957 per 100,000 women
over the age of 65 per year, a small increase in risk suddenly
has implications for a very large population (123).
The roots of this controversy may potentially be traced back
to the 1940s and 1950s. Before the advent of PPIs, total and
partial gastrectomies or vagotomies were performed to control acid-related disorders. It was soon apparent that patients who underwent these radical surgical procedures developed osteoporosis/-malacia (58, 305, 732, 876). A study
that assessed the prevalence of osteomalacia in gastrectomized patients concluded that up to 12% of patients (19%
of females) had histologically overt osteomalacia, although
general disturbances in calcium metabolism were estimated
to occur in up to 28% of patients (208, 368). Other inves-
232
tigations came to lower prevalence results of ⬃5–10%
(1091). The osteomalacia was also shown to translate into
an increased incidence of fractures in these patients (795).
Naturally, gastrectomy represents a radical intervention,
and the reasons for this correlation may be multifactorial,
but reduced acid output may be of significance.
Back in the field of PPIs, the seminal epidemiologic investigation by Yang et al. was subsequently followed up by a
number of studies, which also focused on other types of
fractures, other populations, and other drugs reducing gastric acid output, such as H2 receptor antagonists (202, 228,
252, 394, 400, 559, 868, 923). Although their conclusions
were somewhat controversial, a recent meta-analysis supports the initial hypothesis that a correlation between PPI
intake and fracture risk (hip, spine, and any-site fractures)
exists (1195). The meta-analysis considered 11 studies and
identified an overall odds ratio of 1.30 for all fracture types
combined (1195). There was no association between H2
blocker intake and an increase in fracture risk, although
some single studies supported a link (228, 1195). Another
meta-analysis came to a comparable conclusion with regard
to an increased fracture risk under PPI exposure (616).
B. Gastric Acid and Intestinal
Calcium Uptake
A variety of reasons could theoretically account for the
observation that PPIs increase the likelihood of fractures.
The most prominent hypothesis assumes that the reduced
acidity in the stomach impairs the intestinal absorption of
dietary calcium. This assumption is based on both patient
observations and experimental animal data. Alas, the number of animal studies, which in contrast to investigations in
humans per default allow more radical experimental designs and genetic manipulation, is very small.
1. Effects of gastrectomy, vagotomy, and PPIs on
mineral metabolism in humans
Before discussing the reports that try to correlate PPI use
with calcium uptake, it is worthwhile to examine older
literature on patients that had undergone partial or total
gastrectomy. As discussed previously, these procedures are
known to be linked to bone disease. In contrast to PPIs,
which eliminate the singular factor of acid secretion, gastrectomies also influence gastric emptying, the emulsification of food stuffs, and food habits. It is thus more difficult
to draw conclusions on the influence of acid secretion on
calcium absorption from gastrectomized patients than from
individuals on PPI therapy. A further caveat lies in the type
of gastrectomy, as different surgical procedures are and
were in use. Some surgeries bypass the duodenum (Billroth
II, Roux-en-Y, total gastrectomy), whereas some leave the
duodenal passage intact (Billroth I).
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A common finding among gastrectomized patients is their
low 25(OH)-vitamin D levels, while levels of 1,25(OH)2vitamin D seem to be increased (101, 244, 378, 511, 794,
972, 1081). The pathophysiological reason for this is not
entirely clear. It has been argued that bone disease and low
25(OH)-vitamin D are a result of impaired vitamin D absorption following surgery; however, the consensus seems
to be that uptake rates of vitamin D is not impaired in these
patients (245, 378; contested by Ref. 1081). The vitamin D
insufficiency may also be a byproduct of improper nutrition
(378). As fat and milk intolerance can develop, especially in
surgeries which exclude the duodenum (Billroth II), a
change of dietary habits with insufficient intake of the fatsoluble vitamin D may be an underlying cause. Indeed,
long-term longitudinal studies suggest that maintaining
body weight reduces the risk of developing bone disease
after gastrectomy, which emphasizes the role of adequate
nutrition (667, 668). On the other hand, Billroth I and II
patients show the same loss in bone density, although Billroth II surgery (bypassing of the duodenum) is associated
with a much higher degree of fat malabsorption (110, 651).
A different investigation even concluded that Billroth I patients have a higher loss in bone density than Billroth II
patients (794). A more recent report suggests that the problem underlying bone disease after gastrectomy may be impaired calcium absorption, rather than dietary vitamin D
deficiency (244). It has been shown that high 1,25(OH)2vitamin D levels accelerate the breakdown of 25(OH)-vitamin D (210 –212, 244). The observed low 25(OH)-vitamin
D levels in gastrectomized patients may thus be a byproduct
of increased catabolism, and not insufficient intake,
whereas the high 1,25(OH)2-vitamin D levels may represent
compensatory upregulation due to insufficient calcium absorption or intake (244). Calcium absorption in gastrectomized patients (Billroth I ⫹ II) has been reported to be in the
low-normal range, while 1,25(OH)2-vitamin D levels are
increased (794). In line with these findings, secondary hyperparathyroidism, an indicator of compensatory upregulation due to low serum calcium levels, is a known finding
after gastrectomy (101, 244, 1171). Other investigations on
intestinal calcium absorption in gastrectomized patients
came to very contradictory results, ranging from increased
to impaired absorption (8, 34, 255, 398, 585, 794). Many
of these reports failed to assess 1,25(OH)2-vitamin D and
PTH levels, which means that adaptive mechanisms may
mask the insufficient baseline uptake of calcium in the intestine (8, 34, 255, 398, 585, 794). In conclusion, the exact
pathogenesis of postgastrectomy osteopenia remains somewhat unclear. The disorder may be attributable to vitamin
D insufficiency, impaired calcium absorption, inappropriate diet, or a combination of all factors.
The intrusiveness of gastric surgery makes it difficult to
dissect the influence of gastric acid on these parameters.
This is why vagotomized patients are a somewhat more apt
patient population to study the effects of gastric acid on
calcium uptake, albeit the number of studies on this cohort
is very limited. Vagotomy abolishes the parasympathetic
input to the stomach and thereby decreases the amount of
secreted acid. Although the gross anatomy of the stomach
remains intact, other parameters, such as gastrin levels, are
also deranged given the important role of the vagus nerve in
the regulation of gastric acid secretion (see sect. IIB). While
bone disease is generally not reported in these patients, low
25(OH)-vitamin D levels are common (514, 793). Similarly
to gastrectomy, the 1,25(OH)2-vitamin D levels are concomitantly elevated, suggesting adaptive upregulation potentially to compensate for decreased calcium absorption
(793). As discussed previously, the decreased 25(OH)-vitamin D could be indicative of augmented catabolism of the
vitamin (244). Serum calcium is commonly decreased or in
the lower normal range, while intestinal calcium absorption
is increased, presumably in response to elevated 1,25(OH)2vitamin D (110, 974). Secondary hyperparathyroidism does
not manifest (793, 974).
In general, the disturbance in mineral metabolism is more
pronounced in gastrectomized patients than in vagotomized patients, as evidenced by the higher incidence of bone
disease and secondary hyperparathyroidism. It is challenging to draw clear conclusions on the influence of gastric acid
on calcium absorption in either patient group. Yet, it is
apparent that compensatory mechanisms are in place in
these patients, as evidenced by the increased 1,25(OH)2vitamin D and PTH levels. Less efficient calcium uptake due
to decreased acid output may be one explanation for this,
but without further analysis this conclusion remains speculative.
With the advent of PPIs and H2 blockers, the number of
surgical interventions to control acid-related disorders decreased massively. Given their high specificity, PPIs selectively eliminate gastric acid output. Several investigations
that try to tie PPI intake to a disturbance in mineral metabolism exist. Graziani et al. (396) observed in eight healthy
volunteers that postprandial calcium concentrations did
not increase in subjects on a PPI regime (omeprazole 20 mg
3⫻ daily), whereas control subjects demonstrated a clear
spike in serum calcium levels. Urine calcium excretion was
also reduced compared with the control group (396). A
similar effect of PPIs was later observed by two independent
groups in patients undergoing hemodialysis (395, 423). It
should be noted that neither of these studies directly assessed intestinal calcium absorption, but rather measured
serum calcium as an indirectly related parameter. More
recently, intestinal calcium absorption was measured by
O’Connell et al. (807) using a radiolabeled calcium isotope.
The investigators reported that 7 days of PPI (omeprazole
20 mg 1⫻ daily) intake significantly reduced calcium absorption in elderly women under fasting conditions compared with the placebo group. Although these studies support a role of PPIs in reducing calcium uptake, conflicting
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evidence exists. Several investigations that assessed calcium
absorption using radioactive tracers and a whole gut lavage
technique found no evidence for a decrease in absorption
under short-term PPI treatment (421, 991, 1173). It is not
clear why these discrepancies in the outcomes of the trials
exist. There is, however, variability in the experimental
technique to measure calcium uptake, the cohorts investigated (young vs. postemenopausal women vs. dialysis patients) and the form of calcium administration (calcium
salts vs. whole meals), which may partially account for the
divergent results. Indeed, different calcium salts are absorbed with different effectiveness in acid suppressed individuals, which will be subject of later discussion (see sect.
VC). Furthermore, different populations may have different
capacities for endocrine compensation.
In summary, it is experimentally difficult to unmask the
potential correlation between a reduction in gastric acidity
and calcium absorption, given our body’s high capacity for
compensation. In addition, slight alterations in mineral homeostasis may take years to manifest themselves clinically,
for example, in osteopenia or fractures. Without following
up on test subjects on a long-term basis, snapshot measurements which may still lie within clinically normal range can
be misleading.
2. Effects of gastrectomy, vagotomy, and PPIs on
mineral metabolism in the animal model
To further elucidate the problem of osteopenia following
gastrectomy or PPI use, several animal studies tried to replicate and expand the observations made in human test
subjects.
For example, Axelson et al. (49) measured serum calcium
concentrations in rats who had undergone parathyroidectomy and various surgical procedures to reduce gastric acid
output (vagotomy, antrectomy, gastrectomy). While parathyroidectomy alone predictably reduced serum calcium
levels, the gastric operations (with intact parathyroid
glands) had little to no effect on calcium concentrations
(49). Interestingly, intestinal calcium absorption was even
increased in the latter group. The authors attributed this
observation to a compensatory upregulation of PTH secretion and concomitant 1,25(OH)2-vitamin D production. To
eliminate this factor, gastrectomy or fundectomy was conducted after parathyroidectomy, thereby depriving the animals of their compensatory machinery. This intervention
resulted in massive hypocalcemia and death after a few
days, which led the authors to conclude that acid secretion
is important for the maintenance of calcium homeostasis
(49). Another investigation in rats that had undergone
antrectomy (Billroth I) observed a significantly decreased
absorption of calcium (345). Fundectomy did not affect
calcium absorption (927). However, both studies employed
the balance method to calculate calcium absorption, which
is considered less accurate than using radiotracers. In pigs,
234
total gastrectomy causes massively reduced calcium uptake
and secondary hyperparathyroidism (700). In this study,
the duodenum was surgically bypassed by esophagojejunostomy, thereby eliminating the site of maximal active
calcium absorption and limiting the conclusion that can be
drawn (700).
In addition, several reports of vagotomy in a rat model
are available to us (49, 307, 308, 928). It has been demonstrated that vagotomy alone has no effect on the rate of
intestinal absorption (307, 928). Secondary hyperparathyroidism was observed by one group, while PTH levels
were reported to be unaffected by the other group (307,
928). 1,25(OH)2-vitamin D was not measured, which
would have provided further evidence of compensation
due to decreased calcium bioavailability. However, if vagotomy and parathyroidectomy are performed together,
intestinal calcium absorption is significantly impaired
compared with vagotomy or parathyroidectomy alone
(307). This is in accordance with the low serum calcium
concentrations found in gastrectomized and parathyroidectomized rats (49).
PPIs were also used to relate acid secretion to bone disease in rats. Bone weight did not change in rats that were
treated for 4 wk with omeprazole (841). Alas, calcium
absorption was not measured in these animals, and a
decrease in bone weight represents a very terminal and
long-term outcome. A more recent investigation by
Schinke et al. (963) demonstrates that mice which have
been genetically manipulated to be achlorhydric (CCK2
⫺/⫺) have decreased serum calcium levels as well as develop osteoporosis and secondary hyperparathyroidism
in an effort to maintain calcium balance (963). This study
is especially noteworthy, as acid secretion is knocked out
selectively in this mouse model while the stomach remains intact (stomach morphology). Furthermore, a genetic mutation that has been associated with osteopetrosis (a disease characterized by increased bone density)
due to osteoclast malfunction was also shown to cause
decreased gastric acid secretion. These patients present
with lower serum calcium values. Rather than being a
product of impaired bone resorption (osteoclast defect),
the hypocalcemia may thus be related to impaired intestinal calcium absorption (gastric acid secretory defect)
(963).
C. Calcium Salts
Many investigators have employed calcium salts to determine the efficacy of intestinal calcium absorption. Furthermore, calcium salts are in wide clinical use as a dietary supplement. As will be discussed in this section,
calcium salts differ in their bioavailability, which not
only represents a potential source of error in experimental designs, but more importantly, has extensive clinical
implications.
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Calcium salts represent the most common supplementation
form of calcium for individuals who do not meet their adequate daily intake. The indications for supplementation
can be diverse, but mostly include conditions such as osteoporosis/-penia or preventative intake after menopause, during glucocorticoid intake or if lactose intolerant. In the year
2000, the National Health Interview Survey concluded that
11% of Americans ingest calcium supplements on a daily
basis (739). Females account for 80% of this population,
mostly to ensure supply after menopause (739). Calcium
salts exist in multiple formulations. The most commonly
used salts are calcium carbonate, calcium citrate, calcium
lactate, and calcium gluconate. Calcium carbonate is the
most widely used formulation, because it contains the highest percentage of elemental calcium per weight (40%),
which equates to small tablet size and easier ingestion (997).
In comparison, calcium citrate contains 21% elemental calcium, calcium lactate 14%, and calcium gluconate 9%
(997). However, the calcium salts do not only differ in their
calcium fraction, but are massively divergent with regard to
their solubility in water. Calcium carbonate is the least water-soluble salt at a neutral pH. For example, calcium citrate
dissolves 17 times more readily in water than calcium carbonate (997). Very fundamental in vitro solubility experiments have shown that after 1 h in 500 ml of water only 1%
of the initial 500 mg of calcium carbonate are dissolved at
37°C (997). The solubility of calcium carbonate can be
greatly improved by an acidic environment (390, 997). Adjusting the pH to 5.5 in the same experiment dissolves 86%
of the calcium carbonate; further lowering it to 2.5, a value
that can be expected in the stomach, increases the dissolved
fraction to 100%. Given these differences in solubility, a
plethora of studies have investigated the bioavailability of
the various calcium salts, mostly focusing on the difference
between calcium carbonate and calcium citrate. Again, the
conclusions are heterogenic. Several studies suggest that
calcium carbonate is absorbed less effectively than the more
soluble calcium citrate (422, 438, 439, 789), while others
conclude that there is no difference in bioavailability (432,
433, 520, 547, 831, 832, 891, 997, 1038). A detailed analysis of the individual trials is beyond the scope of this review. It suffices to say that there is strong variability in the
experimental methods (direct absorption measurements vs.
measurement of postprandial serum calcium vs. urine excretion) and design of the studies (populations; administration in the fasting state vs. with a meal). A confounding
factor to the results of these studies may be the gastric pH at
the time of the measurement. As discussed earlier, calcium
carbonate is not very soluble at more alkali pH values,
which may have implications for patients using these supplements while on PPI therapy (997). Furthermore, meals
dramatically affect gastric pH and may change the bioavailability of the supplements. Indeed, there seems to be a correlation between gastric pH and the absorbability of calcium carbonate. The first observation indicative of this association was made by Ivanovich et al. in the late 1960s
(520). The group reported that absorption of calcium carbonate was severely impaired in four male patients suffering
from achlorhydria (520). Compared with five control subjects, who absorbed between 9 and 18% of the ingested
calcium carbonate, these patients only absorbed 0 –2%. Interestingly, when gastric acid secretion of one of these patients was stimulated by administration of betazol hydrochloride (a histamine analog), calcium carbonate absorption rose from 2 to 10% (520). This investigation somewhat
spawned the entire controversy of whether gastric acid is
necessary to absorb calcium effectively from the intestine. A
similar investigation was conducted later by Recker
(891) in a larger sample of achlorhydric patients. The
investigator concluded that 1) control subjects absorb
calcium carbonate and calcium citrate equally well,
2) but that achlorhydric patients lose their capability to
absorb calcium carbonate, while calcium citrate absorption is increased (presumably through compensatory upregulation of the absorption via vitamin D) (891). It is
important to mention that these absorption assays were
conducted in the fasting state. When the achlorhydric
patients ingested the calcium carbonate salt together with
a meal, their calcium uptake normalized. It cannot be
conclusively answered which factor of the meal was responsible for the increase, as several food components,
such as fiber and protein, are known to affect calcium
uptake. However, the authors speculated that the pH
(5.8) of the meal was sufficiently low to dissolve the
ingested calcium carbonate (891). Furthermore, a previously cited study that demonstrated decreased calcium
absorption under PPI therapy employed calcium carbonate as source of calcium for the conducted measurements
(807). Patients with gastric bypass surgery also absorb
calcium carbonate less effectively than calcium citrate
(1089). The importance of acid for the absorption of
calcium carbonate was also demonstrated in the previously discussed achlorhydric CCK2 (⫺/⫺) mouse model
(963). The osteoporotic phenotype and secondary hyperparathyroidism in these achlorhydric mice could only be
fully rescued by a high-calcium gluconate (2%) diet, but
not by a high-calcium carbonate (2%) diet (963). In the
light of these reports, it is evident that although the bioavailability of calcium salts in the healthy individual may
be equal, an impairment of acid secretion has a negative
effect on the bioavailability of calcium carbonate, presumably because of decreased solubility. Given the fact
that calcium carbonate is the most commonly used formulation for calcium substitution therapy, these findings
may partially account for the statistical correlation between PPI use and the increased risk of sustaining fractures.
Another factor that needs to be taken into consideration
when assessing solubility of calcium salts is the PCO2
(390). In the local milieu of the duodenum, the PCO2 can
reach values of up to 300 mmHg, resulting from the
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SASCHA KOPIC AND JOHN P. GEIBEL
pancreatic secretion of bicarbonate (929). Solubility experiments of calcium carbonate have shown that a high
PCO2 negatively affects its solubility, as the carbonate
enters equilibrium with CO2 (390). Compared with other
calcium salts, the particularly low bioavailability of calcium carbonate in acid suppressed patients may thus be a
compounded effect of reduced acid secretion and a high
duodenal PCO2.
D. The Endocrine Stomach and
Calcium Homeostasis
Apart from being a mere acid secretory organ, the stomach
also plays an important role as an endocrine organ. It
should be noted that all of the aforementioned surgical or
pharmacological interventions, i.e., gastrectomies, vagotomies, or pharmacological acid suppression therapy, will inevitably impact the endocrine functions of the stomach. It is
therefore plausible that not only the changes in intragastric
pH affect the absorption of calcium, but that the dysregulation of the endocrine stomach is responsible for changes in
calcium homeostasis. The following section will address
how hormones that are secreted by the stomach may impact
calcium and bone homeostasis.
1. Ghrelin
Ghrelin has been discovered fairly recently (1999) by Kojima and colleagues and is mainly implicated in regulating
food intake in the hypothalamus (588, 777). Ghrelin levels
are inversely correlated with body mass and elevated in
conditions of fasting, such as anorexia nervosa (377). Ghrelin is mainly synthesized and secreted in a pulsatile manner
by special neuroendocrine cells (P/D1 cells) in the fundic
region of the stomach (242, 777). The influence of ghrelin
on gastric acid secretion is discussed in a separate section
(see section IIB5E). A few years after its discovery, it was
shown that ghrelin can also directly affect osteoblasts (254,
357, 576, 691). Ghrelin induces osteoblast proliferation
and differentiation and inhibits their apoptosis (357, 576,
691, 1141). It is not entirely clear whether this effect is
mediated via the ghrelin surface receptor, the growth hormone secretagogue receptor 1a (GHS-R1a), or not. While
the receptor is expressed in rat and murine osteoblasts and
its pharmacological inhibition abolishes the effects of ghrelin on differentiation and proliferation, no GHS-R1a
mRNA could be detected in a human osteoblast cell line
(254, 357, 691). It should be noted that this effect is independent of growth hormone (GH). Ghrelin serves as a potent stimulator of GH secretion from the pituitary gland,
which in turn acts as an activator of osteoblasts through the
GH/IGF-I axis. However, the observations that 1) pharmacological inhibition of GHS-R1a attenuates the effects of
ghrelin and that 2) GH-deficient rats are still sensitive to
ghrelin, suggest a direct effect on osteoblasts (357). In vivo,
the activation of osteoblasts translates to an increase in
236
bone mineral density (BMD) in rat and murine models (261,
357). Ghrelin also promotes the formation of new bone
following injury (261). For example, mice that received a
standardized bone injury demonstrated 1.6 times more new
bone surface if treated with ghrelin compared with control
animals (261).
Several studies aimed to identify a link between serum ghrelin levels and BMD in human populations. The most recent,
and one of the largest (n ⫽ 707 subjects), investigation
assessed BMD with peripheral quantitative computed tomography (pQCT). This technique allows for separate
analysis of trabecular and cortical bone. The results showed
a positive correlation between ghrelin and trabecular BMD
in elderly men and women (775). A different large-scale
study, investigating a similar cohort (n ⫽ 977) found no
association using dual-energy X-ray and single-photon absorptiometry (1157). These techniques, however, do not
permit a discrimination between cortical and trabecular
bone. Other small-scale studies also came to contradictory
conclusions (388, 811). The reason for these discrepancies
is elusive. Since the formation of trabecular bone represents
a more dynamic process, its direct measurement may be
more sensitive to subtle changes than overall bone density
measurement (775). Baseline plasma ghrelin levels were
also shown to be inversely correlated to type 1 collagen ␤
C-telopeptide (␤CTX), a marker for bone resorption (501).
The source of ghrelin represents another potential caveat. In
vitro studies suggest that osteoblasts can also synthesize
ghrelin (254, 357). Ghrelin was identified on the mRNA
and protein level by two investigations (254, 357). A different group did not find evidence for ghrelin in osteoblasts
(214). This has important implications, as ghrelin may be
secreted in an auto-/paracrine fashion, which would make
plasma ghrelin levels less significant for osteoblast activation. On the other hand, (partial) gastrectomy significantly
decreases plasma ghrelin concentrations, which could contribute to postgastrectomy osteopenia, although these may
just be two independent factors. Total gastrectomy causes a
drop in plasma ghrelin levels by as much as 70% (528).
Partial gastrectomy also severely decreases plasma ghrelin;
however, levels normalize depending on the type of resection to 48 – 88% of the preoperative levels due to compensatory production in the remaining gastric mucosa (528).
This recovery already occurs after 7days. It thus remains to
be elucidated if the slightly decreased ghrelin levels after
small gastric resections can account for the long-term phenomenon of postgastrectomy osteopenia. Furthermore, in
mice, the reduction of bone mass after gastrectomy cannot
be rescued by exogenous administration of ghrelin (277).
Ghrelin administration did also not affect markers of bone
resorption in gastrectomized patients, although these parameters were only measured very acutely 4 h after ghrelin
infusion (501). So far, no data on the effect of chronic
ghrelin treatment on BMD are available.
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Although decreased ghrelin levels could in theory be a contributing factor to the reduction of bone mass following
gastrectomy, it is unknown if PPIs can directly affect ghrelin
levels. A potential link between PPI-related fractures and
ghrelin levels remains to be investigated. An indirect association may be present in patients with Helicobacter pylori
infections. These infections represent a common indication
for PPI intake and were suggested to coincide with reduced
ghrelin in plasma and the gastric mucosa (527). Given the
recent discovery of ghrelin’s impact on osteoblast function,
many questions still remain to be answered. It is, however,
clear that our view of the stomach as a mere acid secretory
pouch needs to be expanded to a new level.
2. Gastrin
Gastrin represents one of the main acid secretagogues (see
sect. IIB2). It is secreted by specialized G-cells in the antrum
of the stomach and the duodenum. The released gastrin
enters the circulation and induces acid secretion in gastric
parietal cells via the CCK2 receptor. It has been hypothesized fairly early that plasma gastrin may have an impact on
bone metabolism. Injection of gastrin and its synthetic analog pentagastrin was shown to decrease plasma calcium
levels in pigs and rats in the 1970s (222, 980). This effect
was attributed to gastrin-stimulated release of calcitonin
from the thyroid gland. Indeed, pentagastrin was shown to
be a potent stimulator of calcitonin secretion in various
species and is still in clinical use to evaluate thyroid C-cell
hyperplasia and medullary carcinomas (155, 156, 226, 441,
829). Although a clinical correlation between plasma gastrin levels and plasma calcitonin has been demonstrated by
one study in patients with Zollinger-Ellison syndrome (hypergastrinemic patients) and in pigs, it is unclear if native
gastrin, i.e., not pentagastrin, acts as an important secretagogue for calcitonin in humans (224, 1020). In fact, other
investigations found no association between gastrin and
calcitonin levels in other cohorts (122, 454).
At least in the rat, the hypothesis of the gastrin-calcitonin
axis has been severely challenged. Although gastrin decreases plasma calcium levels in rats, the same effect occurs
in (para)thyroidectomized animals, suggesting that calcitonin is not involved in this process (980). Furthermore,
cultured rat thyroid cells could not be stimulated to release
calcitonin if incubated with gastrin (225). Fundectomy- and
omeprazole-induced hypergastrinemia also did not affect
calcitonin levels in rats (843, 927). Interestingly, hypocalcemia after gastrin injection could not be induced in rats
that had been (para)thyroidectomized and gastrectomized
(844, 981). This observation led to the conclusion that gastrin may stimulate the release of an unknown substance
from the rat stomach, which in turn exerts calcitropic activity. In accordance with this hypothesis, mucosal extracts
from rat stomachs were shown to have the same hypocalcemic effects as gastrin and to stimulate uptake of radiolabeled calcium into the bone (844). These findings were also
replicated in chicken (842). The unknown hormone was
tentatively named ”gastrocalcin“ (844). When ghrelin was
discovered, it was speculated that it might represent a candidate hormone for gastrocalcin. However, unlike gastrocalcin, ghrelin is not under gastrin control, making this
proposition unlikely (276). Subsequent investigations suggested that the origin of gastrocalcin were the gastric ECL
cells. ECL extracts can indeed trigger a calcium second messenger response in osteoblast (629, 630). Yet, functional
evidence for gastrocalcin-mediated osteoblast activation is
still lacking. A recent report postulates that parathyroid
hormone-like hormone (PTHLH) may in fact be gastrocalcin (676). PTHLH exerts similar physiological effects as
PTH by sharing a common receptor and is commonly elevated in paraneoplastic syndromes (1056). PTHLH has
been identified in ECL cells, and its transcription has been
shown to be inducible by gastrin in parietal cells (523, 676).
Of note, PTH causes effects opposite to those assigned to
gastrin and gastrocalcin, namely, hypercalcemia. Further
studies will thus be needed to corroborate this hypothesis.
Whatever the exact effector hormone of gastrin may be,
changes in gastrin levels cannot entirely explain the clinical
phenomenon of postgastrectomy osteopenia and PPI-related fractures. Although vagotomy and PPIs undoubtedly
increase serum gastrin levels through a negative-feedback
mechanism, most partial and all total gastrectomies result
in hypogastrinemia. Yet both hyper- and hypogastrinemic
conditions have similar outcomes, i.e., osteopenia and increased risks of fractures. It is, of course, plausible that
different factors contribute to this outcome in each individual group. Gastrin may be involved in certain pathologies,
but given that its true impact on bone metabolism is somewhat elusive, this assumption remains speculative.
Acid suppression
Calcium solubility
Gastrin
?
Intestinal absorption
Pancreastatin
Calcitonin
PTH
FIGURE 9. Model summarizing the potential impact of acid suppression on calcium homeostasis.
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SASCHA KOPIC AND JOHN P. GEIBEL
3. Pancreastatin
Pancreastatin is a cleavage product of chromogranin A that
was initially isolated from porcine pancreas (1072). Gastric
ECL cells are also known to harbor significant amounts of
chromogranin A and pancreastatin. Pancreastatin is secreted together with histamine from ECL cells in response
to their neuroendocrine stimulation (see sect. IIB3) (176). In
rat, it has been shown that the serum pancreastatin levels
correlate with the secretory status of ECL cells. States that
enhance ECL cell secretion, such as gastrin infusion, resulted in elevated serum pancreastatin levels (411). In accordance with this hypothesis, and of special relevance for
the topic of this review, PPI therapy also resulted in increased serum pancreastatin levels (the ECL is stimulated by
gastrin, which in turn is released in response to high gastric
pH) (411). These observations led the investigators to conclude that ECL cells are a major contributor to the serum
levels of pancreastatin in the rat and that these levels change
in parallel with ECL cell secretion (411). This is also corroborated by the observation that gastrectomy reduces pancreastatin levels in rats (644).
review we have focused on the important role calcium plays
as a first and second messenger in the maintenance of bone
health. By relying on a complex series of receptors, channels, and transport proteins, calcium is tightly controlled at
the cellular and tissue level to ensure its bioavailability to
bone. Modulations to any of these pathways by disease,
mutation, or pharmaceutical perturbation can lead to clinical changes in bone health.
ACKNOWLEDGMENTS
S. Kopic is a Howard Hughes Medical Institute International Student Research Fellow. Special thanks to Sashka
Dimitrievska for her untiring support and critical editing of
the manuscript.
Address for reprint requests and other correspondence: J. P.
Geibel, Yale School of Medicine, 310 Cedar St., BML 238,
New Haven, CT 06510 (e-mail: John.geibel@yale.edu).
DISCLOSURES
Pancreastatin exerts a variety of metabolic effects. Apart
from influencing energy metabolism, pancreasstatin was
shown to affect the secretion of PTH from the parathyroid
gland. In isolated bovine and porcine parathyroid cells,
pancreastatin has a clear inhibitory effect on PTH secretion
(282, 317, 911). The suppression of PTH functions on a
transcriptional level (1211). Reduced PTH secretion in turn
would have a potential impact on calcium and bone metabolism. Whether the same observations are valid for humans
is less clear, as pancreastatin failed to inhibit PTH secretion
from isolated human parathyroid cells (911). Regardless,
the volume of data on pancreastatin and its influence on the
parathyroid gland is very small, and further investigations
would be necessary to establish this intriguing link. Apart
from potential modulation of parathyroid secretion, pancreastatin has also been shown to have an inhibitory effect
on gastric acid secretion (655).
In summary, it should be noted that the stomach secretes
not only acid, but also hormones that have been shown to
directly alter calcium and/or bone homeostasis. The secretion of these hormones depends on the neuroendocrine machinery that also regulates acid secretion. It is therefore
plausible that the correlation between states of impaired
acid secretion and impaired bone mineralization is multifactorial by depending on intragastric pH and serum levels
of gastric hormones (FIGURE 9).
No conflicts of interest, financial or otherwise, are declared
by the authors.
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