Acid and Base Transport
Where does it happen?
• VERTEBRATES
• Stomach - gastric acid secretion
• Kidney proximal tubule - urinary
acidification (urinary bladder in lower verts)
• Exocrine pancreas/duodenum - alkali
secretion
• Osteoclasts - bone remodeling
• INVERTEBRATES
• Gut
• Excretory Organs
Vertebrate stomach - the H+/K+ ATPase mechanism
of acid secretion
the mechanisms
of HCl
secretion in
gastric parietal
cells (= oxyntic
cells). The
K+/H+ ATPase is
specifically
inhibited by
omeprazole, a
drug developed
to treat peptic
ulcers.
A complex control system: the vertebrate
stomach
• Effectors: parietal cells, which secrete 3-4
liters/day of fluid with pH < 1.
• 1st order control cells: Enterochromaffinlike (ECL) cells resemble mast cells of the
non-specific immune system - secrete the
monoamine histamine.
2nd order control: 2 kinds of enteroendocrine
cells
G cells which secrete the peptide gastrin
D cells which secrete the peptide somatostatin
The turn-on pathway at the beginning of a meal
Amino acids from
meal
G CELL
Bombesin
Gastrin
CCK-B receptor
Ach
ECL CELL
Parasympathetic
N.S.
Histamine
H2 receptor
PARIETAL CELL
Norepinephrine
Sympathetic
N.S.
(inhibits)
HCl
Turn-off pathways that protect the stomach and
duodenum
Secretin: hormone - from
duodenum, secreted when
duodenal pH falls
D cell
somatostatin
SST 2 receptor
Prostaglandin:
from stomach
tissues secreted
in response to
tissue damage
ECL cell
Histamine
Parietal cell
HCl
Longer term effects of gastrin on ECL
cells
• 30-120 min: Increase in expression of the
gene for histidine decarboxylase - this gene
has Ca++ response elements.
• Days-weeks: elevated gastrin levels cause
mitosis of ECL cells (hyperplasia) caused
by activation of Ca++-sensitive tyrosine
kinase
Stimulus-secretion coupling in ECL cells
• Gastrin binds to CCK-B receptor on ECL
cell
• CCK-B receptor is coupled to G-protein
• G-protein activates Ca++ channels (?) and/or
pathway to intracellular Ca++ release- [Ca++]
increase is detectable within 30 sec of
gastrin binding
• rise in Ca++ stimulates exocytosis of
histamine-containing vesicles within 5 min.
Competition between turn-on and turn-off G
proteins for control of Ca++ channels in ECL
cells
Ca++
CCK-B (gastrin)
Gq
SSTR 2 (somatostatin)
Gi
Sites of action of some relevant drugs
that, until recently, were used for peptic
ulcers
• H2 receptor blocker: ranitidine
• H+/K+ pump blocker: omeprazole
• prostaglandin synthesis blocker: aspirin
The acid-secreting mitochondria-rich cell
Key features: The apical membrane contains
vacuolar-type H+ ATPase; the basolateral
membrane contains Cl-/HCO3- exchanger (AE).
The cytoplasm
contains abundant
carbonic anhydrase
and ER vesicles.
MR cells from rat kidney (left) and toad
urinary bladder (right).
Where membrane proteins are in cells:
immunohistochemistry.
1. Isolate membrane domain by
homogenization/sonication followed by differential
centrifugation
2. Solubilize/purify membrane proteins
3. Make monoclonal antibodies to individual membrane
proteins
4. Get fluorescent-labeled secondary antibodies. For
example, if the monoclonal was raised in mouse cells, get
fluorescent-coupled antibodies against the mouse antibody
raised in goat, rat, rabbit, etc.
5. Treat tissue samples with the primary antibody and then
add the secondary antibody
6. Examine under fluorescence microscope
V-Type H+ATPase histochemistry
This rat kidney tubule has
been stained with antibody to
the V-Type H+ ATPase. The
stain shows up as yellowgreen glow.
Only intercalated cells stain.
The gaps between stained
cells are filled by unstained
principal cells, which are not
involved in acid-base
transport.
Some cells have apical
labeling only (Alpha cells),
some have basolateral stain
only (Beta cells) and some
have both (Gamma cells).
Depending on the homeostatic demand, H+ATPase and AE can be targeted to apical or
basolateral membrane
• Turtle bladder and mammalian nephrons
can secrete either acid or base
• Current evidence suggests that depending
on whether the animal is acidotic or
alkalotic, V-ATPase and AE containing
vesicles may insert in either membrane,
allowing the same cell to convert between
the A and B cell type.
You could read more about this in:
Brown, D. & Breton, S. (1996) Mitochondria-rich,
proton-secreting epithelial cells J. Exp. Biol. 199,
2345-2358.
Bone remodeling and acid
secretion
• Bone is a complex of Ca Carbonate and Ca
Phosphate in an organic matrix,
hydroxyapatite.
• Bone is deposited by osteoblasts, but it is
constantly being remodeled in response to
mechanical stress. Bone demolition is the
responsibility of osteoclasts.
The osteoclast is an honorary A cell
The micrograph shows three osteoclasts dissolving a
bone surface. The brown color is a deposit of antibody
to the V-ATPase. The plasma membrane next to the
bone surface could be thought of as an extracellular
lysosome.
A close encounter of the third kind: an
osteoclast landed here.
This scanning electron micrograph shows an eroded crater
on bone surface resulting from osteoclast activity.
Now for a little relief from acid
digestion - some alkali-secreting
cells.
Pancreatic secretion
Secretion of alkaline pancreatic juice is turned on as food
enters the intestine.The hormone secretin from the duodenum
is the signal for this. In A the secretory cells are inactive, with
a reserve of vesicles containing transporters. The binding of
the hormone with its receptor (B) causes vesicles to fuse with
the target membranes, turning on secretion (C).
The ‘stomach’ of mosquito larvae
Aedes aegypti
Overview of Gut Structure
Acid-base cycle in midgut
•
•
•
•
•
Anterior midgut raises lumen pH to >10
Posterior midgut returns pH to <8
Both processes are driven by V-ATPase
V-ATPase is basal in AMG; apical in PMG
Extreme alkalinization apparently protects the
animal against bacterial colonization.
• Buffered medium can overwhelm the alkali
secretion cycle, and the animals die as a result.
In anterior stomach, V-ATPase is characteristically
concentrated in distinctive patterns at the basal
membrane.
wholemount
sectioned
Intracellular pH is measured using the fluorescent probe BCECF
A: normal light microscope
view
B: fluorescence microscope
view with areas of interest
marked
C: 4 representative tissues’
response to serotonin and
postexperiment calibration
Serotonin-stimulated guts
secrete alkali and the
cytoplasm also becomes very
alkaline.
Comparison of simultaneous responses of an anterior
midgut ‘area of interest’ compared to a posterior
midgut one in the same tissue.
Intracellular alkalinization could be needed to get enough carbonate
for a carbonate transporter; it also increases the upper limit of luminal
alkalinization as determined by the energetics of the V-ATPase. Two
hypotheses are presently being tested.
After this lecture, you should be able to
• Describe two cellular mechanisms for
secreting acid, and give specific examples.
• Describe a general mechanism for secreting
alkali, and give at least one specific
example.