0163-769X/85/0604-0564$02.00/0
Endocrine Reviews
Copyright © 1985 by The Endocrine Society
Vol. 6, No. 4
Printed in U.S.A.
Dopamine: A Prolactin-Inhibiting Hormone*
NIRA BEN-JONATHAN
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana
46223
interactions, in particular with gonadal steroids, occur at
the level of the lactotroph.
The last decade has seen an explosion in the number
of publications on the DA-PRL interactions at the whole
body, organ, and cellular levels. Any attempt at a comprehensive treatise would be impractical. Therefore, this
review will focus on four topics: 1) the organization and
properties of DA neurons in the hypothalamus and posterior pitutary and alterations in their activity and secretion under various endocrine conditions; 2) central
and peripheral interactions affecting DA and/or PRL
secretions, including the enigma of what causes the massive rise in PRL during the suckling stimulus; 3) anterior
pituitary DA receptors, their structure, regulation, and
presence in PRL-secreting tumors; and 4) DA action at
the level of the lactotroph, with survey of the current
knowledge of cellular electrical activity, intrinsic PRL
pulsatility, and the three major intracellular mediators
of DA action, namely cAMP, calcium, and phospholipids.
A. Introduction
D
OPAMINE (DA) is an interesting and versatile
compound. In the central nervous system (CNS)
it is involved with the control of fine movements and
mental processes. Its association with disorders such as
Parkinsonism and schizophrenia is well recognized. In
the hypothalamo-hypophysial axis, DA is the primary
physiological inhibitor of PRL secretion. Currently, this
catecholamine represents the only nonpeptidergic hypothalamic agent with a well defined hypophysiotropic
function. By virtue of its dual role as a neurotransmitter
and a hormone, DA provides perhaps the best example
of neuroendocrine interactions. It also possesses a more
universal property. In all hormonal systems studied thus
far, whether at the hypothalamic, posterior pituitary, or
anterior pituitary level, DA functions as an inhibitor.
Perhaps the common denominator to these diverse cells
is the presence of D2 DA receptors, which are negatively
linked to the adenylate cyclase system.
PRL is a pituitary hormone which is prevalent in all
vertebrates from fish to man. It is involved with numerous reproductive and nonreproductive processes and is
an essential hormone for the initiation and maintenance
of lactation in mammals. In human, it is also the most
conspicuous hormone secreted by pituitary adenomas.
PRL hypersecretion is one of the major causes of neuroendocrine-related infertility in women and impotence
in men. Unlike the majority of endocrine cells, the pituitary lactotroph requires constant inhibition to keep its
secretory activity under control. Given that PRL does
not have a specific peripheral hormone to relay feedback
information to the lactotroph, the major inhibition is
provided by DA. Several neuropeptides and neurotransmitters as well as PRL itself impinge upon the DA
neurons and alter their activity and secretory rate. Other
B. The Hypothalamic and Posterior Pituitary
Dopaminergic Systems
1. Organization and neural properties
There are several distinct DA pathways within the
CNS that differ in distribution, organization, and function. Two major systems with relatively long projections,
the nigrostriatal and the mesolimbic, originate in the
substantia nigra. The first sends terminals to the striatum with dense innervation in the caudate-putanum, and
the second extends projections to various limbic and
cortical areas. The hypothalamus contains two major DA
pathways, the incertohypothalamic and the tuberoinfundibular (TIDA), and also receives projections from
periventricular DA neurons. The perikarya of the incertohypothalamic neurons are located in the caudal hypothalamus, zone incerta, and rostral periventricular hypothalamus and project to the dorsal hypothalamus,
preoptic area, and septum (1, 2). Although most of these
systems have some neuroendocrine functions, it is the
TIDA system which directly participates in the regulation of PRL secretion.
Address all correspondence and requests for reprints to: Dr. Nira
Ben-Jonathan, Department of Physiology and Biophysics, Indiana
University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana 46223.
* This work was supported in part by Grants NS-13234 and Research
Career Development Award NS-219 from the NIH.
564
Fall, 1985
DA, A PRL-INHIBITING HORMONE
The perikarya of the TIDA neurons are located in the
arcuate and periventricular nuclei of the medial basal
hypothalamus (MBH). These medium size neurons are
scattered among other, more numerous neurosecretory
neurons (3, 4). Because of their suborganization and
anatomical projections, they are further subdivided into
two groups: the TIDA, with terminals in the median
eminence and pituitary stalk, and the tuberohypophysial,
with terminals in the neural and intermediate lobes of
the pituitary. Except for a few fibers along blood vessels
which disappear after sympathectomy (4), the anterior
pituitary is not innervated.
DA axon terminals are especially abundant in the zona
externa of the median eminence, where they comprise as
much as a third of all terminals. They are found in close
proximity to other monoaminergic terminals, ependymal
cells, and precapillary spaces of the portal vessels (5).
The terminals are characterized by dense core vesicles,
15-120 nm in diameter (6), but do not appear to form
true synaptic connections (5, 6). DA projections to the
intermediate and neural lobes originate from the anterior
and central portions of the arcuate nucleus, respectively.
In the neural lobe, they are found in close proximity to
pituicytes, magnocellular axon terminals, and precapillary spaces. In the avascular intermediate lobe, they
make close contacts with melanocytes.
The relative concentration of dihydroxyphenylacetic
acid (DOPAC) is lower in the median eminence than in
other DA-rich brain areas. Since DOPAC levels represent the amount of DA released and then recaptured by
nerve terminals, Demarest and Moore (7) compared the
DA uptake capabilities of several brain regions. They
concluded that TIDA neurons lack a high affinity transport system for DA. Similar observations were made by
Annunziato et al. (7A), and are consistent with the
absence of synapses in these terminals and with their
close proximity to hypophysial portal capillaries. Thus,
the released DA is quickly transported by the blood away
from the terminals, and less is available for reuptake.
This might also explain the resistance of TIDA neurons
to destruction by the neurotoxin 6-hydroxydopamine,
which requires an active uptake mechanism (8).
The DA concentration in the posterior pituitary (neurointermediate lobe) is similar to that in the MBH, but
is lower than that in the median eminence (9-11). The
posterior pituitary contains tyrosine hydroxylase and
dihydroxyphenylanine (DOPA) decarboxylase, but not
DA /?-hydroxylase (11). Isolated posterior pituitaries can
synthetize DA de novo from tritiated tyrosine, with negligible production of norepinephrine (NE) (12, 13). The
source of these biosynthetic enzymes appears to be central, rather than local, since within 7 days after pituitary
stalk transection, the posterior pituitary DA concentration is reduced by more than 80% (14). A portion of local
565
NE, however, originates from the superior cervical ganglia (4, 14). There are diurnal variations in the concentrations of DA and NE, with the highest levels during
daylight hours (15). Both DA concentration (16) and
synthesis (17) in the posterior lobe increase significantly
after dehydration, whereas DA in the median eminence
remains unaffected.
There is further diversity within the TIDA system.
Injection of DA agonists increases and injection of DA
antagonists decreases DOPA accumulation in the posterior lobe, but not in the median eminence (18). This
suggests an autoreceptor regulation in the posterior pituitary, but its absence in the median eminence. Indeed,
well defined DA receptors are present in the intermediate
lobe (19), but not in the median eminence (20). Additional evidence has been obtained using the neurotoxin
monosodium glutamate (MSG), which causes a selective
destruction of retinal and arcuate nuclear neuronal perikarya (21). As shown in Fig. 1, MSG reduces DA in the
MBH by more than 60%, but does not affect the posterior
pituitary DA concentration (21-23). The unchanged levels of NE in the two sites are consistent with its origin
in extrahypothalamic neurons (4, 14). In similar studies,
Morgan et al. (24) reported that DA levels in the median
eminence of female Snell dwarf mice are severely reduced, but DA concentrations in the posterior pituitary
are unaffected. These observations suggest that the perikarya or axon terminals of the two TIDA branches
might have different susceptibilities to pharmacological
agents or genetic defects.
Dopamine secretion into hypophysial portal blood
Blood flow to the pituitary gland is one of the highest
in any tissue and is maintained at a fairly constant rate
MBH
50-
•
Control
POSTERIOR
PITUITARY
-20
-15
40-
S.E
U
CD
u
30-
- 10
20-
-5
10-
DA
NE
DA
rl
NE
FIG. 1. Effect of treatment with MSG on DA and NE concentrations
in the MBH and posterior pituitary. The bars represent the means ±
SEM of eight observations.
566
BEN-JONATHAN
under many conditions (25). The hypophysial portal
vasculature consists of primary capillaries in the median
eminence, pituitary stalk, and neural lobe. These are
supplied by the superior, middle, and inferior hypophysial arteries, respectively. Blood reaches the anterior
pituitary from the median eminence via the long portal
vessels, which run along the pituitary stalk, and from the
neural lobe via the short portal vessels, which bridge the
avascular cleft of the pars intermedia (26-28). Approximately 20-30% of the total blood flow to the rat anterior
pituitary is supplied by the short portal vessels (29). The
secondary capillary plexus within the anterior pituitary
is linked to the systemic venous circulation by the Yshaped pituitary veins.
Until recently, it was believed that blood flows undirectionally from the hypothalamus to the pituitary gland.
Retrograde blood flow was first observed by Torok (30)
and later confirmed by physiological (26, 31) observations. The prevailing notion to date is that blood can
flow in several directions depending upon the state of
vasoconstriction in any particular vascular bed (32, 33).
This versatility of blood flow is well suited to the transfer
of information between the components of the hypothalamopituitary system. For instance, it allows for delivery of hypothalamic or posterior pituitary substances to
the anterior pituitary, and it provides a route for anterior
pituitary hormones to reach the hypothalamus.
Given the lack of direct innervation of the anterior
pituitary, the portal vasculature serves as the only link
between hypothalamic hormones and the anterior pituitary. To validate a substance as a hypophysiotropic hormone, its presence in portal blood should be demonstrable, and its concentration in portal blood should reflect
the anticipated changes in the target pituitary hormone.
A variety of surgical techniques are available for portal
blood collection in rats, rabbits, sheep and monkeys.
These include single vessel cannulation, whole stalk cannulation, and collection of blood accumulating around a
severed stalk (34-37). In spite of some drawbacks resulting from anesthesia and surgical stress, these techniques
are indispensable for studying certain aspects of the
hypothalamo-pituitary interactions. Unfortunately, the
short portal vessels connecting the two lobes of the
pituitary are extremely minute and technically inaccessible for cannulation. Hence, direct determination of
substances in these vessels is virtually impossible, and
indirect methods must be employed.
After the discovery and subsequent isolation of the
first peptidergic hypothalamic releasing/inhibiting hormones in the early 1970s, many investigators searched
for a peptide PRL-inhibiting hormone (PIH). Although
pharmacological and in vitro studies had implicated DA,
its presence in hypophysial portal blood had to be demonstrated to establish its physiological importance. This
Vol. 6, No. 4
required the development of sensitive assays capable of
quantifying catecholamines in minute volumes of blood.
Using a radioenzymatic assay (10), Ben-Jonathan et al.
(38) first showed that the DA level, but not that of NE
or epinephrine, is. significantly higher in portal blood
than in the general circulation. Subsequently, Gibbs and
Neill (39), using an HPLC method, confirmed that DA
concentration in portal blood is sufficiently high to cause
PRL inhibition in vivo.
DA secreted into portal blood is derived primarily from
a newly synthetized pool rather than from a storage pool
(40). Administration of tyrosine hydroxylase or DOPA
decarboxylase inhibitors causes a prompt reduction in
DA levels in portal blood and a subsequent rise in circulating PRL levels (40, 41). Infusion of DA into rats
treated with a-methyltyrosine results in suppression of
circulating PRL to near-normal levels. Reymond and
Porter (41) estimated that about 15-20% of the total
amount of DA synthetized in the median eminence is
released into portal blood. In addition to continued synthesis, DA release into portal blood depends upon intact
hypothalamic storage and monoamine oxidase activity
(42). Although there are no apparent differences in the
distribution or activity of DA terminals within the median eminence, twice as much DA is found in portal
vessels located in the middle of the stalk than in lateral
vessels (43). Since lactotrophs appear to be distributed
evenly within the anterior pituitary, the physiological
significance of this observation remains obscure.
3. Alterations in TIDA neuronal activity under various
endocrine conditions
DA appears to be the main substance of hypothalamic
origin involved in the inhibition of PRL secretion at the
level of the anterior pituitary. However, even in restricted
sites such as the median eminence or posterior pituitary,
DA is involved in a complex interplay with other hormones or neurotransmitters besides PRL. In addition,
PRL levels at any given time are also influenced by
several other inhibiting, releasing, and/or modulating
hormones. These considerations imply that a simple
inverse relationship does not always exist between TIDA
neuronal activity or DA concentration in portal blood
and circulating PRL levels.
PRL secretion is, in general, much lower in males than
in females. This could imply a more effective DA inhibition in males. However, DA synthesis and turnover in
the median eminence and the DA concentration in portal
blood are also lower in males (38, 44, 45). This sexrelated difference is not androgen dependent. Neither
orchiectomy of neonatal males nor treatment of neonatal
females with testosterone affects the DA concentration
in portal blood. On the other hand, DA secretion into
Fall, 1985
DA, A PRL-INHIBITING HORMONE
portal blood decreases after estradiol treatment of adult
rats and decreases after ovariectomy of neonatal rats
(45). Since the estrogen-induced increase in DA turnover
in the median eminence is abolished by hypophysectomy
(44, 46), the effect of estradiol appears to be mediated by
PRL. Indeed, TIDA neurons in the female are more
responsive to activation by PRL than they are in males
(44). Thus, the low TIDA activity in the male may be
due to the lack of estrogens. In addition, the low circulating PRL levels are attributable to fewer lactotrophs
in the male pituitary or to their reduced sensitivity to
substances with PRL-releasing activity rather than to
increased DA inhibition.
Aging in both male and female rats is characterized by
a rise in plasma PRL levels and a decline in DA concentrations in the median eminence, posterior pituitary, and
portal blood (47, 48). Thus, it appears that the primary
defect causing the elevated PRL concentration is at the
level of the hypothalamus. There is no significant loss of
TIDA neurons with advancing age, since the numbers of
tyrosine hydroxylase-containing perikarya in the arcuate
nuclei of aged and young female rats are essentially the
same. However, tyrosine hydroxylase activity is lower in
old rats (49). The inability of the TIDA neurons to
produce sufficient DA may be the result, rather than the
cause, of hyperprolactinemia, since the onset of elevated
PRL precedes the decline in TIDA neuronal activity
(50). Paradoxically, the anterior pituitary of old rats
contains more DA than does that of young rats (51).
There is also a marked difference in the subcellular
distribution of anterior pituitary DA between young and
old rats. Arita et al. (51) concluded that an impaired
ability of the aged anterior pituitary to process DA might
be the primary cause of age-related hyperprolactinemia.
During the estrous cycle, PRL secretion is generally
low, except on the afternoon of proestrus, when a surge
of PRL coincides with the preovulatory gonadotropin
rise. This PRL surge is attributable to the preceding rise
in estradiol, which acts either directly on pituitary lactotrophs or indirectly on the TIDA neurons. Most investigators report a decrease in DA turnover rate in the
median eminence during the afternoon of proestrus (5254). Indeed, the DA concentration in portal blood is
significantly lower on proestrus than throughout the
remainder of the cycle (Fig. 2) (38) and is inversely
related to plasma PRL concentrations. A 50% reduction
in the portal blood DA concentration occurs during the
afternoon of proestrus (55). A decrease in the portal
blood DA concentration is also seen during the last day
of pregnancy, coincidental with the marked rise in PRL
levels before parturition (56).
4. Posterior pituitary contribution to the control of PRL
release
The recognition of posterior pituitary participation in
the regulation of anterior pituitary hormone secretion is
567
slow in coming. This probably results from an adherence
to the neurohumoral hypothesis, which assigns an exclusive regulatory role to the hypothalamus. Many investigators still believe that the posterior pituitary is just an
anatomical extension of the hypothalamus and does not
subserve an independent function. However, the posterior pituitary is independent of the hypothalamus in the
production and processing of proopiomelanocortin
(POMC)-related peptides (57), and its participation in
the regulation of PRL (58-60), LH (60-62), GH (63),
and ACTH (64-66) secretion is now well documented.
Although the posterior pituitary is often treated as a
single unit, several of its hormones are unevenly distributed between the neural and intermediate lobes. For
example, more than 95% of total vasopressin is contained
in the neural lobe, whereas 88% and 70% of MSH and
0-endorphin, respectively, are present in the intermediate lobe (67). Such a clear segregation, however, is not
evident with respect to biogenic amines. The concentrations of serotonin and histamine are similar in the two
lobes (11), and the DA concentration has been reported
to be higher (11), similar (68), or lower (67) in the neural
lobe. Basal DA activity appears to be the same in the
two lobes, as judged by similar turnover rates (67), but
electrical stimulation of the pituitary stalk evokes a
higher release of DA from the intermediate lobe than
from the neural lobe (69). In postnatal rats, nerve fibers
containing catecholamines appear earlier in the intermediate lobe than in the neural lobe (68), although fiber
density is higher in the neural lobe of adult rats.
The postulate that the posterior pituitary participates
in the regulation of PRL secretion is based on the following premises: 1) DA is a physiological inhibitor of PRL
secretion; 2) DA is present at high concentration in the
posterior pituitary; and 3) vascular channels exist between the two lobes. Given that the short portal vessels
are inaccessible to cannulation, the above hypothesis can
only be tested indirectly. This has led us to propose that
removal of the posterior lobe (posterior lobectomy)
should result in an elevation of plasma PRL levels.
Indeed, we have shown that posterior lobectomy in urethane-anesthetized male rats induces a significant 2- to
3-fold elevation of plasma PRL, but no change in the
plasma LH concentration (59). Peak PRL levels are
observed 30 min after lobectomy, and PRL levels remain
elevated for at least 3 h. Injection of DA via a cannulated
internal carotid artery results in an immediate reversal
of the lobectomy-induced rise in plasma PRL (Fig. 3)
(59).
The profile of hormone secretion after posterior lobectomy in anesthetized female rats differs significantly
from that in males. For example, the PRL elevation is
more rapid, but is of shorter duration; it also depends on
the presence of certain gonadal steroids. Posterior lobec-
BEN-JONATHAN
568
4 —
FIG. 2. DA in hypophysial portal plasma
and PRL in systemic plasma of female
rats on each day of the estrous cycle and
in male rats. PRL levels were determined
in blood collected from rats not subjected
to portal blood collection. The bars represent the mean ± SEM of 12-13 observations. P, Proestrus; E, estrus; D-l,
diestrous day 1; D-2, diestrous day 2.
-5
Vol. 6, No. 4
I
p-q Dopamine in Portal
kill Plasma
•
PRL in Systemic
Plasma
3
-
120
-
IOO
- 80
I
- 60
I
- 20
*"
D-1
PRL
&•—-O
120 _
80-
Sham
Lob ex
40-
30
60
90
180
Time (min)
FlG. 3. Effect of posterior pituitary lobectomy (Lobex) on plasma LH
(lower panel) and PRL (middle panel) in urethane-anesthetized male
rats. Upper panel, Thirty minutes after Lobex, rats were injected with
either DA (150 ng into an internal carotid artery) or vehicle. Each
value is a mean ± SEM of 15-18 determinations.
tomy results in a rapid rise in PRL during estrus and a
smaller and slower rise on diestrous day 1, but no change
on either diestrous day 2 or proestrus (61); ovariectomy
completely abolishes the lobectomy-induced rise in PRL
(61 A). In addition to PRL, posterior lobectomy in cycling
female rats induces a marked rise in plasma LH concentrations, which is slower in onset, higher in amplitude,
and of longer duration than that of PRL (61). Indirect
evidence suggests that the substance(s) responsible for
D-2
Males
the inhibition of LH secretion works by inhibiting the
secretion of LHRH, rather than by suppressing basal LH
secretion or changing pituitary sensitivity to LHRH (62).
Although the chemical identity of the LH inhibitor is as
yet unknown, the opioids are potential candidates, since
they have been shown to inhibit LHRH secretion (70,
71).
Inhibition of PRL release by the posterior pituitary
can also be demonstrated in vitro in the absence of neural
and vascular influences. A methanol-HCl extract of the
posterior pituitary causes a dose-dependent inhibition of
PRL, but not LH, secretion from cultured anterior pituitary cells (12, 59). This is not likely caused by vasopressin or oxytocin, both of which are ineffective in
changing PRL release from the cells (58). The PRLinhibiting activity of the posterior pituitary can be accounted for by the endogenous DA in the extract. Coincubation of pituitary cells with the specific DA receptor
antagonist (+)butaclamol prevents the reduction in PRL
secretion caused by posterior pituitary extracts alone
(Fig. 4) (12, 59).
Recently, the profile of PRL secretion was examined
in conscious cycling rats subjected to chronic posterior
pituitary lobectomy (72). As seen in Fig. 5, within 2-3 h
after lobectomy, plasma PRL levels increase 2- to 3-fold
and remain elevated for 3 days before declining to nearcontrol levels on the fourth day. This decline is attributable to a compensatory activation of hypothalamic DA
by the elevated PRL level. Although there is partial
regeneration of the posterior pituitary, this occurs only
after 3-4 weeks (73) and cannot explain the early decline.
Indeed, daily water consumption, which serves as an
index for vasopressin deficiency, remains elevated in
lobectomized rats for at least 2 weeks (72). Fagin and
Neill (61A) also reported that 4 days after lobectomy in
ovariectomized rats, the plasma PR1 level does not differ
DA, A PRL-INHIBITING HORMONE
Fall, 1985
100-
C. Central and Peripheral Interactions Affecting
DA and PRL
I
X
75-
—
X
50-
X
25 -
o-J
III
C
BU
DA
DA
BU
PP
PP
BU
FIG. 4. Inhibition of PRL secretion from cultured anterior pituitary
cells by DA (10~7 M) and posterior pituitary extracts (PP) and its
reversal by the DA receptor antagonist (+)butaclamol (BU; 10"7 M).
Each value is a mean ± SEM of four determinations.
• — • Lobex
O--O Sham
=
75-
50-
25-
0
Hour
569
09 13 17 09 13 17 09 13 17 09 13 17 09 13 17
Day
FIG. 5. Plasma PRL concentration during the 4 days following chronic
posterior pituitary lobectomy (Lobex) performed on estrus. Dotted
columns between each day represent dark time (light off, 1900-0700 h).
Note the proestrous PRL surge on the fourth day in Sham rats. Each
value is a mean ± SEM of 8-14 determinations (Murai, I., and N. BenJonathan, unpublished observations).
from that in controls. Posterior lobectomy in cycling rats
also results in an interruption of cyclicity for about 7-10
days, which might be caused by the initial hyperprolactinemia. Posterior lobectomy does not cause prolonged
damage to the reproductive capacity, as evidenced by the
fact that upon resumption of cyclicity, lobectomized rats
have a normal number of tubal ova (72).
Collectively, these data are consistent with the hypothesis that the dopaminergic inhibition of PRL secretion involves two interdependent systems. One consists
of the hypothalamus-long portal vessels, and the second
is comprised of the posterior pituitary-short portal vessels. Depending upon the endocrine conditions and the
nature of hormonal or neuronal stimuli, either one or
both systems may be activated.
1. Neuropeptides and neurotransmitters
PRL secretion is regulated in a complex manner by a
variety of neurotransmitters and neuropeptides. The
main substances that regulate PRL release at the hypothalamic level are opioid peptides and serotonin. TRH
and vasoactive intestinal peptide (VIP) act directly on
the anterior pituitary, whereas -y-amino butyric acid
(GABA) has both central and peripheral actions. Other
neuropeptides (e.g. angiotensin, substance P, neurotensin, and oxytocin) and certain neurotransmitters (e.g.
NE, histamine, and acetylcholine) also effect PRL release, but their sites of action and physiological significance are yet undetermined.
The opioids are widely distributed in the brain, pituitary gland, adrenal medulla and other peripheral organs.
Based on their precursor molecules, they are classified
into three groups. POMC gives rise to 0-endorphin,
ACTH, and MSH; proenkephalin is the precursor for
met-enkephalin; and prodynorphin yields dynorphin and
leu-enkephalin (74). Within the hypothalamus, met-enkephalin is found primarily in the preoptic area and the
anterior and lateral hypothalamus. Leu-enkephalin accompanies the mangocellular neurons, with perikarya in
the supraoptic and paraventricular nuclei and terminals
in the neural lobe of the pituitary (75). The concentration
of /3-endorphin is highest in the intermediate lobe, where
it is produced from POMC; because of posttranslational
acetylation, it is largely devoid of opioid activity (57). A
nonacetylated form is present in the median eminence,
periventricular nuclei, and arcuate nuclei, but there are
conflicting reports of whether its levels change after
hypophysectomy (76, 77).
The report that morphine stimulates lactation in estrogen-primed rats (78) predated by 10 yr the isolation
of endogenous opiates and provided the first indirect
evidence that they stimulate PRL release. Opiates or
their synthetic agonists markedly increase plasma PRL
levels, an effect abolished by pretreatment with opiate
antagonists such as naloxone (79, 80). ,3-Endorphin is
500-2000 times more potent than met-enkephalin in
eliciting a rise in PRL (81). This contrasts with its lower
binding affinity to the opiate receptor (82) and is probably due to higher resistance to degradation (83).
Whether naloxone alone affects basal PRL release is
controversial (84, 85). Endogenous opiates may not regulate PRL release under normal conditions, but they
probably participate in stress-induced PRL release (86).
Opioids do not stimulate PRL release directly from the
anterior pituitary, but at high concentrations they transiently reverse the DA inhibition (87). Their main target
is the hypothalamic DA system. Morphine- and opiate-
570
BEN-JONATHAN
Vol. 6, No. 4
2. Modulation of hypothalamic DA by PRL
information. This is normally achieved by negative feedback and results in a reciprocal relationship between the
signal and the hormone. Under special circumstances, a
positive feedback system, aimed at amplifying hormone
release, is also operative. The regulation of anterior
pituitary hormone secretion may involve several types of
feedback loops. One is a long loop through the peripheral
circulation. Target organ hormones influence the pituitary hormone and/or its hypothalamic releasing/inhibiting hormone. A second type is a short loop involving
the hypophysial portal vasculature. In this case, the
pituitary hormone directly modulates the activity/release
of a hypothalamic hormone. A third possibility, which
appears to be of minor importance, is the ultrashort loop,
which does not require vascular transport. Here, the
pituitary hormone exerts a local influence over its own
synthesis and secretion.
Unlike most pituitary hormones, PRL has multiple
peripheral target sites and no identifiable target organ
hormone(s). It follows, then, that its main feedback
regulation must be exerted via the short loop. The existence of a short loop was first postulated when implantation of PRL into the hypothalamus resulted in suppression of PRL release (103). PRL is a normal constituent
of the cerebrospinal fluid, where its levels parallel the
plasma concentrations (104). Specific PRL-binding sites
are present in the median eminence and choroid plexus
(105). Since the median eminence and possibly its adjacent arcuate nuclei have an incomplete blood-brain barrier (106), uptake from the general circulation may also
be a route by which PRL can reach the brain. Another
is by retrograde transport via the portal vessels (30-33).
Injections of PRL increase DA synthesis (107, 108)
and turnover rate (109) in the median eminence, but not
in the posterior pituitary. This activation leads to increased DA secretion, as shown by a number of in vitro
and in vivo studies. PRL augments DA release from
superfused MBH fragments in a dose-dependent fashion
(110). DA levels in portal blood are elevated in rats
bearing PRL-secreting tumors (111) and after intraventricular injections of PRL (112). Treatment of rats with
haloperidol to increase plasma PRL levels induces a rise
in the portal plasma DA concentration; this is abolished
by pretreatment with antiserum to PRL (112). Finally,
the rate of DA synthesis in the median eminence is
reduced by hypophysectomy and restored to normal levels by PRL (44, 46). Collectively, these reciprocal relations between PRL and DA provide very strong evidence
that PRL regulates its own release via a short loop
feedback mechanism involving the TIDA system.
A central dogma in endocrinology is that a hormone
functions best when presented to the target cell within a
specific range of concentrations. To maintain optimal
conditions, the secreting cell has to receive feedback
The time course of PRL action has long been a subject
of dispute. The latency between PRL administration and
changes in TIDA neuronal activity varies from 16-24 h
in intact rats (44) to 2-4 h in hypophysectomized rats
like peptides decrease DA synthesis and turnover rate in
the median eminence (79, 80, 88, 89), but not in the
posterior pituitary (89), and reduce the DA concentration
in portal blood (88, 90). In addition to suppressing DA,
opioids may induce the release of a PRL-releasing hormone (PRH). Injections of /3-endorphin or morphine
elicit a marked increase in plasma PRL levels in spite of
DA infusion (90).
Serotonin has a proven and important control over
PRL secretion. It is primarily associated with the suckling-induced PRL rise, which will be discussed in a later
chapter. Most of the serotonergic innervation of the
hypothalamus originates from the midbrain dorsal raphe
nucleus, with the highest concentration in the superchiasmatic and arcuate nuclei (91). There are also intrinsic serotonergic fibers with cell bodies in the dorsal
medial nucleus (92). Axon terminals containing serotonin and its biosynthetic enzymes are present in the
median eminence and in the anterior and posterior pituitaries (11). Systemic injection of the precursor 5hydroxytryptophan (93), intraventricular administration
of serotonin (94), and treatment with drugs that elevate
endogenous brain serotonin (93), markedly stimulate
PRL release. These effects appear to be modulated by
gonadal steroids. Inhibition of serotonin synthesis with
para-cholorophenylalanine (PCPA), reduces PRL secretion in intact males (95), has no effect on gonadectomized
rats (96), and blocks the estrogen-induced surge in PRL
(97). At present, the mechanism by which serotonin
stimulates PRL secretion is speculative, but there is
little, if any, evidence that it has a direct effect on the
anterior pituitary. It is generally believed that serotonin
stimulates PRH release, but evidence also exists for
interposing DA and opiate mechanisms (98).
Similar to the TIDA system, there is also a tuberoinfundibular GABAergic pathway (99). Unlike DA, GAB A
exerts a dual control over PRL secretion: one component
is a stimulatory effect on a CNS site, and the other is an
inhibitory effect on the anterior pituitary (100). The
central stimulatory component can be ascribed, at least
in part, to an inhibition of TIDA function (101). The
pituitary inhibitory component is exerted via specific
GABAergic receptors (102). The extent of GABA participation in the normal regulation of PRL secretion remains to be determined. The contribution of TRH and
VIP to the regulation of PRL secretion will be discussed
in the section on lactation below.
Fall, 1985
DA, A PRL-INHIBITING HORMONE
(113). Such a sluggish response is at variance with the
prompt stimulation of DA release by PRL from isolated
hypothalamic tissue (110). Demarest et al. (108) recently
postulated the existence of two interdependent components of PRL action. One is a rapid tonic component,
which mediates short term changes in TIDA neuronal
activity in response to acute alterations in circulating
PRL. The second is an induction delayed component,
which responds to prolonged increases in plasma PRL
and alters the capacity of the tonic component. Although
this model consolidates several conflicting observations,
its experimental basis is still fragmentary and awaits
further investigation.
Chronic hyperprolactinemia, in contrast to short term
elevations in plasma PRL, appears to have several deleterious effects. It exerts a neurotoxic effect on the TIDA
neurons and induces a marked fall in the hypothalamic
DA concentration. In old rats, a progressive loss of TIDA
neurons is associated with the development of prolactinomas (114). Young rats with estrogen-induced prolactinomas have less DA in portal blood, show a lower
response to DA-releasing drugs, and exhibit a reduction
in DA release from median eminence in vitro compared
to controls (115). The mechanism responsible for the
neurotoxicity of PRL or the reduction of its effectiveness
in activating the TIDA neurons is not well understood.
Nonetheless, clarification of this mechanism is of interest clinically in light of the high incidence of human
prolactinomas with an apparently normal response to
DA agents (116, 117).
3. Interactions with ovarian steroids
Although there is agreement that ovarian steroids play
a very important role in the economy of PRL release,
their primary mode of action remains controversial. Several factors complicate the interpretation of experimental studies. First, steroids have an integrated action at
the brain and the pituitary, preventing a clear distinction
between primary and secondary effects. Second, they
regulate the secretion of other reproductive hormones
and markedly influence sexual behavior. Steroid-induced
changes in brain neurochemistry may be unrelated to
alterations in PRL. Third, estradiol and progesterone
have synergistic and/or antagonistic actions, depending
on the rate of change and the ratio of their plasma
concentrations. And fourth, in addition to promoting
secretion, ovarian steroids are involved with PRL synthesis and also have mitotic activity. Hence, acute and
chronic steroidal effects may be implemented by different
cellular mechanisms. Most studies to date have used in
vivo steroidal treatment and so cannot distinguish between a primary steroidal effect and one that is secondary
to alterations in hypothalamic DA secretion.
571
The proestrous rise in PRL is preceded by a rapid
increase in serum estradiol levels and can be prevented
by pretreatment with an antiserum to estradiol. Ovariectomy suppresses plasma PRL below basal levels, and
proestrous-like surges can be initiated in ovariectomized
rats treated with estradiol. When a large dose of estradiol
is administered on estrus, it initiates the daily nocturnal/
diurnal surges of PRL that characterize pseudopregnancy; progesterone appears to provide a positive feedback to sustain this secretory pattern (118). Although
there are no clear cyclic changes in PRL levels during
the human menstrual cycle, there is a marked estrogendependent rise in circulating PRL toward the end of
pregnancy (119).
At the level of pituitary lactotrophs, estrogens are
known to have multiple and divergent actions. These
include stimulation of PRL synthesis, storage, and secretion (120-122); modification of DA and TRH activities
(122, 123); an increase in the number of lactotrophs
(124); and induction of pituitary tumors (125). Estrogen
increases de novo synthesis of PRL (126). It acts via
specific nuclear binding sites (127) and induces transcription of the PRL gene by increasing the synthesis of
PRL mRNA (120). Estrogens increase intracellular PRL
storage capacity (121), but do not have an acute effect
on exocytosis. Therefore, the estrogen-induced rise in
basal PRL release is probably secondary to an increased
synthesis and storage. Estradiol increases the binding of
TRH to GH3 cells and enhances TRH-induced PRL
secretion in normal pituitary cells (123). It also directly
antagonizes the inhibitory action of DA. Preincubation
of rat pituitary cells with estradiol leads to almost complete reversal of the DA inhibition (122,128). In contrast
to that in the rat, the stimulation of PRL secretion by
estradiol is more modest in primates, and estrogen does
not decrease the effectiveness of DA inhibition in monkey pituitary cells (128A).
Ovarian steroids exert a profound influence on virtually every aspect of reproductive function of the brain.
In the hypothalamus, tyrosine hydroxylase-containing
neurons in the arcuate nucleus are targets for estradiol
(129). Although this provides an anatomical basis for a
direct action on the TIDA system, steroidal effects are
often mediated by PRL. Short term estrogen treatment
increases hypothalamic DA synthesis (Fig. 6) (12, 130)
and turnover rate (131, 132) as well as the DA concentration in portal blood (133,134). A combined estrogenprogesterone regimen potentiates this effect (134, 135).
However, activation of TIDA neurons does not occur in
hypophysectomized rats (44,46), indicating an obligatory
role of PRL. Long term treatment with estradiol reduces
DOPA accumulation in the median eminence, in part by
attenuating TIDA neuronal responsiveness to PRL
(130).
Vol. 6, No. 4
BEN-JONATHAN
572
5
400-
aT
200-
ANTERIOR PITUITARY
DOPAMINE RECEPTORS
0
150-
PLASMA PRL
100 —
50-
20 -
DOPAMINE
SYNTHESIS
Posterior
Pituitary
MBH
10 -
EB
EB
FIG. 6. Effect of treatment of ovariectomized rats with estradiol benzoate (EB) on DA synthesis (lowerpanel), plasma PRL (middlepanel),
and anterior pituitary DA receptor density (upper panel). Rats were
treated with vehicle (C) or with 5 or 25 jig/kg BW EB for 5 consecutive
days. Each value is a mean ± SEM of four to eight determinations.
4. Lactation and the suckling stimulus
PRL is essential for the initiation and maintenance of
lactation. It acts directly on the mammary alveoli to
promote synthesis and secretion of milk proteins. Lactation is initiated by elevated PRL levels together with
the removal of inhibition by estrogens and progesterone,
levels of which decline after parturition. PRL secretion
is maintained during lactation by the frequent application of suckling. Suckling is the most powerful natural
stimulus for PRL release; within a few minutes after its
initiation, PRL increases 30- to 50-fold and decreases
shortly after removal of the young (136). The quantity
of PRL release depends upon the intensity and duration
of suckling and the time interval between suckling episodes. The suckling stimulus represents a classical neuroendocrine reflex. It triggers nerve impulses from sensory receptors in the nipple, which ascent in the spinal
cord and pass through the midbrain to the hypothalamus.
In addition to PRL, suckling stimulates the secretion of
oxytocin from the posterior pituitary. Oxytocin causes
contraction of the myoepithelial cells in the mammary
gland, resulting in increased intramammary pressure and
milk ejection (137).
The identity of the neural substance(s) that mediates
suckling-induced PRL secretion has become a central
issue of PRL research. Logically, the acute PRL response
to suckling could result from a decrease in an inhibitor
(DA), an increase in a stimulator (PRH), or both. The
evidence that DA is the major neural component is
inconclusive. Most investigators report a reduction in
DA concentration in the median eminence during suckling (138, 139) and a decrease in TIDA activity (140,
141), but others find no changes (142). The DA concentration in portal blood rises after prolonged pup separation (Fig. 7) (56). Electrical stimulation of the mammary
nerve to simulate suckling, evokes a marked increase in
plasma PRL and a transient, though significant, fall in
portal blood DA levels (143, 144). Although an abrupt
withdrawal of DA induces a rise in PRL (136, 145), the
brevity of DA reduction appears insufficient to fully
account for the massive rise in PRL during suckling.
Nonetheless, DA appears to be important for the rapid
decline in PRL immediately after removal of the pups
(146) and for maintaining low plasma PRL levels during
nonsuckling intervals (56).
Grosvenor et al. (147, 148) have proposed another
model for suckling in which two interdependent processes generate a biphasic response. The first phase involves a brief removal of DA inhibition. This, in turn,
activates the second phase, which involves stimulation
of PRH release and/or increased responsiveness of pituitary lactotrophs to PRH action. This model consolidates a variety of observations, but does not identify
PRH. Of the numerous substances with PRL-releasing
activity, attention has focused on TRH and VIP. Both
are present in hypophysial portal blood and stimulate
PRL release in vitro at relatively low concentrations
NOREPINEPHRINE
DOPAMINE
1 "
3 -
l.vV.j Portal Plasma
I
|
| Arterial Plasma
2 -
1 -
0
II
Control
Separated
Control
Separated
FIG. 7. DA and NE concentrations in hypophysial portal and arterial
plasma in lactating rats. Rats were separated from pups for 30 min
(control) or for 24 h (separated). The bars represent the mean ± SEM
of 10-11 rats.
Fall, 1985
DA, A PRL-INHIBITING HORMONE
(149-151). Mammary nerve stimulation increases TRH
in portal blood (149), and TRH is more effective in
stimulating PRL release after removal of DA inhibition
(145). On the other hand, the ability of TRH to induce
a significant rise in PRL during lactation is questioned
(152), and the lack of a synchronous rise in PRL and
TSH during suckling is of concern (152, 153). VIP antiserum completely blocks the PRL response to ether, but
only delays the PRL response to suckling (154). Thus,
in spite of numerous indirect observations suggesting the
existence of a distinct entity with PRL-releasing activity,
its identity remains elusive.
There is a strong evidence that serotonin-containing
neurons participate in the suckling-induced rise in PRL.
Although serotonin itself does not function as a PRH, it
provides a neural link between sensory input reaching
the brain from the mammary gland and hypothalamic
DA and/or PRH, which directly affect anterior pituitary
PRL secretion. Depletion of serotonin by PCPA blocks
the suckling-induced rise in PRL, an effect that is reversed by repletion with 5-hydroxytryptophan (93).
Suckling decreases hypothalamic serotonin levels, with
reciprocal changes in its metabolite 5-hydroxyindolacetic
acid (138). Lesions of the dorsal raphe nucleus, which
projects to the hypothalamus (155), or hypothalamic
deafferentation (156) significantly reduce hypothalamic
serotonin levels and impair suckling-induced PRL release as well as maternal behavior. Recent evidence
indicates the existence of a short loop feedback mechanism between PRL and hypothalamic serotonin (46).
Several intriguing observations were recently made
when lactating rats were subjected to posterior pituitary
lobectomy (Murai, I., and N. Ben-Jonathan, unpublished
observations). As shown in Fig. 8, basal PRL levels are
elevated 2- to 3-fold after lobectomy compared with those
after sham operation, indicating a partial removal of DA
inhibition. However, suckling does not increase PRL
levels in lobectomized rats. Oxytocin replacement does
not change the pattern of PRL secretion in lobectomized
rats, but does allow for milk release, as judged by an
increase in the pups' weights during suckling. The synthesis of DA and serotonin in the MBH of lobectomized
and sham-operated rats are similar, suggesting that these
hypothalamic systems are not impaired. Furthermore,
the absence of response to suckling is selective, since
exposure to ether results in significant rises in plasma
PRL levels in both lobectomized and sham-operated
lactating rats. These data suggest that in addition to DA,
the posterior pituitary contains a PRH. The coexistence
of both substances at a variable ratio may explain our
previous observation (Fig. 4) (59) of a net PRL-inhibiting
activity in posterior pituitary extracts from male rats.
Figure 9 depicts a model which conceptualizes these
findings. Two interdependent routes that govern PRL
573
9-0 000-0
~
i*^
c
IOO
80-
c
o
•E 60-
40o
o
20o
o
SUCKLING
Time
( hr)
FIG. 8. Effect of suckling on blood PRL levels in posterior lobectomized lactating rats with or without oxytocin injections (0.1 IU at 8-min
intervals during suckling). Pups were separated at the time of surgery
(16 h before the experiment) and then reunited with the mothers. Pups
of sham-operated (SHAM) and lobectomized (LOBEX) and oxytocintreated rats gained 0.9 g each in body weight during suckling, but those
from LOBEX alone lost 0.1 g each. Three representative rats are shown
from a pool of eight rats in each group with similar results (Murai, I.,
and N. Ben-Jonathan, unpublished observations, 1985).
secretion are envisioned. One includes the TIDA DA
neurons, PRH, and the long portal vessels. The second
involves the tuberohypophysial DA neurons, PRH, and
the short portal vessels. PRL and other modulators, i.e.
opioids and gonadal steroids, exert their major effects on
the hypothalamus-long portal vessel route. The suckling
stimulus, mediated by serotonergic neurons impinging
upon the hypothalamus, activates the posterior pituitary
route. Suckling also increases oxytocin secretion from
the posterior pituitary. The synchronous release of PRL
and oxytocin during suckling is thus regulated at a common site, the posterior pituitary. Verification of this
model must include the demonstration of PRH activity
in the posterior pituitary.
D. Anterior Pituitary Dopaminergic Receptors
1. Characterization of pituitary dopaminergic receptors
Catecholamines interact with target cells by binding
to specific recognition sites, designated receptors, which
are complex proteins located in the cell membrane. Receptors are not rigidly fixed at a particular site in the
membrane, but instead are capable of lateral mobility,
aggregation, coupling to membranous transducers or ion
channels, internalization, and recycling back into the cell
membrane (157). Receptor activation can lead to opening
or closing of ion channels, phospholipid synthesis, or
modulation of adenylate cyclase (158). These membrane
BEN-JONATHAN
574
PARAVENTRICULAR
NUCLEUS
HYPOTHALMUS
£»
ARCUATE NUCLEUS
Serotonin?
-
•
-
\
A.
Dopamine
MEDIAN EMINENCE
J
Dopamine Oxytocin
P.RH?
Dopamine
ANTERIOR
POSTERIOR
LOBE
LOBE
PRH?
%
Oxytocin
^—-—— Prolactin
MAMMARY GLAND
SUCKLING
STIMULUS
\
MILK
FIG. 9. A model depicting the regulation of PRL secretion during
lactation. PRL release is controlled by two interdependent routes. One
involves the posterior pituitary and short portal vessels, and the second
involves the hypothalamus and long portal vessels. The suckling stimulus, mediated by serotonin, affects the tuberohypophysial (TH) branch
of the DA system. It suppresses DA and stimulates PRH secretion
from the posterior lobe. The second route involves short loop feedback
inhibition by PRL and other modulators (opioids and ovarian steroids)
on the tuberoinfundibular (TI) branch of the DA system. The model
assumes that the suckling-induced release of both PRL and oxytocin
is controlled at a single site, the posterior pituitary.
effects initiate a cascade of reactions which ultimately
result in cellular responses such as action potentials,
enzyme activation, or secretion. Exposure of a receptor
to a ligand often results in a progressive attenuation of
the biological response. This phenomenon, termed desensitization, involves either a reduction in the number of
surface receptors by clustering or internalization or an
uncoupling of the receptor from intracellular transducers
(159).
To establish receptor specificity, several criteria must
be met. These include saturability, binding kinetics, tissue linearity, stereoselectivity, and correlation with
known biological responses. To investigate the molecular
structure of a receptor, it is solubilized from its native
membrane and purified. Retention of binding specificity
and biological activity may be verified by reconstituting
the purified receptor into mutant cells lacking either the
receptor itself or any of the components of its transduction apparatus (158, 159). Once a functional purified
Vol. 6, No. 4
receptor is available, monoclonal antibodies can be used
to identify functional domains.
Although DA is structurally similar to other catecholamines, it does not bind well to either a- or /3-adrenergic
receptors, but has its own distinct set of receptors. According to their anatomical distribution and coupling to
adenylate cyclase, DA receptors were designated Dl and
D2 by Kebabian and Calne (160). The Dl receptors
stimulate adenylate cyclase when occupied by agonists.
They are found primarily in the caudate nucleus and the
parathyroid gland. The D2 receptors either decrease or
have no effect on the formation of cAMP. Their prototype is found primarily in the intermediate and anterior
lobes of the pituitary. Recent studies have indicated the
existence of another subclass, the D3 receptor (161). It
has been termed an autoreceptor and is presumably
located on presynaptic terminals. However, D3 sites,
which are absent in the noninnervated anterior pituitary,
may actually represent the high affinity binding state of
the Dl receptor (162).
High affinity, saturable, and stereoselective DA-binding sites have been characterized in anterior pituitary
membranes from several species (116, 163-165). In contrast to the brain, where multiple DA receptor subtypes
exist, there is only a single receptor subtype (D2) in the
anterior pituitary (166, 167). The rank order of competition with radioligand binding by DA agonists and antagonists agrees closely with their ability to inhibit or
stimulate, respectively, PRL secretion (164). For example, the agonists apomorphine and ergots (bromocryptine, lisuride, and lergotrile) exhibit high affinity binding
to the D2 receptors and only a partial agonist activity at
the Dl receptor. They are also potent inhibitors of PRL
release both in vivo and in vitro (164, 168). The butyrophenones and related antagonists (spiperone, haloperidol, and domperidone) are fairly selective ligands for D2
receptors and have lower affinities for the Dl receptor.
They do not affect PRL secretion in vitro when tested
alone, but increase PRL release when administered in
vivo, presumably by antagonizing endogenous DA (164,
169,170).
The D2 receptor exists in two interconvertible affinity
states, differentially labeled by agonists and antagonists
(162, 166). Antagonist competition curves are monophasic, with Hill coefficients of 1 (171). They best fit the
model of a single homogenous receptor of high affinity.
In contrast, agonist competition curves exhibit heterogenous characteristics, with Hill coefficients less than
unity. Furthermore, agonists label only half as many
sites as antagonists, and all are of high affinity. The two
affinity forms of the receptor are modulated by guanine
nucleotides (172). Under their influence, the agonist high
affinity form is converted to a low affinity form, and the
antagonist low affinity form is converted to a high affin-
Fall, 1985
DA, A PRL-INHIBITING HORMONE
ity form. Two models have been offered to explain these
findings (166). One is that there are two discrete DA
receptors with identical affinity for antagonists but different affinity for agonists. Guanine nucleotides could
inhibit agonist binding to the high affinity receptor in
an allosteric fashion. The second is that a single receptor
exists in two affinity binding states. In this model, guanine nucleotides regulate the interconversion between the
high and low affinity states.
Support for two affinities/one receptor model comes
from studies with dispersed anterior pituitary cells (173).
The binding of antagonists to whole cells and that to
membrane preparations are nearly identical. In contrast,
there are no detectable high affinity 3H-labeled agonist
bindings to whole cells. Upon homogenization, however,
both sites are present in washed membrane preparations.
Addition of exogenous guanine nucleotides to dispersed
cells no longer cause alterations of the agonist competition curves as in membrane preparations. Taken together, these data strongly suggest that in intact cells,
endogenous GTP rapidly reverses the high affinity agonist binding but does not impair antagonist binding.
2. Molecular structure of the D2 receptor
Complete characterization of the structure of the DA
receptor awaits its successful isolation, purification, and
reconstitution. This requires a tissue source with an
abundant receptor population. Given that the D2 receptors are present only on a small fraction of pituitary
cells, large scale purification is difficult. At present, only
initial steps toward isolation have been taken. These
reveal that, as is found with adrenergic receptors, the
link between the DA receptor and the catalytic subunit
of adenylate cyclase is via a guanine nucleotide regulatory protein.
Treatment of anterior pituitary membranes with the
detergent digitonin results in solubilization of the receptor with retention of its binding specificity (174). However, upon solubilization, the receptor loses its ability for
high affinity interaction with agonists and also its sensitivity to guanine nucleotides (175). Nevertheless, a
stable agonist-receptor complex can be preserved by
preoccupying the receptor with a labeled agonist before
solubilization. This results in an increase in the apparent
size of the complex (176). The larger size complex is
converted back to the smaller antagonist-receptor complex upon interaction with guanine nucleotides. The
difference in molecular size (~100,000 daltons) between
the two species approximates the functional size of
known guanine nucleotide-binding proteins (177, 178).
Studies with adrenergic receptors have indicated that
the inhibitory (Ni) and stimulatory (Ns) guanine nucleotide regulatory proteins are distinct (178). The N; is a
575
heterotrimer, consisting of a-, /?-, and 7-subunits of
41,000, 35,000, and 10,000 daltons, respectively (179).
The j8-subunits of N8 and Ni may be the same (178).
There is indirect evidence that DA inhibition of PRL
secretion is associated with a functional coupling between the receptor and Nj. Treatment of dispersed anterior pituitary cells with pertussis toxin attenuates the
ability of DA to inhibit cAMP accumulation and PRL
release, without affecting binding parameters (180). Pertussis toxin acts at a site proximal to the catalytic subunit
of adenylate cyclase by ADP-ribosylation of the 41,000dalton moiety of N5 (181).
Fashioned after other adenylate cyclase-linked receptors (158,178,182), Creese et al. (166) have proposed the
following model of the D2 anterior pituitary DA receptor.
An agonist (A) binds to the receptor (R) to form a ligandreceptor complex (AR). The binding induces a conformational change in the receptor, enabling its coupling to
a guanine nucleotide regulatory-inhibiting protein (Ni).
This ternary complex (ARNj) is responsible for the high
affinity agonist binding state and also serves as the link
for inhibiting the catalytic subunit of adenylate cyclase.
The ARNi complex appears to exist only transiently
because of its rapid dispersal by endogenous GTP.
3. Dopaminergic receptors in PRL-secreting tumors
Hypersecretion of PRL may be caused by a reduction
in DA release, changes in pituitary vascular supply, a
loss of pituitary DA receptors, or postreceptor defects.
About 50-60% of previously classified nonfunctional human pituitary adenomas are now diagnosed as prolactinomas and are the major causes of nonpregnancy related
hyperprolactinemia (183). They are frequently associated
with amenorrhea, oligomenorrhea, and/or infertility in
women (117) and diminished libido and/or impotence in
men (184). In addition to human prolactinomas, several
rat PRL-secreting cell lines are widely used as model
systems for studying the regulation of PRL secretion.
Several lines of evidence suggest that the DA inhibitory mechanism is relatively intact in the majority of
human prolactinomas. Most hyperprolactinemic patients
show a reduction in circulating PRL after treatment with
DA or bromocyrptine (117). Only a small number respond poorly to drug administration. DA agents suppress
PRL secretion by human pituitary adenomas with potencies similar to control values (116,185). Membrane preparations from human prolactinomas have high affinity,
saturable, and stereoselective binding sites characteristic
of DA receptors (186). A functional continuity between
the receptor and intracellular transducers has been verified by the suppression of adenylate cyclase by DA
agonists (187).
It appears that, with the exception of a small subpop-
576
BEN-JONATHAN
ulation of hyperprolactinemic patients which are resistent to DA, most patients show no alteration in responsiveness to DA action. Hence, the etiology of human
PRL-secreting adenomas remains unresolved. In search
of a plausible explanation, Weiner et al. (188) have
proposed that tumors could have escaped DA regulation
by changes in pituitary vascular supply. In support of
this hypothesis, they showed a direct arterial vascularization of the anterior pituitary in estrogen-induced PRLsecreting tumors in rats. This would effectively dilute
the DA concentration reaching the anterior pituitary via
the portal vessels, resulting in an escape from tonic
inhibition of PRL secretion. However, at present, there
is no direct evidence that a similar mechanism is involved
in the formation of, or results from, PRL-secreting adenomas in humans.
In contrast to the human tumor, a variety of receptor
and postreceptor defects have been detected in rat PRLsecreting tumors. Best studied are GH3 cells, derived
from radiation-induced pituitary tumors, which secrete
GH and PRL. GH3 cells increase PRL secretion in response to estradiol and TRH (189), possess specific receptors for TRH (190), and can be indefinitely maintained in culture. For these reasons, they have been
extensively used to study the mechanism of PRL secretion. However, GH3 cells and their subclones are largely
refractory to DA inhibition (191). DA or bromocryptine
decreases PRL secretion only at concentrations 3 orders
of magnitude greater than those required for normal
pituitary cells (192). The inhibitory action of bromocryptine is not reversible with the DA antagonist butaclamol.
This unresponsiveness correlates with a lack of high
affinity binding sites for radiolabeled spiperone in GH3
cells (193). A low affinity saturable site with a dissociation constant at a micromolar level has been observed,
but it lacks stereoselectivity.
Absolute or relative refractoriness to DA has also been
observed in the diethylstilbesterol-induced transplantable rat tumors MtTW15 and 7315a (194). Unlike GH3
cells, these tumors have high affinity, saturable, and
stereoselective DA-binding sites. As in normal cells,
guanine nucleotides cause a decrease in high affinity
agonist binding, but do not affect antagonists. These
data suggest that the DA receptor is intact, and that the
unresponsiveness to DA is probably due to a defect at a
site distal to the receptor. Although the precise biochemical defect responsible for refractoriness to DA is yet
unknown, PRL-secreting tumors are excellent candidates for future reconstitutions with purified DA receptors.
4. Regulation of the D2 anterior pituitary
receptors
The sensitivity of a target cell to hormonal action is
inversely related to the levels of hormone to which the
Vol. 6, No. 4
cell has been exposed. The number of membrane receptors is one of the factors that ultimately regulate cell
sensitivity. The terms down-regulation and up-regulation are used to describe a decrease and an increase,
respectively, in receptor density in response to exposure
to a homologous hormone. Also, the action of one hormone can alter cell sensitivity to another hormone. This
had led to the concept of heterologous hormone regulation. The heterologous hormone can regulate both the
affinity and the number of binding sites of the other
hormone.
Several studies have documented dynamic changes in
anterior pituitary DA receptors which correlate well with
the presumed DA levels reaching the anterior pituitary.
Some of these changes, however, are of a smaller magnitude than anticipated. A plausible explanation is that
DA receptors may be located on cells other than lactotrophs. If so, PRL-related alterations in receptor number
would be attenuated or masked by an unchanged high
background. Although early studies indicated that DA is
a specific inhibitor of PRL only, it now appears that it
also inhibits GH (195) andTSH (196) release. Goldsmith
et al. (197) have used an immunocytochemical method
with labeled haloperidol to vizualize binding sites on
dispersed anterior pituitary cells. Binding sites are present on the majority of lactotrophs, but a significant
number are also evident on somatotrophs and gonadotrophs.
Interruption of the DA supply to the anterior pituitary
by surgical destruction of the MBH (198) or electrolytic
lesions of the median eminence (199) results in a significant increase in pituitary sensitivity to DA. There is
also a 60% increase in the number of dihydroergocryptine-binding sites without a change in affinity (199). We
have used MSG to investigate the relationship among
DA, PRL, and DA receptors (23). As seen in Fig. 10,
MSG causes a 60% reduction in DA concentration in the
MBH, a 3-fold rise in plasma PRL levels, and a 60%
increase in anterior pituitary [3H]spiperone-binding
sites, without a change in binding affinity. The above
data support the view that a reduction in DA reaching
the anterior pituitary results in an up-regulation of DAbinding sites and an enhanced pituitary responsiveness
to DA inhibition. Down-regulation of DA receptors after
chronic treatment with bromocryptine has recently been
reported (200).
Anterior pituitary DA receptors are also regulated
during the estrous cycle. A marked increase in the number of binding sites is observed between 1300 and 1700 h
on proestrus, coincident with the preovulatory PRL
surge. Binding capacity remains elevated during estrous
and declines on diestrous day 1 and day 2 (201). These
results are consistent with the reports that both DA
turnover in the median eminence and DA concentration
DA, A PRL-INHIBITING HORMONE
Fall, 1985
MBH DOPAMINE
60-
PLASMA PRL
ANTERIOR PITUITARY
DOPAMINE RECEPTORS
120—
300u
FIG. 10. Effect of treatment with
MSG on the DA concentration in the
MBH, plasma PRL levels, and anterior
pituitary DA receptor density. C, Control. Each bar represents the mean ±
SEM of four to seven determinations.
a
U —
ao-
80-
20-
40-
™'S 200•o
P
5 aa> E
C MSC
577
i I
C
100
MSC
in portal blood are significantly lower on proestrus than
throughout the remainder of the cycle (38, 54, 55) (Fig.
2). The data also demonstrate that changes in DA receptor density occur rapidly. A similar rapid change in [3H]
spiperone-binding sites during proestrus was reported by
Pasqualini et al. (202) except that they show a decrease,
rather than an increase, in binding sites at the onset of
the preovulatory PRL surge. There is no clear explanation for this discrepancy.
The effects of gonadal steroids on anterior pituitary
DA receptors are controversial. Treatment of ovariectomized rats with estradiol for 5 days causes an increase
in DA biosynthesis by MBH fragments, an elevation in
plasma PRL levels, and a dose-dependent reduction in
anterior pituitary DA-binding sites (203) (Fig. 6). Estradiol and diethylstilbesterol, but not progesterone or dihydrotestosterone, cause a time- and dose-dependent
reduction in DA receptors in a transplantable MtTF 4
tumor (204). On the other hand, neither estradiol nor
progesterone alone affects DA-binding sites; a 2-fold
increase is seen when ovariectomized animals recieve
both (134). An earlier study also failed to find changes
in DA receptors after treatment with estradiol (205).
Whereas only one high affinity binding site is revealed
by [3H]spiperone, high and low affinity sites can be
distinguished with [3H]domperidone (206). The high affinity site is not influenced by ovarian steroids, but
estradiol and progesterone have antagonistic effects on
the low affinity site (207). It appears that depending
upon the concentration, ratio, and length of exposure,
gonadal steroids can affect DA receptors in opposite
ways: one by directly changing pituitary DA receptors
and the other by decreasing hypothalamic DA secretion,
resulting in up-regulation of the receptors.
Changes in DA receptor density have been observed
under other endocrine conditions. For example, the number of [3H]spiperone-binding sites is higher in lactating
than in cycling or male rats. Furthermore, the density of
binding sites decreases significantly after the separation
of pups from lactating mothers (203). Lactation and
~
C
MSC
pregnancy in monkeys are also associated with an increase in the number of anterior pituitary DA receptors
(208). A correlation among DA-, PRL-, and DA-binding
sites has been reported in aging rats. Aged, constant
estrous rats have a reduced concentration of DA in portal
blood (48). They also show a significant increase in [3H]
spiperone-binding sites in the anterior pituitary and an
elevated plasma PRL level (51).
In summary, DA receptors in the anterior pituitary are
subjected to dynamic regulation by both homologous and
heterologous hormones. However, the usefullness of current techniques of receptor determination is limited by
the uncertainty of receptor localization and the alteration in the number of lactotrophs under the mitogenic
influence of estrogens. An ultimate solution may require
the development of methods for studying receptors at
the level of a single cell.
E. DA and Pituitary Lactotrophs
1. Characteristics of lactotrophs
Lactotrophs constitute about 20% and 35-40% of the
total anterior pituitary cell population in male and cycling female rats, respectively (209). A substantial increase in their number occurs during late pregnancy,
presumably under the mitogenic influence of estrogen.
About 5% of pituitary cells appear to secrete both PRL
and GH (209, 210). The somatolactotrophs may serve as
stem cells from which lactotrophs and somatotrophs
differentiate, or they may represent a transient stage in
the normal cell cycle. Lactotrophs contain several morphologically distinct secretory granules, ranging from
500-900 nm in diameter (211). Granule size is often used
to distinguish PRL-producing cells by electron microscopy. However, GH3 cells or lactotrophs from suckled
lactating rats with high basal PRL secretion contain only
a few small granules. When PRL secretion is blocked by
prolonged periods of nonsuckling, the large mature granules reappear (211). This suggests that large PRL-containing granules are not obligatory for hormone secre-
578
BEN-JONATHAN
tion. Instead, they may serve as a form of storage when
cells stop secreting at a rapid rate.
PRL is synthesized on the rough endoplasmic reticulum, initially as a precursor with an amino-terminal
extension which is cleved before the nascent peptide is
completed. PRL is then translocated to the Golgi zone,
where it is packaged into secretory granules. The rate of
PRL synthesis is directly modulated by several hormones. TRH and estradiol stimulate PRL synthesis by
increasing the production of PRL mRNA (126, 212). On
the other hand, DA and its agonists inhibit de novo
synthesis of PRL (126, 213). Detectable changes in the
rate of PRL synthesis may take several hours compared
with the few minutes required to affect secretion. Several
molecular variants of PRL have been detected, which
differ in turnover rate, bioactivity, and immunoreactivity
(214). Preferential release of different variants can occur
under various conditions (215). There is evidence for
posttranslational modification by amidation or chain
cleavage (216, 217) which can alter PRL biological activity by changing its susceptibility for degradation and/or
its receptor-binding properties.
In addition to molecular variability, there is cellular
heterogeneity. Subpopulations of lactrotrophs, differing
in size and cytoplasmic granulation, can be distinguished
cytochemically (218). The sedimentation profiles of lactotrophs on gradients of BSA vary with the endocrine
state of the pituitary donors. Lactotrophs recovered from
the various fractions secrete different amounts of PRL
(219). There is also functional heterogeneity with respect
to the incorporation of tritiated leucine into PRL and its
subsequent release. The newly synthesized PRL is preferentially released during spontaneous secretion from
normal cultured pituitary cells (220, 221). Cells differ,
however, in their responsiveness to TRH, which stimulates the release of older stored PRL without affecting
the release of newly synthetized hormone (221). It is not
known whether the fast and slow releasable pools exist
within the same cell or are present in different subpopulations of lactotrophs.
After binding to the membrane receptor, DA is also
found internalized into lactotrophs, where it appears to
be associated with PRL secretory granules (222) and
lysosomes (223). DA agents increase lysosomal enzyme
activity (224) and enhance PRL degradation (225); estradiol suppresses anterior pituitary responsiveness to
this action of DA (226). The DA-induced stimulation of
lysosomal enzyme activity may be part of a delayed
mechanism by which DA inhibits PRL secretion. However, the functional significance of the association of DA
with PRL-containing secretory granules is yet to be
determined.
Vol. 6, No. 4
2. Electrical activity of lactotrophs
Neurosecretory and endocrine cells share many features. Among them is the presence of membrane-bound
secretory granules and an absolute requirement for calcium ions for exocytosis. Although neurosecretory cells
are traditionally considered electrically excitable, i.e. capable of generating action potentials, endocrine cells
have been assumed to be electrically inactive. This notion
should be reevaluated in view of the accumulating evidence of electrical activity in a variety of endocrine cells,
including pancreatic islets (227, 228), chromaffin cells
(229, 230), and pituitary cells (231-234).
Like all living cells, pituitary cells have a membrane
potential that depends upon the gradients of ions across
the cell membrane and their relative conductances. Resting membrane potentials of pituitary cells are smaller
than those of nerve or muscle cells (—35 to —50 mV
compared with —70 to —90 mV). Several investigators
have shown high amplitude action potentials and low
amplitude potential fluctuations in normal anterior pituitary cells (231, 232). Only a few cells exhibit spontaneous electrical activity, but many discharge in response
to depolarizing or hyperpolarizing current pulses. Extracellular recordings from dispersed cells have shown random action potentials. On the other hand, one study
employing intracellular recording from rat hemipituitaries reported transient hyperpolarizations occurring at
fairly regular intervals (235). This led to the suggestion
that some cells have pacemaker-like properties.
A major difficulty in interpreting the above data is
that the pituitary cells were unidentified. Taraskevich
and Douglas (236) circumvented this problem by taking
advantage of the segregated location of PRL-secreting
cells in the pituitary of the alewife fish. Extracellular
recordings reveal spontaneous action potentials which
are slowed or completely suppressed by DA or NE. Most
other investigators have used the rat GH3 PRL-secreting
cells. Kidokoro (231) was the first to report spontaneously occurring spikes which are calcium dependent.
TRH increases the percentage of cells displaying action
potentials and the frequency of existing spikes in others
(237, 238). Estradiol is also capable of triggering a burst
of electrical activity from GH3 cells (234). Although GH3
cells are largely refractory to DA, the subclone B 6 retains
responsitivity to very high concentration of DA. About
half of these cells are electrically excitable by outward
current pulses, whereas a quarter show spontaneous action potentials. DA decreases the firing rates of active
cells, and haloperidol antagonizes the inhibitory effect of
DA. Exposure of cells to estrogen abolishes the effect of
DA (234, 239).
The most significant data to date on the effect of DA
on the electrical activity of lactotrophs come from a
Fall, 1985
DA, A PRL-INHIBITING HORMONE
recent study using human prolactinoma cells, which retain sensitivity to DA agents (240). Only a few cells show
spontaneous activity, but action potentials could be readily induced by injection of depolarizing currents. Induced
action potentials are blocked by inhibitors of calcium
current, but are insensitive to sodium blockers. DA induces a significant hyperpolarization, cessation of action
potentials, and a concomitant increase in membrane
conductance. This suggests the involvement of potassium
ions. The DA effect is transient and reversible and is
blocked by D2 but not Dl receptor antagonists. It is
noteworthy that in hippocampal pyramidal cells, where
DA functions as an inhibitory neurotransmitter, it also
causes a hyperpolarization, which appears to be mediated
by a calcium-activated potassium conductance (241).
Understanding of the interrelationship between DA
and electrical activity of lactotrophs awaits studies with
normal nontumorous cells. Recently, we used the reverse
hemolytic plaque assay (242) to identify live secreting
normal lactotrophs (Croxton T. L., N. Ben-Jonathan,
and W. McD. Armstrong, unpublished observations).
Dispersed pituitary cells grown in monolayers were exposed to protein A-coupled red blood cells, complement,
and PRL antiserum. PRL-secreting cells are identified
by lysis plaques (Fig 11A). Patch clamp experiments
(Fig. 11B) have demonstrated the presence of potassium
channels. Work is underway to characterize the ionic
selectivities of these channels and the possible modulating effects of secretagogoues on their gating kinetics.
On the basis of existing data, albeit limited, several
postulations can be made regarding the interactions between DA and ions in the regulation of PRL secretion.
First, lactotrophs may have a higher calcium conductance than other pituitary cells, accounting for their
unique ability to secrete large amounts of PRL upon
removal of DA inhibition. Second, DA may function by
causing membrane hyperpolarization, possibly through
activation of potassum channels. A resulting cessation
of action potentials could decrease the influx of calcium
into the lactotroph, thus decreasing calcium available for
exocytosis.
3. Intrinsic PRL pulsatility
Most anterior pituitary hormones are released into the
circulation in a pulsatile manner. Pulse-generating ability is usually associated with neurons or muscle cells
rather than with endocrine cells. For example, the episodic release of LH which occurs at 1- to 2-h intervals is
determined by pulses of LHRH released from hypothalamic neurons (243, 244). Both the amplitude and the
frequency of these pulses are regulated in a complex
manner by gonadal steroids, opioids, and catecholamines.
Several observations have indicated that circulating
579
PRL shows rapid pulsatility. PRL pulsatility persists
and is, in fact, amplified after blockade of DA receptors
by pimozide and butaclamol (245). Anterior pituitaries
transplanted under the kidney capsule of hypophysectomized male rats exhibit a high frequency PRL pulsatility at 8- to 10-min intervals. The pulse magnitude is
greatly magnified by estradiol (246). In a recent study,
monkey hemipituitaries were perifused in vitro, and PRL
and GH secretion were determined at 2-min intervals.
Both hormones displayed pulsatile release with similar
interpulse intervals of 8-10 min, but with a higher pulse
amplitude for PRL than for GH (247). We made a similar
observation in 1973 as a corollary to a study aimed at
determining hypothalamic LHRH secretion (248). As
shown in Fig. 12, individual hemipituitaries superfused
with arterial blood show high frequency pulses of PRL,
but not LH, secretion. Taken together, these data clearly
indicate that the anterior pituitary possesses an intrinsic
interlactotroph communication system permitting synchronized PRL secretion without a direct input from the
hypothalamus.
The intrinsic pulsatility of PRL secretion raises several intriguing questions. 1) Is it unique to lactotrophs
or is it shared by other endocrine cells? 2) Is it triggered
by membrane electrical events? 3) Are all lactotrophs
capable of generating rhythmicity? 4) What is the mechanism for intercellular communication leading to synchronization of secretion? 5) Does DA or do other secretagogoues modulate PRL pulsatility? At present, very
limited information is available to answer these questions, and this aspect of PRL secretion should provide
an interesting objective for future research.
Rapid oscillations of hormone secretion may represent
a more generalized phenomenon than is presently appreciated. Several hormones, including insulin, glucagon,
somatostatin, and PTH, exhibit fast cyclic fluctuations
(249-251). The rapid pulses of insulin and glucagon in
monkeys are not affected by administration of neurally
active substances (252, 253). It is noteworthy that a pulse
interval of 8-12 min is common to all hormones studied
thus far. This raises the possibility that most, if not all,
hormones are released into the circulation in a rapid
oscillatory manner. Such a phenomenon could easily
have escaped notice for several reasons. One is infrequent
blood sampling. The second is the masking effect of long
half-lives of many hormones. The third is the use of
dispersed cells in perifusion systems rather than tissue
fragments, a common practice which precludes preservation of intercellular integrity.
The best studied example of pacemaker activity in
endocrine tissue is the /3-cell of the pancreas (228). Some
islet cells display periodic alterations between a polarized
silent phase of membrane potential (at about —50 mV)
and a depolarized plateau phase with calcium-dependent
580
BEN-JONATHAN
Vol. 6, No. 4
FIG. 11. Ionic channel activity in a cultured normal lactotroph. A, Photomicrograph of reverse hemolytic plaque. Cultured anterior pituitary cells
were incubated with protein A-conjugated red blood cells, anti-PRL antiserum, and complement. One lactotroph (arrow) is surrounded by red
blood cell ghosts (a plaque), while two other nonplaque-forming cells are seen. B, Patch clamp recording from indentified lactotroph. The current
record was obtained from a detached inside-out membrane patch (intracellular surface facing bath). With zero applied potential, a difference in
potassium ion concentration across the patch resulted in positive current flow into the pipette (downward deflection). Quantal changes in current
indicate opening or closing of a discrete ion channel. Several similar channels are evident. pA, Picoamperes; ms, milliseconds. (Croxton, T. L., N.
Ben-Jonathan, and W. McD. Armstrong, unpublished observations, 1985).
action potentials, Elevated glucose levels cause a marked
change in the timing of the phase transitions without
affecting the spike amplitude or membrane potential of
the two phases (228). The author suggested that pacemaker activity occurs as a result of complex interaction
among voltage-dependent calcium channels, calcium
fluxes, changes in intracellular calcium buffering capacity, and potassium channels which are sensitive to intracellular free calcium.
It is possible that calcium channels and/or calciumgated potassium channels are involved in the generation
of PRL pulsatility. Using a whole cell patch clamp technique, Armstrong and Matteson (254) recently reported
the existence of two distinct populations of calcium
channels in GH3 cells. One channel is activated in a
relatively negative voltage range, closes slowly on repolarization, and inactivates during long depolarization.
The second channel closes rapidly and is not inactivated.
The authors postulated that fast channels are well suited
for calcium influx, whereas the slow channels could
function in pacemaking. It is tempting to speculate that
selected lactotrophs may function as pacemakers and
generate an oscillatory pulse. There is virtually no information, however, on possible anatomical segregation of
such cells or how this signal might be communicated to
other lactotrophs to synchronize secretion.
4. Intracellular mediators of dopaminergic action
Hormones that have cell surface receptors require a
second messenger system to couple the extracellular
stimulus to specific cellular responses. For more than 2
decades, cAMP has been considered the primary and
universal second messenger for most peptide hormones
and catecholamines. Many, if not all, of its actions are
mediated through cAMP-dependent protein kinase,
which catalyzes the phosphorylation of proteins serving
as effectors or modulators of the various cellular reactions.
Although early studies failed to find consistent effects
of DA on cAMP accumulation in the anterior pituitary,
Fall, 1985
DA, A PRL-INHIBITING HORMONE
581
LH
PRL
#39
- 160
FIG. 12. PRL pulsatility in individual
hemipituitaries superfused at 7 jtl/min
with arterial blood from anesthetized hypophysectomized female rats. Note the
interpulse intervals of 8-10 min of PRL,
but the absence of clear pulses of LH.
(Ben-Jonathan, N, and J. C. Porter, unpublished observations).
^w.
a functional connection between the two is now well
established. DA reduces adenylate cyclase activity and
inhibits cAMP accumulation in primary cultures of pituitary cells (255-258) and in human prolactinomas
(187). Adenylate cyclase activity is more sensitive to DA
inhibition in lactating than in male rats (259). DA at
nanomolar concentrations suppresses intracellular
cAMP within 1-5 min (257). The suppression is GTP
dependent and is reversed by D2 receptor antagonists
(259). The elevation of cAMP and PRL release induced
by cholera toxin is attenuated by DA agonists (260). This
suggests either that DA can override the toxin-induced
activation of adenylate cyclase or that it acts via noncAMP-dependent pathways. In lactotoroph-rich, but not
thyrotroph-rich, populations, DA inhibits the increase in
cAMP induced by TRH (258). Collectively, these data
are in good agreement with the documented coupling
between the D2 receptor and adenylate cyclase (166,176,
180) and support the notion that the suppression of
intracellular cAMP levels comprises one of the mechanisms of DA action.
The involvement of calcium in a broad spectrum of
cell activities has led to its wide acceptance as an intracellular second messenger. According to the stimulussecretion coupling concept (261), a stimulus delivered to
the cell membrane causes an instantaneous 5- to 10-fold
rise in intracellular free calcium from a resting level of
about 0.1 /zM (262). This involves both redistribution
from intracellular stores (mitochondria and endoplasmic
reticulum) and influx from the extracellular fluid via
membrane calcium channels. A variety of calcium-binding proteins (i.e. calmodulin) and calcium-activated enzymes (i.e. protein kinase C) participate, by a yet unknown mechanism, in the induction of exocytosis.
Like other pituitary hormones, the secretion of PRL
is calcium dependent. PRL secretion is enhanced by
increased calcium concentration and is suppressed by
removal of calcium from the medium or addition of
calcium channel blockers (263). Agents that increase
intracellular calcium by activating voltage-dependent
calcium channels, i.e. potassium (264, 265) and maitotoxin (266), or by increasing calcium uptake in a nonspecific manner, i.e. calcium ionophores (267, 268), also
stimulate PRL secretion.
Changes in intracellular calcium concentrations are
difficult to study in a heterogenous cell population. Given
that the PRL-secreting GH3 cells respond well to TRH,
it is not surprising that TRH represents perhaps the best
studied example of a functional link among a ligand,
calcium, and hormone secretion. Using intracellularly
trapped Quin 2 (269) or aequorin (270) as probes, TRH
was shown to induce a biphasic change in cytoplasmic
free calcium. The initial rapid rise is independent of
changes in extracellular calcium, whereas the smaller
and prolonged second rise is greatly attenuated by blockers of calcium influx. This biphasic response corresponds
temporally with the pattern of PRL secretion induced by
TRH in a perifusion system (265,269). The model emerg-
582
BEN-JONATHAN
ing from these studies shows that after ligand binding to
its receptor, there is an initial mobilization of calcium
from intracellular stores. This is followed by an effect on
calcium fluxes across the cell membrane. It is noteworthy, however, that normal pituitary cells and GH3 cells
differ significantly in their calcium requirement for secretion (271). This and the absence of DA receptors in
GH3 cells (193) raise the question of whether PRL secretion by tumor cells truely reflects the situation in normal
cells.
Unlike TRH, the understanding of the precise relationship between DA and the calcium messenger system
is still fragmentary. Electrophysiological studies indicate
that DA inhibits calcium-dependent action potentials
(231, 236, 239). However, there are conflicting reports of
whether DA agents may act by decreasing the cytoplasmic calcium concentration (255, 256, 264). Some of
this controversy can be attributed to the extensive use
of bromocriptine, which acts irreversively (272) and
therefore may not duplicate the action of the native
compound. In one study, DA was shown to cause a rapid
fall in intracellular calcium in a lactotroph-enriched population (273). Nonetheless, it remains to be determined
whether DA regulates calcium fluxes across the cell
membrane, mobilizes intracellular calcium, or both.
A step after calcium mobilization is also involved.
Many of the regulatory functions of calcium are mediated
by its binding to calmodulin. The calcium-calmodulin
complex governs both the synthesis and degradation of
cAMP, albeit at different concentrations (274). Several
neuroleptic agents which act as DA receptor antagonists
at nanomolar concentrations inhibit PRL secretion at
micromolar concentrations (275). This inhibition appears to involve a direct antagonistic action on calmodulin (276). Several lines of evidence now suggest that
in addition to affecting calcium mobilization, DA modulates calcium-calmodulin-dependent cAMP formation
(256, 267).
The polyphosphoinositides now appear to function as
second messengers by mediating signal transmission for
a wide variety of hormones and neurotransmitters (277).
After a hormone-receptor interaction, phospholipase C
is activated and catalyzes the hydrolysis of phosphatidylinositol 4,5-biphosphate (PIP2) into inositol triphosphate and diacylglycerol. Both compounds interact with
calcium; inositol triphosphate causes a transient rise in
intracellular free calcium by releasing bound calcium
from intracellular stores, and diacylglycerol activates
protein kinase C by increasing its affinity for calcium
(277). Although there is increasing evidence that TRH
activates the phospholipid pathway (278, 279), only limited information is presently available with respect to
DA. DA as well as bromocriptine inhibit PIP 2 hydrolysis
both in vivo and in vitro, and this effect is counteracted
Vol. 6, No. 4
by DA receptor antagonists (280, 281). Because PIP 2
turnover is tightly linked to calcium mobilization, this
may represent yet another pathway by which DA affects
calcium metabolism of lactotrophs.
In summary, all three second messenger systems,
namely cAMP, calcium, and phospholipids, appear to
mediate DA action. Their relative importance, however,
is still undetermined. Nonetheless, it is now becoming
clear that these systems do not function in a parallel
independent manner. Instead, they interact in a synergistic or sometimes antagonistic manner and complement each other with respect to the response time and
its duration. Much more research is necessary to accurately define the multifaceted mechanisms by which DA
modulates PRL secretion.
F. Concluding Remarks and Perspectives .
The purpose of this review was to consolidate the
enormous literature that exists in support of DA as a
major physiological regulator of PRL secretion. Practical
considerations dictated that only selected topics were
covered, but the interested reader could further explore
the many excellent reviews by previous authors which
are cited herein. Although progress in this field has been
impressive, many questions remain unanswered. In the
live animal, the sequence of events leading to the suckling-induced rise of PRL remains enigmatic. In the area
of pathophysiology, the etiology of PRL-secreting tumors
is yet to be determined. In the brain, the roles of extrahypothalamic sites and various neuromodulators in the
regulation of PRL release should be examined. In the
anterior pituitary, intercellular communication and synchronization of secretion are largely unexplored phenomena. At the level of the cell membrane, the interactions
among hormones, electrical activity, and ionic channels
are yet to be placed in perspective. Finally, there is much
to be learned about the coupling between the receptor
and several second messenger systems in translating
hormonal signals into cellular responses. It is the hope
of this author that with the development of new techniques and the opening of new directions for investigation, some of these questions will be resolved.
Acknowledgments
I wish to express my gratitude to Drs. William McD. Armstrong and
Thomas L. Croxton for critical reading of the manuscript. I am indebted
to my past and present mentors, collaborators, and students for their
continuous enthusiasm and support.
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