Inhibition of protein phosphatase 2A (PP2A) mimics suckling-induced sensitization of mammotropes: involvement of a pertussis toxin (PTX) sensitive G-protein and the adenylate cyclase (AC).

In lactating rats, suckling renders mammotropes more responsive to prolactin (PRL)-releasing stimuli and less responsive to PRL-inhibiting secretagogues. We have previously shown that a decrease in the activity of protein phosphatase 2A (PP2A) may be responsible for the decrease in responsiveness to the inhibitory secretagogue dopamine (DA). In our present experiments, we have studied the involvement of the adenylate cyclase (AC), stimulatory and inhibitory GTP-binding proteins and also the role of PP2A in the sensitization phenomenon. Pituitary cells obtained from mother rats separated from their pups for 4 h prior to dispersion (non-suckled), suckled for 10 or 30 min after the separation period (suckled) and without separation (continual suckling) were incubated in the presence of different doses of forskolin to activate AC and DA. In a further study, pituitary cells of non-suckled rats were pretreated with cholera toxin (CTX) or pertussis toxin (PTX) and tested for the stimulatory action of forskolin or TRH on PRL release. Ocadaic acid (OA) pretreatment has been used to investigate the involvement of PP2A. Hormone secretion was measured by the reverse hemolytic plaque assay (RHPA). Our results have shown that cells from non-suckled rats were unresponsive to forskolin. A 10-min suckling stimulus sensitizes pituitary mammotropes to respond with a PRL release to a dose-dependent activation of AC by forskolin. This sensitization of AC becomes a permanent feature of the cells when suckling continues for an additional 20 min. We have also found that pituitary mammotropes from non-suckled dams respond to forskolin or TRH with PRL release when they were preincubated with either PTX or the PP2A inhibitor OA. It clearly indicates that the non-responsive pituitary can be shifted to the responsive stage by uncoupling of inhibitory G-protein from its receptor as well as by inhibition of PP2A. This latter finding, consonant with our previous results, suggests that suckling may cause selective changes in the function of G(i) of mammotropes due to a rapid phosphorylation which can remove tonic, GTP-dependent inhibitory function.     

Suckling increases the proportions of mammotropes responsive to various prolactin-releasing stimuli

It is well established that the suckling stimulus sensitizes or primes the anterior pituitary to PRL-releasing stimuli. It is also recognized that PRL-secreting cells from a given animal are not all alike but instead exhibit a considerable degree of functional heterogeneity. The goal of the present study was to determine whether the suckling-induced priming phenomenon is manifest at the cellular level by shifts in the relative abundance of various mammotrope subpopulations. This was accomplished by using reverse hemolytic plaque assays to evaluate the secretory characteristics of individual PRL secretors derived from lactating rats either before or after the transient application of a suckling stimulus. Groups of day 10 lactating rats separated from their litters for 4 h were either killed immediately or were reunited briefly (10 min) with their pups before death. Adenohypophyseal cells obtained after trypsin dispersion were then subjected to plaque assays for PRL. Mammotropes derived from suckled rats were, on average, considerably more responsive to the stimulatory actions of TRH and angiotensin II and less susceptible to inhibition by dopamine. Mammotropes from nonsuckled rats exhibited a bimodal frequency distribution in which plaques from the second mode were roughly 6-8 times larger (released considerably more PRL) than those from the first. Superimposition of suckling (or in vitro treatment with dopamine) caused the second mode to disappear. Suckling also enhanced greatly the fraction of PRL cells that shifted from the first to the second mode (i.e. released more hormone) after treatment with TRH or angiotensin II. Taken together, our results demonstrate that the suckling-induced sensitization of pituitary tissue to PRL-releasing stimuli is manifest at the cellular level as proportional shifts toward those cells most responsive to stimulatory secretagogues and away from those most susceptible to inhibition by dopamine.     

Functional variations among prolactin cells from different pituitary regions

Reverse hemolytic plaque assays were used to compare the responsiveness of cells from different pituitary regions to the modulatory effects of human pancreatic GH-releasing factor (GRF), TRH, and dopamine (DA). Tissues from the peripheral rim (outer zone) and the central region (inner zone) of adenohypophyses from day 10 lactating rats were dispersed with trypsin, and the cells were placed into culture. On the following day, these cells were subjected to GH plaque assays (conducted in the presence or absence of GRF) and PRL plaque assays (performed with or without TRH and DA). Cells from both zones responded similarly to GRF with a rapid acceleration of GH plaque formation. However, the rate of PRL plaque formation in response to TRH and DA differed between cells from these regions. For outer zone cells, plaque development increased greatly with TRH treatment, but was only moderately affected by DA. Plaque formation from inner zone cells was influenced slightly by TRH, but markedly inhibited by DA. These results suggest that PRL, but not GH, cells from these pituitary regions are differentially responsive to at least two hypothalamic secretagogues. We then performed fixed sequential plaque assays to determine whether the proportions of cells that released PRL only (classical mammotropes) or those that released both GH and PRL (mammosomatotropes) also differed between the inner and outer zones. Using this approach, we found that the outer zone contained a much larger proportion of dual hormone secretors than did the inner zone. These results, when taken together with the responsiveness differences discussed above, raise the possibility that the release of PRL from mammotropes and mammosomatotropes is regulated differently and that the ratio of these two cell types may dictate, in part, the manner in which a specific region of the pituitary responds to hypothalamic input.     

The stimulatory and inhibitory effects of dopamine on prolactin secretion involve different G-proteins.

The reverse hemolytic plaque assay (RHPA) was used in this study to further characterize the mechanism whereby low concentrations of dopamine (DA) stimulate PRL secretion in vitro. Female Sprague-Dawley rats were used as a source of anterior pituitary cells for the RHPA. Pituitary cells were infused into Cunningham chambers along with a suspension of protein-A-coated ovine red blood cells. Excess cells were rinsed from the chambers leaving a monolayer of cells attached to the glass. The cells were then incubated with solutions containing PRL antiserum (1:40) and various concentrations of DA. After 4 h, a solution containing guinea pig complement (1:60) was infused into the chambers. Thirty minutes later, the cells were fixed and plaques (zones of hemolysis) surrounding PRL-producing cells (lactotrophs) were measured and used as an index of the amount of PRL secreted. Control cells that received no DA had a mean plaque area of 8,000 microns 2 and two distinct subpopulations of plaque sizes. This biphasic population of cells consisted of a small and a large plaque producing population. The mean plaque area surrounding lactotrophs was significantly (P less than 0.05) decreased if 1 microM or 10 microM DA was present (4,500 microns 2 and 3,500 microns 2, respectively). These cells which received inhibitory concentrations of DA demonstrated a monophasic distribution of plaque-forming cells. On the other hand, mean plaque area was significantly (P less than 0.05) increased if 0.1 nM or 1 nM DA was presented to the cells (15,000 microns 2 and 14,500 microns 2, respectively). These cells receiving stimulatory doses of DA exhibited a multiphasic distribution of plaque-forming cells. The possibility that the two physiological opposing actions of DA on PRL secretion might be mediated by different GTP binding proteins was also examined using cholera toxin (CTX) and pertussis toxin (PTX). Anterior pituitary cells were pretreated with either CTX (50 micrograms/ml) or PTX (5 micrograms/ml) for 1 h before initiation of the RHPA. In the RHPA, cells received no DA, a stimulatory dose of DA (0.1 nM), or a inhibitory dose of DA (10 microM). The effects of toxin pretreatment on mean plaque area of DA-treated cells was determined. PTX pretreatment significantly attenuated the inhibitory effects of DA while having no effect on the stimulatory effects of DA on PRL secretion. CTX significantly (P less than 0.05) potentiated the stimulatory effects of DA on PRL secretion and had no effect on inhibition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis by plaque assays of GH and prolactin release from individual cells in cultures of male pituitaries. Evidence for functional heterogeneity within rat mammotrope and somatotrope populations

In this study we evaluated the quantitative influence of GRF and TRH on the rate of hormone secretion from single cells in cultures of male pituitaries. To accomplish this, we dispersed pituitaries from male rats with trypsin and cultured them for 24 or 48 h. Reverse hemolytic plaque assays for GH and prolactin were then performed on retrypsinized cultures to identify individual cells that secreted these hormones. Mammotropes and somatotropes were found to comprise 31.4 +/- 1.8 and 32.2 +/- 0.9% (mean +/- SE, n = 3 experiments), respectively, of all cells in 24-hour cultures. Immunocytochemical staining of different batches of cells from the same dispersions corroborated the proportions of these two cell types. Differences in the rate of basal hormone secretion were observed within each of these cell populations as evidenced by the gradual appearance of prolactin and GH plaques over a 4-hour period when incubations were conducted in the absence of stimulatory secretagogues. Addition of increasing concentrations of GRF (1 X 10(-10) -1 X 10(-7) M) or TRH (1 X 10(-9) -1 X 10(-6) M) to these incubations resulted in dose-related increases in the rate of GH and prolactin plaque formation, respectively. Maximal plaque development by somatotropes could be induced within 30 min of administering large doses of GRF, indicating that most, if not all somatotropes are responsive to this secretagogue. In contrast, approximately one third of all mammotropes could not be stimulated to form plaques acutely when subjected to similar treatment with TRH. This observation suggests that mammotropes are heterogeneous with respect to TRH responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)     

The suckling stimulus increases the responsiveness of mammotropes located exclusively within the central region of the adenohypophysis

Studies from several groups, including our own, have shown that the suckling stimulus increases the responsiveness of pituitary cells to PRL-releasing stimuli. These findings, when viewed in light of differences in PRL cell responsiveness from one pituitary region to another, raised the possibility that suckling may influence responsiveness of cells in only a specific portion of the gland rather than in the entire pituitary. To address this issue, we evaluated cell responsiveness by performing plaque assays [with and without TRH, Angiotensin II (AII), and dopamine] on cells from two different regions of pituitaries from suckled and nonsuckled rats. These pituitary regions consisted of the inner zone, which is a central area proximate to the neurointermediate lobe, and an outer zone, which encompasses the remaining peripheral area of the anterior lobe. We found that inner zone cells from nonsuckled animals were highly responsive to dopamine and relatively unresponsive to TRH and AII. However, after suckling, a complete shift occurred with inner zone cells becoming sensitive to TRH and AII and resistant to dopamine. In contrast to these inner zone alterations, outer zone cells did not change after suckling, but remained responsive to TRH and AII and unresponsive to dopamine. Our results demonstrate clearly that suckling-induced alterations in PRL cell responsiveness to certain modulatory agents can be attributed to a discrete subpopulation of cells located in a specific region of the pituitary.     

Effects of epinephrine, norepinephrine, and dopamine on gonadotropin-releasing hormone-induced secretion of luteinizing hormone in vitro

Recent evidence indicates that catecholamines may directly alter anterior pituitary function. In the present study, an in vitro perifusion system was used to investigate whether catecholamines affect the gonadotrope. Pituitary tissue from castrated ram lambs was incubated in the presence of epinephrine (EPI), norepinephrine (NE), or dopamine (DA). During a 2-h treatment period, neither DA (10(-8) M), NE (10(-7) M), nor EPI (10(-7) - 10(-9) M) significantly affected basal LH secretion. In contrast, the LH response to a subsequent 10(-10) M GnRH challenge was significantly potentiated by NE and EPI. NE increased the amount of LH secreted in response to GnRH 14 +/- 1.1% (P less than 0.01). Likewise, 10(-7), 10(-8), and 10(-9) M EPI resulted in 22 +/- 1.4% (P less than 0.001), 13 +/- 1.2% (P less than 0.001), and 6 +/- 1.3% (P less than 0.03) increases, respectively. The stimulatory effect of 10(-7) M EPI was blocked by pretreatment with propranolol (a beta-adrenergic blocker), but not with phentolamine (an alpha-adrenergic blocker). The beta-adrenergic agonist isoproterenol enhanced GnRH-induced LH secretion 46 +/- 1.5% (P less than 0.001), but had no effect on basal LH release. DA had no effect on LH secretion; however, it inhibited PRL release 24 +/- 0.9% (P less than 0.001). Neither NE, EPI, nor isoproterenol had any effect on PRL secretion. These results suggest that EPI, acting by a beta 2-adrenergic receptor, modulates the pituitary gonadotrope's response to GnRH.     

Effect of posterior pituitary lobectomy on in vivo and in vitro secretion of prolactin in lactating rats

The effect of removing the posterior and neuro-intermediate lobes (PLX) of the pituitary gland of lactating rats was determined on both suckling-induced release and transformation of prolactin (PRL), and on regionalization of PRL release. Sixteen hours, or 1 or 4 d after either PLX or sham surgery, acute (15-min) suckling was applied. Also, regionalization of PRL release was analyzed by incubating the central and peripheral regions of APs from nonsuckled rats. Plasma PRL was analyzed by radioimmunoanalysis (RIA), whereas anterior pituitary (AP) PRL content and in vitro released PRL were determined by polyacrylamide gel electrophoresis. Plasma PRL increased 25- to 30-fold after suckling in intact and sham, and 10- to 15-fold in 1- and 4-d PLX rats, but no change occurred on either 16-h PLX nonsuckled and suckled rats. Also, PRL transformation occurred in intact, sham, and 4-d PLX suckled rats, but not in 16-h sham, or in 16-h and 1-d PLX suckled rats. Finally, the higher secretion of PRL shown in vitro by the central region of APs from intact and sham was not observed in APs from PLX rats. These results show that PLX transiently depresses the suckling-regulated PRL transformation and release. Likewise, influences from the posterior and/or neuro-intermediate lobes may determine regionalization of PRL release.     

Regional mechanisms within anterior pituitary of lactating rats may regulate prolactin secretion

Prolactin (PRL) release was compared after incubating the central and peripheral regions of the anterior pituitary of lactating rats, either nonsuckled or suckled in conditioned medium obtained by incubating medium with the same anterior pituitary regions from nonsuckled or suckled rats. To collect conditioned media, anterior pituitary regions were incubated in Earle’s medium for 4 h, and conditioned medium was filtered and employed double concentrated. Each anterior pituitary was incubated in conditioned medium for 30 min. PRL released in vitro was determined by polyacrylamide gel electrophoresis. As a control, anterior pituitary regions from lactators were incubated in medium conditioned by male rat anterior pituitary regions, and they showed no changes of PRL release compared with those cultured in Earle’s medium. In general, conditioned media from both anterior pituitary regions of nonsuckled and suckled rats inhibited PRL release in peripheral anterior pituitary regions, whereas PRL release was stimulated in central regions of both nonsuckled and suckled rats. A higher number of stimulatory effects was provoked by conditioned media from suckled than from nonsuckled rats, and most of these effects were from conditioned media of the peripheral region of suckled rats. Together, these results suggest the existence within anterior pituitary regions of factors that regulate PRL secretion and that their action depends on the physiologic condition of the animal.     

Effect of suckling on prolactin receptor immunoreactivity in the hypothalamus of the rat

Previous studies showed that expression of prolactin (PRL) receptor is increased in numerous hypothalamic nuclei in mid-lactating rats. The increase in PRL receptor expression could be initated by neurohormonal changes during proestrus or pregnancy, or by the suckling stimulus during lactation. The present study investigated whether the PRL receptor expression in numerous hypothalamic nuclei is altered by the suckling stimulus. Three groups (n = 4) of rats on lactation day 10 were used: a continuously suckled group, a nonsuckled group (pups removed for 12 h) and a resuckled group (pups removed for 12 and then resuckled for 9 h). Animals were perfused with 2% paraformaldehyde and brains were sectioned (20 microm) for the immunofluorescence study. Immunoreactivity was semiquantitatively analyzed by counting the immunoreactive cells and measuring the immunostaining intensity in a specific area. Neurons expressing PRL receptors were observed in numerous hypothalamic areas with the highest number being in the arcuate, paraventricular and supraoptic nuclei. The PRL receptor immunofluorescence in several nuclei was significantly decreased in the nonsuckled group, and recovered in the resuckled group. These areas included the ventromedial preoptic, ventrolateral preoptic, lateroanterior hypothalamic, ventrolateral hypothalamic and ventromedial hypothalamic nuclei. PRL receptor immunoreactivity in other areas was not significantly altered by the suckling stimulus. These results demonstrate that expression of PRL receptor in hypothalamic nuclei was differentially affected by the suckling stimulus. PRL receptors in those nuclei which were significantly altered by suckling stimulus may play more critical roles during lactation than those areas which were less sensitive to the suckling stimulus.     

Ovine prolactin (PRL) and dopamine preferentially inhibit PRL release from the same subpopulation of rat mammotropes

Autoregulation of PRL release was studied at the single cell level by the use of a reverse hemolytic plaque assay. Monodispersed pituitary cells from adult male rats were first preincubated with test substances and then coincubated with antirat PRL antiserum before development of plaques with complement. At the conclusion of the assay, the percentage of all pituitary cells in culture that formed plaques was evaluated microscopically, and the rate of plaque development was used as an index for the rate of hormone release. In controls, the maximal percentage of pituitary cells formed PRL plaques within a 1.5-h antibody incubation period, and addition of TRH (1 X 10(-7) M) did not increase this proportion. Treatment with ovine PRL (oPRL, 100 ng/ml) or dopamine (1 X 10(-7) M), either alone or in combination, caused a comparable suppression of the rate of PRL plaque development, which was reversed by the presence of TRH. Pretreatment of cells with the lysosomotropic agent chloroquine (1 X 10(-5) M) overrode dopamine inhibition of PRL plaque development, but did not influence oPRL inhibition. Taken together, these results demonstrate that oPRL inhibits basal, but not TRH-induced, PRL release from rat pituitary cells and support the view that PRL can act at the pituitary level to inhibit its own secretion. Moreover, the equipotency and lack of additivity exhibited by oPRL and dopamine coupled with the differential effects of chloroquine suggest that these factors both act upon the same subpopulation of mammotropes to inhibit PRL release, but by separate intracellular mechanisms.     

Antagonist of pituitary adenylate cyclase activating polypeptide suppresses prolactin secretion without changing the activity of dopamine neurons in lactating rats

Pituitary adenylate cyclase activating polypeptide (PACAP) is a relatively new neuropeptide, and it has a potent stimulatory effect on adenylate cyclase activity in rat pituitary cells. However, the role of PACAP in the physiological control of prolactin (PRL) secretion is still unclear. In the present study, we investigated the physiological significance of endogenous PACAP on PRL secretion in lactating rats. On lactation days 7-8, pups were separated from their mother rats for 5 h before the onset of suckling and PACAP6-38 (16 microg), a receptor antagonist, was injected through the lateral ventricle cannula just after the removal of pups. The effects of PACAP6-38 on PRL and oxytocin secretion, and on the activity of tyrosine hydroxylase (TH), were examined after the onset of suckling. Administration of PACAP6-38 inhibited PRL levels in response to suckling, but it did not affect the activity of TH, as measured by DOPA accumulation at 15 min after administration of NSD 1015 (25.0 mg/kg), an L-aromatic amino acid decarboxylase inhibitor, or the plasma concentrations of oxytocin in lactating rats. Injection of alpha-methyl-p-tyrosine (alpha-MT; 50 mg/kg), an inhibitor of dopamine synthesis, increased PRL levels, and suckling caused a further increase in the plasma concentrations of PRL. An injection of PACAP6-38 (i.c.v.) also inhibited the PRL response to suckling under dopamine depletion. These results suggest that endogenous PACAP acts as a neurotransmitter or neuromodulator within the hypothalamus and plays an important role for PRL secretion in lactating rats. Endogenous PACAP may regulate PRL secretion, possibly mediated by PRL-releasing factors such as vasoactive intestinal polypeptide or vasopressin.     

Suckling-induced change in oxytocin but not in alpha-MSH concentrations of the median eminence, the neural-, intermediate- and anterior lobes of the pituitary gland

Previous reports have implicated that pituitary-derived prolactin (PRL) is secreted from two distinct zones of mammotropes within the anterior lobe (AL). The inner zone (AL-IZ), located adjacent to the neuro-intermediate lobe (NIL), is supposed to be involved in the rapid and massive discharge of PRL from the pituitary gland due to suckling stimulus. Whereas the outer-zone (AL-OZ) gives the basal secretion and it does not play a role in the acute secretory response during nursing. Anatomically, the AL-IZ has an intimate contact with the NIL because the blood passing through the short portal vessels (SPV) bathes it first. Based on this fact it would be hypothesized that locally released and/or produced compounds, like OXY and alpha-MSH, can be delivered to the AL-IZ. In conjunction, OXY and alpha-MSH have already been implicated to play a role in the regulation of PRL release during suckling. Therefore, the purpose of this study was to examine the possible local transportation of these hormones into the median eminence and various regions of the pituitary gland of lactating rats. We have measured the concentrations of OXY and alpha-MSH from tissue samples of nonsuckled (NS) and 10 or 30 min after suckling (S) was initiated using specific RIAs. It has been shown that there are no changes in the concentration of OXY and alpha-MSH in theAL-IZ and AL-OZ due to suckling stimulus. In contrast, our data provide compelling evidence that OXY is transported into the IL, which can be further increased by suckling stimulus. These data suggest that blood transfusing NL passes through the IL before it is drained into the cavernous sinus, which opens the road for OXY into the general circulation. In addition, our data have unequivocally shown a lack of local delivery of either alpha MSH or OXY into the AL that raises serious doubt about their possible role in PRL secretion during suckling stimulus.     

Modulation of pituitary gonadotropin-releasing hormone receptors during lactation in the rat

Lactation is associated with a suppression of pituitary GnRH receptors (GnRH-R), and removal of the suckling stimulus for 24 h causes a 4- to 5-fold increase in GnRH-R. These studies were designed to examine the time course of recovery of GnRH-R after pup removal and to determine the roles that GnRH and PRL may play in modulating GnRH-R during lactation and after pup removal. All studies were performed on day 10 postpartum using ovariectomized rats suckling eight or zero pups. GnRH-R had more than doubled by 8 h after pup removal and had increased 4-5 times 16 h after pup removal to reach levels observed in nonsuckled controls. The increase in GnRH-R after pup removal resulted in a significant increase in pituitary responsiveness to GnRH. Maintenance of hyperprolactinemia after pup removal, by injecting ovine PRL, reduced the increase in GnRH-R by about 50%. Simultaneous administration of GnRH with ovine PRL restored GnRH-R to control levels. Administration of a potent antiserum to GnRH at the time of pup removal completely blocked the up-regulation of GnRH-R 24 h later. In the presence of the suckling stimulus and hyperprolactinemia, administration of pharmacological doses of GnRH caused a complete restoration of GnRH-R to levels observed in nonsuckled controls. Inhibition of suckling-induced PRL secretion with CB-154 caused a 2-fold increase in GnRH-R, and this effect could be completely reversed by simultaneous treatment with ovine PRL. These studies show that the suppression of pituitary GnRH-R during lactation appears to be due primarily to inhibition of GnRH secretion. After pup removal, recovery of GnRH-R occurs very rapidly, with recovery (4- to 5-fold increase) being completed by 16 h. Endogenous GnRH secretion is absolutely necessary for the up-regulation of GnRH-R to occur. The decrease in PRL levels after pup removal contributes to this process, most likely by causing an increase in GnRH secretion.     

Effects of age and long term ovariectomy on prolactin secretion, as assessed by the reverse hemolytic plaque assay

PRL secretion from pituitary lactotrophs was assessed using the reverse hemolytic plaque assay in young (2- to 3-month-old), middle-aged (10- to 12-month-old), and middle-aged long term ovariectomized (LT-OVX) rats to investigate whether 1) a change in the percentage of pituitary cells secreting PRL is detectable in middle-aged animals, 2) the amount of PRL secreted per cell changes with age, 3) aging involves a change in responsiveness to TRH and/or dopamine (DA), and 4) LT-OVX can prevent any of these changes. Young and middle-aged rats were OVX for 1 week. LT-OVX rats were OVX at 2-3 months of age and used when they were 10-12 months old. All animals were implanted with Silastic capsules containing estradiol (E2) in sesame oil and killed 3 or 8 days later. Anterior pituitaries were collected, and cells were dispersed and prepared for the reverse hemolytic plaque assay. Three days after E2 was implanted, the percentage of anterior pituitary cells that secrete PRL was higher in middle-aged compared to young rats. LT-OVX prevented this increase; the percentage of cells secreting PRL was significantly lower in LT-OVX than in both young and middle-aged rats. Basal secretion of PRL per cell was not different in young compared to middle-aged rats and was significantly lower in LT-OVX than in either young or middle-aged rats. TRH induced similar increases in plaque size in young and middle-aged rats, but had no effect in LT-OVX rats. DA (10(-7) M) inhibited plaque size only in LT-OVX rats; however, higher concentrations of DA were equally effective in the three experimental groups. Eight days after E2 was implanted, the percentage of cells that secrete PRL increased in LT-OVX rats, but was still significantly lower than that in middle-aged animals. After 8 days of E2 treatment, PRL release was similar in the three experimental groups under basal conditions. In LT-OVX rats TRH produced a small increase in PRL secretion (30-40%); DA suppressed PRL release in a similar manner in the three groups. These data demonstrate that middle-aged rats exhibit an increase in the percentage of cells secreting PRL without a concomitant detectable change in the amount of PRL released by single cells and/or a change in responsiveness to TRH or DA. Long term estrogen deprivation prevents this age-related change, suppresses responsiveness to TRH, and enhances sensitivity to DA.     

Prolactin stimulates the release of oxytocin in lactating rats: evidence for a physiological role via an action at the neural lobe

The present studies were designed to investigate whether prolactin (PRL) influences the secretion of oxytocin (OT) in lactating rats, and to test whether the previously reported inhibitory and stimulatory effects of dopamine-2 (D-2) agonists and antagonists, respectively, on OT release might be secondary to their respective inhibitory and stimulatory effects on the release of PRL. Intravenous administration of either rat (r) or ovine (o) PRL to lactating, nonsuckled rats increased basal plasma concentrations of OT. rGH was ineffective, but administration of oGH did produce some stimulation of OT release. Both oPRL and rPRL significantly enhanced the electrical stimulation-induced release of OT from isolated stalk-neurointermediate lobes, in vitro, without affecting the basal release of the peptide. oGH was ineffective on basal or stimulated in vitro OT release, and neither hormone altered basal or stimulation-induced release of vasopressin from these tissues. Both rPRL and oPRL reversed the inhibitory effect of the D-2 dopamine agonist bromocriptine. Immunoneutralization of circulating PRL with a highly specific antiserum abolished the increases in OT in response to either suckling or to administration of the D-2 dopamine antagonist domperidone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoactive intestinal peptide is a physiological mediator of prolactin release in the rat

To determine whether VIP functions as a physiological PRL-releasing factor, the effects of immunoneutralization of endogenous vasoactive intestinal peptide (VIP) on the PRL secretory response to suckling and ether stress were assessed. Using a porcine VIP-thyroglobulin conjugate as antigen, a peptide-specific antiserum was generated in a rabbit which bound porcine VIP with a Kd of 5.1 X 10(-11) M and a maximum binding capacity of 1830 ng/ml. In a RIA, this antiserum demonstrated immunoreactive VIP in tissue extracts of various regions of the brain and gastrointestinal tract. IR VIP in extracts of cerebral cortex and hypothalamus coeluted with synthetic porcine VIP on Bio-Gel P-30 column chromatography. Using chronically implanted right atrial catheters for blood sampling to avoid effects of stress and anesthesia, PRL blood levels in normal controls began to rise almost immediately after initiation of suckling from basal values of 3.0 +/- 0.9 ng/ml to reach a plateau of 158.1 +/- 33.5 ng/ml after 40 min. When the VIP antiserum was administered immediately before initiation of suckling, the onset of the PRL response was delayed by 40 min, but PRL levels then rose at a slower rate to reach the plateau level of normal animals approximately 80 min later. When VIP antiserum was administered to rats who had been suckling for at least 1 h, PRL levels fell from a mean basal elevated level of 152.7 +/- 16.0 ng/ml to a nadir of 50.4 +/- 9.1 ng/ml 80 min after injection and then gradually returned to basal levels. The effect of VIP antiserum was studied in rats in whom PRL secretion was increased by exposure to ether, a stimulus that acts on the release phase of PRL secretion. In rats in whom the depletion-transformation of PRL was induced by a prior brief period of suckling, subsequent exposure to ether caused a rise in serum PRL levels. The response was completely blocked in rats given VIP antiserum, whereas animals given nonimmune serum showed a significant increase in serum PRL to 38.6 +/- 17.3 ng/ml. We conclude from these studies that VIP mediates the acute PRL response to suckling and is required for maintenance of PRL levels in continuously suckling animals but is not the only factor causing PRL elevation. Complete abolition by the VIP antiserum of the PRL response to ether indicates that the effect of the anesthetic is mediated entirely by the release of VIP. These findings are consistent with the view that VIP is a physiological PRL-releasing factor in the rat.