Mechanism(s) by which the transient removal of dopamine regulation potentiates the prolactin-releasing action of thyrotropin-releasing hormone

The transient removal of dopamine (DA) selectively potentiated the prolactin (PRL) releasing action of thyrotropin-releasing hormone (TRH) but not vasoactive intestinal peptide (VIP). Consistent with these findings, the PRL-stimulating actions of agents which activated the Ca2+/protein kinase C second messenger pathway but not the adenylate cyclase system were also potentiated. In the current study we have extended these findings to determine the second messenger system mediating the potentiating action of the removal of DA. Dispersed anterior pituitary cells from E2-treated Sprague-Dawley rats were cultured on plastic coverslips. Cells tonically superfused with DA (500 nM were challenged with TRH (100 nM) 20 min after no additional treatment or a 10-min treatment with 8-Br-cyclic adenosine monophosphate (8-Br-cAMP), the Ca2+ ionophore A23187,12-O-tetradecanoyl-phorbol-13-acetate (TPA), TRH, or VIP. The potentiation of the TRH response was compared to the 4- to 5-fold potentiation observed following the removal of DA for 10 min 8-Br-cAMP at the concentration used (500 microM) was unable to alter the basal rate of PRL release, but, as VIP (500 nM), potentiated 2- to 3-fold the PRL-releasing action of TRH. A prior administration of TRH (100 nM) did not affect the responsiveness of the cells to a second challenge with TRH 20 min later. Both A23187 (20 microM) and TPA (5 or 50 nM) induced a sustained rise in the rate of PRL release. TPA-treated cells showed an increased responsiveness to TRH, whereas A23187-treated cells did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dopamine withdrawal on activation of adenylate cyclase and phospholipase C in enriched lactotrophs

In order to define the roles of cAMP and inositol phosphates (IPx) in the mechanisms governing dopamine (DA)-regulated PRL release, we have carried out studies with enriched lactotrophs from dispersed anterior pituitaries of estrogen-treated rats. Changes in the intracellular levels of cAMP as well as IPx were determined in response to the acute addition or removal of DA. The withdrawal of DA from the incubation medium in cells cultured with DA (500 nM) for 24 h resulted in a rapid and significant increase in cAMP concentration from 1.29 +/- 0.098 to 3.89 +/- 0.199 pmol/dish. On the other hand, the administration of DA for 10 min to cells cultured without it resulted in a significant decrease in intracellular cAMP (from 3.04 +/- 0.208 to 1.62 +/- 0.057 pmol/dish). Similarly, PRL released into the medium was stimulated (95.1 +/- 9%) or inhibited (46.9 +/- 6%) after DA withdrawal or addition, respectively. Cells incubated 24 h with [3H]inositol and DA (500 nM) responded to DA withdrawal with a significant increase in the concentration of [3H]IPx (5148 +/- 199 vs. 8,376 +/- 164 cpm/dish), whereas acute DA administration had no effect on the level of [3H]IPx. The administration of 8-Br-cAMP (0.5 and 2.5 mM) and forskolin (10 microM) for 10 min to cells tonically cultured in the presence of DA had no effect on the intracellular concentration of [3H]IPx, although they decreased the relative proportion of [3H]IP3 fraction from 22.1% to 11.6%, 13.6%, and 9.6%, respectively. The administration of TRH (100 nM), either under tonic DA inhibition or 10 min after the transient removal of DA inhibition, resulted in a similar stimulation of IPx formation (from 5,625 +/- 155 to 21,830 +/- 100 and 24,870 +/- 80 cpm/dish, respectively). However, the release of PRL induced by TRH was potentiated 6-fold (38.2 +/- 2.17 vs. 227 +/- 41 ng/dish) by the transient removal of DA. These findings support the conclusions that: 1) DA receptors in lactotrophs are negatively coupled to adenylate cyclase as seen during the addition or removal of DA. 2) DA receptors are negatively coupled to phospholipase C; however activation is only seen upon the removal of DA. 3) The effect of DA withdrawal on the formation of IPx is not secondary to the activation of adenylate cyclase. 4) The potentiation of the PRL response to TRH after DA withdrawal does not involve increased production of IPx.     

Dopamine withdrawal and addition of thyrotropin-releasing hormone stimulate membrane translocation of protein kinase-C and phosphorylation of an endogenous 80K substrate in enriched lactotrophs

The biochemical mechanisms underlying the direct stimulatory action of dopamine (DA) withdrawal on PRL release and on the potentiation of TRH stimulation are not known. These actions can be mimicked by pretreatment of lactotrophs with the protein kinase-C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate. Previous studies have shown that administration of TRH or withdrawal of DA stimulates polyphosphoinositide breakdown, although to different degrees. We have tested whether the acute withdrawal of DA activates PKC and determined if the prior removal of DA modifies the activation of PKC by TRH. Primary cultures of dispersed anterior pituitaries from estradiol-treated rats consisting of approximately 80% lactotrophs were maintained overnight in 500 nM DA. Activation of PKC was assayed immunochemically as translocation of PKC to a membrane fraction and by in situ phosphorylation of an acid-soluble heat-stable 80K substrate. Acute withdrawal of DA induced translocation of immunoreactive PKC to the membrane fraction (25-250%) and enhanced phosphorylation (40-100%) of an 80K protein. These effects were detected within 5-15 sec of DA withdrawal and were prolonged (10-30 min). TRH induced a rapid and transient activation of both parameters. The duration and magnitude of the action of TRH were increased by prior removal of DA. These results are consistent with a role for PKC activation in transduction of the stimulation of PRL release by the withdrawal of DA. The longer lasting activation of PKC may explain at least in part the potentiation of the PRL-releasing action of TRH by the withdrawal of DA.     

Potentiation of prolactin secretion following lactotrope escape from dopamine action. I. Dopamine withdrawal augments l-type calcium current.

Hypothalamic dopamine (DA) tonically inhibits prolactin (PRL) release from the anterior pituitary gland. Transient escapes from this DA tone elicit a pronounced potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). Previous evidence has suggested that modulation of Ca(2+) channels can be involved in this potentiation. With a lactotropic cell line (GH(4)C(1)) expressing human D(2)-DA receptors, we tested the hypothesis that a brief escape from the tonic inhibitory action of DA triggers a facilitation of Ca(2+) influx through Ca(2+) channels. We initially found that in these cells, DA effectively and reversibly inhibited PRL secretion, and reversibly enhanced an inwardly rectifying K(+) current. The effects of DA administration and withdrawal on Ca(2+) currents were examined using the patch-clamp technique in the whole-cell configuration and Ba(2+) as a divalent charge carrier through Ca(2+) channels. Macroscopic Ba(2+) currents were significantly decreased by short term (1-10 min) applications of DA (500 nM), which further declined following 24 h of constant exposure to DA. After DA removal, a biphasic facilitation of the density of Ba(2+) currents was observed. An initial 2-fold enhancement of conductance was detected between 10 and 40 min, followed by a second facilitation of the same magnitude observed 24 h after DA withdrawal. The present results directly demonstrate that dissociation of DA from D(2)-receptors expressed in GH(4)C(1) lactotrope cells causes an increase of high-voltage-activated Ca(2+) channel function, which may play an important role in the cross-talking amplification of endocrine cascades such as that involved in the TRH-induced PRL-release potentiating action of DA withdrawal.     

Bradykinin parallels thyrotropin-releasing hormone actions on prolactin release from rat anterior pituitary cells

Bradykinin (BK), a nonapeptide, originally discovered in blood, is also present in neurons and fibers of the hypothalamus. We tested the putative releasing factor properties of BK on prolactin (PRL) release from anterior pituitary cells in vitro. BK stimulated the release of PRL in a dose-dependent manner, the threshold concentration being in the range. 0.1-1.0 nM. The release of PRL induced by BK at 1 nM concentration was about 2-fold, delayed and sustained over many minutes. Higher concentrations of BK stimulated PRL release in two phases. The shape of the BK-induced PRL release was superficially similar to that induced by thyrotropin-releasing hormone (TRH). 10 nM BK and 10 nM TRH induced about a 4-fold increase in PRL release within 5 min, followed by a gradual recovery to basal secretion. These results indicate that this peptide can act directly at the anterior pituitary gland to release PRL. Phorbol ester also promoted PRL release over the range of 1-10 nM, but the time course of the release was somewhat different.     

The dynamics of arachidonic acid liberation and prolactin release: a comparison of thyrotropin-releasing hormone, angiotensin II, and neurotensin stimulation in perifused rat anterior pituitary cells

The dynamics of arachidonic acid (AA) liberation and PRL release were highly correlated in perifused rat anterior pituitary cells during stimulation by three different neuropeptides: TRH, angiotensin II (AII), and neurotensin (NT). After preincubation of these cells with 1 microCi [3H]AA, a 20-min perifusion with AII (100 nM), TRH (100 nM), or NT (1 microM) elicited a sharp initial increase in PRL release and [3H]AA efflux, which rapidly subsided (within 6 min) to less elevated levels of PRL release and AA liberation. The plateau responses were sustained throughout the remainder of the 20-min treatment period; after the cessation of neuropeptide perifusion, the responses rapidly returned to basal levels. AII and TRH elicited a greater initial stimulation of PRL release and AA liberation, whereas NT resulted in less pronounced initial responses and a greater plateau of sustained PRL release and AA liberation. Dopamine (DA; 500 nM) or calcium-depleted medium (containing 60 microM EGTA) evenly attenuated the stimulation of PRL release throughout exposure to the neuropeptides; however, the initial stimulation of AA efflux by AII and TRH was relatively resistant to inhibition by DA or calcium-depleted medium. In contrast, the stimulation of AA liberation by NT was abolished by DA or calcium-dependent medium. These results establish that the time course of AA liberation is complimentary to that of PRL release during stimulation by AII, TRH, and NT and support a possible role for AA liberation and metabolism as one of the mechanisms that participates in the regulation of PRL release. A lesser ability of NT to elicit functional and biochemical responses to intracellular calcium mobilization is postulated as an explanation for the observed differences among AII, TRH, and NT effects on PRL release and AA liberation.     

The preovulatory prolactin surge: an evaluation of the role of dopamine

This study examined the contribution of dopamine (DA) to the control of PRL secretion during the preovulatory PRL surge. Immature female rats were injected with PMSG on day 28. At selected times during the periovulatory period, rats were injected with different pharmacological agents, and jugular blood was collected at frequent intervals. Blood PRL levels in vehicle-treated rats were low on the morning of day 30, rose 15- to 20-fold to peak levels from 1400-1500 h, were maintained at a plateau from 1900-2300 h, and were reduced to basal levels on the morning of day 31. Haloperidol, a DA antagonist, induced a 20-fold rise in PRL before the surge, a 2-fold rise above peak PRL levels at 1500 h, and a 50-fold rise on the morning of day 31. In contrast, haloperidol failed to alter PRL release during the plateau phase. Apomorphine, a DA agonist, reduced PRL levels when injected during either the peak or the plateau phase. Injection of 5-hydroxytryptophan, a serotonin precursor, increased PRL levels at all times examined. Anterior pituitary PRL content was reduced to 30% and 10% of the presurge level during the peak and plateau phases, respectively, but increased on the morning of day 31. Basal PRL release by hemipituitaries incubated in vitro paralleled the anterior pituitary PRL content, with markedly less PRL secreted during the peak and plateau phases compared to the presurge period. However, the percent inhibition of PRL release by hemipituitaries incubated with 50 nM DA was similar at all times tested. These data indicate that the peak PRL surge occurs in spite of DA input to the anterior pituitary, a continued responsiveness to DA inhibition, and a diminishing pituitary PRL content. We conclude that a nondopaminergic mechanism, possibly involving a PRL-releasing factor, is responsible for the peak. The plateau phase probably results from an absence of DA input to the anterior pituitary together with a reduction in the releasable pool of PRL. The termination of the PRL surge is caused by the restoration of DA input.     

Calcitonin inhibits basal and thyrotropin-releasing hormone-induced release of prolactin from anterior pituitary cells: evidence for a selective action exerted proximal to secretagogue-induced increases in cytosolic Ca2+

Salmon calcitonin (sCT)-like peptide is present in the central nervous system and pituitary gland of the rat, and this peptide inhibits basal and TRH-stimulated PRL release from cultured rat anterior pituitary (AP) cells. The present studies were designed to examine further the inhibitory actions of sCT on basal and TRH-stimulated PRL release and investigated 1) the temporal dynamics of the responses, 2) the effects of sCT on PRL release induced by other secretogogues, and particularly those acting via elevations of cytosolic Ca2+, and 3) the selectivity of sCT action on basal and stimulated AP hormone release. The inhibition of basal PRL release by sCT (0.1-10 nM) was dose-dependent and was characterized by a rapid onset with a gradual recovery to normal rates of release after the period of sCT inhibition. The inhibitory effect of sCT on basal PRL release was reversed by treatment with either the Ca2+ ionophore A23187 or with the phorbol ester, phorbol myristate acetate (PMA). sCT infusion did not affect the basal release of GH, TSH, FSH, or LH by perifused AP cells. When administered in short pulses, TRH, at concentrations from 1-100 nM, elicited a dose-dependent increase in PRL release. When coadministered with short 10 nM TRH, sCT (1-100 nM) inhibited TRH-induced PRL release in a dose-dependent manner, with a maximal inhibition of 78% at a concentration of 10 nM, and an ED50 concentration of approximately 3 nM. During longer (30 min) pulses of TRH (100 nM), PRL release increased sharply over 4-fold within 2 min, followed within 12 min by a rapid decline to a level 1.5-2-fold higher than basal, and this level was maintained for the remainder of the stimulation period. sCT pretreatment inhibited the overall PRL response to TRH. In contrast to its inhibition of TRH-induced PRL release, sCT failed to prevent the stimulation of PRL release by either ionophore A23187, PMA, vasoactive intestinal peptide, or forskolin. In addition, sCT failed to block TRH-induced TSH release or GnRH-induced LH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Angiotensin II receptors and prolactin release in pituitary lactotrophs

Logical properties of angiotensin II receptors in the rat adenohypophysis were analyzed in cultured rat pituitary cells incubated with angiotensin II and known stimuli of pituitary hormone secretion. PRL release during incubation for 3 h with 3 nM angiotensin II was consistently increased by 68 +/- 5%, comparable with that elicited by TRH (63.1 +/- 4%). The ED50 of 0.5 nM for PRL release by angiotensin II was significantly lower than that of TRH (2.9 nM) in the same cell cultures. The antagonist analog [Sar1,Ala8]angiotensin II prevented the angiotensin-induced rise in PRL production but not that evoked by TRH, whereas dopamine and SRIF inhibited basal, angiotensin, and TRH-stimulated PRL release. Angiotensin II also caused a small increase in ACTH release but had no effect on the release of LH, TSH, and GH. Angiotensin II binding and PRL release were measured in partially purified lactotrophs prepared by elutriation, by which the initial cell suspension was separated into seven fractions. Most of the lactotrophs were present in the two fractions eluted at flow rates of 15.7 and 19.8 ml/min, as indicated by their immunoreactive PRL content. The 2.5- to 3.2-fold enrichment of lactotrophs was accompanied by a 2- to 3.5-fold increase in angiotensin II receptor concentration, with no change in binding affinity (Ka = 3.5 x 10(9) M-1). In the same fractions, angiotensin II-induced PRL release was similarly increased by 1.6- to 3.5-fold above basal, compared with values of less than 1 in the initial cell suspension and other fractions. The preferential location of angiotensin II receptors in the lactotroph-containing fractions and the close correlation between angiotensin II binding sites and stimulation of PRL release indicate the functional importance of the pituitary angiotensin II receptor sites. These findings also suggest that angiotensin II could contribute to the physiological regulation of PRL secretion.     

Rat anterior pituitary cells maintained on artificial capillaries: responses of thyrotrophs and lactotrophs to depolarization,TRH and dopamine

Rat anterior pituitary cells have been maintained over an 18-day period in a perfusion system designed around artificial capillaries. Using novel methodology the cells have been visualized by light microscopy and appear as aggregates, closely attached to and sometimes stretching around the capillaries. Their morphology is consistent with previous histology at the level of light microscopy. The techniques described are compatible with immunohistochemistry and electron microscopy. The functional integrity of thyrotrophs and lactotrophs maintained in the system has been examined by measuring the dynamics of TSH and PRL secretion in response to depolarization, TRH and dopamine (DA). TSH and PRL were significantly and reproducibly released by TRH over a 7-day period. On each day the release was dose-dependent with a threshold of at least 28 pg. Qualitatively the responses were rapid in onset (within minutes) for both hormones. Similar responses were measured in response to high K⁺ depolarization. Basal secretion of TSH and PRL was rapidly and significantly inhibited by DA in a dose-dependent manner (ED₅₀ 20 ± 25 nM for TSH and 70 ± 40 nM for PRL). Inhibition was dependent on the continued presence of DA and could be mimicked by bromocriptine and stereospecifically prevented by the active but not the inactive isomer of the DA receptor antagonist butaclamol. Simultaneous administration of 10^?6 M DA with 10^?8 M TRH prevented the release of TSH and PRL. The effect of DA was transient, subsequent TRH responses being qualitatively and quantitatively normal. The method described has proven sensitivity and precision of response' to established secretogogues in a dynamic setting and is compatible with morphological analysis. It enjoys the advantages of an extended period of cell culture and allows repeated experimentation.     

Role of calcium and sodium ions in the inhibitory control of baseline and stimulated prolactin release

The mechanism of dopamine (DA) inhibition of pituitary PRL release is still unclear. To study it, we utilized enzymatically dispersed anterior pituitary cells obtained from adult female Sprague-Dawley rats. The cells were incubated in media with or without Na+ and in the presence or the absence of various drugs for 30 min for evaluating the secretion of PRL under baseline and experimental conditions. In some experiments, 45Ca2+ (1 microCi/ml) was added after 30 min of incubation and the latter prolonged for an additional minute to determine Ca2+ uptake. DA inhibited baseline PRL release and 45Ca2+ uptake in a dose-dependent manner only in the presence of Na+ and was totally inactive in its absence. The inhibitory effects of Nifedipine and Nicardipine, two Ca2+ channel antagonists, on PRL release were also found to be Na+ dependent. BAY K 8644, a Ca2+ channel agonist, stimulated PRL release and Ca2+ uptake in a dose-dependent manner, and these effects were enhanced by Na+-free media. DA antagonized the stimulatory actions of BAY K 8644 on PRL release in a similar dose-dependent manner both in the presence and the absence of Na+. However, on stimulated 45Ca2+ uptake DA was less effective in the absence of Na+. The stimulatory action of TRH on PRL release was enhanced by the absence of Na+. DA antagonized the effect of TRH in a dose-dependent manner both in the presence and in the absence of Na+ but appeared more effective in the absence of the ion. The PRL-releasing effects of phorbol ester and of the Ca2+ ionophore A23187 were antagonized by DA in a Na+- independent manner. These results suggest the existence of two mechanisms of DA inhibitory action: one exerted on baseline PRL release which is Na+ dependent, receptor linked, and probably implicates potential operated Ca2+ channels; the other is exerted on stimulated PRL release, is Na+ independent, and appears to be a postreceptorial intracellular event probably involving protein kinase C and/or cytosolic Ca2+ levels.     

Selectivity of melatonin pituitary inhibition for luteinizing hormone-releasing hormone

The pineal indole melatonin suppresses the neonatal rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH) responses to LH-releasing hormone (LHRH), as shown in previous studies from this laboratory. We show in this study that the melatonin inhibition is a selective effect and is not due to general inhibition of pituitary function. The effects of the indole on the responses to thyrotropin-releasing hormone (TRH) and somatostatin (SRIF) and on basal pituitary hormone secretion were examined with cells in culture. Neonatal rat anterior pituitary cells dissociated with collagenase and hyaluronidase were cultured overnight and distributed to 35-mm dishes at the time of use. For examination of melatonin effects on the response to releasing hormones, the cells were incubated for 3 h in control medium or medium containing LHRH (10-9-10-6 M), TRH (10-10-10-6 M), or SRIF (10-9-10-6 M), either alone or in the presence of melatonin (10-8 or 10-6 M). For examination of basal hormone secretion, the cells were incubated for 1.5, 3, 6, 15, or 24 h in either medium alone or medium containing melatonin (10-6 M). Medium and cell lysate concentrations of LH, FSH, thyroid-stimulating hormone (TSh), prolactin (PRL) and growth hormone (GH) were determined by double antibody RIA. As previously, melatonin (10-8 M) significantly suppressed LH and FSH release by all concentrations of LHRH. This concentration of the indole produced maximal suppression of both LH and FSH responses to LHRH. By contrast, melatonin at a 100-fold greater concentration (10-6 M) had no effect on TRH stimulation of TSH or PRL release or on SRIF inhibition of GH release. Similarly, melatonin had no effect on basal release of TSH, PRL, or GH at the times examined. These findings show that melatonin inhibition of the gonadotroph response to LHRH is a selective effect.     

Gender-biased activity of the novel prolactin releasing peptides: comparison with thyrotropin releasing hormone reveals only pharmacologic effects

The prolactin- (PRL) releasing activities of the newly described PRL-releasing peptides (PrRPs) were compared to that of thyrotropin-releasing hormone (TRH) in dispersed, rat anterior pituitary cell cultures. A dose-related stimulation of PRL release by TRH was observed in cells harvested from both intact male and random cycle female pituitary donors. The minimum effective dose of TRH ranged from 1 to 10 nM. Neither PrRP-20 nor PrRP-31 significantly altered PRL secretion in cells from male donors even at doses as high as 1 microM. In cells harvested from females, only the highest doses of PrRP-20 and PrRP-31 tested (0.1 and 1.0 microM) significantly stimulated PRL secretion. The PRL-releasing action of TRH was observed already at 15 min of incubation, whereas those of PrRP-20 and PrRP-31 appeared only after 1 and 2 h of incubation, and the magnitude of PRL release in the presence of 1 microM PrRPs was significantly less than that of a similar dose of TRH. These data do not suggest a physiologically relevant role for the PrRPs in the neuroendocrine regulation of PRL secretion in intact male and nonlactating, random-cycle female rats.     

Evidence for paracrine interaction between gonadotrophs and lactotrophs in pituitary cell aggregates

Pituitary cell aggregates prepared from 14-day-old male or female rats and maintained for 4-5 days in culture were superfused with LHRH during periods of 20 or 90 min. LHRH provoked a rapid and sustained rise of PRL release at concentrations similar to those stimulating LH release (10(-11)-10(-8) M). Dopamine, at a concentration inhibiting PRL release for 90%, weakened but did not prevent this stimulation. LHRH also stimulated PRL release in aggregates prepared from adult male rat pituitary cells, but the effect was weaker and seen only after a more prolonged period in culture. There was no PRL response to LHRH in aggregates of lactotroph-enriched populations, obtained by gradient sedimentation at unit gravity, in which only few and small gonadotrophs are present. When a lactotroph-enriched/gonadotroph-poor population was coaggregated with a highly enriched population of large gonadotrophs, LHRH very effectively stimulated PRL release, the extent of stimulation being dependent on the proportional number of gonadotrophs in the coculture. Superfusion of lactotroph-enriched/gonadotroph-poor aggregates with medium in which the gonadotroph-enriched aggregates had previously been incubated for 3 h with 1 nM LHRH (gonadotroph-conditioned medium) also provoked a clear-cut rise in PRL release. This effect was not due to LH, FSH, or the small amounts of PRL present in the gonadotroph-conditioned medium. The LHRH antagonist [D-Phe2-D-Ala6]LHRH was capable of blocking the PRL response to LHRH but not that to the gonadotroph-conditioned medium. In the lactotroph-gonadotroph coaggregates TRH stimulated PRL release but had no effect on LH release. TRH was also ineffective in releasing LH or FSH in populations containing both gonadotrophs and thyrotrophs. The present data suggest that gonadotrophs can activate the secretory activity of the lacotrophs through the release of a paracrine humoral factor.     

Effects of repeated stimuli on prolactin release in vitro from normal and adenomatous rat lactotrophs

It is now well known that dopamine (DA) plays a major role in the inhibitory control of prolactin (PRL); however, the mechanisms that are physiologically involved in the stimulation of PRL release are still under investigation. Indeed, although suppression of DA inhibitory tonus, administration of thyrotropin-releasing hormone (TRH) or vasoactive intestinal peptide (VIP) are all known PRL releasers, it is not clear whether they interact during physiological periods of PRL release such as suckling and estrus. No clear indications exist, furthermore, on whether they all act upon a same pituitary pool that may become depleted following repeated exposure to stimuli. Refractoriness to a single or a repeated stimulus has been reported to occur in prolactinoma-bearing or normal humans, respectively, the mechanism of which is still matter for discussion. Our present studies performed by perifusing normal or adenomatous rat lactotrophs attached to Cytodex I microcarrier beads was undertaken to try and answer some of these questions. The experimental period consisted in perifusing the cells for 1 h with Dulbecco's modified Eagle's medium (DMEM) containing DA 10(-5) M, then for 2 h with either DMEM, DMEM and TRH 10(-8) M, DMEM and VIP 10(-7) M, then again with DA in DMEM for 1 h, and finally with DMEM, DMEM and TRH, or DMEM and VIP. Three experiments of various combinations were performed. Lower PRL levels were observed under DA, while two periods (first and second) of PRL release followed the suppression of DA infusion with or without the addition of either one of the two peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Normal and adenomatous human pituitaries secrete thyrotropin-releasing hormone in vitro: modulation by dopamine, haloperidol, and somatostatin

TRH is present in human normal pituitaries and in pituitary adenomas. In this study we demonstrated that the same tissues can release TRH in vitro. Fragments from seven normal pituitaries (10-15 mg/syringe) and dispersed cells from eight prolactinomas, four GH-secreting and two nonsecreting adenomas (1-3 x 10(6) cells/syringe) were perifused using a Krebs-Ringer culture medium. After 1 h of equilibration the perifusion medium was collected every 2 min (1 mL/fraction) for 3 h. TRH, PRL, and GH were measured by RIA under basal conditions and in the presence of 10(-10) to 10(-6) mol/L dopamine (DA), alone or concomitant with haloperidol, or in the presence of 10(-10) or 10(-6) mol/L somatostatin. Both normal pituitary fragments and pituitary adenomatous cells (from all types of adenomas studied) spontaneously released TRH in vitro. TRH was detected in the perifusion medium either immediately after the end of the equilibration period or 30-60 min later. The molecular identity of TRH was assessed by high pressure liquid chromatography. There was no difference in the profile and the rate of TRH secretion between normal and tumoral tissues, and no correlation was found between the level of TRH release and that of PRL or GH secretion. DA stimulated TRH release from normal pituitaries and from PRL- and GH-secreting adenomas at doses as low as 10(-10) mol/L. A concomitant decrease in PRL and GH release was observed from adenomatous cells and in one case of normal tissue. Haloperidol (10(-7) mol/L) antagonized the effect of 10(-8) mol/L DA on both TRH and PRL secretion in normal pituitary and in prolactinomas. DA had no effect on TRH release from two nonsecreting tumors. The amounts of TRH released during 1 h of perifusion were 60-1640 pg/2 mg wet wt tissue in normal pituitaries and 54-2174 pg/10(6) cells in adenomas; these values were very high compared to those precedently reported within the tissues. These results indicate that pituitary cells can release TRH in vitro and suggest that TRH might be synthesized in situ. We suggest that TRH could act on pituitary hormone secretion and/or cell proliferation via a paracrine and/or an autocrine mechanism.     

Epidermal growth factor decreases the concentration of thyrotropin-releasing hormone (TRH) receptors and TRH responses in pituitary GH4C1 cells.

Treatment of pituitary GH4C1 cells with epidermal growth factor (EGF) caused up to a 60% reduction in the amount of [3H]MeTRH bound to specific TRH receptors. The effects of EGF were first detectable after a 2-h incubation and maximal by 24-72 h. EGF elicited a half-maximal response at 0.03 nM. Equilibrium binding analysis was performed on intact cells that had been incubated with or without 10 nM EGF for 96 h. EGF decreased the apparent number of TRH receptors (maximum binding = 0.36 vs. 0.58 pmol/mg protein for EGF-treated and control cells, respectively) without altering the apparent affinity (dissociation constant = 6.4 vs. 7.4 nM). The effects of EGF on TRH receptors were reversible. When EGF was removed from the medium, TRH receptors returned to control levels within 48 h. To assess whether the reduction of TRH receptors was functionally important, the ability of TRH to stimulate phospholipid turnover was measured in cells with a normal complement of TRH receptors and in cells that had been treated with EGF for 72 h to reduce TRH receptor density. EGF significantly blunted the ability of TRH to stimulate release of inositol phosphates from metabolically labeled cells. TRH increased inositol monophosphate accumulation 6.3-fold in control cultures and 2.0-fold in EGF-treated cells. These data show that EGF regulates the concentration of TRH receptors on pituitary GH4C1 cells and the responsiveness of the cells to TRH.     

Evidence for dopamine receptor-mediated inhibition of prolactin cell function in the female Syrian hamster

Since the exact nature of the hypothalamic regulation of prolactin (PRL) cell activity in the photosensitive female Syrian hamster is unknown, the present investigation was designed to determine whether dopamine (DA), a physiological PRL-inhibitory hormone in the rat, inhibits the synthesis and release of female hamster PRL in vitro via a DA receptor-mediated mechanism. Anterior pituitary glands from long photoperiod-exposed adult female Syrian hamsters were incubated in the presence of increasing concentrations of DA (5 nM, 500nM and 50 microM) in Kreb's Ringer Bicarbonate medium for 2 hours following a preincubation period of 1 hour in medium not containing DA. While PRL synthesis, as measured by the incorporation of 3H-leucine into PRL, was unaffected by DA, the release of immunoreactive (RIA)-PRL into the medium was inhibited by 55% and 53% by 500 nM and 50 microM DA, respectively; however, these same concentrations of DA inhibited the release of 3H-PRL into the medium by only 25% and 23%, respectively. The DA receptor blocker, pimozide (PIM) was effective in blocking the PRL-inhibitory effects of DA (500 nM) on both RIA- and 3H-PRL release in vitro. These are the first data suggesting that DA directly inhibits PRL release from female hamster anterior pituitary glands via a DA receptor-mediated mechanism.     

The effects of transient dopamine antagonism on thyrotropin-releasing hormone-induced prolactin release in pseudopregnant rats

The effectiveness of TRH in releasing PRL after transient dopamine (DA) blockade was investigated in female rats between days 3 and 11 of pseudopregnancy (PSP). At 0930 h on the morning of the experiment, each animal was injected with the DA antagonist domperidone (0.01 mg/rat, iv) or vehicle (acetic acid in saline); 5 min later, the DA agonist 2-bromo-alpha-ergocryptine maleate (CB-154; 0.5 mg/rat, iv) was administered. Sixty minutes later, TRH (1.0 micrograms/rat, iv) was administered. Blood samples were withdrawn via indwelling catheters before, 5, 20, 40, and 70 min after domperidone or vehicle administration, and 5 and 10 min after TRH administration. On day 3 of PSP, TRH-induced PRL release was significantly enhanced by the domperidone-CB154 treatment compared to that in vehicle-treated control rats. By day 9 of PSP, the effectiveness of TRH in stimulating PRL release after domperidone treatment was decreased by 50% compared to that on day 3 of PSP. This reduction in PRL response to TRH was not due to decreased progesterone levels, as no difference was observed in plasma progesterone between days 3 and 9. Rats that were given domperidone on day 11 of PSP did not exhibit a significant increase in sensitivity to TRH; however, the effectiveness of TRH was enhanced by domperidone on day 11 of PSP in animals that were hysterectomized on day 2 of PSP. Since DA receptor blockage increased the sensitivity to a putative PRL-releasing factor (TRH) and this mechanism was eliminated around the time that the PRL surges of PSP disappear, we suggest that this pituitary mechanism is a critical component of the PRL release mechanism during the surges of PSP. Further, the observed loss of the mechanism between days 9 and 11 of PSP may be due to the direct influence at the anterior pituitary of a uterine PRL inhibitory factor which has been recently described.     

The influence of oestrogen administration in vivo on in vitro prolactin release. Interaction between dopamine and thyrotrophin releasing hormone.

The influence of oestrogen administered to the ovariectomized rat on the interaction between dopamine (DA) and thyrotrophin releasing hormone (TRH) on the release of radioimmunoassayable (RIA) and [3H]leucine incorporated into prolactin ([3H]PRL) was examined in vitro. Dopamine had a more marked suppressing effect on newly synthetized PRL (80%), as determined [3H]PRL, than on total PRL (50%), as determined by RIA-PRL. The administration of 5 micrograms of oestradiolbenzoate (OeB) for 7 days resulted in blocking the suppressing effect of DA when RIA-PRL was measured but not when [3H]PRL was measured. The administration of 5 micrograms of OeB enabled TRH to partially override the suppressing effect of DA and the degree of response was more marked when RIA-PRL was measured than when [3H]PRL was measured. The administration of 50 micrograms of OeB for 3 days enabled TRH to override the DA blockade of prolactin release to levels comparable to that of the control when RIA-PRL was measured but had little to no effect on [3H]PRL. The results are discussed in relation to the two storage pools of PRL in the pituitary and the data suggest that DA acts predominantly to suppress the newly synthetized, rapidly releasable pool. Oestrogen acts to block DA action on the older more stable PRL pool. The ability of TRH to override the DA blockade of PRL release depends upon the presence of oestrogen; here TRH acts predominantly on the older more stable pool of PRL. Oestrogen's action on disrupting the DA suppression of PRL release appears to be related to the time of day the hormone is administered subsequent to when the pituitary is exposed to DA in vitro.