Somatostatin partially impedes the stimulatory effects of thyrotrophin-releasing hormone and dibutyryl cyclic AMP on prolactin release: prolactin release through multiple routes.

Patterns of prolactin release were examined using stimulating and inhibiting agents. Primary cultured pituitary cells primed with oestrogens were used for perifusion experiments. TRH (100 nmol/l) increased the peak prolactin concentration to 360% of the basal concentration, while TRH, under inhibition by 1 nmol somatostatin/l, raised the peak prolactin concentration to 185% of the basal levels. When the somatostatin concentration was increased to 10, 100 and 1000 nmol/l, TRH still stimulated prolactin release to 128%, 121% and 140% respectively, indicating that concentrations of somatostatin of 10 nmol/l or higher did not further suppress the stimulatory effect of TRH. TRH (1 mumol/l) stimulated prolactin release under the influence of 0 (control), 1, 10, 100 and 1000 nmol dopamine/l (plus 0.1 mmol ascorbic acid/l) to 394, 394, 241, 73 and 68% of the basal concentration respectively, showing that the dopamine concentrations and peak prolactin concentrations induced by TRH have an inverse linear relationship in the range 1-100 nmol dopamine/l. The stimulatory effect of dibutyryl cyclic AMP (dbcAMP) on prolactin release was also tested. The relationship between dbcAMP and somatostatin was similar to that between TRH and somatostatin. When adenohypophyses of male rats were used for perifusion experiments, somatostatin (100 nmol/l) did not inhibit basal prolactin release from the fresh male pituitary in contrast with the primary cultured pituitary cells, but dopamine (1 mumol/l) effectively inhibited prolactin release. In conclusion, (1) oestrogen converts the somatostatin-insensitive route into a somatostatin-sensitive route for basal prolactin release, (2) TRH-induced prolactin release passes through both somatostatin-sensitive and -insensitive routes, (3) dopamine blocks both somatostatin-sensitive and -insensitive routes and (4) cAMP activates both somatostatin-sensitive and -insensitive routes.     

In vitro study of frog (Rana ridibunda Pallas) neurointermediate lobe secretion by use of a simplified perifusion system. IV. Interaction between dopamine and thyrotropin-releasing hormone on alpha-melanocyte-stimulating hormone secretion

The interaction between dopamine and TRH on alpha-melanocyte-stimulating hormone (MSH) release from the intermediate lobe of amphibian pituitary has been studied in vitro using the perifusion model. Dopamine (10(-10) to 10(-6) M) was responsible for a dose-related inhibition of alpha-MSH secretion. The inhibitory effect of dopamine (10(-8) and 3.16 X 10(-8) M) was completely abolished in the presence of haloperidol (10(-5) and 10(-6) M, respectively). It has been previously established that, in amphibians, TRH stimulated alpha-MSH release in vitro and that the action of TRH was not mediated via an inhibition of the release of endogenous dopamine (M. C. Tonon, P. Leroux, M. E. Stoeckel, S. Jégou, G. Pelletier, and H. Vaudry, 1986, Endocrinology 112, 133-141). In the present study we demonstrate that TRH (10(-7) M) reverses the inhibitory effect of dopamine (for concentrations ranging from 3.16 X 10(-8) to 10(-6) M) on alpha-MSH secretion and that the effects of TRH and dopamine are additive. Thus, these results indicate that the intracellular events associated with TRH-induced stimulation and dopamine-induced inhibition of alpha-MSH release are not linked together.     

Prolactin secretion from human prolactinomas perifused in vitro: effect of TRH, prostaglandin E1, theophylline, dopamine and dopamine receptor blockers

To clarify the functional characteristics of prolactin (Prl)-producing adenoma cells, the effect of TRH, prostaglandin E1 (PGE1), theophylline, dopamine and dopaminergic antagonists on Prl secretion was examined in vitro in perifused pituitary adenoma tissues obtained at surgery from 8 patients with prolactinoma. Perifusion with TRH at a concentration of 10(-6) to 10(-5) M resulted in a significant increase in effluent Prl levels in 3 of the 8 adenoma tissues. In the remaining 5 adenomas, TRH produced no effect on Prl release in vitro. On the other hand, PGE1 (10(-5) M) stimulated Prl secretion in 2 of the 4 adenomas examined. Addition of theophylline (5.5 mM) caused a marked increase of effluent Prl levels in all 8 prolactinomas regardless of the reactivity to TRH or PGE1. Dopamine (5 X 10(-7) M) suppressed Prl secretion from adenoma tissue in 5 of 7 patients tested but had no effect in the remaining two adenomas. When perifused simultaneously with dopamine, sulpiride (D2-selective dopamine receptor blocker, 5 X 10(-7) M) blocked the inhibitory effect of dopamine on Prl release in 3 of the 4 dopamine-sensitive prolactinomas. In one adenoma responsive to dopamine but resistant to sulpiride, YM-09151-2 (relatively specific D1-dopamine receptor blocker, 5 X 10(-7) M) antagonized the dopaminergic inhibition of Prl release. When perifused alone, neither sulpiride nor YM-09151-2 affected Prl release from any of the adenoma tissues tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of the adenosine 3',5'-monophosphate and the Ca2+ messenger systems together reverse dopaminergic inhibition of prolactin release

Dopaminergic inhibition of PRL release stimulated by agents that affect cytosolic Ca2+ concentrations, C-kinase activity, and cAMP levels was studied in perifused rat anterior pituitary cells cultured on cytodex beads. We used A23187 (20 microM) to increase intracellular Ca2+, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 50 nM) to stimulate C-kinase, forskolin (10 microM) to increase intracellular cAMP, and 8-bromo-cAMP to mimic cAMP. Dopamine (10 microM) inhibited PRL release to 20-60% of the basal release within 10 min. After 30 min of preincubation with dopamine, the absolute amount of release stimulated by 100 nM TRH was strongly inhibited, although the pattern of release, a quick burst followed by sustained release at a lower rate, was the same in the presence or absence of dopamine. A23187 (20 microM) caused a rapid burst of PRL release that subsided within 10 min, and TPA (50 nM) caused a sustained release that began within 4 min and continued for at least 30 min. TPA and A23187 combined caused a rapid burst of release followed by a sustained phase of release similar to that caused by TRH. Preincubation with dopamine inhibited the absolute amount of PRL release caused by A23187 alone, TPA alone, or the two combined, although, as with TRH, the pattern of release remained the same. Forskolin (1 or 10 microM) or 8-bromo-cAMP (3 mM) induced a 1.5- to 2-fold increase in PRL release, and this was completely prevented by dopamine. Preincubation with both dopamine and 8-bromo-cAMP or forskolin restored the amount of release stimulated by TPA alone or TPA and A23187 in the presence of dopamine to the level of release stimulated by these agents in the absence of dopamine. Therefore, activating either the cAMP messenger system or the Ca2+ system alone will not abolish dopaminergic inhibition, but activating the two together will. These results suggest that dopamine blocks release by inhibiting both adenylate cyclase and a step in the Ca2+ messenger system.     

Involvement of calcium ions in dopamine inhibition of prolactin secretion from sheep pituitary cells

The involvement of calcium in the regulation of prolactin secretion and a possible inhibitory mechanism of action for dopamine have been investigated. Basal prolactin secretion from cultured ovine pituitary cells was dependent on the concentration of calcium ions (Ca²⁺) in the medium and was inhibited by the presence of verapamil (10 μM). The divalent cation ionophore A23187 (1 μM) caused a rapid stimulation of prolactin release from the cells. The effect was essentially complete within 10 min and subsequently secretion of prolactin occurred at close to the basal rate. A23187 had no effect on cell cyclic AMP levels. Dopamine (0.1 μM) but not verapamil (10 μM) inhibited the A23187 (10 μM) induced release of prolactin. Inhibition of basal and A23187 (1 μM) stimulated prolactin secretion occurred over a similar range of dopamine concentrations. The dopamine receptor antagonist haloperidol (1 μM) reversed the inhibitory effect of dopamine (0.1 μM) on A23187-stimulated prolactin release. These results provide evidence to support the concept that control of Ca²⁺ handling by lactotrophs may be of fundamental importance in the regulation of prolactin secretion.     

Effect of vasoactive intestinal polypeptide on growth hormone secretion in perifused acromegalic pituitary adenoma tissues

The effects of vasoactive intestinal polypeptide (VIP), dopamine, and somatostatin (SRIF) on GH secretion were examined in vitro in perifused pituitary adenoma tissues obtained at surgery from seven patients with acromegaly. The perifusion of VIP at 5 x 10(-8) M resulted in a significant increase in effluent GH levels in five of the seven adenomas. A dose-related GH response was observed from 5 x 10(-9) to 5 x 10(-7) M VIP in two adenomas examined. SRIF at 5 x 10(-8) to 10(-7) M suppressed not only baseline secretion of GH but also inhibited GH rises elicited by VIP in six of the seven adenomas. Dopamine at 5 x 10(-7) to 5 x 10(-6) M decreased the baseline secretion of GH in six of the seven adenomas. In four of the six adenomas responsive to dopamine, dopamine suppressed VIP-induced GH release when perifused simultaneously. In the remaining two dopamine-sensitive adenomas in which VIP alone failed to affect GH release, the inhibition by dopamine of GH release was blocked by VIP perifused concomitantly with dopamine. Synthetic TRH or theophylline perifused at the end of the experiment stimulated GH release in all of the adenomas, indicating the viability of tumor cells throughout the study. These results suggest that VIP stimulates GH release by its direct action on pituitary adenoma cells of acromegalic patients and that VIP, SRIF, and dopamine interact at the pituitary level in modulating GH secretion from these adenomas.     

Mechanisms involved in the effects of TRH on GHRH-stimulated growth hormone release from ovine and bovine pituitary cells

Incubation of cultured ovine pituitary cells with growth hormone-releasing hormone (GHRH) (10(-12)-10(-7) M) stimulated growth hormone secretion up to 3-fold. At a maximal stimulatory concentration of GHRH (10(-10) M), thyrotropin-releasing hormone (TRH) (10(-7) M) caused an inhibition of growth hormone release to approx. 50% of the response obtained with GHRH alone (during a 15 min incubation period). TRH also caused a small inhibition of the GHRH-stimulated cellular cyclic AMP level but this effect was only significant at a relatively high concentration of GHRH (10(-9) M). Incubation of cultured bovine pituitary cells with GHRH (10(-11)-10(-8) M) plus TRH (10(-7) M) caused a significant stimulation of growth hormone release by up to 40%, compared with the response obtained with GHRH alone (at all concentrations of GHRH). TRH (10(-7) M) had no effect on GHRH (10(-8) M)-stimulated cellular cyclic AMP levels in a partially purified bovine pituitary cell preparation. The effects of varying extracellular [Ca2+] (0.1-10 mM) on intracellular [Ca2+] and on the responsiveness to releasing hormones were also determined using ovine pituitary cells. GHRH (10(-10) M)-stimulated growth hormone release was inhibited when cells were incubated at both high (10 mM) and low (0.1 mM) [Ca2+] (compared with 1 mM or 3 mM Ca2+) with or without TRH (10(-7) M). At 1 mM Ca2+, TRH produced a synergistic effect with GHRH to stimulate growth hormone release. However, at 3 mM Ca2+ TRH inhibited GHRH-stimulated growth hormone release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine and somatostatin inhibit forskolin-stimulated prolactin and growth hormone secretion but not stimulated cyclic AMP levels in sheep anterior pituitary cell cultures

Forskolin, an activator of adenylate cyclase, has been used to investigate the effects of raising pituitary cell cyclic AMP concentrations on prolactin and growth hormone secretion and to examine the role of cyclic AMP in the inhibitory actions of dopamine and somatostatin. Incubation of cultured ovine pituitary cells with forskolin (0.1-10 microM; 30 min) produced a modest dose-related increase in prolactin release (120-140% of basal) but a much greater stimulation of growth hormone secretion (170-420% of basal). Cellular cyclic AMP concentrations were only increased in the presence of 1 and 10 microM forskolin (2-5.5 times basal). A study of the time course for forskolin (10 microM) action showed that stimulation of prolactin (1.5-fold) and growth hormone (4.7-fold) secretion occurred over 15 min; subsequently (15-60 min) the rate of prolactin secretion from forskolin-treated cells was equivalent to that measured in controls, while growth hormone release remained elevated. Cellular cyclic AMP concentrations were also rapidly stimulated by forskolin (10 microM); they reached a maximum (12 times control) within 15 min, and then declined (15-60 min) but remained elevated relative to those in untreated cells (4.9 times control at 60 min). Dopamine (0.1 microM) inhibited basal secretion of both prolactin and growth hormone. In the presence of forskolin (0.1-10 microM), dopamine (0.1 microM) inhibited prolactin secretion to below the basal level and considerably attenuated the stimulation of growth hormone secretion. Similarly, somatostatin suppressed both basal and forskolin-induced prolactin and growth hormone secretion. However, neither dopamine nor somatostatin significantly decreased the stimulatory effect of forskolin on cellular cyclic AMP accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of release of prolactin from fowl anterior pituitary glands incubated in vitro: effects of calcium and cyclic adenosine monophosphate

Fowl anterior pituitary glands were bisected and each half was pretreated in either Medium 199 or medium containing EGTA to deplete endogenous calcium (Ca2+) stores, after which they were incubated in Medium 199, or Ca2+-free medium, containing prolactin release-stimulating agents and verapamil, a Ca2+ channel blocker. High K+ concentrations, hypothalamic extract, synthetic thyrotrophin-releasing hormone (TRH) and dibutyryl cyclic AMP (dbcAMP) all stimulated release of prolactin from control (non EGTA-treated) hemianterior pituitary glands. The effects of TRH and dbcAMP were not additive, but the response to submaximal concentrations of TRH was augmented by theophylline, a phosphodiesterase inhibitor. Reduction of Ca2+ availability with EGTA or verapamil reduced basal release of prolactin, prevented the prolactin-stimulating effects of high K+ concentrations and TRH, and markedly attenuated responses to hypothalamic extract and dbcAMP, EGTA being more effective than verapamil. Increasing the Ca2+ concentration of the medium did not augment basal or stimulated release of prolactin. These results suggest that both Ca2+ and cyclic AMP may act as intracellular mediators in the release of prolactin. Both basal and stimulated release of prolactin depend upon the presence of Ca2+. Although influx from the medium may be the major source of Ca2+, endogenous stores of Ca2+, perhaps mobilized by dbcAMP, may be able to maintain some release of prolactin. The prolactin-stimulating effects of TRH may be mediated by cyclic AMP.     

Differential inhibition of dopamine and bromocriptine on induced prolactin release: multiple sites for the inhibition of dopamine.

Effects of dopamine and bromocriptine on TRH- or dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP)-induced prolactin release from primary cultured rat pituitary cells were studied using a perifusion system. TRH (100 nmol/l) stimulated prolactin release from basal concentrations of 33.8 +/- 0.5 to 151.2 +/- 28.0 ng/ml (net increase) or 447% increase. Dopamine inhibited the basal release of prolactin throughout the experiment, but TRH (100 nmol/l) was still able to stimulate prolactin release under the influence of dopamine. The increment in prolactin release was inversely proportional to the dopamine concentration. When TRH (100 nmol/l) was introduced during a perifusion period with bromocriptine 1 nmol/l, the prolactin concentration was increased to 110.9% of basal levels. The stimulatory effect of TRH under the influence of bromocriptine (1 nmol/l) was significantly lower than that without bromocriptine (control), although the higher concentrations of bromocriptine (10 and 100 nmol/l) did not further reduce the peak concentration of TRH-induced prolactin release. During a perifusion period with a low concentration of dopamine (1 nmol/l plus 0.1 mmol/l ascorbic acid), introduction of dbcAMP (3 mmol/l) stimulated prolactin release to 48% of basal concentration. A higher concentration of dopamine further reduced the stimulatory effect of prolactin release. Bromocriptine impeded the stimulatory effect of dbcAMP (3 mmol/l) on prolactin release in a similar manner as dopamine. Since a higher concentration of bromocriptine (10 and 100 nmol/l) did not further inhibit the TRH-induced prolactin release whereas a higher concentration of dopamine did, it is concluded that dopamine acts through additional mechanism(s) other than the D2 receptor transduction system.     

Actions of dopamine on prolactin secretion and cyclic AMP metabolism in ovine pituitary cells

Prolactin secretion from cultured sheep pituitary cells was inhibited by low concentrations of dopamine (0.1 nM-0.1 microM) with a half-maximal effect at 3 nM. At a maximally effective dose (0.1 microM) dopamine significantly inhibited prolactin secretion within 5 min. with an 80% inhibition of basal secretion over 2 h. Basal prolactin secretion was stimulated by the addition of methylisobutylxanthine (MIX) (0.3-1.0 mM) and 8-bromo-cyclic AMP (2 mM), but cholera toxin (3 micrograms/ml) and prostaglandin E2 (0.1-1.0 microM), which also raised cellular cyclic AMP levels, had no effect on prolactin release. The inhibition of prolactin release by dopamine (0.1 microM) was not affected by any of these compounds. Dopamine inhibited MIX-induced cyclic AMP accumulation over a similar concentration range to the inhibition of secretion, but had no effect on the changes in cyclic AMP concentration produced by cholera toxin and prostaglandin E2. Overall the results with sheep pituitary cells suggest that lowered cyclic AMP levels do not mediate the inhibitory effects of dopamine on basal prolactin secretion, but that changes in cellular cyclic AMP levels may alter the secretion of this hormone, and dopamine may affect pituitary cell cyclic AMP concentrations in some circumstances.     

Dopaminergic inhibition of anterior pituitary adenylate cyclase activity and prolactin release: the effects of perturbing calcium on catalytic adenylate cyclase activity

The dopaminergic inhibition of anterior pituitary adenylate cyclase activity, cAMP accumulation, and prolactin release was studied in the presence of the Ca2+ channel activator, maitotoxin. In isobutylmethylxanthine (IBMX)-treated cells, maitotoxin stimulated prolactin secretion within 30 s and cAMP accumulation within 1 min. Although dopamine reduced cAMP accumulation and prolactin release, the effectiveness of the catecholamine was reduced in the presence of maitotoxin. When hemipituitary glands were exposed for 10 min to 100 ng maitotoxin/ml, their membranes showed increased adenylate cyclase activity. The hypothesis that maitotoxin stimulates adenylate cyclase activity by increasing Ca2+ availability was supported by the observation that, at concentrations up to 100 microM, Ca2+-stimulated anterior pituitary adenylate cyclase activity. Although dopamine decreased basal and maitotoxin-stimulated pituitary cAMP accumulation, via changes in adenylate cyclase activity, the decrement in cyclic nucleotide production, but not prolactin release, can be ascribed to the effect of the catecholamine on the basal activities of these parameters. These data provide additional evidence that an increased Ca2+ flux is stimulating to cAMP generation and prolactin release, whereas dopamine is inhibitory to these processes.     

Dehydroepiandrosterone (DHEA) modulates GHRH, somatostatin and angiotensin II action at the pituitary level

In view of the present controversy related to the potential beneficial effects of clinical dehydroepiandrosterone (DHEA) treatments, and considering our own previous results that reveal an influence of this steroid in pituitary hyperplasia development in vivo in rats, we decided to evaluate the role of DHEA in prolactin and GH secretion, as well as in second messengers involved, in cultured rat anterior pituitary cells. DHEA (1 x 10(-5) to 1 x 10(-7) M) did not modify basal GH or prolactin release, and a prolactin inhibitory effect was observed only for androstenediol, a metabolite of DHEA. DHEA partially prevented dopamine (1 x 10(-6) M)-induced prolactin inhibition and facilitated the prolactin-releasing effect of 10(-8) M Ang II, without modifying the resulting Ca2+(i) mobilization. Furthermore, DHEA potentiated the GH release and cAMP production induced by 1 x 10(-8) M GHRH. Finally, DHEA partially reversed the inhibitory effect of 1 x 10(-8) M somatostatin on GH, but not prolactin, release. We conclude that DHEA in vitro, directly or indirectly through conversion into metabolites, is able to modulate the hormonal response of the pituitary to hypothalamic regulators. It can enhance pituitary prolactin release and induce GH secretion. These effects could help explain some of the side effects observed in prolonged DHEA treatments in vivo and should be taken into account when considering its use in human clinical trials.     

Pituitary vasoactive intestinal peptide regulates prolactin secretion in the hypothyroid rat

In this study, we demonstrated that the cell content and basal secretion of vasoactive intestinal peptide (VIP) in primary rat pituitary cell cultures were increased in hypothyroidism. VIP release from hypothyroid pituitary cells in vitro was stimulated by thyrotropin releasing hormone (TRH 10(-8) to 10(-6) M) and growth hormone (GH)-releasing hormone (GHRH 10(-9) to 10(-8) M) but not by corticotropin-releasing hormone or luteinizing hormone-releasing hormone in concentrations up to 10(-6) M. In the presence of anti-VIP antisera, there was a significant decrease in basal prolactin secretion from cultured hypothyroid pituitary cells (p less than 0.005) indicating that VIP exerts a tonic stimulatory effect on prolactin (PRL) secretion. The increment in PRL secretion following TRH was not affected by exposure to anti-VIP indicating that PRL release after TRH is not mediated by VIP at the pituitary level. In contrast to changes in PRL, exposure to anti-VIP had no effect on basal GH secretion, indicating that the PRL changes are hormone specific. Similarly, GHRH-induced GH release was unaffected by VIP immunoneutralization.     

Evidence for a possible role for Ca++ in the 3,5,3'-triiodothyronine inhibition of thyrotropin-releasing hormone-induced secretion of thyrotropin by rat anterior pituitary in vitro

The present study was undertaken to determine whether T3 could modify anterior pituitary Ca++ metabolism under basal conditions and in the presence of TRH. Paired hemipituitaries from hypothyroid rats were preincubated with T3 (10(-7) M) for 2 h, allowed to accumulate 45Ca++ for the third hour, and repeatedly washed in static incubations or superfused for the next 2 h with isotope-free medium and finally with TRH (10(-8) M) for 10 min. T3 treatment had no effect on the basal pattern of isotope loss throughout the 2-h wash period. TRH stimulated 45Ca++ fractional efflux from a basal value of 0.76 +/- 0.10% to 1.66 +/- 0.38% min-1 (P less than 0.02; mean +/- SD; n = 9). T3 reduced TRH-stimulated efflux to 1.30 +/- 0.20% min-1, while basal values were unaltered (0.70 +/- 0.10% min-1). Similarly, T3 inhibited the TSH response to TRH from 480% to 200% of basal secretion. T3 pretreatment also inhibited the basal uptake of 45Ca++ when a La+++ displacement protocol was employed from 1751 +/- 36 to 1400 +/- 61 cpm/mg pituitary (mean +/- SEM; n = 13; P less than 0.001). Similar data for 45Ca++ efflux were obtained in experiments where tissue was superfused. rT3 did not affect basal or stimulated 45Ca++ efflux, and inhibition of stimulated secretion and 45Ca++ efflux by T3 was dependent on preincubation. The data indicate that T3 is capable of altering anterior pituitary Ca++ homeostasis. Such a mechanism could be involved in the T3-induced inhibition of TRH-stimulated TSH release which appears to require a redistribution of cellular Ca++.     

Inhibition of adrenocorticotropin secretion by somatostatin in pituitary cells in culture

AtT20/D16v is a clonal strain of mouse pituitary tumor cells which synthesizes and secretes ACTH. Somatostatin, a hypothalamic tetradecapeptide, has been shown to inhibit the release of PRL, GH, and TSH from the pituitary gland. We have characterized specific binding sites for somatostatin on AtT20/D16v cells and demonstrate that somatostatin inhibits stimulated ACTH release by these cells. Equilibrium binding studies with [125I]Tyr1]somatostatin showed the presence of a single class of noninteracting binding sites on AtT20/D16v cells. Half-maximal binding of somatostatin occurred at 1.7 X 10(-9) M, and there were 26,300 binding sites/cell. The binding of [125I]Tyr1]somatostatin was not significantly inhibited by the hypothalamic peptides TRH, LHRH, and substance P. Somatostatin had no consistent effect on basal ACTH secretion by AtT20/D16v cells, but it inhibited ACTH secretion stimulated with either 50 mM KCl or a hypothalamic extract. Half-maximal inhibition occurred with 4 X 10(-10) M somatostatin. TRH, LHRH, and substance P at concentrations of 10(-7) M were without effect. Somatostatin had no effect on either basal or stimulated hormone secretion by GH12C1 or F4C1 cells, two cell strains which lack specific somatostatin-binding sites. A critical concentration of extracellular calcium was required for the stimulation of ACTH secretion in AtT20/D16v cells. No response to 50 mM KCl occurred in the presence of EGTA or cobalt. Increased extracellular calcium overcame the inhibition of stimulated hormone secretion by EGTA, cobalt, and somatostatin. Therefore, we conclude that the inhibition of stimulated ACTH secretion by somatostatin involves the interaction of the peptide with specific binding sites on AtT20/D16v cells and the inhibition of stimulus-elicited calcium influx.     

Interaction between substance P and TRH in the control of prolactin release

Substance P (SP) may participate as a paracrine and/or autocrine factor in the regulation of anterior pituitary function. This project studied the effect of TRH on SP content and release from anterior pituitary and the role of SP in TRH-induced prolactin release. TRH (10(-7) M), but not vasoactive intestinal polypeptide (VIP), increased immunoreactive-SP (ir-SP) content and release from male rat anterior pituitary in vitro. An anti-prolactin serum also increased ir-SP release and content. In order to determine whether intrapituitary SP participates in TRH-induced prolactin release, anterior pituitaries were incubated with TRH (10(-7) M) and either WIN 62,577, a specific antagonist of the NK1 receptor, or a specific anti-SP serum. Both WIN 62,577 (10(-8) and 10(-7) M) and the anti-SP serum (1:250) blocked TRH-induced prolactin release. In order to study the interaction between TRH and SP on prolactin release, anterior pituitaries were incubated with either TRH (10(-7) M) or SP, or with both peptides. SP (10(-7) and 10(-6) M) by itself stimulated prolactin release. While 10(-7) M SP did not modify the TRH effect, 10(-6) M SP reduced TRH-stimulated prolactin release. SP (10(-5) M) alone failed to stimulate prolactin release and markedly decreased TRH-induced prolactin release. The present study shows that TRH stimulates ir-SP release and increases ir-SP content in the anterior pituitary. Our data also suggest that SP may act as a modulator of TRH effect on prolactin secretion by a paracrine mechanism.     

Calcium control of growth hormone release from chicken pituitary glands in vitro

The influence of calcium on the basal and stimulated release of growth hormone (GH) from chicken pituitary glands has been determined in vitro. Basal GH release occurred in Ca2+ deficient media, although it was increased in proportion to the medium Ca2+ concentration. Growth hormone release was stimulated by 10(-7)-10(-9) M thyrotrophin-releasing hormone (TRH), maximal stimulation being observed in the presence of 10(-8) M TRH and 1.5 mM Ca2+. Decreases in the Ca2+ concentration (to 0.75, 0.375, or 0 mM) suppressed the GH response to 10(-8) M TRH, as did increases (to 3.0 and 6.0 mM) in the Ca2+ concentration. These results suggest that GH release in chickens is regulated by Ca2+-dependent mechanisms.     

Effects of bromocriptine on prolactin release, electrical membrane properties and transmembrane Ca2+ fluxes in cultured rat pituitary adenoma cells

The effects of the dopamine (DA) agonist bromocriptine on prolactin (Prl) release, electrical membrane properties and transmembrane Ca2+ fluxes have been studied in a clonal strain of rat pituitary adenoma cells (GH3). These cells generate Ca2+ dependent action potentials, and produce and secrete spontaneously both Prl and growth hormone. Prl release stimulated by thyroliberin (TRH) and elevated extracellular K+ concentration was completely blocked by bromocriptine, whereas the basal release was only moderately affected. The TRH and K+ evoked Prl release were half maximally inhibited by bromocriptine at 5-10 and 10-50 microM, respectively. The normal biphasic membrane response to TRH and the depolarizing effect of elevated K+ concentration were not altered by bromocriptine, whereas the Ca2+- spikes in Na+-free solution were suppressed by the drug. We therefore suggest that bromocriptine blocks the voltage sensitive Ca2+-channels of GH3 cell. In agreement with this notion, bromocriptine also suppressed the basal and TRH induced 45Ca2+ efflux from preloaded cells. We conclude that the inhibitory effect of bromocriptine on the voltage dependent Ca2+- channels is an important mechanism responsible for suppression of Prl release.     

Dihydropyridine-sensitive calcium channel activity related to prolactin, growth hormone, and luteinizing hormone release from anterior pituitary cells in culture: interactions with somatostatin, dopamine, and estrogens

In the present work, we determined the activity of voltage-dependent dihydropyridine (DHP)-sensitive Ca2+ channels related to PRL, GH, and LH secretion in primary cultures of pituitary cells from male or female rats. We investigated their modulation by 17 beta-estradiol (E2) and their involvement in dopamine (DA) and somatostatin (SRIF) inhibition of PRL and GH release. BAY-K-8644 (BAYK), a DHP agonist which increases the opening time of already activated channels, stimulated PRL and GH secretion in a dose-dependent manner. The effect was more pronounced on PRL than on GH release. BAYK-evoked hormone secretion was further amplified by simultaneous application of K+ (30 or 56 mM) to the cell cultures; in parallel, BAYK-induced 45Ca uptake by the cells was potentiated in the presence of depolarizing stimuli. In contrast, BAYK was unable to stimulate LH secretion from male pituitary cells, but it potentiated LHRH- as well as K+-induced LH release; it had only a weak effect on LH secretion from female cell cultures. Basal and BAYK-induced pituitary hormone release were blocked by the Ca2+ channel antagonist nitrendipine. Under no condition did BAYK affect the hydrolysis of phosphoinositides or cAMP formation. Pretreatment of female pituitary cell cultures with E2 (10(-9) M) for 72 h enhanced LH and PRL responses to BAYK, but was ineffective on GH secretion. DA (10(-7) M) inhibited basal and BAYK-induced PRL release from male or female pituitary cells treated or not treated with E2 (10(-9) M). SRIF (10(-9) and 10(-8) M) reversed BAYK-evoked GH release to the same extent in cell cultures derived from male or female animals. It was ineffective on BAYK-induced PRL secretion in the absence of E2, but antagonized it after E2 pretreatment. The effect was dependent upon the time of steroid treatment and was specific, since 17 alpha-estradiol was inactive. In addition, DA and SRIF decreased the 45Ca uptake induced by the calcium agonist. These data demonstrate that DHP-sensitive voltage-dependent calcium channels of the L type present on different pituitary cells are not equally susceptible to BAYK activation under steady state basal conditions, indicating that their spontaneous activity and/or distribution vary according to the cell type; their activity is modulated by sex steroids. In addition, these data suggest that Ca2+ channels represent a possible site of DA and SRIF inhibition of PRL and GH release, respectively, by gating calcium entry into the corresponding cells.