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.     

Somatostatin inhibits basal and vasoactive intestinal peptide-stimulated hormone release by different mechanisms in GH pituitary cells

Somatostatin (SRIF) inhibits both basal and vasoactive intestinal peptide (VIP)-stimulated hormone secretion by the GH4C1 clonal strain of rat pituitary tumor cells. We have previously shown that SRIF inhibits cAMP accumulation stimulated by VIP but does not alter basal cAMP levels in this cell line. To determine the importance of changes in cAMP accumulation in the mechanism of SRIF action, we have compared the effect of SRIF on hormone release stimulated by VIP and two other secretagogues which increase effective intracellular cAMP concentrations: forskolin and 8-Bromo-cAMP (8-Br-cAMP). VIP stimulated GH and PRL secretion to the same maximal extent (220% of control) with similar ED50 values (0.37 +/- 0.03 and 0.43 +/- 0.08 nM, mean +/- SE, respectively). SRIF (100 nM) reduced maximal VIP-stimulation of both GH and PRL release from 220 to 140% of control; however, it did not significantly change the ED50 values for VIP. The effect of SRIF on VIP-stimulated hormone release parallels its action on VIP-stimulated cAMP accumulation. Furthermore, the concentrations of SRIF required to produce half-maximal inhibition of VIP-stimulated GH and PRL release (0.8 +/- 0.2 nM and 0.7 +/- 0.1 nM, respectively) were similar to its potency to inhibit VIP-stimulated cAMP accumulation (1.2 +/- 0.1 nM). These data indicate that changes in cAMP levels mediate inhibition of VIP-stimulated hormone secretion by SRIF. Forskolin increased cAMP accumulation with an ED50 value of 2.4 +/- 0.5 microM. A maximal concentration of forskolin (100 microM) stimulated cAMP accumulation to a greater extent than 100 nM VIP (34 +/- 4-fold vs. 9 +/- 1-fold). Together, forskolin (100 microM) and VIP (100 nM) stimulated cAMP accumulation by more than 50-fold. However, PRL secretion in response to maximal concentrations of VIP or forskolin individually or together were the same (approximately 200% of control). These results support the conclusion that both compounds stimulate PRL secretion by a cAMP-mediated mechanism which can be fully activated by either one alone.(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.     

The role of adenosine 3',5'-monophosphate in dopaminergic inhibition of prolactin release in anterior pituitary cells

The relationship between cAMP stimulation and dopaminergic inhibition of PRL release was studied in primary cultures of rat anterior pituitary cells. Bromocriptine, a dopaminergic agonist, and cholera enterotoxin, isobutylmethylxanthine, theophylline, and 8-bromo-cAMP, agents which mimic cAMP action or cause increases in cAMP, were used. Short term PRL release (that which occurs within 1 or 2 h) was stimulated 1.5- to 2-fold by all of the cAMP agents. Bromocriptine (5 nM) decreased basal release and completely abolished any short term stimulation above basal caused by cholera enterotoxin and 8-bromo-cAMP. Isobutylmethylxanthine and theophylline did cause some stimulation of PRL release above basal, but the magnitude of the increase was half that in the absence of bromocriptine. The short term release stimulated by 8-bromo-cAMP was dependent on extracellular calcium. PRL accumulation in the medium for 1-3 days was increased by all of the cAMP agents. A long term increase caused by these agents was also observed in the presence of bromocriptine. The magnitude of the increase in release above basal was the same with and without bromocriptine, but the total PRL in the medium was always less in the presence of bromocriptine, since basal release was reduced. These results show that the short term inhibition of PRL release by bromocriptine cannot be completely reversed by agents which increase cAMP.     

Is calcium or cyclic AMP involved in the inhibitory effect on pituitary hormone secretion of the tripeptide aldehyde proteinase inhibitors?

The mechanism by which tripeptide aldehyde proteinase inhibitors decrease prolactin (PRL) and growth hormone (GH) secretion was studied. Agents known to modify the intracellular levels of cyclic adenosine monophosphate (cAMP) or cytosolic free calcium were used in monolayer cultures of the rat anterior pituitary gland. The phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), 8-bromo-cAMP and forskolin all stimulated PRL release. Boc-D-Phe-Pro-arginal (Boc-DPPA) at 1 mmol/l concentration was a potent inhibitor of basal PRL release and significantly decreased the effect of 8-Br-cAMP, forskolin or IBMX (0.5 mmol/l). Forskolin (1 mumol/l) stimulated ACTH, PRL and GH release and all these effects were decreased by 100 mumol/l of Boc-D-Phe-Phe-lysinal (Boc-DPPL). Neither tripeptide aldehyde affected the forskolin-induced rise in intracellular cAMP. Growth hormone releasing factor (hpGRF, 1 nmol/l) stimulated both GH release and intracellular cAMP generation; Boc-DPPL (100 mumol/l) significantly decreased stimulated GH release without affecting cAMP accumulation. Increasing medium K+ to 10 times normal level stimulated PRL release presumably by enhancing Ca2+ entry into the cells and 1 mmol/l Boc-DPPA decreased high potassium-stimulated PRL release. The ionophore A-23187 stimulated PRL release at 10 mumol/l but not at 1 mumol/l. At 1 mumol/l A-23187 prevented the Boc-DPPA-induced inhibition of PRL release. These findings suggest that the tripeptide aldehyde proteinase inhibitors inhibit PRL and GH release at a site beyond cAMP formation.     

Nongenomic actions of testosterone on a subset of lactotrophs in the male rat pituitary

Rapid, nongenomic effects of testosterone on PRL release in vitro were investigated. Anterior pituitary tissue from adult male rats was stimulated in vitro for 5 or 20 min with testosterone (T; 1 or 100 nM) or testosterone-BSA (T-BSA; 1 or 100 nM) with or without 1.2 mM tannic acid, which enables visualization of secretory granule exocytosis. Within 5 min, both concentrations of T and T-BSA stimulated exocytosis from type 2 lactotrophs (characterized by small spherical granules), but not from type 1 lactotrophs (characterized by large polymorphic granules). The effects of T on type 2 lactotrophs could be blocked by preincubation with dopamine (500 nM), but were not time or concentration dependent, and could not be inhibited by 1) removal of extracellular Ca2+, 2) the L-type Ca2+ channel blocker nifedipine (100 nM), 3) the Ca2+-adenosine triphosphatase inhibitor thapsigargin (150 nM), 4) the PKC inhibitor retinal (10 microM), or 5) the gamma-aminobutyric acidA chloride channel blocker picrotoxin (100 microM). T-BSA (0.1 nM to 1 microM) for 5 or 20 min also caused an increased release of immunoreactive PRL into the medium compared with control incubations. T and T-BSA did not stimulate exocytosis from gonadotrophs or cause LH release. In conclusion, we report for the first time a rapid, nongenomic effect of T on PRL secretion.     

Mechanism of the inhibitory action of dopamine and somatostatin on prolactin secretion from human lactotrophs in culture

In an attempt to delineate the mechanism(s) of PRL secretion from human lactotrophs, the effects of dopamine and somatostatin on PRL release from adenomatous and nonadenomatous human pituitary cells in culture was studied. High K+ and the divalent cation ionophore A23187 both elevated PRL secretion, which was blocked by dopamine and somatostatin. When the cells were incubated in low calcium medium, PRL secretion was significantly inhibited. The addition of dopamine or somatostatin to low calcium medium further decreased PRL release. The stimulatory action of ionophore A23187 on PRL release was found even in the absence of extracellular calcium. Theophylline and isobutylmethylxanthine, when added to the incubation medium, increased PRL secretion, and dopamine as well as somatostatin again inhibited PRL release induced by phosphodiesterase inhibitors. No qualitative difference in these PRL responses was found in adenomatous and nonadenomatous human lactotrophs. In prolactinoma cells obtained from three different patients, cAMP generation was correlated with hormone release. Exposure of the cells to dopamine or somatostatin resulted in a parallel decrease in intracellular cAMP content and PRL secretion. The inhibitory effect of dopamine on PRL secretion and cAMP accumulation was blocked by coincubation of the cells with haloperidol. These results suggest that an increase in cytosol calcium caused by either mobilization from intracellular calcium pools or influx from the extracellular compartment and intracellular cAMP accumulation may be involved in the mechanism of PRL secretion from human lactotrophs, and dopamine and somatostatin may influence these two messengers to suppress PRL secretion.     

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)     

The actions of forskolin, cholera toxin and iloprost on casein secretion by lactating doe mammary glands

The secretagogue effects of prolactin (PRL) and of various agents acting on cAMP levels, forskolin, cholera toxin and iloprost (a stable analogue of prostaglandin I2) have been assessed in lactating doe mammary gland fragments in vitro. Forskolin (10 microM), cholera toxin (1 microgram/ml) and iloprost (10 mM) stimulated milk casein secretion. The effects of forskolin (10 microM) and cholera toxin (1 microgram/ml) were potentiated by PRL (10 micrograms/ml). Conversely, the action of iloprost (10 microM) was not amplified by PRL (10 micrograms/ml). Forskolin (10 microM) and cholera toxin (1 microgram/ml) stimulated the intracellular accumulation of cAMP. Neither PRL nor iloprost, at concentrations which stimulated casein secretion, modified the accumulation of cAMP. These results demonstrate that PRL does not act directly by any increase in intracellular cAMP levels. However, stimulating effects of forskolin and cholera toxin on casein secretion and intracellular cAMP levels suggest that various transduction signals are effective in the mammary cells.     

Pituitary hormone gene expression and secretion in human growth hormone-releasing hormone transgenic mice: focus on lactotroph function

The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA) levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per microg total RNA) were unchanged. In contrast, total PRL contents were unchanged, whereas the concentrations of PRL (micrograms of PRL per mg total protein) were decreased 3-fold. Hypothalamic tyrosine hydroxylase steady state mRNA levels were not altered in the hGHRH transgenic mice, but hypothalamic tyrosine hydroxylase activity was increased 2-fold in transgenic mice. Dopamine D2 receptor mRNA concentrations in the anterior pituitary were increased 2.5-fold in hGHRH transgenic mice, and total pituitary D2 receptor mRNA levels were increased nearly 10-fold. Furthermore, the basal secretory capacity of lactotrophs from transgenic mice was increased significantly at the level of the single cell, and dopamine inhibited the secretion of PRL to a greater extent in hGHRH transgenic mice. Thus, although the total number of lactotrophs is increased 2-fold in hGHRH transgenic mice, the present data are consistent with the hypothesis that increased hypothalamic dopamine synthesis and release coupled with an increase in D2 dopamine receptor gene expression and functional sensitivity in the pituitary result in normal plasma levels of PRL.     

Growth hormone-releasing factor secretion from fetal hypothalamic cell cultures is modulated by forskolin, phorbol esters, and muscimol

The regulation of GRF secretion was studied using a fetal rat hypothalamic cell culture system. The cells were subjected to short term release experiments on days 10-18 after plating, and GRF secretion was assessed by RIA. The identity of GRF immunoreactivity in the incubation medium was confirmed by reverse phase liquid chromatographic analysis. Depolarization of the cells with 56 mM K+ evoked a 4-fold increase in basal GRF release. When cultures were pretreated for 6 days with the adenylate cyclase activator forskolin, basal GRF release was augmented in subsequent release experiments to levels 2-fold greater than those in the control cultures. In nonpretreated cultures, forskolin (1-100 microM) and the protein kinase C activator phorbol 12-myristate 13-acetate (10 nM-1 microM), stimulated basal GRF release in a dose-dependent fashion. The Ca2+ channel blocker verapamil (100 microM) significantly inhibited the GRF response to both forskolin and phorbol 12-myristate 13-acetate. The gamma-aminobutyric acid (GABA) agonist muscimol (0.1-10 microM) inhibited forskolin-stimulated, but not K+ stimulated, GRF release in a dose-dependent manner. This inhibition was reversed by the GABA antagonists bicuculline and picrotoxinin. Muscimol (10 microM) slightly suppressed basal GRF release. The present findings suggest that GRF secretion can be evoked by agents known to increase intracellular cAMP levels or activate protein kinase-C. They also support a role for GABA in the inhibitory control of GRF secretion.     

Involvement of cyclic adenosine monophosphate in the stimulation of gonadotropin secretion from the pituitary of the teleost fish, tilapia.

The present study examines the involvement of cAMP in the transduction of the short-term effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release in the teleost fish, tilapia. A 5 min pulse of dibutyryl cyclic AMP (dbcAMP; 0.03-3 mM) or forskolin (0.1-10 microM) resulted in dose-dependent surges in tilapia gonadotropin (taGTH) secretion from the perifused pituitary. The initial increase in taGTH in response to dbcAMP (3 mM) occurred within 6 min. The concentration of cAMP in the effluent medium increased about 20-fold after a pulse of [D-Ala6,Pro9-NEt]-luteinizing hormone-releasing hormone (LHRH) (GnRHa; 100 nM). To rule out the possibility that the observed effects were due to stimulation by endogenous GnRH release from intact nerve terminals present in the fragments, further experiments were performed in primary cultures of dispersed pituitary cells. Exposure (30 min) of the cells to forskolin (0.01-1.0 microM) resulted in a dose-dependent increase in taGTH release similar to that achieved by GnRHa (1 pM to 10 nM). Also 8-bromo cAMP (0.01-1.0 mM) evoked a dose-related increase in taGTH release. A 3-fold increase in the release occurred in the presence of isobutylmethylxanthine (IBMX) (0.2 mM), similar to that obtained by GnRHa (1.0 nM) in the absence of IBMX. However, when combined, the increase in taGTH release was 16-fold. Moreover, exposure of the cultured cells to GnRHa (0.1 or 10 nM, 60 min) resulted in a dose-related elevation of intracellular cAMP levels and taGTH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galanin secretion from anterior pituitary cells in vitro is regulated by dopamine, somatostatin, and thyrotropin-releasing hormone.

Lactotrophs, somatotrophs, and thyrotrophs have been shown to contain immunoreactive galanin. Furthermore, estrogen stimulates galanin mRNA and peptide levels in the rat anterior pituitary, particularly within lactotrophs. To determine whether galanin is released from the anterior pituitary in a regulated manner, we used cultured pituitary cells from male and ovariectomized Fischer 344 rats implanted with estrogen-containing capsules. Anterior pituitary cells (5 x 10(5) cells/well) were challenged (0.5-3 h) with hypothalamic factors known to regulate anterior pituitary hormone secretion, and medium galanin levels were measured by RIA. In female pituitary cells, galanin secretion was inhibited by dopamine (10 and 100 nM) and stimulated by TRH (20 and 100 nM). Although galanin release was significantly lower in male pituitary cells, dopamine and TRH inhibited and stimulated galanin secretion, respectively. Medium galanin levels were also significantly reduced by somatostatin (5 nM) in both female and male cells. The pattern of PRL release in response to dopamine, TRH, and somatostatin was similar to that observed for galanin, regardless of the sex of the pituitary donor. Although galanin has been localized in somatotrophs, 5 nM GH-releasing hormone (GRF) failed to alter galanin release in male as well as female pituitary cells; GH secretion was significantly increased by GRF. LHRH (5 nM) and CRF (5 nM) failed to alter galanin release in vitro. We conclude that in estrogen-exposed pituitary cells obtained from male and ovariectomized Fischer 344 rats: 1) galanin secretion is inhibited by dopamine and somatostatin, and stimulated by TRH; 2) GRF, LHRH, and CRF do not regulate galanin release in these cells; and 3) the profile of the regulated pathway for galanin release suggests that the primary location of galanin is the lactotroph, probably within secretory granules.     

Influence of dopamine on the altered release of prolactin, luteinizing hormone, and follicle-stimulating hormone induced by interleukin-2 in vitro.

Interleukin-2 (IL-2) alters the release of anterior pituitary hormones at femtomolar concentrations from hemipituitaries incubated in vitro. This cytokine significantly lowered luteinizing hormone (LH) and follicle-stimulating hormone (FSH) and stimulated prolactin (PRL) release, thus demonstrating a reciprocal action of the lymphokine on lactotrophs and gonadotrophs. Since dopamine (DA) is a powerful inhibitor of PRL release, in the present experiments were evaluated possible dose dependent effects of DA on IL-2-induced alterations of the release of PRL, LH, and FSH. Hemipituitaries were incubated with varying concentrations of DA, a combination of IL-2 plus DA, or a combination of haloperidol (1 x 10(-5) M) with DA for 1 h, followed subsequently by incubation with medium containing only high potassium (K+) to study the effects on depolarization-induced hormone release. DA induced a dose-related, significant lowering of the basal PRL release with a minimal effective dose (MED) of less than 19 nM. The depolarization-induced PRL release was also inhibited, but the MED was 100-fold higher than the MED to inhibit basal PRL release. DA at much higher concentrations (30, 60, and 90 microM) significantly reduced pituitary PRL content. The addition of 0.187, 3.75, 15, or 60 microM DA to IL-2-induced PRL release. IL-2 (10(-15) M) produced a significant decrease in LH and FSH release. The combination of 3.75 or 15 microM DA plus IL-2 failed to alter the IL-2 suppressed LH release, whereas the addition of 0.187 microM DA to IL-2 blocked its suppressive influence, and 60 microM DA added to Il-2 produced an additive inhibitory effect. Thus, the interaction of IL-2 and DA is biphasic on LH release. The significant reduction of FSH release induced by IL-2 was blocked in the presence of 0.187, 3.75, 15, or 60 microM DA. DA alone at relatively high concentrations of 30, 60, and 90 microM suppressed basal LH and FSH release. The effects of DA on PRL, LH, and FSH at all doses tested were blocked by the DA receptor blocker, haloperidol which by itself at the concentration tested (1 x 10(-5) M) had no effect. Thus, the actions of DA at all concentrations tested appear to be mediated via DA receptors. In conclusion, DA was capable of blocking the stimulatory action of IL-2 on PRL release and its inhibitory action on FSH release by a DA receptor mediated action.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vasoactive intestinal polypeptide and alpha 2-adrenoceptor agonists regulate adenosine 3',5'-monophosphate accumulation and melatonin release in chick pineal cell cultures

Vasoactive intestinal polypeptide (VIP) has been shown to stimulate melatonin synthesis in mammalian pineal; however, a regulatory role for VIP in the avian pineal has not been explored. Immunocytochemical and physiological response experiments were performed to investigate whether 1) immunoreactive VIP fibers innervated the avian pineal gland; 2) VIP had a specific effect on melatonin release that was mediated by cAMP stimulation; and 3) alpha 2-adrenergic signal transduction was associated with a reduction in cAMP levels. Immunocytochemical experiments demonstrated the presence of both tyrosine hydroxylase- and VIP-immunoreactive fibers in the avian pineal gland. Treatment of dispersed chick pineal cell cultures with VIP stimulated melatonin release (maximum 6-fold increase; EC50 = 1.8 nM) when administered during the 12-h light period of a 12-h light, 12-h dark cycle. Of the other four peptides tested [porcine VIP-(10-28), porcine peptide histidine isoleucine, porcine secretin, and human glucagon), only peptide histidine isoleucine stimulated melatonin release (EC50 = 30 nM). The effect of VIP was mediated by a time- and dose-dependent increase in cAMP accumulation (maximum 4-fold increase). The specific alpha 2-agonist UK-14,304 reduced cAMP accumulation (maximum 43% reduction) and inhibited melatonin release (EC50 = 19 nM) in the presence of 3 X 10(-8) M VIP. Norepinephrine-induced inhibition of nocturnal melatonin release was blocked by the elevation of cAMP achieved through the administration of forskolin (EC50 = 0.2 microM), isobutylmethylxanthine (EC50 = 112 microM), or 8-bromo-cAMP (EC50 = 166 microM). Collectively, these results demonstrate the presence and functional significance of VIP in the avian pineal gland, and the interaction of VIP and norepinephrine at the level of cAMP in the regulation of melatonin biosynthesis.     

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)     

Effect of forskolin, isoproterenol and IBMX on angiotensin converting enzyme and cyclic AMP production by cultured bovine endothelial cells

The production of angiotensin converting enzyme (ACE) is known to be increased by glucocorticoids, thyroid hormones and converting enzyme inhibitors. We have recently reported that active cAMP analogues also stimulate production of the enzyme. The effect of stimulation of adenylate cyclase in cultured endothelial cells or of phosphodiesterase inhibition on ACE production was therefore evaluated. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (10(-4) M), produced 10.5 +/- 1.3 and 1.3 +/- 0.1 (P less than 0.01 and P greater than 0.1) fold increases in extracellular and cellular cAMP levels and a 1.55 +/- 0.10 (P less than 0.0001) fold increase in ACE accumulation. The adenylate cyclase stimulator, forskolin (0.01-10 microM), acutely stimulated cellular cAMP accumulation in a dose-dependent manner, reaching a 2.8 +/- 0.1-fold increase at 10 microM. After 48 h exposure to 10 microM forskolin, significant increases in cellular (1.90 +/- 0.38-fold increase, P less than 0.0001) and extracellular cAMP (2.35 +/- 0.26-fold increase, P less than 0.0001) were also observed but ACE accumulation was unchanged (108 +/- 10% of control, P greater than 0.5). The beta-adrenoceptor agonist, isoproterenol (1-1000 nM), acutely stimulated cellular cAMP accumulation, with a threshold effect at 10 nM, an ED50 of approximately 30 nM, and a plateau effect of 2.0 +/- 0.13-fold increase by 100 nM. After 48 h exposure to isoproterenol (1 microM), extracellular cAMP levels were increased significantly (1.68 +/- 0.33-fold increase, P less than 0.01) but ACE production was slightly inhibited (83 +/- 7% of control, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of adrenocorticotropin/endorphin-related peptide secretion in neonatal rat pituitary cultures

In neonatal rat anterior and neurointermediate pituitary lobe explant cultures maintained in complete serum-free medium for up to 3 weeks, the ACTH/endorphin-related peptide content rose about 3-fold. The basal secretion rates of the ACTH/endorphin-related peptides were 0.2-0.3% and 0.6-0.7% of the cell content secreted/h in the anterior and neurointermediate pituitary lobe explants, respectively. Acute treatment of anterior pituitary explants with 10 nM CRF or 0.5 mM (Bu)2-cAMP-0.5 mM isobutylmethylxanthine resulted in about 6- and 9-fold increases in the basal secretion rates, respectively. Pretreatment of the anterior pituitary cultures with 100 nM dexamethasone for 3 days did not alter the basal secretion rate, but drastically reduced both CRF- and cAMP-stimulated release of peptides. In the anterior pituitary explants, chronic CRF treatment resulted in a dose-dependent increase in secreted ACTH/endorphin-related peptides measured in the medium, and this elevation in secretion rate was maintained throughout 12 days of chronic exposure to CRF. Total hormone produced by neonatal anterior pituitary explant cultures was increased 2- to 4-fold by chronic CRF treatment (10 and 100 nM CRF) after 12 days in culture. Compared to the anterior pituitary lobe explants, the neurointermediate pituitary lobe explants were not very responsive to acute or chronic CRF treatment; exposure to 100 nM CRF resulted in about a 2-fold stimulation of the basal secretion rate. Acute exposure of the neurointermediate pituitary lobe explants to 1 microM isoproterenol or cAMP resulted in about 2- and 7-fold increases in the basal secretion rate, respectively. Dexamethasone pretreatment had little effect on either the basal or stimulated release of the ACTH/endorphin-related peptides. These studies demonstrated that some of the tissue-specific regulatory mechanisms involved in ACTH/endorphin-related peptide synthesis and secretion were present in developing explant cultures of anterior and neurointermediate pituitary lobes.     

Basal and dopamine-inhibited prolactin secretion by rat anterior pituitary cells: effects of culture conditions

Culture conditions for rat pituitary cells were investigated which would result in high PRL synthesis and secretion with maintenance of dopamine-mediated inhibition of PRL secretion. From five commercially available media, RPMI resulted in the highest PRL content and secretion, but no inhibition of PRL secretion by dopamine was observed. MEM with Earle's salts fulfilled best our requirements for culturing functional PRL-secreting cells. PRL secretion was not affected by variations in the concentration of fetal calf serum, but was positively correlated with increasing horse serum concentrations. TRH-induced PRL release increased with increasing serum concentrations and was positively correlated with the concentration of 17 beta-estradiol in the culture medium (P less than 0.0025). An increase in the sodium bicarbonate concentration from 0.85 to 3.0 g/l resulted in a 4-fold stimulation of PRL synthesis and in a 27-fold stimulation of PRL secretion. However, at bicarbonate concentrations above 2.6 g/l, inhibition of PRL secretion by 500 nM dopamine was lost. The addition of 20 mM Hepes to the culture medium decreased basal PRL secretion by 48 +/- 13% (P less than 0.01), while dopamine inhibition of PRL secretion was reduced from 49 +/- 10% to 24 +/- 8% (P less than 0.05). When an increasing number of pituitary cells was cultured in a constant volume, PRL secretion expressed per cell increased up to 0.3-0.4 X 10(6) cells/dish/2 ml. With higher cell concentrations of up to 1 X 10(6) cells/dish, PRL secretion per cell diminished significantly, which indicates a direct negative feedback of high medium PRL on the PRL-secreting pituitary cells. In this culture system dopamine inhibited PRL secretion over a 4 h period in a dose-dependent manner (IC50 20 nM), while no paradoxical stimulation of PRL secretion was observed with low dopamine concentrations. However, a 25% stimulation (P less than 0.05) of PRL secretion by 0.1 nM dopamine could be obtained by addition of 0.01% ascorbic acid, which by itself decreased basal PRL secretion by 49% (P less than 0.01). Thus, tissue culture conditions that result in high PRL production are not necessarily the best choice, since dopamine-mediated inhibition of PRL secretion is another important parameter for the functioning of lactotrophs in culture. The best compromise is MEM with 2.2 g/l of sodium bicarbonate, without Hepes buffer and supplemented with 10% FCS.     

Ketoconazole inhibits corticotropic cell function in vitro

The effects of ketoconazole (KC) on secretion and biosynthesis of ACTH and generation of cAMP in rat anterior pituitary cells were investigated in vitro. KC inhibits CRF-stimulated ACTH release from rat anterior pituitary fragments in a dose-dependent fashion between 1.5 and 100 microM. The effect of CRF as a releaser of ACTH was fully restored after KC was removed from the medium. Similar effects were observed in primary cultures of rat anterior pituitary cells. KC dose-dependently decreased basal and CRF-stimulated ACTH release. In addition, basal and CRF-stimulated mRNA coding for the ACTH precursor were reduced after preincubation with KC. The effects of KC on ACTH release and biosynthesis seem to be mediated by cAMP, since KC inhibits basal and CRF-stimulated cAMP release and content within the same dose range. Since the stimulatory effects of cholera toxin, sodium fluoride, and forskolin were dose-dependently inhibited by KC and since the addition of (Bu)2cAMP abolished the inhibiting effect of KC, it is concluded that KC acts by inhibition of the catalytic component of the adenylate cyclase holoenzyme.