Neuropeptide Y stimulates growth hormone and gonadotropin release from the goldfish pituitary in vitro

The effects of neuropeptide Y (NPY) on release of growth hormone (GH) and gonadotropin (GTH) from the goldfish pituitary in vitro were investigated. Exposure of perifused pituitary fragments, taken from female goldfish at late stages of gonadal recrudescence, to 5-min pulses of human NPY resulted in a rapid dose-dependent stimulation of GH and GTH release, with half-maximal effective dosages of 0.51 +/- 0.24 and 2.37 +/- 1.05 nM for GH and GTH, respectively. Repeated treatments with pulses of NPY (10 nM for GH, 5 nM for GTH) at 55-min intervals did not significantly alter the responsiveness of pituitary fragments to NPY; however, prior exposure of pituitary fragments to pulses of higher doses of NPY (50 nM GH, 10 nM for GTH) significantly reduced the subsequent hormone responses. When given at 85-min intervals repeated treatment with NPY did not blunt hormone responses to the second and third stimulations at these higher dosages. These results indicate that NPY acts at the pituitary level to stimulate GH and GTH secretion in female goldfish. The GTH response and, to a lesser extent, the GH response become desensitized to further stimulation by NPY in dose- and time-dependent manners. NPY should be considered as one element in the multifactorial systems regulating the GH and GTH secretion in goldfish.     

Effects of serotonin on gonadotropin and growth hormone release from in vitro perifused goldfish pituitary fragments

The effects of serotonin (5-HT) on gonadotropin and growth hormone release from perifused goldfish (Carassius auratus, L.) pituitary glands were studied. Serotonin, at micromolar concentrations, caused a dose-related release of gonadotropin and an inhibition of growth hormone release in pituitaries from goldfish at different sexual stages. At lower concentrations 5-HT continued to inhibit growth hormone release, but had no effects on gonadotropin release. The stimulatory effects of 5-HT on gonadotropin release could be blocked by ketanserin and cyproheptadine; however, these two antagonists had no effects on 5-HT inhibition of growth hormone release. Perifusion with melatonin had no effect on the release of gonadotropin or growth hormone release. These results demonstrate that 5-HT has a stimulatory effect on gonadotropin secretion, probably through a 5-HT2 receptor type, and an inhibitory effect on growth hormone through an unidentified receptor type. We hypothesize that the effects on gonadotropin release are due to direct actions on gonadotrophs, whereas the effects on growth hormone release may be due to stimulation of somatostatin release from neurosecretory terminals in the pituitary.     

Differential involvement of nitric oxide signaling in dopamine and PACAP stimulation of growth hormone release in goldfish

Previous studies in goldfish pituitary cells have shown that nitric oxide synthase (NOS)/nitric oxide (NO) signaling is involved in mediating the growth hormone (GH) release response to gonadotropin-releasing hormones. In this study, the involvement of this signaling pathway in mediating the action of two cAMP-mobilizing neuroendocrine stimulators of GH release, pituitary adenylate cyclase-activating polypeptide (PACAP) and dopamine (DA), was investigated in cell column perifusion experiments with primary cultures of dispersed pituitary cells. GH responses to PACAP were unaffected by three NOS inhibitors, aminoguanidine hemisulfate, 1400W and 7-nitroindazole (7-Ni). PACAP-stimulated GH release was also not reduced by two NO scavengers, rutin hydrate and PTIO, but NO-donor sodium nitroprusside (SNP)-elicited GH release was additive to the GH response to PACAP. In contrast, DA-induced GH secretion was reduced by 7-Ni, rutin hydrate and PTIO while not being additive to the GH response induced by SNP. These results indicate that although both PACAP and DA stimulation of acute GH release involve activation of adenylate cyclase/cAMP, DA- but not PACAP-signaling also utilizes the NOS/NO second messenger system.     

Dual action of dopamine on growth hormone release in vitro

The effect of dopamine (DA) on growth hormone (GH) release was studied in perifused freshly dispersed rat anterior pituitary cells. Pulses of DA (0.01-100 nmol/l), each applied for 30 min, resulted in a prompt rise in GH release. This effect was reversible, concentration-dependent and partially antagonized by metoclopramide, a DA antagonist. The effect of DA was further tested on GH-stimulated secretion by human GH-releasing factor (hGRF). Perifusion with hGRF (6.25 pmol/l) for 2 min elicited an immediate rapid increase in GH release which lasted 20 min. Pretreatment of cells with DA (100 nmol/l) for 10 min and a subsequent hGRF challenge during continuation of DA perifusion significantly reduced the effect of hGRF pulses on GH release. The present data suggest that DA has direct opposite actions at the somatotroph level, stimulating the basal GH release and inhibiting the hGRF-induced GH secretion, and may thus be an important modulator of GH release.     

Possible involvement of protein kinase C in gonadotropin and growth hormone release from dispersed goldfish pituitary cells

Static incubation with tumor-promoting 4 beta-phorbol esters, activators of the Ca2(+)- and phospholipid-dependent protein kinase C enzyme (PKC), caused dose-dependent increases in gonadotropin (GTH) and growth hormone (GH) secretion in primary cultures of dispersed goldfish pituitary cells. The estimated half-maximal effective doses (ED50) for stimulating GTH and GH release were 0.35 +/- 0.17 and 0.32 +/- 0.13 nM 12-O-tetradecanoyl phorbol 13 acetate (TPA), 3.71 +/- 1.30 and 1.37 +/- 0.76 nM 4 beta-phorbol 12,13-dibutyrate, 6.90 +/- 4.84 and 1.89 +/- 0.25 nM 4 beta-phorbol 12,13-dibenzoate, and 455 +/- 258 and 311 +/- 136 nM 4 beta-phorbol 12,13-diacetate, respectively. In contrast, treatments with up to 10 microM of the inactive 4 alpha-phorbol 12,13-didecanoate ester did not alter GTH and GH release. Additions of the synthetic diacylglycerol, dioctanoyl glycerol, also enhanced GTH and GH secretion in a dose-dependent manner and with ED50s of 1.73 +/- 0.83 and 1.73 +/- 1.19 microM, respectively. The GTH and GH responses to stimulation by TPA were attenuated by incubation with Ca2(+)-depleted medium containing EGTA or by treatment with the Ca2+ channel blocker verapamil. Coincubation with the PKC inhibitor H7 reduced the GTH and GH responses to TPA. As in previous studies, additions of salmon gonadotropin-releasing hormone (sGnRH) or chicken GnRH-II (cGnRH-II) induced GTH and GH release; these hormone responses to sGnRH and cGnRH-II were also decreased by the addition of H7. These results indicate that activation of PKC may stimulate GTH and GH release in goldfish and suggest that sGnRH and cGnRH-II actions on goldfish pituitary GTH and GH secretion are also mediated, at least partially, by PKC.     

Effects of dopamine and apomorphine on gonadotropin release from the transplanted pars distalis in goldfish

Male goldfish bearing pars distalis transplants from other male goldfish have increased serum gonadotropin (GtH) levels, due to the high spontaneous release rate of GtH by the transplants. Intraperitoneal injection of dopamine or its agonist, apomorphine, each reduced the elevated serum GtH levels caused by the release from the transplanted pars distalis. These results suggest that dopamine has GtH-release-inhibitory activity and acts directly on gonadotrophs to inhibit spontaneous secretion of GtH.     

Participation of arachidonic acid metabolism in gonadotropin-releasing hormone stimulation of goldfish gonadotropin release

Two intraperitoneal injections of a mammalian gonadotropin-releasing hormone (GnRH) analog, [D-Ala6, Pro9-N-ethylamide]-GnRH (mGnRHa; 0.1 micrograms/g), at 12-hr intervals increased serum gonadotropin (GTH) levels in sexually mature and sexually regressed female goldfish 2 and 6 hr after the second injection. This serum GTH response was decreased by the coinjection of a lipoxygenase enzyme inhibitor, nordihydroguaiaretic acid (NDGA: 0.1 to 10 micrograms/g) at the time of the second mGnRHa application. In static cultures of dispersed goldfish pituitary cells, 1-100 microM arachidonic acid (AA) and 0.1-1000 nM [Trp7, Leu8]-GnRH (salmon GnRH, sGnRH) and [D-Arg6, Pro9-N-ethylamide]-sGnRH (sGnRHa) caused dose-dependent increases in GTH release. Additions of 1-40 microM NDGA reduced the sGnRH-stimulated GTH release in a dose-dependent manner, and completely inhibited the GTH response to increasing concentrations of AA. NDGA 40 microM also decreased the elevated GTH levels induced by sGnRHa treatment. Exposure to 10 microM 5,8,11,14-eicosatetraynoic acid, an inhibitor with mixed action on lipoxygenase and cyclooxygenase enzymes, reduced the dose-dependent GTH response to sGnRH and AA. In contrast, coincubation with another cyclooxygenase blocker, indomethacin, at 10 microM, did not alter AA and sGnRH-induced GTH release. These results provide in vivo and in vitro evidence for the participation of AA metabolism in mediating GnRH-stimulated GTH release in the goldfish. The importance of AA metabolism through the lipoxygenase pathway is also indicated.     

Dopamine inhibition of gonadotropin and alpha-melanocyte-stimulating hormone release in vitro from the pituitary of the goldfish (Carassius auratus)

The purpose of the present investigation was to examine the receptor specificity of dopamine inhibition of gonadotropin (GtH) and alpha-melanocyte-stimulating hormone (alpha-MSH) release from the goldfish (Carassius auratus) pituitary in vitro. Pars distalis (PD) and neurointermediate lobe (NIL) fragments of the goldfish pituitary were superfused in vitro under various experimental paradigms; eluate from PD and NIL fragments was analyzed for (GtH) and (alpha-MSH), respectively. Spontaneous GtH release from PD fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited spontaneous GtH release with an estimated ED50 of 10(-4.4) M. Domperidone, a specific D-2 receptor antagonist, reversed the inhibitory action of dopamine and increased spontaneous GtH release. Acute treatment of PD fragments with salmon GnRH (sGnRH) stimulated GtH release; dopamine inhibited GtH release from similarly treated fragments with an ED50 of 10(-7.5) M. The spontaneous release of alpha-MSH from NIL fragments was relatively constant over 6 hr; continuous superfusion with dopamine reversibly inhibited this release with an ED50 of 10(-7.2) M. Acute treatment of NIL fragments with thyrotropin-releasing hormone (TRH) caused acute dose-related increases in alpha-MSH release with an ED50 of 10(-8.2) M; dopamine reversibly inhibited alpha-MSH release from similarly treated fragments with an ED50 of 10(-7.7) M. Both stereoisomers of apomorphine, a dopamine agonist, inhibited GtH release from PD fragments treated with sGnRH; in contrast, alpha-MSH release from NIL fragments treated with TRH was stereospecifically inhibited by (-)-apomorphine, but not by (+)-apomorphine. Domperidone reversed (ED50 = 10(-6.6) M) dopamine (10(-6.3) M) inhibition of GtH release from PD fragments treated with sGnRH. In NIL fragments, the inhibitory action of dopamine (10(-6.3) M) was reversed by domperidone (ED50 = 10(-5.5) M), which restored the acute alpha-MSH release response to TRH. These results suggest the involvement of a low-affinity dopamine/neuroleptic receptor in dopamine inhibition of GtH and alpha-MSH release from the pituitary of the goldfish.     

Differential effects of somatostatin on the release of bioactive and immunoactive rat growth hormone in vitro

The effects of linear (L) or cyclic (C) somatostatin (SRIF) on the release of growth hormone (GH) as measured both by bioassay and by radioimmunoassay (RIA) were studied in a series of incubation experiments with rat adenohypophyses. Although both forms of SRIF inhibited release of RIA-GH in all experiments, only C-SRIF appeared to inhibit release of bioactive GH. L-SRIF even enhanced release of bioassayable GH in one study. Addition of C-SRIF to medium containing secreted GH strongly inhibited the tibial growth response of the bioassay animals, while it had no effect on the measurement of GH by the RIA. Thus, the apparent inhibitory effect of C-SRIF on release of bioactive GH may be an artifact. The conclusion was supported by experiments in which the SRIF was not removed from the medium prior to assay by dialysis or by neutralization with anti-SRIF antiserum. After these manipulations, the inhibition by C-SRIF of the secretion of bioactive GH was eliminated, whereas the inhibitory effect on RIA-GH release was not affected. These results support other evidence for the differential release of rat GH with varying immuno-and bioactivities by different stimuli.     

Differential actions of arachidonic acid and melittin on luteinizing hormone release and synthesis

We studied the actions of arachidonic acid (AA) on luteinizing hormone (LH) release versus synthesis (translation or glycosylation) by cultured rat anterior pituitary cells. Monolayer cells were incubated for 3-4 h with secretagogues (AA, melittin, gonadotropin-releasing hormone; GnRH) which either increase endogenous AA levels or release AA. LH translation and glycosylation were monitored by measuring the incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell plus medium) immunoprecipitable LH. Immunoreactive (IR) LH was measured by radioimmunoassay. Nonlytic doses of AA increased (p less than 0.01) IR-LH release without increasing total [3H]glucosamine-LH, [14C]alanine-LH, or [3H]glucosamine-protein. AA either had no effect or slightly increased (p less than 0.05) [3H]glucosamine uptake, but decreased (p less than 0.01) [14C]alanine uptake and incorporation into total [14C]alanine protein. AA at 250 microM lysed cells, thus increasing medium IR-LH and decreasing (p less than 0.01) [3H]glucosamine and [14C]alanine uptake and incorporation into total LH and protein. Melittin, which releases AA by activating phospholipase A2 (245 and 490 nM), increased medium IR-LH (p less than 0.01) without affecting any other parameter measured. GnRH at 1 nM enhanced (p less than 0.01) both LH (IR-LH, [3H]glucosamine-LH and [14C]alanine-LH) release and total [3H]glucosamine-LH, but had no effect on total [14C]alanine-LH. In summary, AA and melittin at doses which stimulated LH release did not stimulate either LH glycosylation or translation. This suggests that increased LH release by nonspecific secretagogues is not necessarily accompanied by increased LH glycosylation or translation.     

Influences of catecholamines on growth hormone release in female goldfish, Carassius auratus

The influence of catecholamines on growth hormone (GH) release in female goldfish was investigated by monitoring serum GH levels following injections of drugs known to alter catecholamine synthesis and neural activities. Intraperitoneal (i.p.) injection of 6-hydroxydopamine, a catecholaminergic neurotoxin, or alpha-methyl-p-tyrosine, a catecholamine synthesis inhibitor, decreased serum GH levels. Intraperitoneal injection of L-beta-dihydroxyphenylalanine (L-dopa) increased serum GH concentrations in a dose-dependent manner. The L-dopa-induced increase in serum GH was potentiated by i.p. injection of carbidopa, which would increase the availability of L-dopa to brain tissues by blocking the peripheral conversion of L-dopa to dopamine (DA). These results suggest that L-dopa or one of its catecholamine metabolites acts centrally to increase GH release. Intraventricular (i.v.t.) injection of DA and i.p. injection of apomorphine, a DA agonist that crosses the blood-brain barrier, increased serum GH. Intraperitoneal injection of DA did not alter circulating GH levels in normal fish or fish bearing preoptic lesions that abolish an inhibitory hypothalamic influence on GH release; however, DA increased serum GH in fish which had their blood-brain barrier destroyed by sham operation procedures. These results indicate that DA acts centrally to stimulate GH secretion, possibly by inhibiting the release and/or synthesis of GH release-inhibitory factor. Serum GH concentrations were decreased in a dose-dependent manner by i.p. injection of norepinephrine (NE), whereas i.v.t. injection of NE did not alter serum GH levels. These results indicate that NE acts outside of the blood-brain barrier to decrease serum GH levels in the goldfish, possibly by directly influencing pituitary GH cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of porcine follicular fluid, inhibin-A, and activin-A on goldfish gonadotropin release in vitro.

Inhibin and activin are important reproductive regulators in mammalian species and have been demonstrated to be highly conserved in structure. The present study examines the effects of porcine follicular fluid (pFF; a crude inhibin and activin preparation) and purified porcine inhibin-A and activin-A on goldfish gonadotropin-II (GTH-II) release. In studies using primary cultures of dispersed goldfish pituitary cells in static incubation, treatments with pFF, inhibin-A, and activin-A for 10 h caused dose-dependent increase in GTH-II release. In perifusion studies using goldfish pituitary fragments, basal GTH-II release was significantly elevated after 12-h exposure to 500 micrograms/ml pFF. Furthermore, GnRH-induced GTH-II secretion was potentiated by pretreatment with pFF. When pFF was applied in the form of 5-min pulses, a rapid dose-related stimulation of GTH-II was observed. Similarly, challenges with 2-min pulses of 15, 150, and 1500 pM inhibin-A and activin-A stimulated GTH-II release by goldfish pituitary fragments in a rapid and dose-dependent manner. This acute stimulatory action of inhibin on goldfish GTH-II release was completely abolished after pretreatment with specific inhibin antibodies. The acute actions of inhibin and activin on GTH-II release are probably not due to the release of endogenous GnRH from nerve terminals in the pituitary fragments or binding to the GnRH receptors. First, a specific GnRH antagonist did not block the actions of inhibin and activin. Second, dopamine, a potent inhibitor of GnRH-stimulated GTH-II secretion in goldfish, was only partially effective in decreasing inhibin- and activin-induced GTH-II release. Third, the stimulatory effects of inhibin and GnRH on GTH-II release were additive. These lines of evidence also indicate that the mechanisms mediating inhibin and activin stimulation of goldfish GTH-II release may be somewhat different from those of GnRH. These results demonstrate that in contrast with the usual inhibitory effects of inhibin on GTH release in mammals, both inhibin and activin exert long term and acute stimulatory actions on GTH-II release in the goldfish.     

Differential inhibition of growth hormone secretion by analogs selective for somatostatin receptor subtypes 2 and 5 in human growth-hormone-secreting adenoma cells in vitro

Somatostatin (SRIH), a cyclic tetradecapeptide hormone originally isolated from mammalian hypothalamus, is a potent suppressor of pituitary growth hormone (GH) secretion. SRIH acts through a family of G-protein-coupled membrane receptors containing seven transmembrane domains. Five genes encoding distinct SRIH receptor (SSTR) subtypes have so far been cloned in human and other species and termed SSTR1-5. In human somatotrophe pituitary adenomas GH secretion is controlled by both SSTR2 and SSTR5. However, in clinical practice only somatostatin analogs selective for SSTR2 (octreotide and lanreotide) are available. This may explain why clinical and in vitro responses to these analogs in acromegaly are only partial. In this study, we investigated the inhibitory effect of two new SRIH analogs with high selectivity for SSTR2 (NC-4-28B) and SSTR5 (BIM-23268) and compared it to that of native somatostatin (SRIH-14) on a large number of GH-secreting adenomas obtained by transphenoidal neurosurgery. Tissues from 16 adenomas were enzymatically dispersed and plated in 24-well dishes at 50,000 cells/well. After 3 days, groups of three wells were incubated for 4 h with medium alone, SRIH-14 or analogs NC-4-28B or BIM-23268, at the concentrations of 0.01, 0.1 and 1 microM. Our results show that 9 out of 16 adenomas were responsive (GH suppression: 20-40% vs. control, p < 0.05) to SRIH. In this group only 4 adenomas showed similar responses to both selective analogs, with 2 nonresponders (expression of other SRIH receptor subtypes) and 2 responders (concomitant expression of SSTR2 and SSTR5) to both analogs. GH release was selectively inhibited by NC-4-28B in 3 adenomas and by BIM-23268 in the remaining 2 adenomas, suggesting predominant expression of SSTR2 and SSTR5, respectively. SRIH failed to inhibit GH release in 7 adenomas (43%). Interestingly, in that group a better inhibitory effect was obtained with BIM-23268 (5 out of 7 adenomas) than with NC-4-28B, suggesting expression of a few SSTR5 receptors only, or of both SSTR2 and SSTR5, respectively. We conclude that the availability of somatostatin analogs selective for SSTR5 will enhance the treatment potency and spectrum in acromegaly.     

Influence of different serotonin receptor subtypes on growth hormone secretion

The role of serotonin (5-HT) in the regulation of growth hormone (GH) secretion remains unclear due to the existence of many different receptors that mediate the 5-HT actions, and the lack of suitable specific agonist and antagonist drugs. In the present work we have taken advantage of the recent development of new selective 5-HT drugs in order to clarify the role played by different 5-HT receptor types and subtypes on GH secretion. The experiments were carried out on beagle dogs. GH-releasing hormone (GHRH) increased basal canine GH (cGH) levels from 0.8 +/- 0.2 to 8.8 +/- 1.7 microg/l at 15 min. Administration of 5-HT(1D) receptor agonist sumatriptan (SUM) induced a cGH peak at 30 min of 12.9 +/- 2.7 microg/l. The combined administration of GHRH plus SUM strikingly potentiated cGH release with a peak of 36.9 +/- 6 microg/l at 30 min (p < 0.05). Pretreatment with the muscarinic receptor antagonist atropine completely abolished the cGH response to SUM, while the cholinergic agonist pyridostigmine (PYR) did not modify this response (15.3 +/- 5 microg/l PYR plus SUM vs. SUM alone 12.9 +/- 2. 7 microg/l). On the other hand, administration of drugs with activity at 5-HT(2A/C) receptors showed a stimulatory role for the 5-HT(2C) receptor subtype, since LY-53857 (antagonist 5-HT(2A/C)) and DOI agonist (5-HT(2A/C)) both modified the GH response stimulated by GHRH (AUC 88.5 +/- 30.4 and 400 +/- 64.6 vs. 267.3 +/- 52.6 respectively), while ketanserin (antagonist 5-HT(2A)) did not modify this response. The 5-HT(3) antagonist ICS-205-930 failed to modify either basal or GHRH induced GH responses. In conclusion, our data show that 5-HT(1D) receptors play a stimulatory role on GH secretion in the dog, possibly by acting through a decrease in hypothalamic somatostatin release. Similarly, the 5-HT(2C) receptor subtypes also appear to play a stimulatory role. However, 5-HT(2A) and 5-HT(3) receptors do not appear to be involved in the control of basal and GHRH-induced GH secretion.     

In vitro effect of dopamine and L-dopa on prolactin and growth hormone release from human pituitary adenomas.

To determine the site of action of dopaminergic drugs on human PRL and GH release from pituitary adenomas, five PRL-and five GH-secreting adenomas were incubated with and without dopamine and L-dopa. Bromocriptine was also tested in order to compare its effect to that of the other drugs. In all of the experiments except one, a decrease of PRL, which was often statistically significant, was observed. When pooling the results of the PRL-secreting adenomas, the mean levels of PRL with dopamine, L-dopa, and bromocriptine were, respectively, 49%, 55%, and 60% of the control levels. In the GH-secreting adenomas, they were 60%, 67%, and 55% of that of the control. For GH, a decrease of the release was observed in four out of five GH-secreting adenomas. When pooling the results from these tumors, the mean levels of GH with dopamine, L-dopa, and bromocriptine were, respectively, 63%, 76%, and 64% of the control levels. In one case, a significant increase of GH was observed with the three dopaminergic drugs. This study produced the following conclusions. 1) Dopamine acts directly on PRL and GH release from human pituitary adenomas; in vitro, L-dopa effects are similar (its action probably occurs after conversion to catecholamines). These observations strongly suggest the presence of dopaminergic membrane receptors on human lactotroph and somatotroph adenomatous pituitary cells. 2) In vitro hormonal results are in good agreement with in vivo dynamic tests using L-dopa and bromocriptine. 3) The paradoxical effect of dopaminergic drugs on GH secretion in acromegalic patients may be attributed to modified dopamine membrane receptors. However, the paradoxical response is not a constant feature in acromegaly, and its mechanism needs further investigations.     

Involvement of protein kinase C and intracellular Ca in goldfish brain somatostatin-28 inhibitory action on growth hormone release in goldfish

Goldfish brain somatostatin-28 (gbSS-28) is present in brain and pituitary tissues of goldfish. We assessed whether gbSS-28 targets Ca²⁺ and/or protein kinase C (PKC)-dependent signaling cascades in inhibiting growth hormone (GH) release. gbSS-28 decreased basal GH release from primary cultures of dispersed goldfish pituitary cells and intracellular free calcium levels ([Ca²⁺]_i) in goldfish somatotropes. gbSS-28 partially reduced [Ca²⁺]_i and GH responses induced by two endogeneous gonadotropin-releasing hormones (GnRHs), salmon (s)GnRH and chicken (c)GnRH-II. Furthermore, gbSS-28 reduced GH increases and abolished [Ca²⁺]_i elevations elicited by two PKC activators, tetradecanoyl 4β-phorbol-13-acetate and dioctanyl glycerol. The PKC inhibitors Gö6976 and Bis II abolished [Ca²⁺]_i responses to PKC activators, but only attenuated GnRH-induced increases in [Ca²⁺]_i and did not alter basal [Ca²⁺]_i. In cells pretreated with Bis II, gbSS-28 further reduced basal [Ca²⁺]_i. Our results suggest that gbSS-28 inhibits GnRH-induced GH release in part by attenuating PKC-mediated GnRH [Ca²⁺]_i signals. gbSS-28 reduces basal GH release also via reduction in [Ca²⁺]_i but PKC is not involved in this regard.     

Calcium-dependent actions of gonadotropin-releasing hormone on pituitary guanosine 3',5'-monophosphate production and gonadotropin release

The effects of gonadotropin-releasing hormone (GnRH) on cGMP production and LH release in cultured rat pituitary cells are markedly dependent upon the extracellular calcium concentration. The absence of calcium from incubation media caused almost complete loss of the GnRH effects on cGMP production and LH release but did not change the stimulation of cAMP accumulation by GnRH in the pituitary of the adult male rat. In female rat pituitary cells, reduction of the extracellular calcium concentration increased the concentration of GnRH required to produce half-maximal LH release and decreased the maximal gonadotropin output but had no significant effect on basal LH release. The divalent cation ionophore A23187 stimulated LH release, and this action was dependent on extracellular calcium. Both GnRH and A23187 were found to have maximal effects when the calcium concentration was 0.6 mM, and their actions were not additive. The calcium antagonists, verapamil and lanthanum, caused concentration-dependent inhibition of the actions of GnRH, with half-maximal blockade values of 10(-5) and 3 X 10(-6) M, respectively, and had no effect on basal LH release. The binding of a radioiodinated GnRH analog, [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide, to pituitary GnRH receptors was unchanged in the absence of extracellular calcium. These observations demonstrate that stimulation of pituitary cGMP production and LH release by GnRH is dependent on extracellular calcium. The site at which calcium is required during GnRH action is at a postreceptor locus before cGMP formation.     

Mechanism of action of dopamine on the in vitro release of gonadotropin-releasing hormone

Controversy exists on whether dopamine (DA) stimulates or inhibits GnRH secretion and whether its effects are mediated via alpha-adrenergic receptors or dopaminergic receptors. As a means to examine this conflict, we have utilized an in vitro superfusion system to study the effects of DA, norepinephrine (NE), phentolamine (alpha-antagonist), pimozide (DA antagonist), and two DA agonists (apomorphine and bromocryptine) on GnRH release from isolated mediobasal hypothalami from adult male rats. In this dynamic system, graded concentrations of both NE and DA (2.0 nM to 2.0 microM) led to a dose-dependent increase in GnRH output during the 10 min interval that followed each pulse dose of NE (P less than 0.02) or DA (P less than 0.05). The DA-induced GnRH release was reproducible, consistent, and significant over five successive pulses (20 microM) at 30-min intervals (P less than 0.02). Coinfusion of phentolamine (20 microM) prevented the DA (20 microM) induced release of GnRH (P less than 0.03), but pimozide (20 microM) had no significant effect on DA-induced GnRH release (P greater than 0.3). The two DA agonists, apomorphine and bromocryptine, at doses up to 2.0 microM and 200 nM, respectively, had no significant effect on GnRH release. To determine whether DA was causing a direct stimulation of alpha-adrenergic receptors or being enzymatically converted to NE which could then stimulate alpha-receptors to induce GnRH release, rats were injected with sodium diethyldithiocarbamate (DDC) (550 mg/kg BW) ip, 1 h before death. DDC blocks the enzymatic conversion of DA to NE, and this was reflected by a 37% decrease in hypothalamic NE efflux during the superfusion. However, pulses of DA, even in the presence of DDC, were associated with a marked dose-dependent increase in hypothalamic NE efflux, and DDC failed to prevent the subsequent stimulation of GnRH release. We conclude that the apparent DA-induced release of GnRH is most probably attributable to DA-induced release of hypothalamic NE which, in turn, acts through alpha-adrenergic receptors on peptidergic neurons to stimulate GnRH release.     

Calcitonin gene-related peptide mimics calcitonin actions in brain on growth hormone release and feeding

We have examined the capacity of calcitonin gene-related peptide (CGRP) to exert two biological actions in the central nervous system (CNS): modulation of GH release and alteration of food intake. These effects were compared with those of calcitonin (CT). Intracerebroventricular administration of both rat (r) CGRP (10 micrograms/10 microliter) and salmon (s) CT (250 ng/10 microliter) to chronically cannulated freely-moving rats reduced the frequency and amplitude of spontaneous GH secretory pulses; however, the suppressive effect was of longer duration with sCT than with rCGRP. Both peptides also significantly reduced spontaneous food intake over a 6-h period when administered centrally, although the decrease with sCT was again more extensive than that with rCGRP. The results demonstrate that CGRP can simulate two of the effects of CT in brain. Furthermore, the findings suggest that CGRP may function as an endogenous ligand for CT receptors in the CNS, and participate centrally in the modulation of GH release and the control of satiety.     

Role of Ca2+ stores in dopamine- and PACAP-evoked growth hormone release in goldfish

Growth hormone (GH) secretion, evoked by either pituitary adenylate cyclase-activating polypeptide (PACAP) or dopamine (DA), is dependent on both voltage-sensitive calcium channels (VSCC) and cAMP signaling in goldfish. We further characterized the involvement of Ca2+ in evoked release by PACAP and DA, by examining the sensitivity of evoked GH release to perturbations of Ca2+ signaling. Both VSCC and calmodulin/calmodulin-dependent kinase are involved in PACAP signaling as had been shown for DA. In spite of this apparent dependence on VSCC, blockade of TMB-8 but not ryanodine-sensitive intracellular Ca2+ stores inhibited both PACAP- and DA-evoked GH release. Using sarcoplasmic/endoplasmic reticulum Ca-ATPases (SERCA) inhibitors, we found BHQ blocked, whereas thapsigargin (Tg) enhanced stimulated GH release, suggesting that Tg-sensitive SERCA may counteract these cAMP-mobilizing neuroendocrine regulators by sequestering [Ca2+]i. As GH secretion stimulated by two endogenous gonadotropin-releasing hormones is not affected by Tg, it appears that distinct multiple Ca2+ stores mediate the hormone releasing response to different neuroendocrine regulators.