Signal transduction mechanisms mediating secretion in goldfish gonadotropes and somatotropes.

The intracellular signal transduction mechanisms mediating maturational gonadotropin and somatotropin secretion in goldfish are reviewed. Several major signaling mechanisms, including changes in intracellular [Ca2+], arachidonic acid cascades, protein kinase C, cyclic AMP/protein kinase A, calmodulin, nitric oxide, and Na+/H+ antiport, are functional in both cell types. However, their relative importance in mediating basal secretion and neuroendocrine-factor-regulated hormone release differs according to cell type. Similarly, agonist- and cell-type-specificity are also present in the transduction pathways leading to neuroendocrine factor-modulated maturational gonadotropin and somatotropin release. Specificity is present not only in the actions of different regulators within the same cell type and with the same ligand in the two cell types, but this also exists between isoforms of the same neuroendocrine factor within a single cell type. Other evidence suggests that function-selectivity of signaling may also result from differential modulation of Ca2+ fluxes from different sources. The interaction of different second messenger systems provide the basis by which regulation of maturational gonadotropin and somatotropin release by multiple neuroendocrine factors can be integrated at the target cell level.

Agonist-specific Ca2+ signaling systems, composed of multiple intracellular Ca2+ stores, regulate gonadotropin secretion.

Ca2+ signals regulate many cellular functions, including hormone secretion. Agonist-specific Ca2+ signaling may arise from the differential mobilization of multiple Ca2+ stores. Although they act through the same receptor subtype, two gonadotropin-releasing hormones (sGnRH and cGnRH-II) generate quantifiably different Ca2+ signals in goldfish gonadotropes, suggesting that their Ca2+-dependent signaling cascades may differ. We combined electrophysiology, Ca2+ imaging, and radioimmunoassay detection of gonadotropin (GTH-II) secretion to determine the role of intracellular Ca2+ stores in GnRH-stimulated exocytosis. Our findings suggest that voltage-gated Ca2+ channels do not mediate acute GnRH-signaling. Instead, both sGnRH- and cGnRH-II-stimulated GTH-II releases are dependent on Ca2+ mobilized from TMB-8/CPA-sensitive compartments. However, sGnRH, but not cGnRH-II, utilizes intracellular stores sensitive to caffeine and xestospongin C. We also identified a homeostatic mechanism where reduced extracellular Ca2+ availability increase GTH-II release by mobilizing Ca2+ stores. Our results are the first to suggest that several classes of intracellular Ca2+ stores differentially participate in agonist signaling and homeostasis in gonadotropes.

Wortmannin-sensitive and -insensitive steps in calcium-controlled exocytosis in pituitary gonadotrophs: evidence that myosin light chain kinase mediates calcium-dependent and wortmannin-sensitive gonadotropin secretion.

In cultured rat pituitary cells, increases in the cytosolic calcium concentration ([Ca2+]i) and LH release are induced by activation of GnRH receptors as well as by nonreceptor-mediated stimuli. Treatment of pituitary cells with the myosin light chain kinase (MLCK) inhibitor, wortmannin, attenuated GnRH-induced LH release. Wortmannin also reduced the LH responses to nonreceptor-mediated elevation of [Ca2+]i by ionomycin and activation of voltage-sensitive Ca2+ channels by Bay K 8644 or high K+, as well as Ca2+-induced LH release in permeabilized pituitary cells. The [Ca2+]i responses to these stimuli were unaltered in wortmannin-treated pituitary cells, indicating that this compound inhibits a Ca2+-dependent step in exocytosis without affecting Ca2+ signaling. In perifused pituitary cells, the GnRH-induced early spike phase of LH release was not affected by wortmannin, whereas the subsequent plateau phase was almost completely inhibited. No significant changes in GnRH-induced phospholipase D activity and diacylglycerol production were observed in wortmannin-treated pituitary cells during the sustained phase of agonist stimulation. Wortmannin also had no effect on LH responses to the protein kinase C activator, phorbol 12-myristate 13-acetate, further indicating that the attenuation of agonist-induced LH release is not related to inhibition of the diacylglycerol/protein kinase C pathway. In addition, agonist-induced LH release was attenuated by two other MLCK inhibitors, MS-347a and KT5926. These data suggest that MLCK mediates the downstream effects of Ca2+ on exocytosis, an action supported by the finding of wortmannin-sensitive phosphorylation of a 20-kDa protein in pituitary cells and alphaT3-1 gonadotrophs treated with GnRH, K+, and Bay K 8644. This protein was coprecipitated from pituitary extracts with a specific antibody to nonmuscle myosin IIB and comigrated with 20-kDa smooth muscle myosin light chain on SDS-PAGE. These results demonstrate that Ca2+ controls exocytosis through an initial wortmannin-insensitive step and a sustained wortmannin-sensitive step and suggest that the latter event in the cascade of cellular responses is dependent on phosphorylation of nonmuscle myosin IIB light chain by MLCK.

Expression of purinergic receptor channels and their role in calcium signaling and hormone release in pituitary gonadotrophs. Integration of P2 channels in plasma membrane- and endoplasmic reticulum-derived calcium oscillations.

The role of ATP as a positive feedback element in Ca2+ signaling and secretion was examined in female rat pituitary gonadotrophs. ATP and ADP, but not AMP or adenosine, induced a dose- and extracellular Ca2+-dependent rise in [Ca2+]i in identified gonadotrophs in a Mg2+- and suramin-sensitive manner. ATP, adenosine-5'-O-(3-thiotriphosphate), adenosine-5'-O-(1-thiotriphosphate), 2-methylthio-ATP, and 3'-O-(4-benzoyl)benzoyl-ATP were roughly equipotent in rising [Ca2+]i in gonadotrophs, while ADP was effective only at submillimolar concentration range, and none of these compounds permeabilized the cells. On the other hand, alpha,beta-methylene-ATP, beta,gamma-methylene-ATP, and UTP were unable to induce any rise in [Ca2+]i. This pharmacological profile is consistent with expression of P2X2 and/or P2X5 purinergic receptor channels. Patch-clamp experiments showed that ATP induced an inward depolarizing current in gonadotrophs clamped at -90 mV, associated with an increase in [Ca2+]i. The ATP-induced [Ca2+]i response was partially inhibited by nifedipine, a blocker of voltage-sensitive Ca2+ channels (VSCC), but was not affected by tetrodotoxin, a blocker of voltage-sensitive Na+ channels. Thus, the P2-depolarizing current itself drives Ca2+ into the cell, but also activates Ca2+ entry through VSCC. In accord with this, low [ATP] induced plasma membrane-dependent [Ca2+]i oscillations in quiescent cells, and increased the frequency of spiking in spontaneously active cells. ATP-induced Ca2+ influx also affected agonist-induced and InsP3-dependent [Ca2+]i oscillations by increasing the frequency, base line, and duration of Ca2+ spiking. In addition, ATP stimulated gonadotropin secretion and enhanced agonist-induced gonadotropin release. ATP was found to be secreted by pituitary cells during agonist stimulation and was promptly degraded by ectonucleotidase to adenosine. These observations indicate that ATP represents a paracrine/autocrine factor in the regulation of Ca2+ signaling and secretion in gonadotrophs, and that these actions are mediated by P2 receptor channels.

Interactions between signaling pathways in mediating GnRH-stimulated GTH release from goldfish pituitary cells: protein kinase C, but not cyclic AMP is an important mediator of GnRH-stimulated gonadotropin secretion in goldfish.

In the goldfish, it has been proposed that gonadotropin (GTH) release induced by GTH-releasing hormone (GnRH) involves Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC), protein kinase C (PKC) activation, and arachidonic acid (AA) metabolism, but not cyclic AMP (cAMP) action. However, cAMP appears to mediate GnRH action in other teleosts. In this study, the relative importance of PKC and cAMP in mediating GnRH action in goldfish was studied using primary cultures of dispersed pituitary cells. Consistent with an involvement of PKC in GnRH action, the GTH responses to the PKC activating tetradecanoyl phorbol acetate (TPA), salmon (s)GnRH, and chicken (c)GnRH-II were inhibited by two selective PKC inhibitors, calphostin C, and staurosporine. Furthermore, GTH release responses induced by sGnRH or cGnRH-II were not additive to responses stimulated by the PKC-activating diglyceride DiC8, in either long-term static incubation or acute perifusion experiments. In static incubation studies, the GTH responses to sGnRH and DiC8 were potentiated by the VSCC agonist Bay K 8644, suggesting that VSCC participates in both PKC and GnRH action. Concentrations of K+ < 100 mM did not elicit GTH secretion when tested alone, but were effective in stimulating GTH release in the presence of subthreshold doses of DiC8 or TPA. This suggests that minimal activation of PKC greatly enhances the effectiveness of Ca2+ influx to increase GTH secretion. Taken together, these results indicate that PKC is an important mediator of GnRH-induced, VSCC-dependent GTH release. In contrast to the involvement of PKG, cAMP-dependent mechanisms showed no evidence of direct participation in GnRH-induced GTH release in goldfish. In static incubation studies, the GTH responses to sGnRH and cGnRH-II were not affected by H89, a cAMP-dependent protein kinase (PKA) inhibitor. Furthermore, the GTH release stimulated by cAMP was additive to the response to sGnRH, cGnRH-II, DiC8, TPA, or AA. However, compared to the response to forskolin or TPA alone, combinations of forskolin and TPA resulted in a potentiated increase in GTH release. The acute GTH response to forskolin was also enhanced by DiC8. Thus, cAMP-dependent mechanisms may constitute an independent pathway that interacts positively with GnRH-dependent mechanisms in the regulation of GTH release.

GnRH signaling in goldfish pituitary cells.

In goldfish, maturational gonadotropin (GTH) and growth hormone (GH) release are stimulated by two native GTH-releasing hormones (sGnRH and cGnRH-II). Both GnRHs stimulate GTH and GH release via activation of phospholipase C, protein kinase C, Ca2+ entry through voltage-sensitive channels and calmodulin. However, sGnRH-induced GTH release also involves arachidonic acid and intracellular Ca2+ components absent from its action on GH, as well as from cGnRH-II action on GTH and GH secretion. The relative roles and interactions of these signaling pathways in mediating sGnRH and cGnRH-II action on acute and prolonged GTH and GH release are compared. How two GnRHs bind to similar receptors but induce similar and dissimilar transduction mechanisms in two cell types and within one cell type is unknown.

Roles of calcium and calmodulin in the mediation of acute and sustained GnRH-stimulated gonadotropin secretion from dispersed goldfish pituitary cells.

The possible involvement of extracellular Ca2+ ([Ca2+]o) in mediating the acute gonadotropin (GtH) response to salmon gonadotropin-releasing hormone (sGnRH) and chicken gonadotropin-releasing hormone-II (cGnRH-II) in goldfish was examined using dispersed pituitary cells in perifusion. Perifusion with Ca(2+)-deficient medium reduced the GtH responses to 5-min pulses of either GnRH, indicating the participation of [Ca2+]o in acute GnRH action. Using a 10-min GnRH pulse application protocol, the dependence of the acute GtH responses to the two GnRHs on [Ca2+]o entry through voltage-sensitive Ca2+ channels (VSCC) was examined using the dihydropyridine VSCC blocker nifedipine and the cation Co2+. Treatment with nifedipine consistently reduced the acute GtH response to either sGnRH or cGnRH-II. Similarly, perifusion with CoCl2 reduced the sGnRH-induced GtH release. In contrast to its effects on sGnRH, CoCl2 abolished the cGnRH-II-induced GtH release. These results indicate that [Ca2+]o entry through VSCC participates in the acute GtH response to both native GnRHs; however, the cGnRH-II-stimulated acute release is relatively more dependent on [Ca2+]o and VSCC functions than sGnRH-induced secretion. The involvement of calmodulin (CaM) in mediating GnRH action was also examined. Treatment with a CaM antagonist, calmidazolium, or with a Ca2+/CaM-dependent protein kinase II inhibitor, KN62, reduced GtH responses to sGnRH and cGnRH-II in 2-hr static incubation, but not in perifusion studies with dispersed goldfish pituitary cells using 5- or 10-min GnRH pulses. These results suggest that CaM-dependent mechanisms participate in mediating the long-term, but not the acute, GtH response to GnRH. Compared to sGnRH, cGnRH-II-induced GtH release was more sensitive to inhibition by KN62, indicating a higher degree of dependence of cGnRH-II action on CaM. These results extend our understanding of the differential involvement of [Ca2+]o and CaM in mediating the short-term and long-term actions of the two native GnRH peptides on GtH release in goldfish.

Gonadotropin-releasing hormone-induced sensitization of calcium-dependent exocytosis in pituitary gonadotrophs.

Agonist-induced increases in cytoplasmic calcium concentration ([Ca2+]i) play a pivotal role in regulated exocytosis by promoting the fusion of secretory vesicles with the plasma membrane. In permeabilized and ATP-primed pituitary cells, increases in ambient [Ca2+]i stimulated the release of LH from gonadotrophs with an EC50 of 2-3 microM. In contrast, the responses of intact gonadotrophs to agonist stimulation by GnRH were characterized by transient [Ca2+]i elevations of up to 1.5 microM, followed by a plateau of 300-400 nM. The sensitivity of the exocytotic response of permeabilized cells to [Ca2+]i was significantly increased by GnRH, which reduced the EC50 for [Ca2+]i to the submicromolar concentration range. The stimulatory action of GnRH on LH release in permeabilized cells was not a consequence of intracellular Ca2+ release, but was associated with increased cytidine diphosphate diacylglycerol production. Activation of protein kinase C by phorbol esters caused a similar increase in the Ca2+ sensitivity of LH release from permeabilized cells, and this effect was not additive to that of GnRH. Furthermore, the GnRH-induced increase in the sensitivity of the exocytotic response to Ca2+ was attenuated by inhibitors of protein kinase C. These findings indicate that although elevated [Ca2+]i per se can promote LH release from permeabilized gonadotrophs, concomitant activation of protein kinase C is necessary to support exocytosis at the physiological [Ca2+]i levels that prevail in GnRH-stimulated intact cells. Such sensitization of the Ca(2+)-dependent secretory mechanism by protein kinase C may be an important step in the agonist-induced release of LH from pituitary gonadotrophs.

Interactions of cyclic adenosine 3',5'-monophosphate, protein kinase-C, and calcium in dopamine- and gonadotropin-releasing hormone-stimulated growth hormone release in the goldfish.

In the goldfish, GH release is under the stimulatory control of multiple neuroendocrine factors, including dopamine (DA) and GnRH. We have previously shown that DA-stimulated GH release in the goldfish is mediated via D1 receptors and is dependent on cAMP, whereas the GH response to GnRH involves protein kinase-C (PKC) and extracellular Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC). In this study, the interactions of PKC, cAMP, and Ca2+ in the regulation of GH release responses to DA and GnRH were examined using dispersed goldfish pituitary cells. The GnRH-induced GH secretion was unaffected by a protein kinase-A (PKA) inhibitor, but was additive to the GH responses to 8-bromo-cAMP (8-Br-cAMP), the phosphodiesterase inhibitor isobutylmethylxanthine, or the D1 agonist SKF38393. Similarly, the GH responses to PKC activators were additive to those to 8-Br-cAMP, forskolin, SKF38393, or the general DA agonist apomorphine. In contrast, depletion of cellular PKC content inhibited the GH responses to GnRH and PKC activators, but not those to DA, SKF38393, 8-Br-cAMP, or forskolin. Furthermore, the GH response to 8-Br-cAMP or SKF38393 was unaffected by the selective PKC inhibitor H7. Taken together, these results suggest that GnRH and DA D1 mechanisms stimulate GH release in the goldfish independently via PKC- and cAMP-dependent intracellular signaling pathways, respectively. However, both the cAMP (D1) and PKC (GnRH) mechanisms also interacted with VSCC. The VSCC agonist Bay K8644 potentiated, whereas the VSCC blocker verapamil reduced, the GH responses to GnRH, PKC activators, SKF38393, and activators of the cAMP pathway. A hypothesis is proposed based on these findings. GnRH and DA initiate GH release independently by activation of PKC- and cAMP-dependent mechanisms. The secondary activation of VSCC by either PKC or cAMP/PKA further enhances the GH response.

Signal transduction pathways in GnRH- and dopamine D1-stimulated growth hormone secretion in the goldfish.

In goldfish, growth hormone (GH) secretion is regulated by multiple neuroendocrine factors. Among these regulators, gonadotropin-releasing hormone (GnRH) and dopamine (DA) are effective stimulators of GH release. The stimulatory actions of GnRH and DA are mediated by GnRH and DA D1 receptors on somatotropes, respectively. In this article, results from recent in vitro pharmacological and electrophysiological studies examining the possible involvement of extracellular Ca2+, protein kinase C, voltage-sensitive Ca2+ channels (VSCC) and phospholipase A2 in mediating GnRH-induced GH release are presented. Results from experiments investigating the possible interactions of cyclic adenosine 3',5'-monophosphate (cAMP), and extracellular Ca2+ entry through VSCC in mediating the DA D1-elicited GH response are also reported. These data were discussed in conjunction with other information available in the literature on the signal transduction mechanisms mediating GH secretion in goldfish. Based on these findings, a model for the transduction pathways integrating the initiation and maintenance of the distinct GnRH-induced and DA D1-elicited GH responses was proposed. GnRH and DA stimulate GH release via separate PKC- and cAMP-dependent mechanisms, respectively. These signalling mechanisms appear to act on distinct GH pools. PKC and cAMP subsequently activate VSCC. Ca2+ entry through VSCC plays a role in the sustained GH release response by enhancing the PKC- and cAMP-induced GH release.

Downregulation of protein kinase C levels leads to inhibition of GnRH-stimulated gonadotropin secretion from dispersed pituitary cells of goldfish.

We have previously shown that the abilities of the two native goldfish GnRHs, salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), to stimulate gonadotropin (GtH) secretion and elevate intracellular Ca2+ levels are mimicked by the protein kinase C (PKC) stimulators, 1,2-dioctanoylglycerol (DiC8) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). The ability of PKC inhibitors to attenuate GnRH-stimulated GtH secretion was also demonstrated. In the present study, the involvement of PKC was examined through the reduction of cellular PKC levels by prolonged preincubation of the cells with TPA (TPA desensitization). TPA pretreatment reduced the levels of PKC in fish pituitary cells as measured by immunoblotting (Western blot). Pretreatment of dispersed goldfish pituitary cells in static culture with TPA abolished the GtH responses to sGnRH, cGnRH II and ionomycin, and drastically reduced TPA- and DiC8-stimulated GtH release, but had no major effect on forskolin-induced GtH release. TPA pretreatment also reduces the cell content of GtH in goldfish pituitary cells. Interestingly, treatment with all of the pharmacological secretagogues tested led to a decrease in cellular contents of GtH, however, the two native GnRHs had no such effect. In rapid column perifusion experiments (1-min fractions), the GtH responses induced by both native GnRHs were characterized by an initial acute increase in hormone secretion followed by a 'plateau' phase which is smaller in magnitude relative to the initial phase. TPA pretreatment of perifused cells greatly reduced both the peak and plateau phases of sGnRH- and cGnRHII-stimulated GtH secretion;TPA-induced GtH release is also greatly attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular mechanisms mediating gonadotropin and growth hormone release in the goldfish, Carassius auratus.

Evidence for the involvement of Ca(2+), protein kinase C, cAMP, and arachidonic acid metabolism in mediating gonadotropin (GTH) and growth hormone (GH) release in the goldfish is reviewed. Models for the signal transduction pathways mediating GTH-releasing hormone (GnRH) and dopamine actions on GTH and GH secretion are postulated. A novel hypothesis that two GnRHs which bind to the same receptor type activate different transduction cascade in two different cell types (GTH vs. GH) as well as within the same cell type (GTH) is presented.

Involvement of protein kinase C in the modulation of gonadotropin and growth hormone secretion from dispersed goldfish pituitary cells.

It has been established that secretion of gonadotropin (GtH) and growth hormone (GH) release in goldfish are both stimulated by GtH-releasing hormone (GnRH); in addition GtH secretion is inhibited by dopamine D2 mechanisms. In the present study, depletion of protein kinase C (PKC) in goldfish pituitary cells reduced the GtH and GH responses to GnRH and an activator of PKC in static culture. In perifusion studies, GtH released in response to sGnRH analog was greatly attenuated in PKC-depleted cells, however, hormone responses to forskolin were enhanced. Stimulation of dopamine D2 receptors reduced the GtH, but not the GH, responses elicited by PKC activators. These results indicate that PKC participates in the GtH and GH responses to natural neuroendocrine regulators in the goldfish.

Actions of two native GnRHs and protein kinase C modulators on goldfish pituitary cells. Studies on intracellular calcium levels and gonadotropin release.

Previous results indicate that the two native gonadotropin (GtH)-releasing hormones of the goldfish, sGnRH and cGnRHII, stimulate GtH secretion in an extracellular Ca2+ ([Ca2+]o) dependent manner. In the present study, sGnRH, cGnRHII, KCI and the protein kinase C (PKC) activators TPA and DiC8, stimulated increases in intracellular Ca2+ ([Ca2+]i) levels in goldfish pituitary cells. Testing in Ca(2+)-deficient medium abolished the [Ca2+]i responses to cGnRHII, TPA and KCI and attenuated responses to sGnRH and DiC8. These results are the first to demonstrate that in teleost pituitary cells both native GnRHs stimulate increases in [Ca2+]i levels via [Ca2+]o entry. sGnRH- and DiC8-stimulated increases in [Ca2+]i also appear to be partially due to mobilization of Ca2+ from intracellular stores. Other results are consistent with a role for PKC in mediating GnRH action especially extracellular Ca2+ entry. Firstly, the PKC inhibitor staurosporine decreased GnRH- and TPA-induced [Ca2+]i responses. Secondly, incubation with Ca(2+)-deficient medium attenuated TPA- and DiC8-stimulated GtH release. Thirdly, GtH release responses to PKC activators were enhanced and reduced by an agonist and an antagonist of Ca2+ channel function, respectively. However, differences in the sensitivity of DiC8- and TPA-elicited responses to manipulations of [Ca2+]o entry indicate that these two PKC activators may have different actions in the goldfish pituitary. A difference in action of the two GnRHs on mobilization of Ca2+ from intracellular stores is also indicated.

Differences in extracellular calcium involvement mediating the secretion of gonadotropin and growth hormone stimulated by two closely related endogenous GnRH peptides in goldfish pituitary cells.

Two endogenous gonadotropin-releasing hormone (GnRH) peptides, salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), stimulate gonadotropin (GtH) and growth hormone (GH) secretion in the goldfish. The extracellular calcium (e-Ca2+) dependence of the GtH and GH response to the two GnRH peptides were compared using static incubations of dispersed goldfish pituitary cells. Incubation with Ca(2+)-depleted medium (without the addition of Ca2+ salts and in the presence of EGTA) did not alter basal GtH secretion, but reduced the GtH response to sGnRH, and abolished the cGnRH II-induced GtH release. Blockade of e-Ca2+ entry by low concentrations of CoCl2 had no effect on basal GtH secretion but reduced cGnRH II and sGnRH stimulated GtH release when applied at 0.1 and 0.5 mM concentrations, respectively. In general, treatments with voltage-sensitive Ca2+ channel (VSCC) antagonists, verapamil, nifedipine and nicardipine, did not alter basal GtH release but attenuated GnRH-stimulated GtH responses. cGnRH II-induced GtH release was decreased by 10 nM verapamil and 1 nM nifedipine, whereas the reduction of GtH responses to sGnRH required 100 times higher concentrations of these VSCC antagonists. cGnRH II but not sGnRH stimulation of GtH secretion was also abolished by 10 microM nicardipine. In contrast to GtH release, exposure to Ca(2+)-depleted medium reduced basal GH release and abolished the GH responses to both GnRH peptides. sGnRH and cGnRH II-stimulated GH responses were both abolished by 0.1 mM CoCl2, decreased by 1 nM verapamil, and reduced by 10 nM nicardipine. Addition of 0.1 and 10 microM nifedipine inhibited the GH responses to sGnRH and cGnRH II, respectively. Basal GH release was not affected by the VSCC antagonists tested. Results from this study indicate that entry of e-Ca2+, in part through VSCC, is involved in GnRH stimulation of GtH and GH release from goldfish gonadotropes and somatotropes; however, the e-Ca2+ dependence of the GtH and GH responses to the two endogenous GnRHs differ. The stimulatory effects of cGnRH II on GtH secretion is more dependent on and sensitive to e-Ca2+ than sGnRH. Whereas the sensitivity of GH responses to manipulations of e-Ca2+ availability is, in most instances, similar for both GnRH peptides. These results further suggest that basal secretion of GH is more sensitive to e-Ca2+ than basal GtH release; however, VSCC are not involved in the maintenance of basal release of either hormone.

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.