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 mMdid 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 PKC, 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.     

Ghrelin-induced growth hormone release from goldfish pituitary cells involves voltage-sensitive calcium channels

Ghrelin (GRL) is a stimulator of growth hormone (GH) release in many organisms, including goldfish. As a first study to examine the signalling mechanisms mediating GRL action on GH release in goldfish, we tested the hypothesis that GLR induces GH release from goldfish pituitary cells by enhancing Ca²⁺ entry through L-type voltage-sensitive Ca²⁺ channels (LVSCCs) using perifusion GH release and fura-2/AM Ca²⁺-imaging experiments. Goldfish (g)GRL₁₉ at 1 nM elicited reversible and repeatable GH responses from dispersed goldfish mixed pituitary cultures. However, the lack of a dose-response relationship in sequential treatments with decreasing concentrations of gGRL₁₉ (ranging from 10 to 0.01 nM) implicated rapid desensitization of the GH response. Sequential applications of gGRL₁₉ (1 nM) and salmon GnRH (100 nM), a known Ca²⁺-dependent stimulator of GH release, increased intracellular free Ca²⁺ levels ([Ca²⁺]_i) from the same identified somatotropes, suggesting co-expression of GRL and GnRH receptors on single cells. In contrast, 1 nM gGRL₁₉ failed to elicit GH release and elevation in [Ca²⁺]_i when the cells are incubated with nominally Ca²⁺-free media. When GH release and [Ca²⁺]_i increases were already stimulated by the LVSCC agonist Bay K8644 (10 μM), addition of 1 nM gGRL₁₉ did not further elevate these responses. Finally, the LVSCC inhibitors nifedipine (1 μM) and verapamil (1 μM) abolished 1 nM gGRL₁₉-induced GH release responses while nifedipine eliminated gGRL₁₉-induced [Ca²⁺]_i increase. Taken together, the results of this study provide evidence that entry of extracellular Ca²⁺ through LVSCCs is a key component of the GRL signalling pathway leading to GH release in the goldfish pituitary.     

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.     

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.     

Kisspeptin-1 directly stimulates LH and GH secretion from goldfish pituitary cells in a Ca(2+)-dependent manner

It has been established that kisspeptin regulates reproduction via stimulation of hypothalamic gonadotropin-releasing hormone (GnRH) secretion, which then induces pituitary luteinizing hormone (LH) release. Kisspeptin also directly stimulates pituitary hormone release in some mammals. However, in goldfish, whether kisspeptin directly affects pituitary hormone release is controversial. In this study, synthetic goldfish kisspeptin-1((1-10)) (gKiss1) enhances LH and growth hormone (GH) release from primary cultures of goldfish pituitary cells in column perifusion. gKiss1 stimulation of LH and GH secretion were still manifested in the presence of the two native goldfish GnRHs, salmon (s)GnRH (goldfish GnRH-3) and chicken (c)GnRH-II (goldfish GnRH-2), but were attenuated by two voltage-sensitive calcium channel blockers, verapamil and nifedipine. gKiss-induced increases in intracellular Ca(2+) in Fura-2AM pre-loaded goldfish pars distalis cells were also inhibited by nifedipine. These results indicate that, in goldfish, (1) direct gKiss1 actions on pituitary LH and GH secretion exist, (2) these actions are independent of GnRH and (3) they involve Ca(2+) signalling.     

Serotonin interferes with Ca2+ and PKC signaling to reduce gonadotropin-releasing hormone-stimulated GH secretion in goldfish pituitary cells

In goldfish, two endogenous gonadotropin-releasing hormones (GnRH), salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), are thought to stimulate growth hormone (GH) release via protein kinase C (PKC) and subsequent increases in intracellular Ca²⁺ levels ([Ca²⁺]_i). In contrast, the signaling mechanism for serotonin (5-HT) inhibition of GH secretion is still unknown. In this study, whether 5-HT inhibits GH release by actions at sites along the PKC and Ca²⁺ signal transduction pathways leading to hormone release were examined in primary cultures of goldfish pituitary cells. Under static incubation and column perifusion conditions, 5-HT reduced basal, as well as sGnRH- and cGnRH-II-stimulated, GH secretion. 5-HT also suppressed GH responses to two PKC activators but had no effect on the GH-releasing action of the Ca²⁺ ionophore ionomycin. Ca²⁺-imaging studies with identified somatotropes revealed that 5-HT did not alter basal [Ca²⁺]_i but attenuated the magnitude of the [Ca²⁺]_i responses to the two GnRHs. Prior treatment with 5-HT and cGnRH-II reduced the magnitude of the [Ca²⁺]_i responses induced by depolarizing levels of K⁺. Similar inhibition, however, was not observed with prior treatment of 5-HT and sGnRH. These results suggest that 5-HT, by direct actions at the somatotrope level, interferes with PKC and Ca²⁺ signaling pathways to reduce the GH-releasing effect of GnRH. 5-HT action may occur at the level of PKC activation or its downstream signaling events prior to the subsequent rise in [Ca²⁺]_i.. The differential Ca²⁺ responses by depolarizing doses of K⁺ is consistent with our previous findings that sGnRH and cGnRH-II are coupled to overlapping and yet distinct Ca²⁺-dependent mechanisms.     

In vitro release of gonadotropin-releasing hormone from the brain preoptic-anterior hypothalamic region and pituitary of female goldfish

In vitro release of gonadotropin releasing hormone (GnRH) from slices of the preoptic-anterior hypothalamic (P-AH) region and fragments of the pituitary of goldfish was studied using a static incubation system. Release of GnRH from both tissue preparations was stimulated by depolarizing concentrations of extracellular potassium ions (K+). Other putative secretagogues, calcium ionophore A23187 (1 microM), forskolin (100 microM), and prostaglandin E2 1 microM) also stimulated release of GnRH from both tissue preparations. Omission of Ca2+, or chelating the remaining remaining Ca2+ by EGTA (0.1 mM), abolished the release of GnRH stimulated by high K+ concentrations (60 mM), but did not reduce spontaneous release. Verapamil (1 microM), a voltage-sensitive calcium channel blocker, abolished the release of GnRH stimulated by high K+ or A21387 from both tissue preparations. The GnRH released in vitro from both the P-AH region and pituitary was concentrated by Sep-Pak and then separated by high-performance liquid chromatography. The major peak of the GnRH immunoreactivity was found to coelute with synthetic salmon GnRH [( Trp7,Leu8]-GnRH) and the minor peak with chicken GnRH-II [( Gln8]-GnRH). Dopamine (10 and 100 microM) inhibited GnRH release from both P-AH slices and pituitary fragments, while serotonin (1-100 microM) stimulated release from both. Norepinephrine (10-100 microM) stimulated GnRH release from P-AH slices but not from pituitary fragments. The results demonstrate that the release of GnRH from goldfish P-AH slices and pituitary fragments in vitro in response to various secretagogues and monoamines can be studied using a static incubation system.     

In vitro goldfish growth hormone responses to gonadotropin-releasing hormone: possible roles of extracellular calcium and arachidonic acid metabolism?

Two hours of incubation of primary static cultures of dispersed goldfish pituitary cells with 0.01 nM to 1 microM [Trp7,Leu8]-gonadotropin-releasing hormone (sGnRH) increased growth hormone (GH) secretion in a dose-dependent manner with an ED50 estimate of 0.13 +/- 0.04 nM. Addition of calcium ionophores, 1 to 100 microM A23187 and 5 to 100 microM ionomycin, significantly elevated GH release with ED50s of 0.84 +/- 0.38 and 4.34 +/- 1.02 microM, respectively. Replacement of normal calcium-containing media with calcium-deficient media (prepared without the addition of calcium salts) significantly depressed basal GH secretion, attenuated the A23187- and ionomycin-stimulated GH release, and completely abolished the GH response to sGnRH. Arachidonic acid (AA) at 1 to 50 microM also enhanced GH secretion with an ED50 of 4.72 +/- 1.52 microM. Coincubation with 1 and 10 microM of a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), 10 microM of the cyclooxygenase inhibitor, indomethacin, and 10 microM of eicosatetraynoic acid, an enzyme blocker with mixed activities on both the lipoxygenase and cyclooxygenase pathways, did not alter basal, AA-, and sGnRH-induced GH release. However, at 100 microM concentration, NDGA increased AA- and sGnRH-stimulated, as well as basal GH, responses. These results confirm the direct stimulatory action of GnRH on goldfish somatotropes and indicate the importance of extracellular calcium in mediating basal and GnRH-induced GH responses. Although AA stimulates GH secretion, its lipoxygenase and cyclooxygenase metabolites probably do not mediate sGnRH action on somatotropes.     

Evidence that gonadotropin-releasing hormone also functions as a growth hormone-releasing factor in the goldfish

The present study examined the influence of GnRH on the in vivo and in vitro secretion of GH in the goldfish (Carassius auratus). Intraperitoneal injection of several GnRH peptides, including a form native to goldfish, salmon GnRH (sGnRH), elevated circulating GH levels in female goldfish. An analog of mammalian GnRH (mGnRH), [D-Ala6,Pro9-NEt] mGnRH (mGnRH-A), at a dosage of 0.1 microgram/g BW increased serum GH levels for up to 48 h after a single ip injection. Goldfish receiving a series of injections of this dose of mGnRH-A also displayed an increased rate of body growth, indicating that the mGnRH-A-induced increase in the circulating GH level was sufficient to accelerate body growth. In vitro experiments using perifused pituitary fragments found that sGnRH stimulated the secretion of GH from the goldfish pituitary in a potent, dose-dependent, and reversible manner. The time course of response and half-maximally effective dose of sGnRH were very similar for both GH and gonadotropin (GTH) secretion in vitro, suggesting that the mechanism(s) mediating the stimulatory actions of GnRH in the goldfish may be similar for both GH and GTH secretion. However, GnRH-induced GH and GTH secretion from the goldfish pituitary can occur independently of each other, as demonstrated by the finding that somatostatin inhibited the GnRH stimulation of GH secretion in vitro, without influencing the GTH response, whereas the dopamine agonist apomorphine inhibited GnRH-induced GTH secretion in vitro, without influencing the GH response. Furthermore, the dopamine antagonist pimozide did not influence serum GH levels, although pimozide potentiated the stimulatory effect of GnRH on GTH secretion in vivo by blocking the endogenous GTH release inhibitory action of dopamine. Results of the present study suggest that the secretion of GH and GTH in the goldfish are regulated, at least in part, through a common releasing factor, GnRH, whereas somatostatin and dopamine appear to act independently as GH and GTH release inhibitory factors, respectively.     

Gonadotropin-releasing hormone stimulates luteinizing hormone secretion by extracellular calcium-dependent and -independent mechanisms

The dependence of LH responses to GnRH on extracellular calcium was investigated in cultured rat pituitary cells exposed to GnRH for 3 h in static culture or for 2 min during column perifusion. During static culture in normal medium, LH release was stimulated by GnRH with an ED50 of 0.3 nM and by K+ with an ED50 of 32 mM. Incubation in Ca2+-deficient (no added Ca2+) or Ca2+-free medium (containing 100 microM EGTA) substantially decreased, but did not abolish, the LH responses to 10 and 100 nM GnRH, whereas K+-induced LH release was almost completely abolished in Ca2+-deficient medium. The Ca2+ channel agonist (BK 8644) and antagonists (nifedipine, nicardipine, verapamil, and Co2+) respectively enhanced or reduced the LH responses to both GnRH and K+. However, the calcium antagonists completely abolished the LH response to depolarization by K+, but only partially inhibited the LH response to GnRH, confirming the existence of a significant component of GnRH action that is not dependent on extracellular Ca2+. In perifused pituitary cells, exposure to Ca2+-deficient medium or normal medium containing 5 mM EGTA or 5 mM EDTA, reduced the initial rapid LH response to 2-min pulses of 10 nM GnRH and abolished the second phase of LH release. Reintroduction of Ca2+-containing medium at the end of the GnRH pulse caused recovery of the second phase of LH secretion, demonstrating that influx of extracellular Ca2+ is not required for the early phase of the LH response to GnRH but, rather, appears to be essential for its prolongation. The release of LH in response to arachidonic acid, which has been implicated in the mechanism of the secretory action of GnRH, was completely independent of extracellular Ca2+ and unaffected by addition of 10 nM BK 8644. These observations indicate that the initiation of the secretory response to GnRH is largely independent of calcium entry, whereas the prolongation of gonadotropin secretion is maintained by calcium influx, in part through voltage-sensitive calcium channels. The role of arachidonic acid metabolites in GnRH action is probably related to the calcium-independent component of GnRH-induced LH secretion. Since GnRH is secreted episodically and for short periods, much of its physiological action on pulsatile gonadotropin release could be independent of calcium influx from the extracellular fluid.     

Ghrelin-induced growth hormone release from goldfish pituitary cells is nitric oxide dependent

Ghrelin (GRLN) is an important neuroendocrine regulator of growth hormone (GH) release in vertebrates. Previous studies show goldfish (g)GRLN₁₉-induced GH from the goldfish pituitary involves voltage sensitive Ca²⁺ channels, increases in intracellular Ca²⁺ and the PKC signalling pathway. We set out to examine the role of the nitric oxide (NO) pathway in gGLRN₁₉-induced GH release from primary cultures of goldfish pituitary cells using pharmacological regulators in cell column perifusion systems. The NO scavenger PTIO abolished gGRLN₁₉-induced GH release and co-treatment with the NO donor SNP and GRLN did not produce additive GH release responses. Nitric oxide synthase (NOS) inhibitors 1400 W and 7-Ni abolished GRLN-induced GH release while treatment with another NOS inhibitor, AGH, had no significant effect. Taken together, these results demonstrate that the NOS/NO is an integral component of gGRLN₁₉-induced signalling within the goldfish pituitary cells, and given the relative specificity of AGH for inducible NOS and endothelial NOS isoforms, suggests that neuronal NOS is the likely NOS isoform utilized in goldfish somatotropes by this physiological regulator.     

Differential involvement of signaling pathways in the regulation of growth hormone release by somatostatin and growth hormone-releasing hormone in orange-spotted grouper (Epinephelus coioides)

Somatostatin is the most effective inhibitor of GH release, and GHRH was recently identified as one of the primary GH-releasing factors in teleosts. In this study, we analyzed the possible intracellular transduction pathways that are involved in the mechanisms induced by SRIF and GHRH to regulate GH release. Using a pharmacological approach, the blockade of the PLC/IP/PKC pathway reversed the SRIF-induced inhibition of GH release but did not affect the GHRH-induced stimulation of GH release. Furthermore, SRIF reduced the GH release induced by two PKC activators. Inhibitors of the AC/cAMP/PKA pathway reversed both the SRIF- and GHRH-induced effects on GH release. Moreover, the GH release evoked by forskolin and 8-Br-cAMP were completely abolished by SRIF. The blockade of the NOS/NO pathway attenuated the GHRH-induced GH release but had minimal effects on the inhibitory actions of SRIF. In addition, inhibitors of the sGC/cGMP pathway did not modify the SRIF- or GHRH-induced regulation of GH release. Taken together, these findings indicate that the SRIF-induced inhibition of GH release is mediated by both the PLC/IP/PKC and the AC/cAMP/PKA pathways and not by the NOS/NO/sGC/cGMP pathway. In contrast, the GHRH-induced stimulation of GH secretion is mediated by both the AC/cAMP/PKA and the NOS/NO pathways and is independent of the sGC/cGMP pathway and the PLC/IP/PKC system.     

Free intracellular Ca2+ concentration and growth hormone (GH) release from purified rat somatotrophs. III. Mechanism of action of GH-releasing factor and somatostatin.

GH-releasing factor (GRF)-stimulated GH release is dependent on a biphasic increase in free intracellular Ca2+ concentration [( Ca2+]i), resulting from an influx of Ca2+ into somatotrophs, while the inhibitory action of somatostatin (SRIF) on basal and GRF-induced GH release results from its ability to lower [Ca2+]i by inhibiting Ca2+ influx. This study was carried out to investigate the mechanism by which GRF and SRIF regulate [Ca2+]i to control GH release. The roles of ion channels, cAMP-dependent processes, and protein kinase-C (PKC) were investigated by measuring changes in [Ca2+]i, 45Ca influx, and GH release when purified rat somatotrophs were exposed to high K+, cAMP analogs, prostaglandin E2, as well as the PKC activators 1,2-dioctanoyl-glycerol and phorbol 12-myristate 13-acetate. High K+ depolarization produced a rapid and transient increase in [Ca2+]i, while cAMP and prostaglandin E2 led to a sustained elevated [Ca2+]i. PKC activators produced a transient increase in [Ca2+]i, followed by a decrease to below baseline. All secretagogues tested raised [Ca2+]i by stimulating Ca2+ influx through L-type voltage-sensitive Ca2+ channels (VSCC), since the increases in [Ca2+]i were blocked by incubation in Ca2(+)-free medium and by the dihydropyridine Ca2+ antagonist nifedipine. SRIF lowered [Ca2+]i by blocking the Ca2+ influx stimulated by all of these GH secretagogues except high K+. These results are consistent with the model in which GRF initiates its action by increasing Na+ conductance to depolarize the somatotroph via cAMP. This depolarization would stimulate Ca2+ influx through VSCC, which would result in the first phase of the GRF-dependent increase in [Ca2+]i. This increase in [Ca2+]i would stimulate Ca2+ removal from the cytosol by activating Ca-ATPase via Ca-calmodulin and/or PKC. This would result in the lowering of [Ca2+]i to the plateau level of the second phase of the GRF response. SRIF prevents the GRF-induced increase in [Ca2+]i by increasing K+ conductance and, thus, hyperpolarizing the cell. Hyperpolarization would close VSCC, leading to a decrease in Ca2+ influx, with a subsequent drop in [Ca2+]i.     

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.     

Inhibition of hypothalamic somatostatin release by beta-adrenergic antagonists

A number of in vivo studies suggest that hypothalamic somatostatin (SRIF) tone is stimulated by the beta-adrenergic system. Employing dispersed adult male rat hypothalamic cells, we studied the effects of beta-adrenergic antagonists on the release of hypothalamic SRIF. Propranolol, at concentrations of 1-100 microM, had no detectable effect on basal SRIF release, but caused dose-dependent inhibition of SRIF release stimulated by ouabain. Two other beta-adrenergic antagonists, labetolol and metoprolol, also caused inhibition of ouabain-stimulated SRIF release. The alpha 2-agonist clonidine was without effect on SRIF release under basal or stimulated conditions. GH secretion from monolayers of dispersed rat anterior pituitary cells was also examined. Propranolol (1-100 microM) had no significant effect on basal GH secretion or GH secretion stimulated by rat GRF. In conclusion, 1) beta-adrenergic antagonists caused inhibition of stimulated SRIF release; 2) clonidine had no detectable effect on SRIF release; and 3) propranolol did not affect GH secretion in vitro. These findings support the hypothesis that beta-adrenergic antagonists augment GH responsivity by inhibiting hypothalamic SRIF release.     

Somatostatin stimulates GH secretion in two porcine somatotrope subpopulations through a cAMP-dependent pathway

Somatostatin (SRIF) inhibits GH release from rat somatotropes by reducing adenylate cyclase (AC) activity and the free cytosolic calcium concentration ([Ca(2+)](i)). In contrast, we have reported that SRIF can stimulate GH release in vitro from pig somatotropes. Specifically, 10(-7) and 10(-15) M SRIF stimulate GH release from a subpopulation of high density (HD) somatotropes isolated by Percoll gradient centrifugation, whereas in low density (LD) somatotropes only 10(-15) M SRIF induces such an effect. To ascertain the signaling pathways underlying this phenomenon, we assessed SRIF effects on second messengers in cultured LD and HD cells by measuring cAMP, IP turnover, and [Ca(2+)](i). Likewise, contribution of the corresponding signaling pathways to SRIF-induced GH release was evaluated by blocking AC, PLC, extracellular Ca(2+) influx, or intracellular Ca(2+) mobilization. Both 10(-7) and 10(-15) M SRIF increased cAMP, IP turnover, and [Ca(2+)](i) in HD cells. Conversely, in LD cells 10(-7) M SRIF reduced [Ca(2+)](i), but did not alter cAMP or IP, and 10(-15) M SRIF was without effect. Interestingly, SRIF-stimulated GH release was abolished in both subpopulations by AC blockade, but not by PLC inhibition. Furthermore, SRIF-induced GH release was not reduced by blockade of extracellular Ca(2+) influx through voltage-sensitive channels or by depletion of thapsigargin-sensitive intracellular Ca(2+) stores. Therefore, SRIF stimulates GH secretion from cultured porcine somatotrope subpopulations through an AC/cAMP pathway-dependent mechanism that is seemingly independent of net increases in IP turnover or [Ca(2+)](i). These novel actions challenge classic views of SRIF as a mere inhibitor for somatotropes and suggest that it may exert a more complex, dual function in the control of porcine GH release, wherein molecular heterogeneity of somatotropes would play a critical role.     

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

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

The assessment of growth hormone status in normal young adult males using a variety of provocative agents

OBJECTIVE In adults, there are few data regarding GH responses to provocative stimuli other than insulin-induced hypoglycaemia. We have compared the GH response to four different growth hormone secretagogues and placebo in normal healthy adult males. DESIGN This was a prospective, randomized, placebo-controlled study in 18 normal male subjects. After an overnight fast, an intravenous cannula was inserted into the arm of each subject and a blood sample was taken for GH at ?30, ?15, and 0 minutes. Four provocative agents (intravenous insulin 0.2 IU/kg; intravenous arginine 20 g/m² as an infusion over 30 minutes; oral clonidine, either 100 or 200 μg; intramuscular glucagon 1 mg) and placebo were administered to each subject in a randomized manner on different days. Further blood samples were taken at 15-minute intervals for 180 minutes for GH estimation. RESULTS The median (range) GH peak response for each agent was insulin 107.7 (28.1–200) mU/l; arginine 22.3 (3.1–72.9) mU/l; glucagon 42 (11.8–200) mU/l; 100 μg clonidine 7.2 (<1–22.2) mU/l; 200 μg clonidine 8.2 (1.1–88) mU/l and placebo 2.4 (<1–30.2) mU/l. The peak GH response to insulin-induced hypoglycaemia (ITT) was significantly greater than for any other agent (P<0.0001). The peak GH response to glucagon was significantly greater than for arginine (P<0.05), clonidine at 100 and 200 μg (P<0.01) and placebo (P<0.01). The peak GH response following administration of arginine was significantly greater than for clonidine 100 and 200 μg (P<0.05), and placebo (P<0.01). The peak GH response following clonidine 200 μg was not significantly greater than following clonidine 100 μg (P=0.38) or placebo. On an individual basis two, six and 15 of 18 subjects failed to achieve a peak GH level of >20 mU/l to glucagon, arginine and clonidine respectively. In complete contrast only one subject achieved a peak response of less than 40 mU/l (28.1 mU/l) to ITT. CONCLUSIONS The most profound GH release is seen after insulin-induced hypoglycaemia. Glucagon appears to be more effective at inducing GH release than arginine. Clonidine at a dose of 100 or 200 μg is no more effective than placebo.     

Somatostatin actions on a protein kinase C-dependent growth hormone secretagogue cascade

In mammals, the ability of somatostatin (SS) to block growth hormone (GH) secretion is due, in part, to the inhibition of two key intracellular mediators, cAMP and Ca2+. We examined whether or not inhibition of Ca2+ signaling was mediating SS-induced inhibition basal, as well as gonadotropin-releasing hormone (GnRH; a protein kinase C (PKC)-dependent growth hormone secretagogue)-stimulated growth hormone (GH) release. Although SS reduced basal GH release from populations of pituitary cells, parallel reductions in [Ca2+]i were not observed within single, identified somatotropes. Similarly, application of GnRH and the PKC activator DiC8 elicited increases in [Ca2+]i and GH release, but abolition of the Ca2+ responses did not accompany SS inhibition of the GH responses. Surprisingly, while DiC8 potentiated SS inhibition of GH release, SS paradoxically increased DiC8-stimulated increases in [Ca2+]i. These data establish that abolition of Ca2+ signals is not a primary mechanism through which SS lowers basal, or inhibits GnRH-stimulated hormone release.     

Dioxin (2,3,7,8-tetrachlorodibenzo-p-doxin) enhances triggered afterdepolarizations in rat ventricular myocytes

The effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) on action potential and afterdepolarizations were studied in rat ventricular myocytes using nystatin-perforated whole-cell patch-clamp technique. TCDD treatment, in the concentration range of 1 to 100 nM, significantly prolonged action potential duration measured at 90% of repolarization (APD₉₀). The triggered delayed-afterdepolarizations (DADs) were observed in 6 out of 8 cells after exposure of TCDD (10 nM). In the presence of isoproterenol (ISO, 10 nM) or Bay K 8644 (1 μM), TCDD (10 nM) markedly augmented the amplitude and frequency of the arrhythmogenic DADs and triggered sustained spontaneous firings in ventricular myocytes. Voltage-clamp data indicated that TCDD (10 nM) exposure significantly enhanced the transient inward current (I_ti). The triggered early-afterdepolarizations (EADs) were evoked only in cells simultaneously exposed to TCDD (10 nM) and ISO (or Bay K8644). Further study indicated that TCDD treatment increased l-type Ca²⁺ current. These results indicate that activation of TCDD signaling pathway can prolong action potential duration and cause abnormal triggered afterdepolarizations. These effects may lead to clinically relevant ventricular arrhythmia especially when susceptible individuals are under elevated sympathetic stress or suffering from other myocardiopathies coincided with Ca²⁺-overload. Part of the work was presented at the 42nd Annual Meeting of SOT.