Regulation of growth hormone release in common carp pituitary cells by pituitary adenylate cyclase-activating polypeptide: signal transduction involves cAMP- and calcium-dependent mechanisms

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon/secretin peptide family and its molecular structure is highly conserved among vertebrates. In this study, the role of PACAP in regulating growth hormone (GH) secretion in fish was examined in vitro using common carp pituitary cells under column perifusion. A dose-dependent increase in GH release was observed after exposing pituitary cells to increasing doses of ovine PACAP38 (oPACAP38) and PACAP27 (oPACAP27), but not vasoactive intestinal polypeptide (VIP). A lack of GH response to VIP stimulation is consistent with the pharmacological properties of PAC-1 receptors, suggesting that this receptor subtype may be involved in PACAP-induced GH secretion in carp species. Although the maximal GH responses induced by oPACAP38 and oPACAP27 were similar, the minimal effective dose and ED50 value for oPACAP38 were significantly lower than that for oPACAP27. These results may indicate that common carp PAC-1 receptors are more sensitive to stimulation by oPACAP38 than by oPACAP27. In parallel studies, oPACAP38 and oPACAP27 were also effective in increasing cAMP release, cellular cAMP content, total cAMP production, and intracellular Ca(2+) ([Ca(2+)](i)) levels in common carp pituitary cells. Besides, the rise in [Ca(2+)](i) induced by oPACAP38 was blocked by removing extracellular Ca(2+) ([Ca(2+)](e)) or by treatment with nifedipine, an inhibitor of voltage-sensitive Ca(2+) channels (VSCC). The dose dependence of PACAP-stimulated GH release in common carp pituitary cells was mimicked by activating adenylate cyclase using forskolin, inhibiting cAMP degradation using IBMX, increasing functional levels of intracellular cAMP using CPT-cAMP, or inducing [Ca(2+)](e) entry using the Ca(2+) ionophore A23187. In contrast, the GH-releasing effect of oPACAP38 was suppressed by treatment with the adenylate cyclase inhibitor MDL12330A, protein kinase A inhibitor H89, and VSCC blocker nifedipine, or by perifusion with a Ca(2+)-free culture medium. These results, as a whole, suggest that PACAP functions as a GH-releasing factor in common carp by activating pituitary receptors resembling mammalian PAC-1 receptors. Apparently, the GH-releasing action of PACAP is mediated through the adenylate cyclase/cAMP/protein kinase A pathway and [Ca(2+)](e) influx through VSCC.     

Stimulation of luteinizing hormone release by gamma-aminobutyric acid (GABA) agonists: mediation by GABAA-type receptors and activation of chloride and voltage-sensitive calcium channels

The mechanism by which gamma-aminobutyric acid (GABA) stimulates the release of LH was analyzed in cultured female rat pituitary cells. In 3-h incubations, GABA (1-100 microM) caused a dose-dependent increase in LH release, with the maximal response about 16% of that evoked by 10 nM GnRH. GABA action was independent of the GnRH receptor, since 1 microM GnRH antagonist [( N-acetyl-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10] GnRH), which completely inhibits GnRH action, did not affect the response to GABA. In studies on the effects of GABA receptor agonists and antagonists, 4,5,6,7-tetrahydoisoxazolo-[5,4-c]pyridin-3(2H)-one (THIP) and muscimol (GABAA agonists) gave similar response patterns, with the same maximal stimulation as GABA but much higher potencies. In contrast, the GABAB receptor agonist baclofen did not stimulate LH release. The GABAA receptor antagonist SR95531 caused dose-dependent inhibition of the LH-releasing effects of GABA and muscimol (10 microM), with complete blockade at 10 microM SR95531. T-Butylbicyclophosphorothionate, an inhibitor of the GABAA receptor-associated chloride channel, also dose-dependently reduced the releasing effect of 100 microM GABA. These results indicate that GABA action is mediated by the chloride channel-associated GABAA receptor. However, the other GABAA receptor antagonists, including bicuculline, picrotoxin, and strychnine, did not attenuate the LH-releasing effect of 100 microM GABA in concentrations up to 100 microM, suggesting that GABA action is mediated by nonclassical GABAA receptors. Incubation in the presence of nifedipine (1 microM) or in calcium-free medium inhibited the LH-releasing action of GABA, indicating that calcium influx through voltage-sensitive calcium channels (VSCC) is required for GABA-induced LH release. Such entry of Ca2+ would result from activation of VSCC by depolarization due to the increased Cl- conductance caused by GABAA receptor activation. In cell perfusion studies, the actions of GABA and muscimol were attenuated or abolished after repetitive stimulation, consistent with desensitization of the GABA receptors. These findings have demonstrated that the stimulation of LH release by GABA is independent of GnRH action, occurs via binding to nonclassical GABAA receptors, which rapidly desensitize, and is mediated by the activation of VSCC.     

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

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

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.     

GABA inhibition of immortalized gonadotropin-releasing hormone neuronal excitability involves GABA(A) receptors negatively coupled to cyclic adenosine monophosphate formation

γ-Aminobutyric acid (GABA) has been implicated in the regulation of reproduction, particularly in the developmental modulation of gonadotropin-releasing hormone (GnRH) secretion. GnRH neurons are innervated by GABA-containing processes, and the administration of GABA stimulates and inhibits GnRH secretion in vivo and in vitro. We have previously shown that GABA can exert both of these actions in sequence, by acting directly on immortalized GnRH neurons. While the stimulation is the result of a GABA_A receptor-mediated depolarization of the plasma membrane, the mechanism involved in the delayed inhibition is the subject of the present investigation. GABA (1 nM-10 μM) decreased the intracellular concentration of cyclic adenosine monophosphate (cAMP) in a dose- and time-dependent fashion. This effect was blocked by bicuculline and mimicked by muscimol but not by baclofen. To analyze the effect of GABA on cellular excitability, we used fura-2 loaded GT1-7 cells. Activation of voltage-sensitive calcium channels by high K⁺-induced depolarization (35 mM) increased [Ca²⁺]_i. GABA (10μM) and muscimol (10 μM) reduced the amplitude of K⁺-induced [Ca²⁺]_i transients. This inhibition was blocked by forskolin (20μM) or 8-Br-cAMP (1 mM). Altogether, these results show that GABA_A receptors mediate a sustained inhibitory effect of GABA on GnRH neurons, and suggest the involvement of the cAMP pathway decreasing cellular excitability.     

GABAergic influences on somatostatin secretion from hypothalamic neurons cultured in defined medium.

The effects of GABAergic influences intrinsic to the hypothalamus on the secretion of somatostatin were studied using cultured fetal rat hypothalamic neurons. The existence of GABAergic neurons within the cultures was confirmed by immunocytochemistry. These neurons appeared to be actively secreting GABA as antagonism of GABAA receptors with bicuculline and picrotoxinin caused a dose-dependent increase in the release of immunoreactive somatostatin (SRIF), which was Ca(2+)-dependent. Although exogenous GABA inhibited SRIF secretion at concentrations of 10(-6) M and greater, muscimol, a GABAA agonist, inhibited SRIF release at 10(-8) M, whereas baclofen, a GABAB agonist, required concentrations two orders of magnitude greater to produce an effect. Phaclofen, a GABAB antagonist, was inactive (10(-8)-10(-4) M). A GABA uptake inhibitor, SKF 89976A, produced a dose-dependent inhibition of SRIF release. These results, therefore, support a role for intrahypothalamic GABA neurons in the regulation of SRIF secretion in the neonatal rat, predominantly via a type A receptor, and provide further evidence for a neuroendocrine role for GABA in controlling growth hormone secretion.     

Regulation of the GABA(A) receptor by nitric oxide in frog pituitary melanotrophs

Nitric oxide (NO) is implicated in the regulation of various endocrine functions, but the effect of NO on GABA(A) receptor transmission has never been reported in endocrine cells. In the present study, we have investigated the effects of various agents acting on the NO transduction pathway on GABA(A) receptor function in frog pituitary melanotrophs. Histochemical studies using the NADPH-diaphorase reaction and immunohistochemical labeling with antibodies against neuronal NO synthase (nNOS) revealed that nNOS is expressed in the intermediate lobe of the pituitary and in cultured melanotrophs. Whole-cell patch-clamp recordings showed that the specific substrate of NOS L-arginine (L-Arg, 10(-4) M) or the NO donor sodium nitroprusside (10(-5) M) provoked a long-lasting inhibition of the current evoked by GABA (5 x 10(-6) M). The NOS inhibitor L-nitroarginine (10(-5) M) produced a biphasic effect, i.e. a transient decrease followed by a delayed increase of the GABA-evoked current amplitude. Similarly, the specific nNOS inhibitor 7-nitroindazole and the specific inducible NOS (iNOS) inhibitor aminoguanidine (10(-5) M each) provoked a transient depression of the current followed by a sustained potentiation. Formation of cGMP in neurointermediate lobes was enhanced by L-Arg (10(-4) M) and by the calcium-releasing agent caffeine (10(-4) M), and inhibited by the calmodulin (CaM)/Ca2+ complex blocker W7 (10(-5) M). The GABA-evoked current was potentiated by the guanylyl cyclase inhibitor ODQ (10(-8)-10(-7) M) and inhibited by the protein kinase G (PKG) activator 8pCPT-cGMP (3 x 10(-7)-3 x 10(-5) M). The present data indicate that NO, produced by a CaM/Ca2+-dependent NOS in frog melanotrophs, exerts an autocrine inhibitory effect on the GABA-evoked current. The action of NO on the GABA(A) receptor function is mediated through activation of the cGMP/PKG pathway.     

Gonadotropin-releasing hormone-induced calcium signaling in clonal pituitary gonadotrophs.

In agonist-stimulated clonal pituitary gonadotrophs (alpha T3-1 cells), cytoplasmic calcium ([Ca2+]i) exhibited rapid and prominent peak increases, followed by lower, but sustained, elevations for up to 15 min. The [Ca2+]i response to GnRH was rapidly inhibited by prior addition of a potent GnRH antagonist. In the absence of extracellular Ca2+ the initial peak [Ca2+]i response was only slightly decreased, but the prolonged increase in [Ca2+]i was abolished, indicating that the peak is derived largely from intracellular calcium mobilization and the sustained phase from Ca2+ influx. Application of the endoplasmic reticulum Ca(2+)-ATPase blocker thapsigargin caused progressive and dose-dependent elevation of [Ca2+]i and decreased the peak amplitude of the GnRH-induced Ca2+ response. On the other hand, addition of dihydropyridine calcium channel antagonists before or after GnRH treatment prevented or terminated the plateau phase, respectively, consistent with entry of Ca2+ through L-type voltage-sensitive Ca2+ channels (VSCC) as the major Ca2+ influx pathway during GnRH action. The presence of L-type VSCC in alpha T3-1 cells was further indicated by the ability of elevated extracellular K+ levels and the dihydropyridine calcium channel agonist Bay K 8644 to elevate [Ca2+]i in an extracellular calcium-dependent manner. These actions of depolarization and Bay K 8644 were inhibited by nifedipine, with an IC50 of 10 nM. High extracellular K(+)- and GnRH-induced Ca2+ entry was also attenuated by phorbol esters and permeant diacylglycerols, indicating that protein kinase-C exerts inhibitory modulation of VSCC activity. In contrast to normal pituitary gonadotrophs, in which GnRH induces a frequency-modulated oscillatory [Ca2+]i response, single alpha T3-1 cells exhibited a nonoscillatory amplitude-modulated signal during agonist stimulation. The [Ca2+]i responses observed in alpha T3-1 gonadotrophs indicate that the immortalized cells retain functional GnRH receptors and their coupling to the Ca2+ signaling pathway. Ca2+ influx through L-type channels maintains the plateau phase of the [Ca2+]i response during agonist stimulation and is inhibited by activation of protein kinase-C.     

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

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

Ionophore bromo-A23187 reveals cellular calcium stores in single pituitary somatotropes

Free cytosolic calcium concentration, [Ca2+]i, in single rat pituitary cells can be measured with the fluorescent, calcium-sensitive probe fura-2 and digital image analysis. A reverse hemolytic plaque assay (RHPA) identifies somatotropes in the mixed population of pituitary cells. Previous studies showed that growth hormone releasing factor (GRF) stimulates growth hormone (GH) release from pituitary somatotropes by increasing the influx of calcium into the cell. Somatostatin reduced [Ca2+]i and inhibits hormone release presumably by closing calcium channels in the membrane. The calcium-ionophore bromo-A23187 rapidly increased [Ca2+]i from a baseline of 226 +/- 38 nM to a peak of 842 +/- 169 nM (mean +/- SEM) which was reached 30 s after exposure to the drug. This spike was followed by a sustained phase of elevated [Ca2+]i approximately 370 nM. When somatostatin (SRIF) (10 nM) was combined with ionophore treatment, the initial rise was preserved. However, the second phase was abolished and SRIF lowered [Ca2+]i to 57 +/- 7 nM. Depolarizing the cellular membrane with high extracellular potassium (60 mM) increased cytosolic calcium as well (797 +/- 178 nM); however, this was not affected by the addition of SRIF (988 +/- 71 nM). KCl depolarization in calcium-free medium (+1.5 mM EGTA) provoked no rise in cytosolic calcium. In contrast, after ionophore, the initial spike was preserved while the sustained phase of elevated [Ca2+]i was abolished. We conclude from these data that (1) membrane depolarization and ionophore treatment lead to an influx of calcium into the cytosol of normal pituitary somatotropes. (2) SRIF inhibits calcium influx induced by ionophore but not influx after depolarization with high potassium concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit composition and pharmacological characterization of gamma-aminobutyric acid type A receptors in frog pituitary melanotrophs

The frog pars intermedia is composed of a single population of endocrine cells directly innervated by gamma-aminobutyric acid (GABA)ergic nerve terminals. We have previously shown that GABA, acting through GABA(A) receptors, modulates both the electrical and secretory activities of frog pituitary melanotrophs. The aim of the present study was to take advantage of the frog melanotroph model to determine the relationship between the subunit composition and the pharmacological properties of native GABA(A) receptors. Immunohistochemical labeling revealed that in situ and in cell culture, frog melanotrophs were intensely stained with alpha2-, alpha3-, gamma2-, and gamma3-subunit antisera and weakly stained with a gamma1-subunit antiserum. Melanotrophs were also immunolabeled with a monoclonal antibody to the beta2/beta3-subunit. In contrast, frog melanotrophs were not immunoreactive for the alpha1-, alpha5-, and alpha6-isoforms. The effects of allosteric modulators of the GABA(A) receptor on GABA-activated chloride current were tested using the patch-clamp technique. Among the ligands acting at the benzodiazepine-binding site, clonazepam (EC50, 5 x 10(-9) M), diazepam (EC50, 10(-8) M), zolpidem (EC50, 3 x 10(-8) M), and beta-carboline-3-carboxylic acid methyl ester (EC50, 10(-6) M) were found to potentiate the whole cell GABA-evoked current in a dose-dependent manner. Methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (IC50, 3 x 10(-5) M) inhibited the current, whereas Ro15-4513 had no effect. Among the ligands acting at other modulatory sites, etomidate (EC50, 2 x 10(-6) M) enhanced the GABA-evoked current, whereas 4'-chlorodiazepam (IC50, 4 x 10(-7) M), ZnCl2 (IC50, >5 x 10(-5) M), and furosemide (IC50, >3 x 10(-4) M) depressed the response to GABA. PK 11195 did not affect the GABA-evoked current or its inhibition by 4'-chlorodiazepam. The results indicate that the native GABA(A) receptors in frog melanotrophs are formed by combinations of alpha2-, alpha3-, beta2/3-, gamma1-, gamma2-, and gamma3-subunits. The data also demonstrate that clonazepam is the most potent, and zolpidem is the most efficient positive modulator of the native receptors. Among the inhibitors, 4'-chlorodiazepam is the most potent, whereas ZnCl2 is the most efficient negative modulator of the GABA(A) receptors. The present study provides the first correlation between subunit composition and the functional properties of native GABA(A) receptors in nontumoral endocrine cells.     

Oleic acid glucose-independently stimulates glucagon secretion by increasing cytoplasmic Ca2+ via endoplasmic reticulum Ca2+ release and Ca2+ influx in the rat islet alpha-cells

The effect of long-chain free fatty acids on glucagon secretion from islet alpha-cells has been a controversial issue. This study examined direct effects of oleic acid (OA) on glucagon release from rat pancreatic islets and on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in single alpha-cells by fura-2 fluorescence imaging. OA at 30 microM increased glucagon release from isolated islets in the presence of low (2.8 mM) and elevated (8.3 mM) glucose concentrations. OA at 6-10 microm concentration-dependently increased [Ca(2+)](i) in alpha-cells, irrespective of glucose concentrations (1.4, 2.8, and 8.3 mM). OA at 10 mum increased [Ca(2+)](i) in 90% of alpha-cells. OA-induced [Ca(2+)](i) increases were strongly inhibited by the endoplasmic reticulum Ca(2+)-pump inhibitors cyclopiazonic acid and thapsigargin and by 2-aminoethoxydiphenyl borate, the blocker of both inositol 1,4,5-trisphosphate receptors and store-operated Ca(2+) channels. Furthermore, the amplitude, but not incidence, of OA-induced [Ca(2+)](i) increases was reduced substantially by Ca(2+)-free conditions and mildly by an L-type Ca(2+) channel blocker, nitrendipine, and an ATP-sensitive K(+) channel activator, diazoxide. OA-induced glucagon release was also inhibited mildly by nitrendipine and strongly by 2-aminoethoxydiphenyl borate. These results indicate that OA glucose-independently stimulates glucagon release by increasing [Ca(2+)](i) in rat pancreatic alpha-cells and that the [Ca(2+)](i) increase is triggered by Ca(2+) release from endoplasmic reticulum and amplified by Ca(2+) influx possibly via store-operated channels and via voltage-dependent L-type Ca(2+) channels. The glucose-independent action of OA to stimulate glucagon release from alpha-cells may operate under hypoglycemic conditions when plasma free fatty acids levels are elevated, possibly playing a role in maintaining glucose metabolism.     

Goldfish brain somatostatin-28 differentially affects dopamine- and pituitary adenylate cyclase-activating polypeptide-induced GH release and Ca(2+) and cAMP signals

Dopamine (DA) and pituitary adenylate cyclase-activating polypeptide (PACAP) stimulate goldfish growth hormone (GH) release via cAMP- and Ca(2+)-dependent pathways while DA also utilizes NO. In this study, identified goldfish somatotropes responded to sequential applications of PACAP and the DA D1 agonist SKF38393 with increased intracellular Ca(2+) levels ([Ca(2+)](i)), indicating that PACAP and DA D1 receptors were present on the same cell. A native goldfish brain somatostatin (gbSS-28) reduced SKF38393-stimulated cAMP production and PACAP- and NO donor-elicited GH and [Ca(2+)](i) increases, but not PACAP-induced cAMP production nor the GH and [Ca(2+)](i) responses to forskolin, 8-bromo-cAMP and SKF38393. gbSS-28 might inhibit PACAP-induced GH release by interfering with PACAP's ability to increase [Ca(2+)](i) in a non-cAMP-dependent manner. However, DA D1 receptor activation bypassed gbSS-28 inhibitory effects on cAMP production and NO actions via unknown mechanisms to maintain a normal [Ca(2+)](i) response leading to unhampered GH release.     

Inhibitory control of growth hormone secretion by somatostatin in rat pituitary GC cells: sst(2) but not sst(1) receptors are coupled to inhibition of single-cell intracellular free calcium concentrations

Rat pituitary tumor cells (GC cells) exhibit spontaneous oscillations of intracellular free calcium concentration ([Ca(2+)](i)) that allow continuous release of growth hormone (GH). Of the somatostatin (SRIH) receptor subtypes (sst receptors) mediating SRIH action, sst(1) and sst(2) receptors are highly expressed by GC cell membranes. In the present study, the effects of sst(1) or sst(2) receptor activation on single-cell [Ca(2+)](i) were investigated in GC cells by confocal fluorescence microscopy. In addition, the effects of sst(1) or sst(2) receptor activation on GH secretion were also studied. Our results demonstrate that SRIH decreases [Ca(2+)](i) baseline and almost completely blocks Ca(2+) transients through activation of sst(2) but not of sst(1) receptors. In contrast, SRIH effectively inhibits GH secretion through activation of both sst(1) and sst(2) receptors. Blocking Ca(2+) transients is less efficient than SRIH to inhibit GH release. The cyclic octapeptide, CYN-154806, antagonizes sst(2) receptors at [Ca(2+)](i) since it abolishes the sst(2) receptor-mediated inhibition of [Ca(2+)](i) without affecting single-cell Ca(2+) signals. On the other hand, CYN-154806 alone potently inhibits GH secretion through the involvement of pertussis toxin-sensitive G proteins. In conclusion, the present results demonstrate that SRIH inhibition of GH release in GC cells involves mechanisms either dependent or independent on SRIH modulation of [Ca(2+)](i). The implications of CYN-154806 inhibition of GH secretion are discussed.     

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.     

Receptors for neuropeptide Y, gamma-aminobutyric acid and dopamine differentially regulate Ca2+ currents in Xenopus melanotrope cells via the G(i) protein beta/gamma-subunit

Secretion of alpha-melanophore-stimulating hormone (alphaMSH) from pituitary melanotrope cells of the amphibian Xenopus laevis is under inhibitory synaptic control by three neurotransmitters produced by the suprachiasmatic nucleus: gamma-aminobutyric acid (GABA), neuropeptide Y (NPY) and dopamine (DA). These inhibitory effects occur through G(i)-protein-coupled receptors (G(i)PCR), and differ in strength: GABA(B)-receptor-induced inhibition is the weakest, whereas DA (via a D2-receptor) and NPY (via a Y1-receptor) strongly inhibit, with NPY having a long-lasting effect. Previously it was shown that DA inhibits two (R- and N-type channel) of the four voltage-operated Ca2+ channels in the melanotrope, and that only part of this inhibition is mediated by beta/gamma-subunits of the G(i) protein. We here demonstrate that also the Y1- and GABA(B)-receptor inhibit only part of the total Ca2+ current (I(Ca)), with fast activation and inactivation kinetics. However, GABA(B)-mediated inhibition is weaker than the inhibitions induced via Y1- and D2-receptors (-21 versus -27% and -30%, respectively). Using a depolarizing pre-pulse protocol it was demonstrated that GABA(B)-induced inhibition of I(Ca) most likely depends on Gbeta/gamma-subunit activation whereas Y1- and D2- induced inhibitions are only partially mediated by Gbeta/gamma-subunits. No differences were found between the Y1- and D2-induced inhibitions. These results imply that activation of different G(i)PCR inhibits the I(Ca) through different mechanisms, a phenomenon that may underlie the different potencies of the suprachiasmatic neurotransmitters to inhibit alphaMSH secretion.     

Stimulatory effect of ghrelin on isolated porcine somatotropes

Research on the mechanism for growth hormone secretagogue (GHS) induction of growth hormone secretion led to the discovery of the GHS receptor (GHS-R) and later to ghrelin, an endogenous ligand for GHS-R. The ability of ghrelin to induce an increase in the intracellular Ca(2+) concentration - [Ca(2+)](i) - in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone. Ghrelin increased the [Ca(2+)](i) in a dose-dependent manner in 98% of the cells that responded to human growth hormone releasing hormone. In the presence of (D-Lys(3))-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca(2+)](i) evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca(2+)](i). The stimulatory effect of ghrelin on somatotropes was greatly attenuated in low-calcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na(+) solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca(2+)](i) transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The nonpeptidyl GHS, L-692,585 (L-585), induced changes in [Ca(2+)](i) similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca(2+)](i). Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Simultaneous application of ghrelin and L-585 did not cause an additive increase in [Ca(2+)](i). Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion.     

In vivo and in vitro studies of GABAergic inhibition of prolactin biosynthesis

The inhibitory action of gamma-aminobutyric acid (GABA) on prolactin (PRL) messenger ribonucleic acid (mRNA) levels was studied in vitro in rat anterior pituitary cells in culture and in intact rats in vivo. PRL mRNA levels were determined by hybridization of cytoplasmic RNA with a radiolabelled deoxyribonucleic acid probe complementary to rat PRL mRNA. Incubation of anterior pituitary cultures with GABA (10-100 microM) produced a dose-dependent decrease in PRL mRNA levels with half-maximal inhibition near 1 microM. The effect was time dependent and reversible after drug withdrawal. Inhibition by GABA was antagonized by bicuculline (10 microM) and mimicked by the GABAA receptor agonists muscimol and isoguvacine, but not with the GABAB agonist baclofen, indicating the involvement of GABAA receptors in the accumulation of PRL mRNA. To investigate the role of endogenous GABA on PRL biosynthesis in vivo, GABA levels were raised by using the GABA transaminase blockers vinyl GABA and ethanolamine-O-sulfate. Injection of vinyl GABA into rats (100 or 800 mg/kg every 2nd day) resulted in a dose- and time-dependent decrease in PRL mRNA levels in rat adenohypophysis. Similar results were obtained by addition of ethanolamine-O-sulfate to the drinking water (5 mg/ml, 250 mg/day). This treatment resulted in a rapid decrease of circulating PRL levels. This was followed by a delayed decrease in PRL mRNA concentrations in the adenohypophysis leading to a transient increase in hormone levels in the anterior pituitary. The results indicate that GABA has an inhibitory role on PRL secretion and PRL gene expression by a direct action at GABAA receptors on pituitary lactotrophs.