Dual modulation of K channels by thyrotropin-releasing hormone in clonal pituitary cells.

Transmembrane electrical activity in pituitary tumor cells can be altered by substances that either stimulate or inhibit their secretory activity. Using patch recording techniques, we have measured the resting membrane potentials, action potentials, transmembrane macroscopic ionic currents, and single Ca2+-activated K channel currents of GH3 and GH4/C1 rat pituitary tumor cells in response to thyrotropin-releasing hormone (TRH). TRH, which stimulates prolactin secretion, causes a transient hyperpolarization of the membrane potential followed by a period of elevated action potential frequency. In single cells voltage clamped and internally dialyzed with solutions containing K+, TRH application results in a transient increase in Ca2+-activated K currents and a more protracted decrease in voltage-dependent K currents. However, in cells internally dialyzed with K+-free solutions, TRH produces no changes in inward Ca2+ or Ba2+ currents through voltage-dependent Ca channels. The time courses of the effects on Ca2+-activated and voltage-dependent K currents correlate with the phases of hyperpolarization and hyperexcitability, respectively. During application of TRH to whole cells, single Ca2+-activated K channel activity increases in cell-attached patches not directly exposed to TRH. In contrast, TRH applied directly to excised membrane patches produces no change in single Ca2+-activated K channel behavior. We conclude that TRH (i) triggers intracellular Ca2+ release, which opens Ca2+-activated K channels, (ii) depresses voltage-dependent K channels during the hyperexcitable phase, which further elevated intracellular Ca2+, and (iii) does not directly modulate Ca channel activity.     

Oscillations in KATP channel activity promote oscillations in cytoplasmic free Ca2+ concentration in the pancreatic beta cell.

Pancreatic beta cells exhibit oscillations in electrical activity, cytoplasmic free Ca2+ concentration ([Ca2+](i)), and insulin release upon glucose stimulation. The mechanism by which these oscillations are generated is not known. Here we demonstrate fluctuations in the activity of the ATP-dependent K+ channels (K(ATP) channels) in single beta cells subject to glucose stimulation or to stimulation with low concentrations of tolbutamide. During stimulation with glucose or low concentrations of tolbutamide, K(ATP) channel activity decreased and action potentials ensued. After 2-3 min, despite continuous stimulation, action potentials subsided and openings of K(ATP) channels could again be observed. Transient suppression of metabolism by azide in glucose-stimulated beta cells caused reversible termination of electrical activity, mimicking the spontaneous changes observed with continuous glucose stimulation. Thus, oscillations in K(ATP) channel activity during continuous glucose stimulation result in oscillations in electrical activity and [Ca2+](i).     

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.     

Modulation of cytoplasmic calcium signaling in rat pituitary gonadotrophs by estradiol and progesterone.

The stimulatory action of GnRH on gonadotropin secretion from cultured rat pituitary cells is modulated by estradiol (E) and progesterone (P). Since secretory responses to GnRH are initiated by phosphoinositide hydrolysis and Ca2+ mobilization, the effects of gonadal steroids on the pattern of Ca2+ signaling were analyzed in single pituitary gonadotrophs. Increasing concentrations of GnRH elicited a spectrum of [Ca2+]i signals in single gonadotrophs, ranging from subthreshold to threshold-oscillatory and biphasic (spike & plateau) responses. In E-treated gonadotrophs, short-term P treatment shifted subthreshold [Ca2+]i responses to oscillatory and oscillatory to biphasic responses, whereas long-term P treatment shifted oscillatory to subthreshold [Ca2+]i response profiles. These changes parallel the effects of P on GnRH-induced LH release, and indicate that the modulatory effects of ovarian steroids on gonadotropin secretion include a significant action on the Ca2+ signaling pathway.     

Cell-type specific messenger functions of extracellular calcium in the anterior pituitary.

Calcium can serve not only as an intracellular messenger, but also as an extracellular messenger controlling the gating properties of plasma membrane channels and acting as an agonist for G protein-coupled Ca(2+)-sensing receptors. Here we studied the potential extracellular messenger functions of this ion in anterior pituitary cells. Depletion and repletion of the extracellular Ca(2+) concentration ([Ca(2+)]e) induced transient elevations in the intracellular Ca(2+) concentration ([Ca(2+)]i), and elevations in [Ca(2+)]e above physiological levels decreased [Ca(2+)]i in somatotrophs and lactotrophs, but not in gonadotrophs. The amplitudes and duration of [Ca(2+)]i responses depended on the [Ca(2+)]e and its rate of change, which resulted exclusively from modulation of spontaneous voltage-gated Ca(2+) influx. Changes in [Ca(2+)]e also affected GH and PRL secretion. The PRL secretory profiles paralleled the [Ca(2+)]i profiles in lactotrophs, whereas GH secretion was also stimulated by [Ca(2+)]e independently of the status of voltage-gated Ca(2+) influx. [Ca(2+)]e modulated GH secretion in a dose-dependent manner, with EC(50) values of 0.75 and 2.25 mM and minimum secretion at about 1.5 mM. In a parallel experiment, cAMP accumulation progressively increased with elevation of [Ca(2+)]e, whereas inositol phosphate levels were not affected. These results indicate the cell type-specific role of [Ca(2+)]e in the control of Ca(2+) signaling and secretion.     

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.     

Dopamine inhibits basal prolactin release in pituitary lactotrophs through pertussis toxin-sensitive and -insensitive signaling pathways

Dopamine D2 receptors signal through the pertussis toxin (PTX)-sensitive G(i/o) and PTX-insensitive G(z) proteins, as well as through a G protein-independent, beta-arrestin/glycogen synthase kinase-3-dependent pathway. Activation of these receptors in pituitary lactotrophs leads to inhibition of prolactin (PRL) release. It has been suggested that this inhibition occurs through the G(i/o)-alpha protein-mediated inhibition of cAMP production and/or G(i/o)-betagamma dimer-mediated activation of inward rectifier K(+) channels and inhibition of voltage-gated Ca(2+) channels. Here we show that the dopamine agonist-induced inhibition of spontaneous Ca(2+) influx and release of prestored PRL was preserved when cAMP levels were elevated by forskolin treatment. We further observed that dopamine agonists inhibited both spontaneous and depolarization-induced Ca(2+) influx in untreated but not in PTX-treated cells. This inhibition was also observed in cells with blocked inward rectifier K(+) channels, suggesting that the dopamine effect on voltage-gated Ca(2+) channel gating is sufficient to inhibit spontaneous Ca(2+) influx. However, agonist-induced inhibition of PRL release was only partially relieved in PTX-treated cells, indicating that dopamine receptors also inhibit exocytosis downstream of voltage-gated Ca(2+) influx. The PTX-insensitive step in agonist-induced inhibition of PRL release was not affected by the addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and lithium, an inhibitor of glycogen synthase kinase-3, but was attenuated in the presence of phorbol 12-myristate 13-acetate, which inhibits G(z) signaling pathway in a protein kinase C-dependent manner. Thus, dopamine inhibits basal PRL release by blocking voltage-gated Ca(2+) influx through the PTX-sensitive signaling pathway and by desensitizing Ca(2+) secretion coupling through the PTX-insensitive and protein kinase C-sensitive signaling pathway.     

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

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

Effects of Bay K 8644 and other dihydropyridines on basal and potassium-evoked output of MSH from mouse melanotrophs in vitro

Dihydropyridines that have been shown in studies on other tissues either to facilitate Ca influx (BAY K 8644) or depress it (nimodipine and nifedipine) were examined for their effects on the secretory activity of the melanotrophs. Isolated mouse neurointermediate lobes, cultured for a week or longer to allow the nerves to degenerate, were perifused and output of MSH activity was measured by bioassay. The Ca-channel agonist BAY K 8644 augmented secretion under basal conditions and potentiated secretion evoked by 30 mM K+, a submaximally depolarizing concentration. The stimulant effect on secretion under basal conditions persisted in the presence of tetrodotoxin (which blocks the action potentials, Na spikes, in the melanotrophs) but was lost when Ca2+ was omitted or nimodipine added. The results are considered to support the view that the prominent basal secretory activity in these cells, as well as that evoked by excess K+, involve the inward flux of Ca2+ through dihydropyridine-sensitive Ca channels. If the stimulant and inhibitory effects of the dihydropyridines on basal secretion are to be attributed to actions on voltage-regulated Ca channels, as the results from other tissues would suggest, then such channels in the melanotrophs are different from those previously described in other tissues.     

Ghrelin reduces voltage-gated calcium currents in GH₃ cells via cyclic GMP pathways

Ghrelin is an endogenous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca2+ concentration ([Ca2+]i), which is determined by Ca2+ influx and release from intracellular Ca2+ storage sites. Ca2+ influx is via voltage-gated Ca2+ channels, which are activated by cell depolarization. The mechanism underlying the effect of ghrelin on voltage-gated Ca2+ channels is still not clear. In this report, using whole cell patch-clamp recordings, we assessed the acute action of ghrelin on voltage-activated Ca2+ currents in GH3 rat somatotrope cell line. Ca2+ currents were divided into three types (T, N, and L) through two different holding potentials (-80 and -40 mV) and specific L-type channel blocker (nifedipine, NFD). We demonstrated that ghrelin significantly and reversibly decreases all three types of Ca2+ currents in GH3 cells through GHS receptors on the cell membrane and down-stream signaling systems. With different signal pathway inhibitors, we observed that ghrelin-induced reduction in voltage-gated Ca2+ currents in GH3 cells was mediated by a protein kinase G-dependent pathways. As ghrelin also stimulates Ca2+ release and prolongs the membrane depolarization, this reduction in voltage-gated Ca2+ currents may not be translated into a reduction in [Ca2+]i, or a decrease in GH secretion.     

Neurotensin stimulates both calcium mobilization from inositol trisphosphate-sensitive intracellular stores and calcium influx through membrane channels in frog pituitary melanotrophs

Neurotensin (NT) is a potent stimulator of electrical and secretory activities in frog pituitary melanotrophs. The aim of the present study was to characterize the transduction pathways associated with activation of NT receptors in frog melanotrophs. Application of synthetic frog NT (fNT) increased the cytosolic calcium concentration ([Ca2+]c) and stimulated the formation of inositol trisphosphate (IP3). The phospholipase C inhibitor U-73122 blocked the electrophysiological and secretory effects of fNT. Intracellular application of the IP3 receptor antagonist heparin abolished fNT-induced electrical activity. Suppression of Ca2+ in the incubation medium markedly reduced the effect of NT on [Ca2+]c, firing rate, and alpha-melanocyte-stimulating hormone (alphaMSH) secretion. Similarly, the inhibitor of IP3-induced Ca2+ release and store-operated Ca2+ channels, 2-Aminoethoxydiphenylborane, and the nonselective Ca2+ channel blockers GdCl3 and NiCl2, attenuated the [Ca2+]c increase and the electrical and secretory responses evoked by fNT. Coapplication of the L- and N-type Ca2+ channel blockers nifedipine and omega-CgTx GVIA reduced the effects of fNT on action potential discharge, [Ca2+]c increase, and alphaMSH release. The protein kinase C (PKC) inhibitors, PKC-(19-31) and chelerythrine, reduced the electrophysiological and secretory responses induced by iterative applications of fNT. Collectively, these results demonstrate that, in frog melanotrophs, NT stimulates the phospholipase C/PKC pathway and increases [Ca2+]c. Both Ca2+ release from intracellular stores and Ca2+ influx through L- and N-type Ca2+ channels are involved in fNT-induced alphaMSH secretion. In addition, the present data indicate that PKC plays a crucial role in maintenance of the responsiveness of melanotrophs to NT.     

Galanin inhibits a dihydropyridine-sensitive Ca2+ current in the RINm5f cell line.

Mechanisms of action of the neuropeptide galanin, a putative neuromodulator in the central and peripheral nervous systems, have been evaluated extensively in insulin-secreting cells isolated from pancreas and cell lines derived from pancreatic tumors. Galanin inhibits insulin secretion from these cells through several mechanisms, including activation of ATP-dependent K+ channels and inhibition of adenylyl cyclase leading to a decrease in cAMP. Here we report that galanin also inhibits a dihydropyridine-sensitive Ca2+ current. Both electrophysiological actions by galanin would result in less Ca2+ entry, as the action to increase K+ current would hyperpolarize the cells and the decrease in voltage-gated Ca2+ current would decrease Ca2+ influx at depolarized potentials where these channels are activated. These galanin actions would directly counter the two opposing electrophysiological responses to carbohydrate stimulation in RINm5f cells, which are to inhibit K+ current and to stimulate Ca2+ current. Given that stimulation of presynaptic nerve terminals in pancreas releases galanin, these results suggest that Ca(2+)-dependent insulin release from native pancreatic beta cells may also be regulated by similar neuropeptide effects.     

Hyperpolarization of the membrane potential caused by somatostatin in dissociated human pituitary adenoma cells that secrete growth hormone.

Membrane electrical properties and the response to somatostatin were examined in dissociated human pituitary adenoma cells that secrete growth hormone (GH). Under current clamp condition with a patch electrode, the resting potential was -52.4 +/- 8.0 mV, and spontaneous action potentials were observed in 58% of the cells. Under voltage clamp condition an outward K+ current, a tetrodotoxin-sensitive Na+ current, and a Ca2+ current were observed. Cobalt ions suppressed the Ca2+ current. The threshold of Ca2+ current activation was about -60 mV. Somatostatin elicited a membrane hyperpolarization associated with increased membrane permeability in these cells. The reversal potential of somatostatin-induced hyperpolarization was -78.4 +/- 4.3 mV in 6 mM K+ medium and -97.2 +/- 6.4 mV in 3 mM K+ medium. These reversal potential values and a shift with the external K+ concentration indicated that membrane hyperpolarization was caused by increased permeability to K+. The hyperpolarized membrane potential induced by somatostatin was -63.6 +/- 5.9 mV in the standard medium. This level was subthreshold for Ca2+ and Na+ currents and was sufficient to inhibit spontaneous action potentials. Hormone secretion was significantly suppressed by somatostatin and cobalt ions. Therefore, we suggest that Ca2+ entering the cell through voltage-dependent channels are playing an important role for GH secretion and that somatostatin suppresses GH secretion by blocking Ca2+ currents. Finally, we discuss other possibilities for the inhibitory effect of somatostatin on GH secretion.     

Calcium signaling and episodic secretion of gonadotropin-releasing hormone in hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) is released episodically into the pituitary portal vessels and from hypothalamic tissue of male and female rats in vitro. Perifused primary cultures of rat hypothalamic neurons, as well as the GT1-1 GnRH neuronal cell line, spontaneously exhibited episodic GnRH secretion of comparable frequency to that observed with perifused hypothalami. Such pulsatile GnRH release from GT1 cells indicates that GnRH neurons generate rhythmic secretory activity in the absence of input from other cell types. In primary hypothalamic cultures, the frequency of GnRH pulses increased with the duration of culture. The spontaneous pulsatility in GnRH release was abolished in Ca(2+)-deficient medium and was markedly attenuated in the presence of nifedipine, an antagonist of voltage-sensitive Ca2+ channels. The basal intracellular Ca2+ level of perifused GT1-1 cells cultured on coverslips was also dose-dependently reduced by nifedipine. Conversely, depolarization with high K+ increased intracellular Ca2+ and GnRH release in an extracellular Ca(2+)-dependent and nifedipine-sensitive manner. The dihydropyridine Ca2+ channel agonist Bay K 8644 increased basal and K(+)-induced elevations of intracellular Ca2+ concentration and GnRH secretion. These findings demonstrate that pulsatile neuropeptide secretion is an intrinsic property of GnRH neuronal networks and is dependent on voltage-sensitive Ca2+ influx for its maintenance.     

Glucose stimulates glucagon release in single rat alpha-cells by mechanisms that mirror the stimulus-secretion coupling in beta-cells

In isolated rat pancreatic alpha-cells, glucose, arginine, and the sulfonylurea tolbutamide stimulated glucagon release. The effect of glucose was abolished by the KATP-channel opener diazoxide as well as by mannoheptulose and azide, inhibitors of glycolysis and mitochondrial metabolism. Glucose inhibited KATP-channel activity by 30% (P<0.05; n=5) and doubled the free cytoplasmic Ca2+ concentration. In cell-attached recordings, azide opened KATP channels. The N-type Ca2+-channel blocker omega-conotoxin and the Na+-channel blocker tetrodotoxin inhibited glucose-induced glucagon release whereas tetraethylammonium, a blocker of delayed rectifying K+ channels, increased secretion. Glucagon release increased monotonically with increasing K+ concentrations. omega-Conotoxin suppressed glucagon release to 15 mM K+, whereas a combination of omega-conotoxin and an L-type Ca2+-channel inhibitor was required to abrogate secretion in 50 mM K+. Recordings of cell capacitance revealed that glucose increased the exocytotic response evoked by membrane depolarization 3-fold. This correlated with a doubling of glucagon secretion by glucose in intact rat islets exposed to diazoxide and high K+. In whole-cell experiments, exocytosis was stimulated by reducing the cytoplasmic ADP concentration, whereas changes of the ATP concentration in the physiological range had little effect. We conclude that glucose stimulates glucagon release from isolated rat alpha-cells by KATP-channel closure and stimulation of Ca2+ influx through N-type Ca2+ channels. Glucose also stimulated exocytosis by an amplifying mechanism, probably involving changes in adenine nucleotides. The stimulatory action of glucose in isolated alpha-cells contrasts with the suppressive effect of the sugar in intact islets and highlights the primary importance of islet paracrine signaling in the regulation of glucagon release.     

Structure, function and transforming potential of the epidermal growth factor receptor

We have examined the pharmacology of the voltage-sensitive Ca2+ channels (VSCCs) that mediate gonadotropin secretion from primary cultures of rat pituitary cells, stimulated by either cell depolarization or by binding of gonadotropin-releasing hormone (GnRH). We also measured single-cell [Ca2+]i transients using fura-2 in gonadotropes identified by a reverse hemolytic plaque assay employing an antiserum to luteinizing hormone (LH). Cell depolarization evoked by either 50 mM K+ or 30 microM veratridine induced 2- to 6-fold increases in gonadotropin secretion over basal levels. GnRH caused 6- to 20-fold increases in follicle-stimulating hormone (FSH) and LH secretion, respectively, with maximal stimulation at 100 nM GnRH. K(+)- or GnRH-induced FSH release was largely prevented by co-incubation with 1 mM CdCl. Tetrodotoxin (TTX, 5 microM) prevented the veratridine-, but not the K(+)- or GnRH-induced, stimulation of FSH secretion. Nitrendipine (Ntd, 1 microM) produced 35-50% inhibition (NS) of both FSH and LH release stimulated by either 50 mM K+ or 100 nM GnRH. Ntd also inhibited the K(+)-induced [Ca2+]i rise (greater than 90%), as well as the secondary, plateau phase of the GnRH-induced elevation of [Ca2+]i (100% inhibition). Omega-conotoxin (omega-CgTx, 100 nM) partially suppressed FSH and LH release (NS) due to both K+ (33% each) and GnRH (44% and 18%, respectively). omega-CgTx showed variable effects on [Ca2+]i transients evoked by K+ or GnRH ranging from clear inhibition to no effect. We conclude that influx of extracellular Ca2+ is one of several fundamental events underlying the depolarization- or receptor-activated release of LH and FSH, and that this influx can be inhibited by dihydropyridine-sensitive ('L') Ca2+ channels. Two classes of L-channels may exist in gonadotropes, that differ in their sensitivity to omega-CgTx.     

Glucose regulation of insulin secretion independent of the opening or closure of adenosine triphosphate-sensitive K+ channels in beta cells

Two major pathways are implicated in the stimulation of insulin secretion by glucose. The K+-ATP channel-dependent pathway involves closure of these channels, depolarization of the beta-cell membrane, acceleration of Ca2+ influx, and a rise in cytosolic free Ca2+ ([Ca2+]i). The K+-ATP channel-independent pathway potentiates the stimulation of exocytosis by high [Ca2+]i. To determine whether this second pathway is influenced by the configuration of the channel, we compared the effects of glucose on [Ca2+]i and insulin secretion in mouse islets under three conditions. First, in the presence of 20, 25, and 30 mM K+, i.e. without pharmacological action on K+-ATP channels, [Ca2+]i and insulin secretion were already elevated at 3 mM glucose. High glucose (20 mM) caused a transient decrease in [Ca2+]i followed by an ascent to slightly above control levels, and rapidly stimulated insulin secretion. Second, opening of K+-ATP channels with diazoxide did not influence [Ca2+]i and insulin secretion at 3 mM glucose and high K+. However, high glucose now caused a sustained lowering of [Ca2+]i accompanied by a slow increase in secretion that augmented with the K+ concentration. Third, when K+-ATP channels were blocked and beta-cells depolarized by high concentrations of tolbutamide or glibenclamide, [Ca2+]i and insulin secretion were elevated even in low glucose. High glucose transiently lowered [Ca2+]i, which then increased to or slightly above control levels, while insulin secretion was rapidly stimulated. Under all conditions the correlation between [Ca2+]i and insulin secretion was excellent at low and high glucose levels, and high glucose increased release at all [Ca2+]i. The potentiation of Ca2+-induced exocytosis by glucose is thus independent of the closed or open state of K+-ATP channels. It is only when the channels are opened by diazoxide that the increase in release is a strict amplification of the action of Ca2+. When the channels are closed (sulfonylureas) or still closable (high K+ alone), the effect of glucose on secretion also comprises a slight increase in [Ca2+]i and, in the latter case, is not strictly K+-ATP channel independent.     

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.     

Effects of bromocriptine on prolactin release, electrical membrane properties and transmembrane Ca2+ fluxes in cultured rat pituitary adenoma cells

The effects of the dopamine (DA) agonist bromocriptine on prolactin (Prl) release, electrical membrane properties and transmembrane Ca2+ fluxes have been studied in a clonal strain of rat pituitary adenoma cells (GH3). These cells generate Ca2+ dependent action potentials, and produce and secrete spontaneously both Prl and growth hormone. Prl release stimulated by thyroliberin (TRH) and elevated extracellular K+ concentration was completely blocked by bromocriptine, whereas the basal release was only moderately affected. The TRH and K+ evoked Prl release were half maximally inhibited by bromocriptine at 5-10 and 10-50 microM, respectively. The normal biphasic membrane response to TRH and the depolarizing effect of elevated K+ concentration were not altered by bromocriptine, whereas the Ca2+- spikes in Na+-free solution were suppressed by the drug. We therefore suggest that bromocriptine blocks the voltage sensitive Ca2+-channels of GH3 cell. In agreement with this notion, bromocriptine also suppressed the basal and TRH induced 45Ca2+ efflux from preloaded cells. We conclude that the inhibitory effect of bromocriptine on the voltage dependent Ca2+- channels is an important mechanism responsible for suppression of Prl release.