Sodium-dependent effects of melatonin on membrane potential of neonatal rat pituitary cells.

Melatonin inhibits GnRH-stimulated release of LH from neonatal rat pituitary cells, probably by inhibiting GnRH-induced elevation of intracellular Ca2+. This effect of melatonin seems to involve inhibition of Ca2+ influx through voltage-sensitive channels. Accordingly, it is possible that melatonin could act by hyperpolarizing pituitary cells, which would close these channels. This issue was addressed here by determining if melatonin influences membrane potential. Membrane potential and intracellular Ca2+ were studied in neonatal rat pituitary cells in suspension, using bis-oxonol and Fluo-3 as fluorescent indicators, respectively. It was found that treatment with melatonin alone causes membrane hyperpolarization and that it has a repolarizing effect after GnRH-induced membrane depolarization. This effect on membrane potential appears to be mediated by high affinity melatonin receptors and a pertussis toxin-sensitive Na(+)-dependent mechanism; it is not dependent upon Ca2+, Cl-, or bicarbonate. This may be the molecular basis of action of melatonin in other tissues with high affinity melatonin receptors.     

Maintenance of gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone release despite desensitization of GnRH-stimulated cytosolic calcium responses.

Involvement of ionized cytosolic calcium ([Ca2+]i) and protein kinase-C (PKC) in GnRH-stimulated LH release was assessed by correlating measurable changes in [Ca2+]i and LH release in PKC-depleted and nondepleted gonadotropes. Primary cultures of anterior pituitary cells were loaded with the calcium-sensitive fluorescent dye fura-2 and placed in a perifusion chamber. GnRH pulses were delivered to the cells, and changes in fura-2 fluorescence and LH release were determined. The level of [Ca2+]i (assessed by fura-2) increased rapidly to a maximum within 20-40 sec, followed by a slower decline over the next minute (spike phase) to a sustained intermediate value (plateau phase). GnRH-stimulated LH release was unaffected by loading cells with fura-2. Both LH release and changes in [Ca2+]i were directly dependent on GnRH concentration. Pretreatment with the GnRH antagonist Antide (50 nM; [NAcD2Nal1-DpClPhe2-D3Pal3-Ser4-NicLys5-++ +DNicLys6-Leu7-ILys8-Pro9-DAla10]NH2 ) had no effect on basal [Ca2+]i or basal LH release, but did block both GnRH-stimulated calcium mobilization and GnRH-stimulated LH release. GnRH pretreatment (3.5 nM; 10 min) blocked the calcium spike phase, but not the plateau phase occurring in response to a GnRH pulse (10 nM; 5 min) delivered immediately after pretreatment. Inhibition of the calcium spike phase was transient (recovery within 15 min) and was dependent on pretreatment concentrations of GnRH. Calcium spike phase inhibition by GnRH pretreatment prevented increased LH release from PKC-depleted cells in response to a subsequent pulse of GnRH, but not from gonadotropes with normal levels of PKC. This suggests that initial LH release is dependent on changes in [Ca2+]i, but enhancement of LH release after periods of elevated GnRH concentrations may be dependent on PKC.     

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.     

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.     

Differences in gonadotropin-releasing hormone-induced calcium signaling between melatonin-sensitive and melatonin-insensitive neonatal rat gonadotrophs

The sensitivity of GnRH-stimulated calcium signaling to melatonin, in a subpopulation of neonatal gonadotrophs, is supposed to be attributable to melatonin receptors. However, it is not yet known whether the intracellular pathway for GnRH action in melatonin-sensitive cells is the same as in melatonin-insensitive cells. By monitoring intracellular Ca2+ changes as an outward current carried through apamin-sensitive Ca2+-activated K+ channels, we compared GnRH-induced calcium responses in these two subpopulations of neonatal gonadotrophs. GnRH induced various oscillatory, as well as nonoscillatory, responses in both cell types that was not related to melatonin sensitivity. Melatonin-sensitive GnRH-induced responses could be clearly distinguished according to the pharmacological properties of their latency. The latency increased in zero extracellular Ca2+ or with the addition of nifedipine, staurosporine, and ryanodine. This effect was only rarely observed in melatonin-insensitive cells. This indicates that there are two pathways for initiation of GnRH-induced calcium signaling in neonatal gonadotrophs. The first pathway is mediated by inositol 1,4,5,-trisphosphate production, whereas the second involves extracellular calcium entry through voltage-dependent L-type Ca2+ channels, protein kinase C activation, and Ca2+ release from a ryanodine-sensitive store, which may coactivate Ca2+ release from an inositol 1,4,5,-trisphosphate-sensitive store. Only the second mechanism is accessible to inhibition by melatonin.     

Dual effect of melatonin on gonadotropin-releasing-hormone-induced Ca(2+) signaling in neonatal rat gonadotropes

In neonatal rat gonadotropes, melatonin inhibits gonadotropin-releasing-hormone (GnRH)-stimulated increase in intracellular Ca(2+) concentration ([Ca(2+)](i)); in cells transfected with the Mel1a melatonin receptor, however, melatonin has been shown to potentiate agonist-stimulated [Ca(2+)](i) increase. To elucidate this discrepancy, we investigated the effects of melatonin in neonatal gonadotropes over a wide range of melatonin concentrations. Nystatin perforated patch recording of Ca(2+)-dependent potassium currents was used to monitor GnRH-induced [Ca(2+)](i) changes. In 32% of cells, increasing melatonin concentrations in the range of 1 pM to 100 nM prolonged the latency of, and inhibited GnRH (10 nM)-stimulated [Ca(2+)](i) increases in a concentration-dependent manner. In the remaining 68% of cells, the Ca(2+) increase elicited by exposure to 10 nM GnRH was also inhibited by picomolar concentrations of melatonin, but at nanomolar concentrations the inhibitory effect disappeared and melatonin was only able to prolong the latency of the response. This dual effect of melatonin however was not observed in cells stimulated with lower (2 nM) GnRH concentrations; in that case, melatonin was inhibitory at all concentrations tested with an IC(50) of about 30 pM. In contrast, application of nanomolar concentrations of melatonin resulted in potentiation of the GnRH-induced Ca(2+) increase in a small population of gonadotropes which did not respond by inhibition or prolonged latency. These results indicate that in neonatal gonadotropes, melatonin has both inhibitory and potentiating effects on GnRH-stimulated [Ca(2+)](i) increases. Ranges of concentrations needed to produce either effect suggest that two distinct G proteins may be involved, as already observed in transfected cells.     

Gonadal steroid modulation of signal transduction and luteinizing hormone release in cultured chicken pituitary cells

The mechanism whereby gonadal steroids modulate GnRH-stimulated LH secretion by primary cultures of chicken pituitary cells was investigated. Estradiol (10(-8) M), testosterone (10(-7) M), and progesterone (10(-7) M) inhibited LH release stimulated by GnRH (10(-7) M) by 56%, 61%, and 53%, respectively, and the inhibitory effects required prolonged preincubation (24-48 h) with the steroids. The steroids inhibited the spike (0-3 min) and plateau (9-30 min) phases of LH release to a similar degree. The ED50 values of estradiol, testosterone, and progesterone for inhibition of GnRH-stimulated LH release were 7 x 10(-11), 2 x 10(-9), and 1 x 10(-9) M, respectively. Estradiol, testosterone, and progesterone inhibited the maximal LH response to GnRH, but the ED50 of GnRH (4 x 10(-9) M) was not altered by steroid pretreatment. Steroid pretreatment did not cause a change in cellular LH content, suggesting that the steroids do not inhibit LH synthesis. Combinations of two or three of the steroids were not additive, suggesting that all three steroids affect GnRH-stimulated LH release via the same mechanism. In experiments investigating their mechanism of action, the steroids inhibited LH release stimulated by GnRH and Ca2+ ionophore A23187, but generally had no effect on the responses to phorbol ester (12-O-tetradecanoylphorbol-13-acetate), forskolin, K+, Bay K8644, or veratridine. Estradiol inhibited GnRH-stimulated 45Ca2+ efflux, but its inhibitory effect on GnRH-induced inositol phosphate production was not significant. Estradiol had no effect on binding of 125I-[His5,D-Tyr6]GnRH to a pituitary cell preparation. These findings suggest that the site of steroid modulation of GnRH action is distal to binding of GnRH to its receptor, and that the inhibitory effects are exerted at two intracellular sites: 1) the coupling events linking receptor activation to mobilization of Ca2+, and 2) a site distal to Ca2+ mobilization.     

Cytosolic free calcium levels in cultured pituitary cells separated by centrifugal elutriation: effect of gonadotropin-releasing hormone

The cytosolic concentration of free Ca2+ ([Ca2+]i) in normal rat pituitary cells separated by centrifugal elutriation was monitored with the fluorescent Ca2+ indicator Quin 2. GnRH (10(-7) M) induced a rapid rise (6-8 sec) in the gonadotroph's [Ca2+]i, followed by a plateau phase of prolonged elevated [Ca2+]i which lasted about 15 min. The stimulatory effect of GnRH was dose dependent, with an ED50 of 10(-9) M, and was blocked by the potent antagonist [Dp-Glu1,pclPhe2,DTrp3.6]GnRH. GnRH elevated [Ca2+]i only in gonadotroph-enriched cell fractions, whereas TRH and GH-releasing factor (GRF) elevated [Ca2+]i in mammotroph- and somatotroph-enriched cells fractions, respectively. A rapid increase (first phase) in [Ca2+]i induced by GnRH was observed in Ca2+-free medium containing EGTA, but this rapid phase was terminated within 2 min. Readdition of Ca2+ to the medium induced a second slower rise in [Ca2+]i (plateau phase). Addition of K+ caused a rapid rise in [Ca2+]i, which was dependent on extracellular Ca2+, but was not affected by prior stimulation with GnRH. On the other hand, stimulation of gonadotroph's [Ca2+]i response by GnRH desensitized the cells to a subsequent GnRH challenge within the time frame studied. These findings indicate an elevation of [Ca2+]i induced by GnRH, TRH, and GRF in their respective separated target cells in the rat pituitary. The rise in [Ca2+]i in GnRH-stimulated gonadotrophs originates partly from intracellular Ca2+ pools and partly from influx of Ca2+ across the cell membrane.     

The relationship between gonadotropin-releasing hormone-stimulated luteinizing hormone release and inositol phosphate production: studies with calcium antagonists and protein kinase C activators

The coupling between GnRH-stimulated phosphoinositide (PI) turnover and LH release has been investigated in rat pituitary cell cultures. Accumulation of [3H]inositol phosphates ([3H]IPs) formed by hydrolysis of PIs was measured in cells that had been preloaded with [3H]myo-inositol. GnRH stimulated both LH release and incorporation of [3H]inositol into total [3H]IPs with similar dose and time dependencies. [3H] IP production in response to GnRH could be blocked by a GnRH antagonist, but was stimulated by a compound that provokes receptor microaggregation. GnRH-stimulated IP production persisted in the presence of either the Ca2+ channel blocker D600 or the calmodulin antagonist pimozide at concentrations that reduced LH release to 60% and 20% of control, respectively. Stimulated [3H]IP production was inhibited at higher concentrations of D600. In 1-h incubations, GnRH-stimulated [3H]IP production, but not LH release, was markedly inhibited by the protein kinase C activators phorbol myristate acetate and 1,2-dioctanoylglycerol. These findings indicate that in the gonadotrope, GnRH-stimulated LH release and [3H]IP production are closely coupled to receptor activation by an agonist; Ca2+ antagonists uncouple stimulated LH release from [3H]IP production; and protein kinase C activators uncouple stimulated PI turnover from LH release. Thus, GnRH-stimulated production of PI metabolites, as measured by [3H]IP accumulation, is apparently not sufficient to support LH release in the absence of Ca2+. In addition, GnRH-stimulated LH release is apparently not dependent on full expression of the PI response.     

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.     

A novel mechanism for the melatonin inhibition of testosterone secretion by rat Leydig cells: reduction of GnRH-induced increase in cytosolic Ca2+

The site of inhibition, by melatonin, of GnRH-dependent testosterone secretion was investigated in adult rat Leydig cells cultured in vitro. The various effects downstream of the binding of GnRH to its own receptor were isolated and mimicked by specific drugs. Testosterone secretion was then evaluated after 3 h stimulation with GnRH, thapsigargin (1 microM), phorbol-12-myristate-13-acetate (100 nM), arachidonic acid (20 microM), and ionomycin (1 microM) in the presence or absence of melatonin (215 nM). The effect of melatonin on the GnRH-induced changes in cytoplasmic calcium concentration ([Ca(2+)](i)) was also studied, using Fura-2 as fluorescent Ca(2+) indicator. Melatonin attenuated the increase in [Ca(2+)](i) and inhibited the testosterone secretion induced by GnRH, but not that induced by ionomycin. Both ionomycin and thapsigargin potentiated GnRH-induced testosterone secretion; however, ionomycin, but not thapsigargin, partially prevented the inhibitory effect of melatonin on cells stimulated with GnRH. The effect of melatonin was probably dependent on the binding of melatonin to its Gi-protein-coupled receptor, as the inhibitory effect on GnRH-induced secretion was supressed in cells pretreated with pertussis toxin in a concentration of 180 ng/ml for 20 h. Assay of 17-hydroxy-progesterone showed that, irrespective of the treatment, cells cultured with melatonin secreted greater amounts than controls. We conclude that melatonin reduces GnRH-induced testosterone secretion by 1) decreasing [Ca(2+)](i), through impairment of the GnRH-dependent release of Ca(2+) from intracellular stores and 2) blocking 17-20 desmolase enzymatic activity, an effect that occurs irrespective of changes in [Ca(2+)](i).     

Signal transduction mechanisms of Ca2+ mobilizing hormones: the case of gonadotropin-releasing hormone

Multiple (at least seven) steps are involved in GnRH-induced gonadotropin secretion and gonadotropin gene expression. After binding to specific receptors located exclusively on pituitary gonadotrophs, GnRH stimulates a rapid phosphodiesteric hydrolysis of phosphoinositides for which no rise in [Ca2+]i is required. Activation of PLC is most likely mediated by a pertussis toxin-insensitive GTP-binding protein (Gp). In its activated state (Gp-GTP) the binding affinity of GnRH to is receptor is reduced. Rapid formation of IP3 will enhance Ca2+ release from intracellular sources most likely via a specific IP3 receptor. The transient Ca2+ rise might be responsible for a burst phase of LH release lasting for about 100 sec, which is not dependent on extracellular Ca2+. The backbone moiety of the phosphoinositides, DG, and the elevated [Ca2+]i are most likely responsible for translocation of PKC subspecies from the cytosol to the membrane. The most likely candidates are alpha- and beta II-PKC. The activated PKC subspecies phosphorylate substrate proteins which activate secretory reactions and participate in gonadotropin gene expression. In parallel Ca2(+)-influx via nifedipine-sensitive and insensitive channels further elevates [Ca2+]i, which participates in the sustained phase of gonadotropin secretion in concert with the activated PKCs. GnRH also triggers the release of AA and the formation of lipoxygenase and/or epoxygenase products of the fatty acid which are also involved in the process of the exocytosis. We predict that the continuous supply of DG and AA needed for GnRH action is also provided via activated PLD which will also supply phosphatidic acid, the role of which is as yet unclear. The interaction of the various second messengers involved in GnRH action (IP3, Ca2+, DG, AA) and their relative roles in gonadotropin secretion and gonadotropin gene expression await further investigation. In several aspects GnRH action on gonadotropin secretion is unique when compared to other Ca2(+)-mobilizing ligands: 1) At physiological concentrations GnRH up-regulates its own receptors whereas most ligands down-regulate the respective receptor; 2) PKC up-regulates GnRH receptors whereas in most cases PKC down-regulates the ligand receptor; 3) GnRH stimulation of PLC activity is most likely mediated by Gp whereas some Ca2(+)-mobilizing ligands operate via Gi; 4) Activated PKC does not exert negative feedback upon GnRH-induced inositol phosphate production as is the case with several other peptides; 5) Activated PKC might be responsible for Ca2+ influx whereas in several other systems PKC is inhibitory to Ca2+ influx.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and release of luteinizing hormone in vitro by rat anterior pituitary cells: effects of gallopamil hydrochloride (D600) and pimozide

We compared the role of Ca2+ in regulating GnRH-induced LH synthesis and release from cultured rat pituitary cells. LH synthesis and release were measured after a 4-h treatment of cells with gallopamil hydrochloride (D600; 1 and 100 microM), a Ca2+ channel blocker, or pimozide (0.5 and 5.0 microM), a calmodulin inhibitor, with or without 1 nM GnRH. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell and medium) immunoprecipitable LH. GnRH significantly (P less than 0.01) increased total [3H]LH (glycosylation), but had no effect on total [14C]LH (translation). D600 significantly (P less than 0.01) depressed (1 microM) and completely blocked (100 microM) GnRH-induced LH glycosylation and release of [3H]LH, [14C]LH, and immunoreactive LH. D600 (100 microM) also reduced (P less than 0.05) total basal synthesis of [14C]LH. Neither dose of D600 altered uptake of [3H]glucosamine, but 100 microM D600 significantly (P less than 0.01) depressed its incorporation into total protein. D600 (100 microM) significantly (P less than 0.01) depressed [14C]alanine uptake and incorporation into total protein. Pimozide significantly (P less than 0.01) reduced, in a dose-related manner, GnRH-induced LH glycosylation, and release of immunoreactive LH, [3H]LH, and [14C]LH. Pimozide did not alter LH translation or uptake of radiolabeled precursors or their incorporation into total protein. These results demonstrate that D600 and pimozide inhibit both GnRH-induced LH glycosylation and release. Thus, the actions of GnRH on LH glycosylation and release are both mediated by similar Ca2+-dependent pathways.     

Structure-function relationship of calcium ion channel antagonists at the pituitary gonadotrope

We have compared the potencies of chemically heterogeneous classes of Ca2+ ion channel inhibitors as antagonists of GnRH-stimulated LH release from the rat pituitary. Verapamil and its more potent derivative methoxyverapamil (D600) are effective inhibitors. The 1,4-dihydropyridines, however, had no antagonistic efficacy, and diltiazem had slight inhibitory action, but only when preincubated with cells before GnRH addition. The observation that the potency series of antagonists was identical (verapanoids greater than diltiazem greater than 1,4-dihydropyridines) whether GnRH or veratridine was used to stimulate the release mechanism is consistent with the premise that the same Ca2+ channel is regulated by the receptor and by agents that evoke gonadotropin release by cell depolarization. This potency series is virtually opposite that observed for muscle tissue. Accordingly, these data suggest that the receptor-regulated Ca2+ ion channel of the pituitary gonadotrope is distinct from that described in muscle.     

Dependence of gonadotropin-releasing hormone-stimulated luteinizing hormone release upon intracellular Ca2+ pools is revealed by desensitization and thapsigargin blockade.

Sustained GnRH-stimulated LH release requires extracellular Ca2+, but GnRH transiently increases LH release in Ca(2+)-free medium. Here we have tested the dependence of the transient effect on intracellular Ca2+ pools. In superfused pituitary cells three Ca(2+)-mobilizing stimuli (GnRH, A23187, and endothelin-1) all caused sustained increases in LH release in normal medium (plateau responses), but only transient increases in Ca(2+)-free medium (spike responses). In Ca(2+)-free medium, GnRH (10(-10) or 10(-9) M) increased LH release transiently and desensitized the cells to the LH-releasing effect of subsequent stimulation with 10(-7) M GnRH. This desensitization was reversed by brief exposure to Ca(2+)-containing medium between the two GnRH stimulation periods. Heterologous desensitization between GnRH and A23187 and between GnRH and endothelin-1 also occurred in Ca(2+)-free medium. Thapsigargin, which inhibits the endoplasmic reticulum Ca(2+)-ATPase and thereby elevates cytosolic Ca2+, stimulated LH release (EC50, approximately 20 microM) in static culture, an effect which, unlike those of GnRH and A23187, was not markedly reduced in Ca(2+)-free medium. Low doses of thapsigargin, which had no effect on LH release alone, inhibited both sustained GnRH-stimulated LH release from static cultures in normal medium and transient GnRH-stimulated LH release from cells superfused in Ca(2+)-free medium. These data suggest that the spike phase of GnRH-stimulated LH release is not only associated with but is also dependent upon the mobilization of a GnRH- and thapsigargin-sensitive intracellular Ca2+ pool and that the Ca2+ pool mediating this GnRH effect is identical to or substantially interchangeable with A23187- and endothelin-1-mobilizable intracellular Ca2+ pools. Inhibition of sustained GnRH-stimulated LH release by thapsigargin also suggests the involvement of an intracellular Ca2+ pool in this phase of GnRH action.     

Production of leukotrienes in gonadotropin-releasing hormone-stimulated pituitary cells: potential role in luteinizing hormone release.

Gonadotropin-releasing hormone (GnRH) stimulated the formation of two major metabolites of the 5-lipoxygenase pathway, leukotriene (LT) B4 and LTC4, as well as luteinizing hormone (LH) release in primary cultures of rat anterior pituitary cells. Several lines of evidence suggested the presence of a GnRH-dependent pituitary endocrine system in which LTs act as second messengers for LH release: (i) GnRH-dependent LT formation was observed within 1 min and immediately preceded GnRH-induced LH release, whereas exogenous LTs stimulated LH release at low concentrations; (ii) the dose responses of GnRH-induced LT production and LH release were similar and both effects required the presence of extracellular Ca2+ ions; (iii) GnRH-induced LH release was blocked by up to 45% following the administration of several LT receptor antagonists; (iv) LTE4 action on LH secretion was entirely abolished by LT receptor antagonists; and (v) an activator of protein kinase C acted synergistically with LTE4 to induce LH release. The major source of LT formation in the pituitary cell cultures appeared to be the gonadotrophs, as shown by GnRH receptor desensitization experiments. The results demonstrate the presence of a GnRH-activatable 5-lipoxygenase pathway in anterior pituitary cells and provide strong support for the hypothesis that LTs play a role in LH release in the GnRH signaling pathway.     

Gonadotropin-releasing hormone-stimulated luteinizing hormone secretion by perifused pituitary cells from normal, gonadectomized, and testicular feminized rats

To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King x Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P less than 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P less than 0.01) than that by cells from intact animals, but lower (P less than 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-dependent LH release. Pituitary cells from intact female rats showed an overall greater (P less than 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P less than 0.0001) or intact female (P less than 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRH-stimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P less than 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact females rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens.     

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.     

Different effects of melatonin pretreatment on cAMP and LH responses of the neonatal rat pituitary cells

The rhythm of the pineal hormone melatonin transduces the effect of photoperiod on seasonal functions. Duration of the melatonin pulse provides information about season and the long melatonin pulse induces reproductive involution in the long day breeders such as photoperiodic rodents. The length of melatonin pulse thus carries photoperiodic information, which regulates the function of target cells. Therefore, we have studied the effects of melatonin pretreatment of various lengths on responsiveness of the neonatal rat pituitary cells cultured in vitro to GnRH or forskolin. In these cells, melatonin treatment inhibits the GnRH-induced LH release as well as the forskolin-induced cAMP accumulation. However, long preincubation with melatonin has a paradoxical stimulatory effect on the cellular responsiveness. When the cells are pretreated with melatonin for 16 hr or more, then rinsed thoroughly and treated with forskolin for 30 min, the increase of cAMP is potentiated. Moreover, in the melatonin-pretreated cells. the subsequent melatonin treatment inhibits the forskolin-induced cAMP accumulation relatively more than in the non-pretreated cells. Although melatonin pretreatment does not potentiate the GnRH-induced LH release, it protects the gonadotrophs against the GnRH-induced desensitization: pretreatment with GnRH for 12 hr or more renders the cells insensitive to subsequent GnRH stimulation, while after pretreatment with GnRH and melatonin, the subsequent GnRH treatment induces significant increase of LH release. These observations indicate that long pretreatment with melatonin improves responsiveness of the pituitary cells to the subsequent stimulation, but its effects on cAMP accumulation and LH release are different.     

Inhibitory actions of keoxifene on luteinizing hormone secretion in pituitary gonadotrophs

The effect of keoxifene (LY 156 758) on GnRH-stimulated LH release and its ability to antagonize estrogen actions were investigated in rat anterior pituitary cells. Estrogens exert either stimulatory or inhibitory effects on GnRH-induced LH secretion in rat pituitary cells depending on the incubation time with the steroid. When pituitary cells were treated for 24 h with 10(-9) M estradiol, the LH response to GnRH was clearly enhanced, and this effect was completely inhibited by 300 nM keoxifene. Short term treatment (4 h) of pituitary cells with 10(-9) M estradiol inhibits GnRH-stimulated LH release, and this effect was also blocked by keoxifene in a dose-dependent manner. In the absence of exogenous estrogen the treatment of pituitary cells for 4 h with increasing concentrations of keoxifene reduced the LH response to 10(-9) M GnRH only at very high concentrations (10(-5) M) of the antiestrogen. After treatment for 24 h, the inhibitory effect of keoxifene was evident at concentrations greater than or equal to 10(-8) M, with a reduction of GnRH-induced LH release by up to 60%. The effects of the antiestrogen were also analyzed in a dynamic culture system, in which pituitary cells grown on microcarrier beads were continuously perifused with medium and stimulated with GnRH in a pulsatile fashion. The LH response to a 2 min pulse of 10(-9) M GnRH was reduced in magnitude after 40 min of perifusion with 10(-9) M estradiol. When keoxifene (300 nM) was present at the same time, the LH response was identical to that observed in vehicle-treated cells. At the concentration of 300 nM, keoxifene per se did not change the responsiveness of the pituitary cells to the GnRH stimulus. These findings show that keoxifene is a potent antagonist of both positive and negative estrogen actions in the pituitary gonadotroph. In addition, after short term treatment with high concentrations or after long term treatment, keoxifene itself exerts an inhibitory effect on GnRH-induced LH secretion.