Progesterone modulation of gonadotropin secretion by dispersed rat pituitary cells in culture. I. Basal and gonadotropin-releasing hormone-stimulated luteinizing hormone release

Dispersed, estradiol-treated, rat pituitary cells were cultured to characterize the influences of a physiologic concentration of progesterone (P, 10(-7) M) on gonadotroph responsiveness to gonadotropin-releasing hormone (GnRH). Acute (less than 6 h) P treatment enhanced and chronic (greater than 12 h) treatment suppressed both basal and GnRH-stimulated luteinizing hormone (LH) release. This modulation took place without any change in intracellular LH stores, indicating that the secretory changes are not attributable to changes in LH synthesis, and were not accompanied by similar alterations in basal or thyrotropin-releasing hormone-stimulated prolactin secretion. Moreover, the timing of these responses was fixed since a 10-fold lower P concentration produced only smaller and briefer alterations in LH release. Analyses of the temporal characteristics of effective P stimuli indicated that a brief 6 h exposure to P inhibited GnRH-stimulated LH secretion 18 h later. In contrast, P's acute actions rapidly dissipated following removal of the steroid from the culture medium. Finally, P-induced enhancement and suppression of GnRH-stimulated LH release could be blocked by appropriately timed treatments with protein synthesis inhibitors. Our findings are consistent with the hypothesis that P influences gonadotroph secretory function via the production of specific proteins.     

Progesterone modulation of gonadotropin secretion by dispersed rat pituitary cells in culture. II. Intracellular metabolism and progestin receptors

Dispersed, estradiol (E2)-treated, rat pituitary cell cultures were used to examine the intracellular processing of progesterone (P) associated with its modulation of gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) secretion. Enhancement and suppression of LH release was only observed with acute and chronic exposures to P or other naturally occurring and synthetic progestins avidly bound by pituitary progestin receptors; such responses were inhibited by cotreatment with the antiprogestin RU486 but not with the antiandrogen flutamide, illustrating the importance of the P + receptor interactions. However, cotreatment with a 100-fold molar excess of the 5 alpha-reductase inhibitor 17 beta-N,N-diethyl-carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA) had no effect on the expression of P's modulatory actions. Additional studies using different E2 pretreatments revealed that P enhanced LH release when progestin receptor levels were elevated. Moreover, the magnitude and duration of P's influences on LH release increased in cells with higher receptor levels. However, there were several instances in which progestin receptor level and P modulation of LH release did not correlate. In several instances E2-induced progestin receptor levels stabilized at a maximal level whereas P enhancement of LH secretion continued to increase in size and duration. These findings underscore the importance of progestin receptors for P-induced modulation of LH secretion and illustrate that 5 alpha-reduction and further metabolism of P is not obligatory for the expression of these responses. In addition, our data demonstrate that the important cellular mechanisms underlying E2 priming of gonadotroph responsiveness to P entail the induction of progestin receptor levels and other as yet unidentified cellular processes.     

Characterization of alphaT3-1 cells stably transfected with luteininzing hormone beta-subunit complementary deoxyribonucleic acid

Luteinizing hormone (LH) consists of alpha- and beta-subunits, and synthesis and secretion of LH are regulated by gonadotropin-releasing hormone (GnRH). In order to examine the molecular mechanisms by which GnRH regulates LH secretion, we transfected alphaT3-1 cells with rat LHbeta-subunit cDNA under the control of a constitutive promoter and established a stable cell line of LH2 cells which secreted LH in response to GnRH. Pulsatile and continuous GnRH pretreatments increased gene expression of the alpha-subunit and synthesis of LH, and enhanced the LH secretion by brief treatments with GnRH and 56 mM KCl. The LH secretions were partially blocked by elimination of extracellular Ca2+. GnRH-induced LH secretion was completely inhibited by calphostin C (a protein kinase C inhibitor) and 1 microM wortmannin. In contrast to the GnRH induction, high K+-induced LH secretion was inhibited by KN93, a Ca2+/calmodulin-dependent protein kinase II inhibitor, as well as by 1 microM wortmannin. We also confirmed that activation of cAMP-pathway induced LH secretion, but activation of mitogen-activated protein (MAP) kinase pathway was not involved in LH secretion. These results suggest that GnRH directly regulates LH secretion as well as LHbeta-subunit synthesis, and that LH2 cells are a useful model for the study of LH secretion induced by several secretagogues.     

Repetitive injections of L-glutamic acid, in contrast to those of N-methyl-D,L-aspartic acid, fail to elicit sustained hypothalamic GnRH release in the prepubertal male rhesus monkey (Macaca mulatta).

The purpose of the present study was to examine whether repetitive intravenous injections of L-glutamic acid (Glu), like those of N-methyl-D,L-aspartic acid (NMA), are able to elicit a sustained train of gonadotropin releasing hormone (GnRH) discharges from the hypothalamus of the prepubertal male monkey. In order to utilize pituitary luteinizing hormone (LH) secretion as a bioassay of hypothalamic GnRH release, the responsiveness of the gonadotroph of the prepubertal animals was enhanced prior to the study with a chronic intermittent intravenous infusion of the synthetic decapeptide (0.1 microgram/min for 3 min every h). Sequential intravenous injections of Glu (150 mg/kg BW) were administered at 3-hour intervals for 6 or 24 h. Although the first injection of this acidic amino acid elicited a robust discharge of GnRH, subsequent stimulation with Glu resulted in GnRH discharges with progressively decreasing magnitudes, and by the 9th injection Glu-induced GnRH release was abolished. Peak concentrations of circulating Glu following the 1st and 4th Glu injection were indistinguishable (3,959 +/- 437 vs. 4,139 +/- 72 nmol/ml, respectively). Interestingly, the failure of repetitive intravenous injections of Glu to sustain pulsatile GnRH release was not associated with a loss of responsiveness to NMA administration, nor was it accompanied by a corresponding decrement in Glu induced growth hormone (GH) discharges. As previously demonstrated, repetitive intravenous administration of NMA (2-5 mg/kg BW) every 3 h for 9 h sustained pulsatile GnRH secretion without decrement. A similar intermittent infusion of kainic acid (KA; 1 mg/kg BW every 3 h for 6 h), however, elicited a GnRH response that mimicked that observed in response to intermittent Glu treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased gonadotrophin releasing hormone receptors on pituitary gonadotrophs: effect on subsequent LH secretion

GnRH, high potassium concentrations, and cAMP derivatives have been previously shown to increase GnRH receptor levels (GnRH-R) in cultured rat pituitary cells. However, the effect of these changes in receptor number on subsequent stimulated LH release has not been investigated. In this study pretreatment of pituitary cells with either 1 nM GnRH, 58 mM KCl, or 1 mM dibutyryl cAMP (dbcAMP) resulted in a 70-100% increase in GnRH-R 7-10 h later. Subsequent LH responses to GnRH in those cells pretreated with GnRH and KCl were markedly reduced and the dose-response characteristics altered such that the curves were non-sigmoidal. When corrected for depletion of cellular LH during the pretreatment period these GnRH response curves were similar to control, implying that hormone depletion was the explanation for apparent desensitisation. By contrast, dbcAMP and low-dose calcium ionophore (0.1 microM A23187) pretreatment, which did not deplete cellular LH, neither enhanced nor decreased subsequent sensitivity to GnRH. Thus, 4 agents which all, under these conditions, increased GnRH receptors did not sensitise gonadotrophs to GnRH. By contrast, pretreatment with 10(-9) and 10(-8) M GnRH for either 12 or 16 h rendered cells completely or partially refractory to further GnRH stimulation, despite an increase in GnRH receptors. This desensitisation could not be explained by cellular LH depletion, and was specific to the homologous ligand since dose-responses to the Ca2+ ionophore A23187 and KCl were normal when corrected for LH depletion. Non-receptor-mediated depletion of cellular LH during A23187 pretreatment (10 microM for 10 h) did not alter subsequent GnRH dose-responses, after correction for LH content. These data indicate that, under these in vitro conditions, the increased GnRH receptors are not functionally linked to the secretory apparatus of the gonadotroph. Furthermore, homologous ligand-induced desensitisation is both time- and concentration-dependent and is mediated largely by post-receptor cellular events independent of cellular LH content. Therefore, post-receptor cellular processes may be more important than changes in GnRH receptors in regulating gonadotrophin secretion. It is suggested that an increase in GnRH receptors may represent a cellular response to generalised gonadotroph activation by a variety of agents, and does not necessarily signify enhanced responsiveness to GnRH.     

Role of Neurokinin B and Dynorphin A in pituitary gonadotroph and somatolactotroph cell lines

The role of Neurokinin B (NKB) and Dynorphin A (Dyn) in the regulation of the hypothalamic pituitary axis is an important area of recent investigation. These peptides are critical for the rhythmic release of GnRH, which subsequently stimulates the secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The present study utilized the gonadotroph cell line LβT2 and the somatolactotroph GH3 cell line to examine the possible role of these peptides in pituitary hormone secretion. The NKB receptor (NK3R) and the Dyn receptor (the κ-opiate receptor (KOR)) were both detected in LβT2 cells and GH3 cells. NKB, by itself, failed to increase gonadotropin LHβ and FSHβ promoter activities and did not modulate the effects of GnRH on gonadotropin promoter activity. In GH3 cells, NKB significantly increased TRH-induced PRL promoter activity although NKB alone did not have an effect on basal PRL promoter activity. Dyn had no effect on gonadotropin promoters alone or in combination with GnRH stimulation. PRL promoters stimulated by TRH were not significantly changed by Dyn. TRH-induced PRL promoter activity was further increased in the presence of higher concentrations of NKB, whereas Dyn did not have a significant effect on the PRL promoter even at a high concentration. In addition, TRH-induced ERK (Extracelluar signal-regulated kinase) activation was enhanced in the presence of NKB. Our current study demonstrated that NKB had a stimulatory effect on PRL expression in a PRL-producing cell, but had no effect on gonadotropin secretion from a gonadotroph cell line.     

Calcium antagonists and hormone release. III. Role of calcium in the biphasic release of luteinizing hormone in response to gonadotrophin-releasing hormone in vivo

Recent in vitro studies have demonstrated that Ca2+ plays an essential role in gonadotrophin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) release. In vivo, we have previously shown that verapamil, a substance known to inhibit calcium entry into cells, is capable of inhibiting basal gonadotrophin release as well as the release of luteinizing hormone and follicle-stimulating hormone (FSH) in response to an iv bolus of GnRH. We have examined the effects of calcium antagonists on the two phases of pituitary LH release in response to constant GnRH infusion in normal subjects. In 6 men, constant infusion of GnRH (0.2 microgram/min x 4 h) resulted in the expected biphasic LH response, with an initial rapid release of LH during the first hour of infusion, followed by a second phase release during the subsequent 3 h. When verapamil (5 mg/h) was infused together with GnRH over a 4 h period, a significant decline of the rapid as well as delayed release of pituitary LH occurred. During the calcium antagonist infusion FSH release was also inhibited, indicating that Ca2+ is also important for the release of this hormone. Our data demonstrate that Ca2+ plays an essential role in the mechanism of GnRH action on both phases of LH release and the release of FSH in normal subjects.     

Basal and gonadotrophin-releasing hormone-induced biosynthesis and release of luteinizing hormone: effect of calcium deprivation.

The present study examines the basal and gonadotrophin-releasing hormone (GnRH)-stimulated biosynthesis and release of luteinizing hormone (LH) by pituitary cells in primary culture, and the effect of extracellular calcium deprivation on these events. Pituitaries from ovariectomized adult rats were enzymatically dispersed and cultured for 96 h. The cells were then incubated for 5 h (Expts. 1 and 3) or for different time intervals between 0 and 5 h (Expt. 2), in medium containing [14C]leucine ([14C]leu) and [3H]glucosamine ([3H]gln), with or without GnRH. Total immunoreactive LH (iLH) was measured in the medium and the cell extract by radioimmunoassay. LH translation (as estimated by [14C]leu incorporation into LH; [14C]LH) and LH glycosylation (as estimated by [3H]gln incorporation into LH; [3H]LH) were measured by immunoprecipitation with specific LH beta antiserum in both medium and cell extract. Treating the cells with GnRH caused both time- and dose-dependent increases of iLH in the medium as well as in total (cells plus medium) content, with an approximate ED50 of 0.7 nM. GnRH also stimulated LH biosynthesis by increasing both LH polypeptide chain synthesis and LH glycosylation. The effect of GnRH on LH glycosylation was detected earlier than that on translation, the [3H]LH rates of production and release being higher than those of [14C]LH. These findings suggest that GnRH-induced translation and glycosylation of LH are independently regulated. Removal of extracellular calcium resulted in the loss of cellular responsiveness to GnRH, preventing not only the stimulatory effects of GnRH on total and released iLH but also the GnRH-induced incorporation of both [14C]leu and [3H]gln into newly synthesized LH. These observations suggest that GnRH-stimulated LH glycosylation and LH translation involve calcium-dependent mechanisms. Neither the uptake of radiolabeled precursors nor their incorporation into total protein were affected by GnRH or Ca(2+)-deficient (no added calcium) medium. The results also suggest that the release of newly synthesized LH is regulated differently from previously synthesized stored hormone.     

Development of gonadotrope desensitization to gonadotropin-releasing hormone (GnRH) and recovery are not coupled to inositol phosphate production or GnRH receptor number.

After initial GnRH pretreatment (10 nM, 5 h), subsequent GnRH-stimulated LH release from the gonadotrope was diminished (1 microM GnRH stimulated release of 36.4 +/- 1.4% total cellular LH over 3 h in cells initially pretreated with medium alone compared to 27.4 +/- 1.2% in GnRH-pretreated cells); however, inositol phosphate (IP) production in response to the releasing hormone remained unaffected (1 microM GnRH provoked IP accumulation of 161 +/- 9% above basal levels after 45 min in control cells and 162 +/- 11% in GnRH-pretreated cells). Pretreatment of pituitary cell cultures with NaF (a guanyl nucleotide binding protein activator, 10 mM, 3 h) also decreased subsequent GnRH-stimulated LH release, and in addition, provoked a decrease in GnRH receptor number, an increase in GnRH receptor affinity, reduction of GnRH-stimulated IP production to basal levels, and an increase in the amount of LH released in response to stimulation with the calcium ionophore A23187. In order to determine if the changes in LH release were a result of decreased IP production and/or decreased GnRH receptor binding, the time course of recovery to control levels of these processes was assessed. GnRH receptor binding continued to decrease after NaF pretreatment, reaching a nadir (62% of control) at 6 h after the pretreatment period and recovering at 48 h (90% of control). In contrast, GnRH-provoked IP accumulation did not return to control levels even after 48 h of recovery after NaF pretreatment (1 microM GnRH-stimulated IP accumulation in NaF-pretreated cells was 57% compared to control cells after 48 h of recovery). GnRH-stimulated LH release was inhibited immediately after NaF pretreatment (1 microM GnRH-stimulated LH release in NaF-pretreated cells was 65% of control levels). Cells began to recover within 3 h (80% of control) and were almost completely recovered by 6 h (90% of control). A23187-provoked LH release was enhanced immediately after NaF pretreatment (30 microM A23187-stimulated LH release in NaF-pretreated cells was 170% of control levels). Responsiveness to ionophore was 133% of control by 0.5 h, and complete recovery was measured within 1 h (100% of control). Furthermore, both NaF and GnRH pretreatment still provoked a decrease in gonadotrope responsiveness when IP production was inhibited by the phospholipase C inhibitor U-73122. The results suggest that the development of gonadotrope desensitization (by either NaF or GnRH pretreatment) can be uncoupled from changes in IP production.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct effects of prolactin and dopamine on the gonadotroph response to GnRH

The intrapituitary mechanisms underlying the inhibitory actions of hyperprolactinaemia on the reproductive axis remain unclear. Previous work on primary pituitary cultures revealed combined suppressive effects of prolactin (PRL) and dopamine on the gonadotrophin response to GnRH. However, whether these effects occur directly at the level of the gonadotroph and are accompanied by changes in gene expression is still unresolved. Here, alphaT(3)-1 and LbetaT2 cells were used to investigate the effects of PRL and dopamine on gonadotrophin synthesis and release in gonadotroph monocultures under basal and GnRH-stimulated conditions. PRL receptor and dopamine receptor mRNA expressions were first determined by RT-PCR in both cell lines. Then, PRL and the dopamine agonist bromocriptine (Br), alone or in combination, were shown to block the maximal alpha-subunit and LHbeta-subunit mRNA responses to a dose-range of GnRH. The LH secretory response was differentially affected by treatments. GnRH dose-dependently stimulated LH release, with a 4-5 fold increase at 10(-8) M GnRH. Unexpectedly, PRL or Br stimulated basal LH release, with PRL, but not Br, enhancing the LH secretory response to GnRH. This effect was, however, completely blocked by Br. These results reveal direct effects of PRL and dopamine at the level of the gonadotroph cell, and interactions between these two hormones in the regulation of gonadotrophin secretion. Moreover, uncoupling between LH synthesis and release in both the basal and the GnRH-stimulated responses to PRL and dopamine was clearly apparent.     

Acute inhibitory effect of follicle-stimulating hormone-suppressing protein (FSP) on gonadotropin-releasing hormone-stimulated gonadotropin secretion in cultured rat anterior pituitary cells

Follicle-stimulating hormone (FSH)-suppressing protein (FSP) or follistatin, a novel gonadal glycoprotein hormone, has been shown to have chronic inhibitory effects on the secretion of both FSH and luteinizing hormone (LH) in response to gonadotropin-releasing hormone (GnRH) in vitro. The present study was designed to investigate the acute effects of bovine FSP on GnRH-stimulated gonadotropin secretion and to examine the potential subcellular sites of this action of FSP using cultured pituitary cells. Anterior pituitaries from adult male Sprague-Dawley rats were enzymatically dispersed and cultured for 48 h, after which the cells were treated with bovine FSP for 6 h, followed by a 4 h stimulation with secretagogues in the continued presence of FSP. Results showed that the 35 kDa form of bovine FSP (0.1-3 nM) dose-dependently suppressed GnRH-stimulated FSH and LH secretion, with inhibition of 38 and 25%, respectively, at 3 nM. In addition, FSP suppressed gonadotropin secretion in response to activators of protein kinase C (phorbol 12-myristate 13-acetate (PMA) and mezerein) and a calcium ionophore (A23187). However, FSP had no effect on gonadotropin secretion evoked by melittin, an activator of phospholipase A2. Furthermore, 35 kDa bovine FSP did not compete with GnRH for GnRH binding sites in a direct competition study and treatment of cultured pituitary cells with FSP (0.1-3 nM) for 10 h did not alter the number of GnRH binding sites on the cell membranes. Finally, similar inhibitory effects on gonadotropin secretion in response to GnRH, PMA and mezerein were obtained with the 31 and 39 kDa forms of bovine FSP, each at a concentration of 1 nM. We conclude from the present study that FSP acutely inhibits GnRH-stimulated gonadotropin secretion in cultured pituitary cells, and that FSP exerts its action beyond the GnRH receptor, possibly by affecting the protein kinase C and/or the calcium-calmodulin systems.     

Suppression of pulsatile LH secretion, pituitary GnRH receptor content and pituitary responsiveness to GnRH by hyperprolactinemia in the male rat

To assess whether gonadotropin-releasing hormone (GnRH) release from the hypothalamus might be altered by hyperprolactinemia in the male rat, we measured in chronically hyperprolactinemic rats the pituitary GnRH receptor content and described the pattern of luteinizing hormone (LH) release during the postcastration rise in gonadotropin secretion 24 and 72 h after gonadectomy. In intact rats, the effect of hyperprolactinemia was determined by describing the pattern of LH secretion, pituitary GnRH receptor content and assessment of pituitary responsiveness to small doses of GnRH (1.0 ng). In addition, to determine the role endogenous opioids might play in inhibiting GnRH release in hyperprolactinemic rats, we examined the effect of both a continuous infusion and a bolus injection of the opioid antagonist naloxone on the pattern of LH release. Chronic hyperprolactinemia was achieved by implanting 4 pituitaries under the kidney capsules 3-4 weeks before study. Acute hyperprolactinemia was achieved by injecting rats with 1 mg ovine prolactin every 12 h for 3 days. Control animals were untreated or were chronically hyperprolactinemic rats in which the hyperprolactinemia was transiently reversed by treatment for 3 days with the dopamine agonist 2-alpha-bromoergocryptine. The mean LH concentration was greatly decreased at 24 postcastration in chronically hyperprolactinemic rats relative to controls. This decrease was associated with a decrease in LH pulse height and pulse amplitude and pituitary GnRH receptor content, but not with an increase in the LH interpulse interval. In contrast, the decrease in mean LH concentrations in hyperprolactinemic animals at 72 h postcastration was primarily associated with a significantly longer LH interpulse interval than that observed in control animals. Chronic hyperprolactinemia in intact rats decreased the pituitary GnRH receptor content, in addition to decreasing the mean LH concentrations during pulsatile GnRH administration. Chronic hyperprolactinemia also inhibited LH release relative to controls during the continuous 4-hour infusion of naloxone and in response to a bolus injection of naloxone. However, in acutely hyperprolactinemic intact male rats a bolus injection of naloxone increase LH secretion 20 min later to levels similar to those obtained in control rats. In summary, these results indicate that chronic hyperprolactinemia decreased LH secretion by primarily decreasing GnRH secretion as suggested by a decrease in pituitary GnRH receptor content and a decrease in LH pulse frequency and pulse amplitude.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of action of gonadotropin releasing hormone upon gonadotropin secretion: involvement of protein kinase C as revealed by staurosporine inhibition and enzyme depletion

The role of protein kinase C (PKC) in the mechanism of action of gonadotropin-releasing hormone (GnRH) upon gonadotropin secretion is controversial and therefore was investigated in primary cultures of rat anterior pituitary cells. A relatively selective PKC inhibitor, staurosporine, inhibited both GnRH- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced luteinizing hormone (LH) release with half-maximal inhibition (IC50) of about 80 nM. Inhibition of GnRH action was not complete suggesting also a PKC-insensitive component in GnRH-induced gonadotropin release. Staurosporine had no effect on basal LH release, or on cellular LH content, neither did the drug interfere with the binding of [125I]iodo-[D-Ser(t-Bu)6]des-Gly10-GnRH N-ethylamide to its receptor in pituitary cells. When cultured pituitary cells were incubated with TPA (1 microM) for 24-48 h no measurable cellular PKC activity could be detected. The decrease in total PKC activity was accompanied by an increase in Ca2+, phosphatidylserine (PS), diacylglycerol (DG)-insensitive activity suggesting the release of a portion of the catalytic domain of PKC (M-kinase) by the phorbol ester treatment. TPA-induced LH release was nearly abolished in PKC-depleted cells and the response to GnRH was markedly reduced (40%). The stimulatory effect of the Ca2+ ionophore, ionomycin, was not impaired in PKC-depleted cells. Impaired responses to GnRH in PKC-depleted cells were only noticed at a later phase (2-4 h) of the exocytotic response of the neurohormone. The data strongly suggest a role for PKC during the second phase of GnRH-induced gonadotropin secretion.     

Effects of lithium and phorbol esters on the dynamics of LH release from dispersed sheep pituitary cells

The possible involvement of polyphosphoinositides in the stimulation of LH release was investigated. Dispersed sheep pituitary cells were incubated in test-tubes, or perifused in columns, with gonadotrophin-releasing hormone (GnRH) and Li+, or with a phorbol ester, and the amounts and patterns of LH release over time compared. Treatment with Li+ (10 mmol/l), which is known to increase levels of inositol phosphates in gonadotrophs, was shown to have effects only on the responses of desensitized cells, significantly decreasing the rate at which the cells desensitize (P less than 0.005) and decreasing the response to supramaximal levels of GnRH stimulus (P less than 0.01). It is suggested that these effects could be due to increased levels of inositol monophosphate, inositol bisphosphate or inositol 1,3,4-trisphosphate. Responses to single or repeated pulses of GnRH at 18-, 30- and 60-min intervals were not significantly altered. Phorbol 12-myristate 13-acetate (PMA), an activator of the calcium and phospholipid-dependent protein kinase (protein kinase C), was specifically active in releasing LH with a half-maximal stimulating dose of approximately 3 nmol/l. Phorbol 12,13-diacetate, which is structurally similar to PMA but does not activate protein kinase C, did not release LH, except at high levels in freshly dispersed cells. The timing of PMA-stimulated LH release was similar to that for GnRH-stimulated release, and PMA was able to release greater amounts of LH than could GnRH. This suggests that activation of protein kinase C is likely to be important in the GnRH-stimulated release of LH from gonadotrophs.(ABSTRACT TRUNCATED AT 250 WORDS)     

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-mediated desensitization of cultured rat anterior pituitary cells can be uncoupled from luteinizing hormone release

GnRH stimulates LH release from pituitary gonadotropes. Prolonged exposure of these cells to GnRH results in decreased sensitivity to further stimulation by the releasing hormone both in vivo and in vitro. Chelation of extracellular Ca++ with EGTA blocks GnRH-stimulated LH release but does not prevent subsequent desensitization. Desensitization occurs when cells are preincubated in EGTA containing 10(-7) M GnRH for a variety of times (20 min to 12 h) or when cells are preincubated for 3 h in EGTA with 10(-10), 10(-9), or 10(-8) M GnRH. A GnRH antagonist does not cause desensitization to GnRH and blocks desensitization in response to GnRH in the Ca++-free medium. Preincubation in EGTA containing 10(-7) M GnRH for 3 h did not alter sensitivity of cells to sn 1,2 dioctanoylglycerol (a protein kinase C activator), Ca++ ionophore A23187, or veratridine (an activator of endogenous ion channels). These results suggest that desensitization results from occupancy of the GnRH receptor by an agonist and may be uncoupled from LH release.     

Acute effects of oestradiol and progesterone on melittin- and gonadotrophin-releasing hormone-induced LH secretion.

It is well established that oestradiol and progesterone modulate gonadotrophin-releasing hormone (GnRH)-induced LH secretion from cultured rat pituitary cells. Short-term oestradiol and long-term progesterone treatment exert inhibition, while short-term progesterone and long-term oestradiol treatment lead to enhancement of GnRH-stimulated LH secretion. There are several lines of evidence to suggest that the steroid effects might be mediated via a mechanism involving modulation of the GnRH signal-transduction system. To evaluate the role of arachidonic acid, which serves as an intracellular signal transducer by itself or its lipoxygenase metabolites, in the mediation of oestradiol and progesterone actions, we examined their effects on melittin (activator of phospholipase (A2)-stimulated LH secretion. When pituitary cells from adult female rats were treated for 48 h with 1 nmol oestradiol/l or 1 nmol oestradiol/l plus 100 nmol progesterone/l, GnRH (1 nmol/l)-induced LH secretion was stimulated or inhibited respectively. However, melittin (10-300 nmol/l)-stimulated LH secretion remained unaffected after such treatment. Short-term treatment with oestradiol inhibited GnRH-induced LH secretion while progesterone treatment of oestradiol-primed cells led to a stimulatory effect. Interestingly, melittin-stimulated LH secretion was influenced in the same way after the short treatment paradigm. Perifusion studies were performed to assess the kinetics of these acute steroid actions further. Four separate perifusion chambers were continuously perifused with medium and stimulated for 2 min with 1 nmol GnRH/l or 1 mumol melittin/l every 50 min in a pulsatile fashion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium signaling and gonadotropin secretion.

Agonist activation of pituitary gonadotrophs by gonadotropinreleasing hormone (GnRH) stimulates rapid InsP(3)-dependent peaks of calcium mobilization and luteinizing hormone (LH) release, followed by sustained increases in calcium-influx and hormone secretion. Receptor-mediated calcium entry through L-type and dihydropyridine-itisensitive calcium channels accounts for the sustained elevation of cytosolic calcium during GnRH action, and for most of the gonadotropin secretory response. Protein kinase C contributes to the phase of sustained LH release from GnRH-stimulated gonadotrophs, and also to gonadotropin synthesis. Calcium-dependent inactivation of L channels occurs during GnRH action, and appears to be a primary factor in the onset of desensitization of gonadotropin secretion.     

Interaction of endogenous chicken gonadotrophin-releasing hormone-I and -II on chicken pituitary cells

The presence of two endogenous forms of gonadotrophin-releasing hormone (GnRH) in the chicken hypothalamus (chicken GnRH-I ([Gln8]GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]GnRH)), and the stimulation of gonadotrophins by both forms, suggests the possible existence of GnRH receptor subtypes and gonadotroph subtypes in the chicken pituitary. This question was investigated by assessing the effects of various combinations of the two known forms of chicken hypothalamic GnRH and antagonist analogues of GnRH on LH release from dispersed chicken anterior pituitary cells in both static and perifused systems. The relative inhibition of chicken GnRH-I-stimulated and chicken GnRH-II-stimulated LH release by 12 GnRH antagonists did not differ significantly, suggesting a single GnRH receptor type. Chicken GnRH-II was approximately sixfold more potent than chicken GnRH-I in releasing LH. Release of LH in response to maximal doses of chicken GnRH-I and chicken GnRH-II and to a mixture of both was similar and the two peptides were not additive in their effects, consistent with the presence of a single type of LH gonadotroph and a GnRH receptor which binds both forms of GnRH. Each form of GnRH desensitized cells to subsequent stimulation with the other form, providing additional evidence for a single type of LH gonadotroph. These findings suggest that chicken GnRH-I and -II stimulate gonadotrophin release through a single GnRH receptor type on a single class of LH gonadotroph in the chicken pituitary.     

Interaction between cortisol and arachidonic acid on the secretion of LH from ovine pituitary tissue.

In several species, glucocorticoids act directly on the pituitary gonadotroph to suppress the gonadotrophin-releasing hormone (GnRH)-induced secretion of the gonadotrophins, especially LH. A mechanism for this action of these adrenal steroids has not been established, but it appears that the glucocorticoids influence LH release by acting on one or more post-receptor sites. This study investigated whether glucocorticoids disrupt GnRH-induced LH release by altering the liberation of arachidonic acid from plasma membrane phospholipids, a component of GnRH-induced LH release. Using perifused ovine pituitary tissue, it was established that exposure of gonadotrophs to 1-1000 nmol cortisol/l for 4 h or longer significantly reduced GnRH-stimulated LH release with the maximal inhibitory effect being observed after 6 h of exposure to cortisol. This suppressive effect of cortisol could be reversed by administration of arachidonic acid, which in its own right could stimulate LH release from ovine pituitary tissue. Furthermore, the inhibitory effect of cortisol on GnRH-stimulated LH release could be directly correlated with decreased pituitary responsiveness to GnRH-stimulated arachidonic acid liberation, consistent with our hypothesis that glucocorticoids can suppress GnRH-induced secretion of LH by reducing the amount of arachidonic acid available for the exocytotic response of GnRH.