Stimulation of luteinizing hormone release by melittin and phospholipase A2 in rat pituitary cells

Gonadotropin release in rat pituitary monolayer cultures was stimulated by phospholipase A2, as well as by its activator melittin. A dose-dependent stimulation of luteinizing hormone secretion by melittin was observed in a dose range of 10(-8) to 10(-4) M. A higher dose (1 mM) melittin had a sub-optimal effect. The stimulatory action of melittin was calcium-dependent and blocked by phospholipase A2 inhibitors, chloroquine and quinacrine. Similar to melittin, phospholipase A2 enhanced the effect of LH release in a dose range of 0.1-100 units/ml. The effect of this enzyme was also calcium-dependent with optimal calcium concentrations at 1.5 mM, as obtained also for melittin. In superfusion experiments, the stimulatory action of melittin and phospholipase A2 was reproducible in their effects on LH release in gonadotrophs. In addition, melittin (10(-7) M) stimulated LH and 3H-arachidonic acid efflux in superfused pituicytes following prelabelling with radiolabelled arachidonate. These data suggest that phospholipase A2, which releases arachidonic acid from phospholipids, may participate in controlling gonadotropin secretion in gonadotrophs, since arachidonic acid and its metabolites have previously been found to enhance gonadotropin release.     

Interactions of ovarian steroids with pituitary adenylate cyclase-activating polypeptide and GnRH in anterior pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) releases LH and FSH from anterior pituitary cells. Although this effect is relatively weak, it has a strong sensitizing action on GnRH-induced gonadotropin secretion. Here we investigated the possibility that ovarian steroids, which are well-known modulators of LH secretion, interact with PACAP and GnRH in pituitary gonadotrophs. Rat pituitary cells were treated for 48 h with vehicle, 1 nmol/l estradiol, 1 nmol/l estradiol + 100 nmol/l progesterone or 48 h with 1 nmol/l estradiol and 4 h with 100 nmol/l progesterone. The cells were stimulated for 3 h with 1 nmol/l GnRH or 100 nmol/l PACAP. Estradiol treatment alone enhanced basal as well as GnRH- or PACAP-stimulated LH secretion. LH release was facilitated by additional short-term progesterone treatment. Long-term treatment with estradiol and progesterone led to reduced LH responses to GnRH and PACAP. Neither treatment paradigms affected cAMP production. However, estradiol treatment led to enhanced cAMP accumulation in quiescent or GnRH-stimulated cells. PACAP-induced increases of cAMP production were inhibited by estradiol treatment. After 7-h preincubation with 10 nmol/l PACAP, cells responded with enhanced LH secretion to GnRH stimulation. When steroid pretreatment was performed the responsiveness of gonadotrophs to low concentrations of GnRH was still increased. In contrast, at high concentrations of GnRH the sensitizing action of PACAP on agonist-induced LH secretion was lost in steroid-treated cells. There were no significant differences between the steroid treatment paradigms. It is concluded that estradiol but not progesterone acts as a modulator of adenylyl cyclase in gonadotrophs. The stimulatory effect of estradiol is thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory effect of estradiol on PACAP-stimulated adenylyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.     

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.     

Calcium-dependent actions of gonadotropin-releasing hormone on pituitary guanosine 3',5'-monophosphate production and gonadotropin release

The effects of gonadotropin-releasing hormone (GnRH) on cGMP production and LH release in cultured rat pituitary cells are markedly dependent upon the extracellular calcium concentration. The absence of calcium from incubation media caused almost complete loss of the GnRH effects on cGMP production and LH release but did not change the stimulation of cAMP accumulation by GnRH in the pituitary of the adult male rat. In female rat pituitary cells, reduction of the extracellular calcium concentration increased the concentration of GnRH required to produce half-maximal LH release and decreased the maximal gonadotropin output but had no significant effect on basal LH release. The divalent cation ionophore A23187 stimulated LH release, and this action was dependent on extracellular calcium. Both GnRH and A23187 were found to have maximal effects when the calcium concentration was 0.6 mM, and their actions were not additive. The calcium antagonists, verapamil and lanthanum, caused concentration-dependent inhibition of the actions of GnRH, with half-maximal blockade values of 10(-5) and 3 X 10(-6) M, respectively, and had no effect on basal LH release. The binding of a radioiodinated GnRH analog, [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide, to pituitary GnRH receptors was unchanged in the absence of extracellular calcium. These observations demonstrate that stimulation of pituitary cGMP production and LH release by GnRH is dependent on extracellular calcium. The site at which calcium is required during GnRH action is at a postreceptor locus before cGMP formation.     

Mechanism of release of beta-endorphin from rat pituitary cells. Role of lipoxygenase products of arachidonic acid

We investigated the effects of metabolites of arachidonic acid on the release of beta-endorphin-like immunoreactivity (beta-end-IR) from rat anterior pituitary cells. Anterior pituitary cells from female rats cultured with arachidonic acid released beta-end-IR in a dose- and time-dependent manner. To determine which metabolites of arachidonic acid stimulated the release of beta-end-IR, we examined the effects of an inhibitor of the cyclooxygenase, indomethacin, and an inhibitor of the 5-lipoxygenase, 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA-861). beta-end-IR release from pituitary cells induced by arachidonic acid was inhibited about 37% by AA-861, but was not affected by indomethacin. Other lipoxygenase inhibitors (eicosatetraynoic and nordihydroguaiaretic acid) also reduced the release of beta-end-IR induced by arachidonic acid. The effects of the 5-lipoxygenase products 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and leukotrienes (LTA4, B4, C4, and D4) on the release of beta-end-IR from rat pituitary cells were also examined. 5-HETE (1-50 microM) elicited a dose-dependent release of beta-end-IR from cultured pituitary cells, and 50 microM 5-HETE induced beta-endorphin release time dependently. LTA4 and LTB4 also significantly stimulated the release of beta-end-IR, but LTC4 and LTD4 had no effect. Other lipoxygenase products (12-hydroxy-5,8,10,14-eicosatetraenoic acid, 12-HETE; 15-hydroxy-5,8,10,14-eicosatetraenoic acid, 15-HETE) were also secretagogues at concentrations of above 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic atrial natriuretic factors (ANFs) stimulate guanine 3',5'-monophosphate production but not hormone release in rat pituitary cells: peptide contamination with a gonadotropin-releasing hormone agonist explains luteinizing hormone-releasing activity of certain ANFs

The effects of atrial natriuretic factors (ANFs) on anterior pituitary hormone secretion and cyclic nucleotide production were investigated in cultured rat pituitary cells. ANF had no effect on ACTH, GH, PRL, and TSH release or on cAMP production either on basal hormone levels or during stimulation of their secretion by the appropriate releasing factor. However, ANF markedly stimulated cGMP production in both mixed anterior pituitary cells and enriched anterior pituitary cell populations fractionated by centrifugal elutriation. Unexpectedly, certain ANF preparations, Bachem rat ANF-(5-28) and rat ANF-(5-25), markedly stimulated LH release from cultured anterior pituitary cells and gonadotroph-enriched elutriated pituitary cells. The same ANFs also displaced [125I-D-Lys6]GnRH ethylamide from binding to anterior pituitary membranes with potencies similar to their LH-releasing activities. Immunoprecipitation of ANF with a specific antiserum abolished the effect of ANF on cGMP production, but did not change the effect of ANF on LH release. In conclusion, ANF did not affect anterior pituitary hormone secretion or cAMP production, but stimulated cGMP formation. The effect of certain ANF preparations on LH release appears to be attributable to peptide contamination with a potent GnRH agonist.     

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.     

A possible role for lipoxygenase and epoxygenase arachidonate metabolites in prolactin release from pituitary cells

We studied the effects of selected leukotrienes and hydroxyeicosatetraenoic acids (HETEs) on prolactin release from primary cultures of female rats anterior pituitary cells. Leukotrienes B4, C4, and D4 had no effect on basal prolactin release; however, they did enhance prolactin release that was stimulated by 1 or 5 nM thyrotropin-releasing hormone (TRH). Leukotriene C4 also enhanced prolactin release that was induced by phorbol myristate acetate (a protein kinase C activator) by maitotoxin (a calcium uptake stimulator), and by angiotensin II. 5-HETE, 12-HETE, and 15-HETE stimulated basal prolactin release at high concentrations (1 microM and greater), and 5-HETE and 12-HETE enhanced TRH- and angiotensin II-induced prolactin release at lower (nanomolar) concentrations as well. In order to determine the role of endogenous arachidonate metabolites in prolactin release, pituitary cell cultures were exposed to selected inhibitors of the 5-lipoxygenase enzyme, which metabolizes arachidonate to leukotrienes and 5-HETE, and to those of the epoxygenase enzyme, which metabolizes arachidonate to epoxyeicosatrienoic acids. These inhibitors decreased basal and secretagogue-induced prolactin release. In additional experiments, it was determined that TRH enhances the liberation from pituitary cells of arachidonate metabolites with high-performance liquid chromatography elution profiles similar to those of leukotriene C4 and omega-OH-leukotriene B4 (a metabolite of leukotriene B4) and the HETEs. Therefore, the production of leukotrienes, HETEs, and epoxyeicosatrienoic acids may be necessary for the normal release of prolactin.     

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.     

Epidermal growth factor stimulates luteinizing hormone and arachidonic acid release in rat pituitary cells

Epidermal growth factor (EGF) directly enhanced luteinizing hormone (LH) release from dispersed rat pituitary cells in monolayer cultures as well as in superfusion columns. This 2.3-fold stimulatory effect was dose and time dependent and was also reconfirmed in a superfusion system. Retinal, a protein kinase C inhibitor, counteracted the EGF effect only partially. Further experiments were therefore carried out to investigate alternate EGF mechanisms. Nordihydroguaiaretic acid and chloroquine suppressed the stimulatory effect of EGF in a dose-dependent manner. Moreover, EGF (10(-7) M) stimulated [3H]arachidonate release from pre-labelled rat pituitary cells. This indicates that phospholipase A2 and arachidonic acid may be involved in EGF action on LH release from rat pituicytes.     

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.     

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.     

Role of arachidonate metabolism on the in vitro release of luteinizing hormone and prolactin from the anterior pituitary gland: possible involvement of lipoxygenase pathway

The aim of the present study was to evaluate whether arachidonic acid metabolism may play a role on luteinizing hormone (LH) and prolactin (PRL) release directly at the pituitary level. To this purpose, exogenous arachidonic acid, alone or in presence of inhibitors of cyclooxygenase (indomethacin:IND) and lipoxygenase pathways (nordihydroguaiaretic acid:NDGA), was added to perfused rat anterior pituitary cells. PGE, PGF alpha, LH and PRL levels present in the eluate were assayed with specific RIA methods. Both PGE and PGF alpha show a dose-related response after the addition of increasing doses of arachidonic acid. The addition of 0.05 mM arachidonic acid induces an increase of LH and PRL. The addition of IND to the perfusion medium highly potentiates the stimulatory effects induced by arachidonic acid on LH and PRL release. On the contrary, the addition to the medium of either NDGA or IND plus NDGA completely reverses the stimulatory action induced by arachidonic acid alone. The present results suggest that: adenohypophyseal cells are able to metabolize exogenous arachidonic acid; arachidonic acid induces an elevation in LH and PRL levels; lipoxygenase pathway metabolite(s) are likely involved in these activities, and the site of action of arachidonic acid is at the pituitary level.     

The nature of the gonadotropin-releasing hormone stimulus-luteinizing hormone secretory response of human gonadotrophs in vivo

To examine the stimulus-secretion response of human pituitary gonadotrophs in vivo, we applied a new multiple parameter deconvolution technique to analyze (1) exogenous GnRH-stimulated LH secretory responses in 10 men with isolated hypogonadotropic hypogonadism (IHH), and (2) endogenous and exogenous GnRH-stimulated LH secretory responses in 8 normal men. The GnRH-deficient men were given 4 bolus doses of synthetic GnRH (7.5, 25, 75, and 250 ng/kg) iv at 2-h intervals in randomized order after long term pulsatile GnRH administration. The normal men were studied by sampling blood at 10-min intervals for 12 h basally and after 2 consecutive 10-micrograms iv GnRH doses. The serum LH peaks in both groups were subjected to quantitative deconvolution to resolve underlying LH secretory and clearance rates simultaneously. Such analyses revealed that exogenous GnRH-induced LH secretory episodes in GnRH-deficient men with IHH could be modeled as algebraically Gaussian distributions of instantaneous LH secretory rates with a mean half-duration of 14 +/- 2 min. The simultaneously resolved half-life of endogenous LH disappearance was 71 +/- 5 min. The log dose-response relationship for GnRH dose vs. maximal LH secretory rate or vs. calculated mass of LH released per secretory burst was linear. In contrast, varying GnRH doses did not alter the duration of LH secretory bursts, the half-time of LH disappearance, or the latency of LH secretory bursts after iv GnRH injections (viz. 7.6 min). Deconvolution analysis of the spontaneous (endogenous GnRH-stimulated) LH peaks in normal men revealed a mean half-duration of secretory bursts of 9.9 +/- 1.5 min, and a mean half-time of endogenous LH disappearance of 76 +/- 5 min. These values were not significantly different from those in the GnRH-treated normal or GnRH-deficient men. In summary, deconvolution analysis of LH release in men with IHH revealed a significant linear relationship between iv doses of pulsed GnRH and computer-resolved LH secretory rate and/or the mass of LH released per secretory event. In contrast, varying doses of GnRH did not alter the lag time between the GnRH stimulus and the LH secretory burst, the duration of LH secretion, or the calculated half-life of the LH released. We conclude that GnRH exerts dose-dependent effects on specific attributes of the secretory response of human gonadotrophs in vivo.     

Estradiol differentially modulates the exocytotic proteins SNAP-25 and munc-18 in pituitary gonadotrophs

Long-term treatment with estradiol increases LH secretion from female gonadotrophs. The mechanisms are not fully clarified yet. Our previous data indicated that sexual steroids might affect late steps in GnRH signal transduction such as exocytosis. The secretion of hormones from neuroendocrine cells requires the merger of secretory vesicles with the plasma membrane. This regulated exocytosis is mediated by specific proteins, which are present in the pituitary gland. Here, we examined whether two of these crucial exocytotic proteins, SNAP-25 and munc-18, are affected by estradiol in female gonadotrophs. Female rat anterior pituitary cells and alphaT3-1 cells, derived from a murine immortalized gonadotroph cell line, were treated with 100 pM estradiol for 48 h. LH secretion of anterior pituitary cells, additionally stimulated with eight consecutive pulses of 1 nM GnRH for 15 min at an interval of 1 h, was determined by RIA. Gene expression was measured by quantitative RT-PCR and protein expression by immunoblotting. Additionally, quantitative RT-PCR was performed in single rat gonadotrophs to ascribe effects exclusively to intact gonadotrophs. Pulsatile GnRH enhanced the mRNA expression of SNAP-25 and munc-18 in accordance with the LH secretory response with the greatest increase at the third pulse of GnRH. Estradiol treatment further increased GnRH-induced LH secretion at all GnRH pulses. SNAP-25 gene expression was significantly decreased at the fifth GnRH pulse and unaffected at basal after 48 h of estradiol treatment. In contrast, munc-18 mRNA levels were not significantly affected by estradiol at different GnRH-pulses in mixed anterior pituitary cells, whereas munc-18 gene expression was significantly increased at basal. In alphaT3-1 cells and single gonadotrophs, long-term estradiol treatment significantly reduced SNAP-25 protein and gene expression. In contrast, the protein and gene expression of munc-18 was significantly enhanced in both alphaT3-1 cells and single gonadotrophs. In conclusion, munc-18 and SNAP-25 were oppositionally influenced by estradiol. The results suggest that estradiol modulates the expression of exocytotic proteins in gonadotrophs and thus affects LH secretion.     

Gonadotropin-releasing hormone action in cultured pituicytes: independence of luteinizing hormone release and adenosine 3',5'-monophosphate production.

The secretory response of pituitary gonadotropes to stimulation by gonadotropin-releasing hormone (GnRH) has been extensively studied, but the mechanism by which GnRH evokes gonadotropin synthesis and release has not been clarified. In particular, there has been conflicting evidence about the role of cAMP in GnRH-induced release of LH. To examine this question in more detail, the actions of GnRH on LH release and cAMP production were analyzed in primary cultures of collagenase-dispersed rat pituitary cells. In this system, addition of 10(-10)--10(-6) M GnRh to cultured pituicytes caused rapid release of LH into the incubation medium. In contrast, GnRH caused no significant change in intracellular or extracellular cAMP or in occupancy by cAMP of the regulatory subunit of protein kinse. Neither dibutyryl cAMP nor methyl isobutylxanthine (MIC) stimulated LH production to the same level as GnRH, and neither agent potentiated the effect of the releasing hormone. Cholera toxin and prostaglandin E1 (PGE1), both of which stimulated cAMP production in cultured pituicytes, did not raise LH levels as markedly as GnRH. These results demonstrate the independence of LH release from cAMP accumulation in cultured pituicytes, suggesting that cAMP is not required for stimulation of LH release from these cells and that GnRH acts on LH secretion by a different mechanism.     

Sustained intermittent release of gonadotropin-releasing hormone in the prepubertal male rhesus monkey induced by N-methyl-DL-aspartic acid

The purpose of the present study was to determine whether gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus of the prepubertal monkey may be prematurely provoked into producing a sustained train of intermittent GnRH release N-methyl-DL-aspartic acid (NMA), an analog of the putative excitatory neurotransmitter aspartate, was used to stimulate the hypothalamus. In order to utilize pituitary luteinizing hormone (LH) secretion as a bioassay of hypothalamic GnRH release, juvenile males were castrated and the responsiveness of their gonadotrophs to GnRH was enhanced prior to the study with a chronic intermittent intravenous infusion of the synthetic decapeptide (0.1 microgram/min for 3 min every hour). Treatment with this regimen of GnRH, which appears to provide the pituitary gonadotrophs with a hypophysiotropic stimulus similar to that produced by the hypothalamus of castrated adults, elicited a pattern of pulsatile LH secretion in prepubertal animals similar to that observed in the open-loop situation in adults. This episodic pattern of LH release was sustained without decrement following termination of GnRH priming and initiation of an intermittent intravenous infusion of NMA (4.5-6.5 mg NMA/kg body weight/pulse, administered over 1 min) delivered at a frequency of 1 pulse/1 h for 50 h. In contrast, an intermittent infusion of the vehicle employed to administer NMA (saline) failed to maintain LH secretion. Administration of the same dose of NMA at a slower frequency of 1 pulse/2 h for 52 h, while also sustaining LH secretion without decrement, resulted in an exaggeration in the LH response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone binding and activation of enriched population of pituitary gonadotrophs

The properties of GnRH receptors were analyzed in isolated rat pituitary cells by binding studies with the labeled GnRH agonist, [¹²⁵I]iodo-[d-Ser(tBu)⁶]des-Gly¹⁰-GnRH-N-ethylamide. The concentration of GnRH-binding sites in pituitary cells from immature female rats was twice as high as in cells from adult females. Electron-microscopic irnmunocytochemistry revealed twice as many gonadotrophs in the immature rat pituitary, indicating that gonadotrophs from immature and mature female rats contain the same number of binding sites. An enriched population of gonadotrophs prepared from immature female rat pituitaries by velocity sedimentation at unit gravity contained 77% of the total radioimmunoassayable LH and 71% of the total GnRH receptors. A second population of small, poorly granulated gonadotrophs was distributed among the cells of other fractions of the gradient and could be detected only by irnmunocytochemistry. The smaller, possibly immature, gonadotrophs did not contain a measurable number of GnRH receptors. When incubated with the cells recovered from the gradient, GnRH stimulated LH release only in the mature gonadotroph-enriched fraction and not in other regions of the gradient. These results indicate that GnRH interacts preferentially with gonadotrophs in the pituitary. The findings also suggest that functional heterogeneity exists among pituitary gonadotrophs and that GnRH binds to and activates only the mature gonadotrophs which exhibit the highest LH-storage capacity.     

Interaction between cyclic nucleotide second messenger systems in murine Leydig cells.

Mouse Leydig cell androgen production can be acutely stimulated by atrial natriuretic factor (ANF) via cyclic guanosine 3',5'-monophosphate (cGMP). This stimulation can approach that seen with high concentrations of luteinizing hormone (LH) acting via cyclic adenosine 3',5'-monophosphate (cAMP). To assess the potential for synergistic interaction between LH/cAMP and ANF/cGMP Leydig cells were co-exposed to ANF and LH or ANF/cGMP and site/type-selective cAMP analogues. Co-exposure to 1 nM ANF and 1 ng/ml LH elicited a synergistic increase in androgen production. Both 500 microM 8-bromo-cGMP and ANF (1.0-2.5 nM) synergized with cAMP analogues selective for either of the two major isoenzymes of protein kinase A. Phosphodiesterase (PDE) inhibition was not involved as inclusion of a PDE inhibitor only augmented the response. It appears that ANF/cGMP may interact cooperatively with LH/cAMP in the stimulatory control of androgen production in the mouse Leydig cell and that the site of synergistic interaction may be the activation of the cAMP-dependent protein kinase.