Neuropeptide-Y stimulation of luteinizing hormone-releasing hormone secretion from the median eminence in vitro by estrogen-dependent and extracellular Ca2+-independent mechanisms

The roles of estrogen and extracellular calcium (Ca2+) in neuropeptide-Y (NPY)-stimulated LHRH release from median eminence (ME) fragments in vitro were examined. Ovariectomized (OVX) rats received one or several sc implants of Silastic tubes containing estradiol benzoate (235 micrograms/ml sesame oil) or vehicle. Plasma estrogen concentrations were similar to levels during the estrous cycle. These estrogen treatments were equally effective in reducing the elevated plasma levels of LH in vehicle-treated OVX rats. Animals were killed 3 days after implantation, and ME fragments were incubated in medium for 30 min (control), followed by a second 30-min period (test) in medium containing NPY or potassium chloride (K+). Estrogen treatment increased the basal release of LHRH and the ME concentration of LHRH in a dose-related fashion. NPY (0.1-10 microM) increased LHRH secretion in a dose-related manner from ME fragments obtained from estrogen-treated OVX rats, but had no effect on MEs from hormonally untreated OVX rats. Treatment with higher doses of estrogen enhanced the LHRH secretory response of ME fragments to NPY (1-10 microM). K+-stimulated LHRH release from ME fragments from estrogen-treated rats was completely eliminated in Ca2+-free medium containing EGTA. In contrast, LHRH release elicited by NPY (10 microM) was unchanged in Ca2+-free medium in both the absence and presence of cobalt chloride (Co2+). Decreasing the Ca2+ concentration from 2.5 to 0.25 mM reduced K+-stimulated LHRH release 7-fold, while NPY-stimulated LHRH secretion was not affected. These results indicate that NPY stimulation of LHRH release from the ME in vitro is related to prior circulating levels of estrogen, but does not require extracellular Ca2+ in the incubation medium. NPY may enhance LHRH release in an estrogen-dependent manner during the estrous cycle and before the LH surge on proestrous.     

Examination of a possible mechanism by which anisomycin suppresses pulsatile luteinizing hormone release in ovariectomized rats

An initial study was performed to ascertain the effects of anisomycin, a reversible inhibitor of protein synthesis, on pulsatile luteinizing hormone (LH) release in adult, ovarietomized (OVX) rats. For this experiment, rats OVX 3-4 weeks earlier were fitted with indwelling atrial cannulae. On the next day (approximately 13.00 h), the rats received a subcutaneous injection of either 100 mg/kg body weight (BW) anisomycin or its saline vehicle. Administration of anisomycin significantly suppressed mean plasma LH levels, mean trough values, and both LH pulse frequency (saline: 6.3 pulses/3 h vs. anisomycin: 2.7 pulses/3 h) and amplitude. To determine whether anisomycin affected anterior pituitary LH responses to LH-releasing hormone (LHRH), a second experiment was performed in which saline- and anisomycin-treated OVX rats were given an intravenous injection of 10 ng/100 g BW LHRH 1.5 h later (14.30 h). Rats then were sacrificed and the anterior pituitary and brain removed. Whereas preinjection plasma LH levels were significantly lower in anisomycin-treated rats, they were significantly higher in anisomycin-treated rats 20 min after LHRH. Consequently, mean maximal increments and percent increments were significantly higher in anisomycin-treated rats. AP LH content and content of LHRH in the medial preoptic and suprachiasmatic nuclei were not influenced by anisomycin treatment. However, median eminence (ME) LHRH concentrations in anisomycin-treated rats were almost double the LHRH levels measured in control rats. A third study was conducted to assess the effects of anisomycin on basal and potassium (K+)-stimulated LHRH release from superfused ME explants.(ABSTRACT TRUNCATED AT 250 WORDS)     

The luteinizing hormone surge is preceded by an estrogen-induced increase of hypothalamic progesterone in ovariectomized and adrenalectomized rats

As circulating estrogen levels rise on the afternoon of proestrus, they stimulate the hypothalamo-pituitary axis. This estrogen positive feedback is pivotal to stimulate the luteinizing hormone (LH) surge required for ovulation and luteinization of ovarian follicles. In addition to estrogen, pre-LH surge progesterone is critical for an LH surge as was demonstrated by blocking progesterone synthesis. In ovariectomized (OVX) rats treated with trilostane, a blocker of the enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD) that catalyzes the conversion of pregnenolone to progesterone, estrogen did not induce an LH surge. Further, estrogen induced an LH surge in OVX and adrenalectomized (ADX) rats, indicating that the source of progesterone was neither the ovary nor adrenal gland. This estrogen-only LH surge was inhibited by pretreatment with trilostane, indicating that although the adrenal gland and ovary were not necessary for positive feedback, progesterone synthesis was critical for estrogen-induced positive feedback in an OVX/ADX rat. This suggested that the LH surge is dependent on the pre-LH surge synthesis of progesterone. Estrogen-induced progesterone receptors in the hypothalamus are vital for the LH surge, so a potential location for progesterone synthesis is the hypothalamus. OVX/ADX female rats were treated with 17beta-estradiol (50 microg) and progesterone levels were assayed by RIA. Progesterone levels were elevated in hypothalamic tissue following estrogen treatment. No increases in tissue progesterone levels were found in parietal cortex, cerebellum, medulla, pituitary or plasma. Additionally, male rats that do not have an estrogen positive feedback-induced LH surge were examined. Castrated/ADX male rats had no increase in hypothalamic progesterone levels after estrogen treatment. Together, these data strongly suggest that estrogen enhances neuroprogesterone synthesis in the hypothalamus that is involved in the positive feedback regulating the LH surge.     

Correlation of luteinizing hormone surges with estrogen nuclear and progestin cytosol receptors in the hypothalamus and pituitary gland. II. Temporal estradiol effects

In these studies we examined the temporal parameters of estradiol (E2) priming required before progesterone (P4) amplifies the surge release of luteinizing hormone (LH) in short-term ovariectomized (OVX) rats. Thereafter, we correlated the time of appearance and concentrations of estrogen nuclear (E2Rn) and progestin cytosol (PRc) receptors in brain regions known to be involved in the surge release of LHRH. Steroid receptor concentrations were also measured in the pituitary gland. 1 week after OVX (day 0), Silastic capsules containing E2 (150 micrograms/ml in oil s.c.) were placed at 09.00 h. The serum E2 concentrations produced by these capsules peaked (41 +/- 2.1 pg/ml) at 10.00 h on day 0 and declined thereafter to values which ranged between 19 and 10 pg/ml on days 1 through 4. Some of these E2-treated rats also received 2 P4-containing Silastic capsules (50 mg/ml s.c. in oil) at 09.00 h on either days 0, 1, 2, 3 or 4. Serum P4 concentrations produced by such capsules were 9.3 +/- 0.5 ng/ml. Blood was collected sequentially at 09.00 h and at hourly intervals from 12.00-18.00 h on the day that the animals received the P4 capsules and 10.00 and 15.00 h samples also were taken from each group the next day. LH surges occurred in these E2-treated rats in the afternoons of days 2-4, but not on days 0 and 1. P4 treatment on day 1 elicited an LH surge and on days 2-4 it amplified plasma LH surge concentrations and advanced by 1 h the time of release of this gonadotropin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of progesterone on the pituitary responsiveness to, and priming effect of luteinizing hormone releasing hormone in female rats exposed to constant light

We have investigated the role of progesterone in the mating-induced release of luteinizing hormone (LH) and ovulation in female rats exposed to a 60-day period of constant light (LL). Plasma LH and progesterone concentrations were increased after mating; plasma estradiol concentrations, although not increased after mating, were increased compared with the concentrations in female rats on light-dark (LD) exposure during diestrus, proestrus evening and estrus. Progesterone induced ovulation in about half the number of female rats exposed to long-term LL, and in these animals, there was a significant increase in pituitary responsiveness to luteinizing hormone releasing hormone (LHRH) 5 h after progesterone injection. The magnitude of the priming effect of LHRH was markedly increased 2 h after progesterone treatment. Treatment with sodium pentobarbitone (SP) 15 min before an injection of progesterone, blocked the increase in pituitary responsiveness to LHRH 5 h later, but treatment with SP 4 h before progesterone injection did not block the increase in the magnitude of the priming effect of LHRH. These results suggest that progesterone acts both at the brain and pituitary to facilitate LH release, and that the increase in plasma progesterone produced by mating is at least partly responsible for the LH surge induced by mating in LL rats.     

Morphological characterization of LH secretory granule response to LHRH and calmodulin inhibitor

A quantitative electron microscopy study was carried out to characterize the luteinizing hormone (LH) contained secretory granules in response to exogenous luteinizing hormone releasing hormone (LHRH) alone or in combination with calmodulin inhibitor (W13) in orchidectomized rats pretreated with estrogen. The plasma LH concentration rose quickly 30 min after a single large LHRH injection, and then gradually increased further until 150 min. However, the rise was attenuated by supplemental administration of W13 30 min after LHRH exposure. The mean diameter of secretory granules quickly decreased inversely to the increase of plasma LH concentration after LHRH injection. However, the mean diameter increased significantly in accordance with the complete attenuation of the increase in LH release caused by administration of W13. There was an inverse correlation between the diameter of secretory granules and plasma LH concentration. Small secretory granules with low electron density frequently appeared in castration cells after LHRH injection. It is concluded that reduced size and density of secretory granules is the main morphological standard responsible for extraordinary LH secretion from gonadotrophs.     

Correlation of luteinizing hormone surges with estrogen nuclear and progestin cytosol receptors in the hypothalamus and pituitary gland. I. Estradiol dose response effects

The present studies were designed to answer three questions: (1) how will a progressive increase in serum estradiol (E2) in ovariectomized (OVX) rats affect progesterone (P4)-induced luteinizing hormone (LH) surge concentrations? (2) Can steroid-induced LH surges be correlated with estrogen nuclear receptor (E2Rn) and progestin cytosol receptor (PRc) levels in brain regions known to regulate LH secretion, and (3) do differences in pituitary responsiveness to LHRH in E2- or E2P4-treated OVX rats parallel changes in E2Rn and PRc concentrations in this gland? 1 week after ovariectomy of adult cyclic rats (day 0), Silastic E2 capsules were placed subcutaneously at 09.00 h and produced serum E2 levels of 6-8 (low), 12-19 (medium) and 27-37 (high) pg/ml, respectively. 2 days later (day 2), some rats also received Silastic P4 capsules subcutaneously which elevated serum P4 concentrations to 10-12 ng/ml. In rats with low serum E2, P4 treatment induced peak serum LH levels of 913 ng/ml. When serum E2 was increased to the medium or relatively high physiologic range, P4 treatment resulted in LH surge levels of 4,686 and 5,030 ng/ml. OVX controls and E2-treated OVX rats were sacrificed at 10.00 h on day 2 and E2Rn and PRc were measured concurrently in the preoptic area (POA), mediobasal hypothalamus (MBH), corticomedial amygdala (CMA) and pituitary gland (PIT). Raising serum E2 from OVX levels to the low range significantly increased both E2Rn and PRc in MBH and PIT, but not in the POA or CMA.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibitory effect of anandamide on luteinizing hormone-releasing hormone secretion is reversed by estrogen

Because Delta-9-tetrahydrocannabinol (THC) inhibited luteinizing hormone-releasing hormone (LHRH) in male rats, we hypothesized that the endocannabinoid, anandamide (AEA), would act similarly. AEA microinjected intracerebroventricularly (i.c.v.) decreased plasma luteinizing hormone (LH) at 30 min in comparison to values in controls (P < 0.001). The cannabinoid receptor 1 (CB1-r)-specific antagonist, [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (AM251), produced a significant elevation in plasma LH (P < 0.01). AEA (10(-9) M) decreased LHRH release from medial basal hypothalami incubated in vitro. These results support the concept that endogenous AEA inhibits LHRH followed by decreased LH release in male rats. In ovariectomized (OVX) female rats, AEA i.c.v. also inhibited LH release, but in this case AM251 had an even greater inhibitory effect than AEA. In vitro, AEA had no effect on LHRH in OVX rats. It seems that endogenous AEA inhibits LHRH followed by decreased LH release in OVX rats but that AM251 has an inhibitory action in this case. In striking contrast, in OVX, estrogen-primed (OVX-E) rats, AEA i.c.v. instead of decreasing LH, increased its release. This effect was completely blocked by previous injection of AM251. When medial basal hypothalami of OVX-E rats were incubated, AEA increased LHRH release. The synthesized AEA was higher in OVX-E rats than in OVX and males, indicating that estrogen modifies endocannabinoid levels and effects. The results are interpreted to mean that sex steroids have profound effects to modify the response to AEA. It inhibits LHRH and consequently diminishes LH release in males and OVX females, but stimulates LHRH followed by increased LH release in OVX-E-primed rats.     

Ovarian influence on gonadotropin and prolactin release in mated rabbits

In 17β-estradiol (E)-treated ovariectomized (OVX) rabbits, the coitus-induced luteinizing hormone (LH) surge is only one fourth that in ovarian-intact rabbits. In this study, we determined the pattern of the coitusinduced gonadotropin release, i.e., LH and folliclestimulating hormone (FSH), in OVX + E animals without or with continuous 3-wk treatment of 20-α-hydroxypregn-4-en-3-one (20αP). For positive and negative experimental controls, ovarian-intact rabbits were either mated or sham mated, respectively. The pituitary hormones prolactin (PRL) and growth hormone (GH) were measured to serve as collateral controls for gonadotropins. The addition of continuous 20αP in OVX + E does fail to stimulate a coitus-induced LH surge equal in magnitude and duration to the LH surge in ovarian-intact rabbits. Postcoital levels of FSH were greater in OVX + E + 20αP animals than those in OVX + E rabbits. Coitus induced a PRL surge in ovarianintact and OVX + steroid-treated females, but not in mated males, thereby suggesting a gender difference in this neuroendocrine circuit. Neither coitus nor steroids altered plasma GH values in female or male animals. We conclude that chronic administration of neither E nor E + 20αP can restore full-scale gonadotropin surges in OVX rabbits, whereas replacement of one or both of these steroids is sufficient for a coitusinduced PRL surge. Moreover, the presented observation that activin stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release suggests a possible involvement of ovarian proteins in the production of a full-scale coitus-induced GnRH/LH surge.     

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-induced LH secretion only under conditions leading to preovulatory LH surges.

We recently demonstrated that neuropeptide Y (NPY) potentiates the ability of pulsatile LHRH infusions to restore LH surges in pentobarbital (PB)-blocked, proestrous rats. In the present study we determined if specific endocrine conditions are necessary for the expression of these direct pituitary effects of NPY. Facilitatory actions of NPY were examined in the absence of gonadal feedback [ovariectomy (OVX)], in the presence of negative gonadal feedback (metestrus), after estrogen priming of the pituitary gland [OVX plus 30 micrograms estradiol benzoate (EB) 2 days before experiments], and after treatments which evoke preovulatory-like LH surges (OVX plus EB and 5 mg progesterone or P the morning of experiments). Rats received jugular catheter implants the day before experiments. On the day of experiments, hourly blood samples were taken from 1100-2100 h. At 1330 h, rats received injections of PB to block endogenous LHRH release, or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15 ng/pulse) or saline, along with concurrent pulses of NPY (1 or 5 micrograms/pulse) or saline. Plasma samples were analyzed by LH RIA. In all cases, pulsatile administration of 15 ng LHRH resulted in plasma LH levels that were significantly elevated above saline-treated, PB-blocked controls. Only in the case of EB+P-treated rats did coadministration of 5 micrograms NPY along with LHRH significantly enhance LHRH-stimulated LH secretion (P < 0.001). NPY had no effect on LHRH-stimulated LH secretion in OVX, OVX + EB-treated, or metestrous rats. Pulsatile administration of either dose of NPY alone did not stimulate LH release in any of the four groups examined. These results demonstrate that the facilitatory effects of NPY on LHRH-stimulated LH secretion can be manifest only under the endocrine conditions required to produce full, preovulatory-like LH surges, i.e. after estrogen and P treatment.     

Decreases in mediobasal hypothalamic and preoptic area opioid ([3H]naloxone) binding are associated with the progesterone-induced luteinizing hormone surge

In view of evidence implicating hypothalamic opioid systems in the control of LH release, we have examined the binding of [3H]naloxone (NAL) to slices of mediobasal hypothalamus (MBH) and preoptic area (POA) during the induction of an afternoon LH surge (1630-1700 h) in estradiol benzoate (EB)-primed ovariectomized (OVX; day 0) rats by treatment with progesterone (P; day 2). Such a surge was invariably accompanied by a decrease from early morning (1000 h) values in the number of NAL-binding sites detectable in the MBH, while the affinity of the binding site was not affected over the course of the day. Time-course studies indicated that P injection at 1000 h on day 2 was followed by a transient midday elevation in the amount of NAL bound to slices of MBH; the binding decreased significantly before the onset of and during the LH surge. A similar diurnal change was not observed in MBH slices of either oil-treated OVX rats (controls) or EB-treated OVX rats, which displayed only a 2-fold increase in LH release in the afternoon. Further studies indicated a similar change in NAL binding to slices of the POA of EBP-treated rats. Since hypothalamic opioid systems inhibit LH release, the decrease in opioid binding to MBH as well as POA slices suggests that P may curtail the existing opioid inhibitory influence in these areas before and during the course of the afternoon LH surge.     

Effect of destruction of the dorsal anterior hypothalamus on follicle-stimulating hormone secretion in the rat

The role of the paraventricular nucleus-dorsal anterior hypothalamus (PVN-DAHA) in the control of anterior pituitary gland secretion of FSH and LH in castrated male and female rats was examined. Bilateral radiofrequency lesions of the PVN-DAHA in chronically ovariectomized (OVX) rats lowered plasma FSH levels by 33% (P less than 0.005) compared to values in unoperated and sham-operated control rats; plasma LH concentrations were unaltered. RIA of median eminence (ME) LHRH concentrations in these animals revealed no differences among the three experimental groups. Other categories of diencephalic destruction did not result in this pattern of selectively reduced FSH release. Bilateral radiofrequency destruction of the PVN-DAHA also attenuated by 50% (P less than 0.025 to P less than 0.005) the progesterone-induced surge of FSH in estrogen-primed OVX rats. Progesterone-induced LH release was unaffected by PVN-DAHA lesions. Other lesion categories failed to show the same result. Bilateral ablation of the PVN-DAHA in male rats resulted in a selective diminution of the postcastration rise of plasma FSH beginning 48 h postcastration (P less than 0.05 to P less than 0.005) and persisting for 14 days (P less than 0.005) after orchidectomy, thus revealing the time course and permanence of this procedure on plasma FSH levels. The postcastration rise of plasma LH levels was not affected by PVN-DAHA lesions. The concentration of ME LHRH was the same among orchidectomized male rats whether they bore PVN-DAHA lesions, sham lesions, or no lesions. In summary, destruction of the PVN-DAHA was found to reduce significantly the elevation of plasma FSH, but not LH, in the OVX rat and the estrogen-progesterone-stimulated OVX rat. PVN-DAHA lesions also attenuated the postcastration rise of FSH, but not that of LH, in the male. The failure of lesions of the PVN-DAHA to alter ME LHRH concentrations in the face of decreased FSH release does not prove that LHRH release is totally unaffected by this procedure. This finding is, however, consistent with the concept that diminished FSH secretion could be the result of a deficiency of a hypothalamic releasing factor (FSH-releasing factor?) other than that of the LHRH decapeptide.     

Changes in local cerebral glucose utilization associated with the spontaneous ovulatory surge of luteinizing hormone in the rat

Brain activity during the spontaneous ovulatory surge of luteinizing hormone (LH) has been studied by measuring local cerebral glucose utilization (LCGU) by the [14C]-2-deoxyglucose method. The LCGU was determined in 37 brain areas and the pituitary gland in conscious, freely moving female rats in the morning and the late afternoon of proestrus. No increases in LCGU were detected, but, unexpectedly, there was a significant decrease in the LCGU measured in the afternoon compared with the morning of proestrus in the medial preoptic and anterior hypothalamic areas, the arcuate nucleus, median eminence and amygdala. Significant reductions in LCGU also occurred in the midbrain central grey and reticular formation. These results suggest that the LH and/or the prolactin surge is associated with a significant reduction in the activity of brain areas known to be essential components of the central control of gonadotropin and prolactin secretion. In the case of the arcuate nucleus and median eminence, for example, the results could be explained by a decreased activity of the opioid and dopaminergic neurons which are known to inhibit the release of luteinizing hormone releasing hormone (LHRH). Disinhibition of LHRH neurons would result in the increased release of LHRH into the hypophysial portal vessels. Reduction in the activity of the arcuate dopamine neurons could also play a major role in the prolactin surge. The decreased LCGU of the midbrain central grey may be related to the onset of lordosis behavior which appears to be time-locked to the LH surge.     

Comparison of mechanisms of action of luteinizing hormone-releasing hormone (LHRH) antagonist cetrorelix and LHRH agonist triptorelin on the gene expression of pituitary LHRH receptors in rats

The mechanisms through which luteinizing hormone (LH)-releasing hormone (LHRH) antagonists suppress pituitary gonadotroph functions and LHRH-receptor (LHRH-R) expression are incompletely understood. Consequently, we investigated the direct effect of LHRH antagonist cetrorelix in vitro on the expression of the pituitary LHRH-R gene and its ability to counteract the exogenous LHRH and the agonist triptorelin in the regulation of this gene. We also compared the effects of chronic administration of cetrorelix and triptorelin on the LHRH-R mRNA level and gonadotropin secretion in ovariectomized (OVX) and normal female rats. The exposure of pituitary cells in vitro to 3-min pulses of 1 nM LHRH or 0.1 nM triptorelin for 5 h increased the LHRH-R mRNA level by 77-88%. Continuous perfusion of the cells with 50 nM cetrorelix did not cause any significant changes, but prevented the stimulatory effect of LHRH pulses on the receptor mRNA expression. In OVX rats, 10 days after administration of a depot formulation of cetrorelix, releasing 100 microg of peptide daily, the elevated LHRH-R mRNA level was decreased by 73%, whereas daily injection of 100 microg of triptorelin caused a 41% suppression. In normal female rats, cetrorelix treatment suppressed the LHRH-R mRNA level by 33%, but triptorelin increased it by 150%. The highly elevated serum LH levels in OVX rats and the normal LH concentration of cycling rats were rapidly and completely suppressed by cetrorelix. Triptorelin decreased the serum LH in OVX rats to the precastration level, but had no effect on basal LH in normal rats. Our results confirm that LHRH antagonists, such as cetrorelix, inhibit the gene expression of pituitary LHRH-R indirectly, by counteracting the stimulatory effect of LHRH. A rapid suppression of serum LH by LHRH antagonists would be advantageous in the treatment of sex hormone-dependent tumors and other conditions.     

Prostaglandin D2 induces release of luteinizing hormone from the rat pituitary gland without the modulation of hypothalamic luteinizing hormone releasing hormone

The effect of prostaglandin D2 (PGD2) on release of LH and LH releasing hormone (LHRH) was studied in a sequential double-chamber superfusion system using the medial basal hypothalamus (MBH) and the pituitary gland from female rats at dioestrus. Infusion of PGD2 (5.7 or 57 mumol/l) caused a significant (P less than 0.05) increase in LH release to values 40-60% above the preinjection values from the pituitary gland superfused either alone or in series with the MBH. No release of LHRH in response to PGD2 was observed from the superfused MBH. These data demonstrate that PGD2 causes LH release from the pituitary gland not by inducing release of hypothalamic LHRH but by a direct action on the gland.     

Effect of LHRH on incorporation of [32P]-orthophosphate into phosphatidylinositol by dispersed anterior pituitary cells

Stimulated release of luteinizing hormone (LH) from the anterior pituitary in response to luteinizing hormone releasing hormone (LHRH), is apparently controlled by Ca2+-mediated events. In many tissues, when an involvement of Ca2+ in secretion has been found, there is an associated increased metabolic turnover of phosphatidylinositol. The objective of this investigation was to determine the effect of LHRH on the incorporation of [32P]orthophosphate into phosphatidylinositol by anterior pituitary cells maintained in vitro. When anterior pituitary cells were incubated for 5-40 min in the presence of [32P]orthophosphate, phosphatidylinositol and phosphatidylcholine were the most rapidly radiolabelled phospholipids. The addition of LHRH to the cell culture medium at concentrations previously demonstrated to release LH, increased the incorporation of 32P]orthophosphate into phosphatidylinositol in a dose-dependent manner. Incorporation of [32P]orthophosphate into other phospholipids was unaffected by LHRH at all concentrations employed. These results are consistent with the hypothesis that LHRH causes a receptor-mediated increase in turnover of phosphatidylinositol and this may be among the early metabolic events in the mechanism of LHRH-stimulated LH secretion from the anterior pituitary gland.     

Acute and long-term changes in central and pituitary mechanisms regulating pulsatile luteinizing hormone secretion after ovariectomy in the rat

These experiments examined the time course of changes in the characteristics of pulsatile luteinizing hormone (LH) secretion that occur after ovariectomy (OVX) in the rat, and compared the response of the brain and pituitary to the absence of ovarian steroid negative feedback. The literature indicates that the brain could respond to OVX by altering the frequency and/or amplitude of the pulsatile luteinizing hormone releasing hormone (LH-RH) release which triggers pulsatile LH secretion, while the pituitary could respond by altering basal LH output and/or its response to LH-RH. In vivo experiments examined changes in mean blood LH levels, LH pulse amplitudes and pulse frequencies in control rats on diestrus 1 (D1) and at 7 h, 14 h, 1, 2 or 8 days, or 3 weeks following OVX. In addition, anterior pituitaries from rats on D1 or rats ovariectomized for 1, 2 or 8 days, or 3 weeks, were incubated in vitro with or without LH-RH to examine changes in both basal and LH-RH-induced LH release. Mean blood LH levels increased within 17-24 h following OVX. This acute, rapid elevation was due to increases in both LH pulse frequency and pulse amplitude. Blood LH levels continued to increase over a 3-week period. However, the long-term increase seen at 8 days or 3 weeks was not only due to increases in LH pulse frequency and amplitude, but also to dramatic increases in basal LH secretion. The frequency of pulsatile LH release was maximal within 8 days, with no further increases occurring over the next 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Forskolin-associated luteinizing hormone release by rat anterior pituitary glands: effects of castration and estradiol replacement.

We have previously shown that the diterpene forskolin, a compound which increases intracellular cyclic AMP (cAMP), causes a concentration-dependent release of luteinizing hormone (LH) by continuously perifused anterior pituitary cells from female rats. To test the hypothesis that cAMP-associated LH release is an estrogen-dependent process, we first compared concentration-response relationships between forskolin and both cAMP production and LH secretion by tissue obtained from intact female, intact male and ovariectomized (OVX) rats. Anterior pituitary fragments were perifused with medium alone or medium plus several concentrations of forskolin for 4 h. All three groups demonstrated concentration-dependent cAMP production in response to forskolin. However, while a concentration-dependent release of LH by forskolin was confirmed in pituitary fragments from intact female rats, no such relationship could be identified for tissue from OVX or male rats. Pituitary fragments from OVX rats administered estradiol via silastic capsule for 48 h were next challenged with 3 microM forskolin. In response to this submaximal concentration of the diterpene, a brisk increase in LH secretion was observed. These results demonstrate that, although forskolin stimulates the production of cAMP in intact male, intact female and OVX animals, an associated release of LH can be documented only in the intact female group. These findings, together with the observation that administration of estrogen appears to restore forskolin-associated LH secretion in OVX animals, suggest that estrogen may play a key role in the stimulation of LH release by cAMP.     

Ascorbic acid stimulates gonadotropin release by autocrine action by means of NO

Because high concentrations of ascorbic acid (AA) are found in the adenohypophysis, we hypothesized that it might have an acute effect on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the gland, particularly because we have reported that AA rapidly inhibits stimulated LH-releasing hormone (LHRH) release from medial basal hypothalamic explants. Incubation of anterior pituitary halves from adult male rats with graded concentrations of AA for 1 h induced highly significant release of both FSH and LH with a minimal effective concentration of 10(-5) M. Release remained on a plateau from 10(-5) to 10(-2) M. When both AA and an effective concentration of LHRH were incubated together, there was no additive response to LHRH and the response was the same as to either compound alone. The FSH and LH release in response to AA was blocked by incubation with N(G)-monomethyl-l-arginine (NMMA) (300 microM), a competitive inhibitor of NO synthase. NMMA also inhibited LHRH-induced LH and FSH release and gonadotropin release in the presence of both LHRH and AA, whereas sodium nitroprusside, a releaser of NO, stimulated LH and FSH release. Membrane depolarization caused by incubation in high potassium (K(+) = 28 or 56 mM) medium stimulated release of FSH, LH, and AA that was blocked by NMMA. We hypothesize that AA is released with FSH and LH from secretory granules. AA is transported back into gonadotropes by the AA transporter and increases intracellular [Ca(2+)]-activating NO synthase that evokes exocytosis of gonadotropins and AA by cGMP.