Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-stimulated LH surges in pentobarbital-blocked proestrous rats

Recent evidence suggests that hypothalamic neurosecretion of neuropeptide Y (NPY) may be required for the preovulatory LH surge in female rats. Results of immunoneutralization and portal blood collection studies have suggested that NPY may serve to enhance the response of gonadotropes to the stimulatory action of LHRH. To directly test this hypothesis, the effects of NPY on LHRH-stimulated LH secretion were assessed in proestrous rats that were anesthetized with pentobarbital (PB) to block endogenous LHRH neurosecretion. Female rats were fitted with atrial catheters on diestrus. On proestrus, hourly blood samples were collected from 0900-2100 h. At 1330 h, rats received PB (40 mg/kg BW) or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15, 150, or 1500 ng/pulse) or saline along with concurrent pulses of NPY (1 or 10 micrograms/pulse). Plasma samples were analyzed by LH RIA. In PB-treated rats receiving vehicle pulses only, LH surges were completely blocked. Pulsatile LHRH treatments at 15, 150, and 1500 ng/pulse produced subphysiological, physiological, and supraphysiological LH surges, respectively. Simultaneous administration of NPY pulses with 15 ng/pulse LHRH produced significant dose-related potentiations of LHRH-stimulated LH surges (P less than 0.0001). Administration of NPY pulses with 150 ng LHRH/pulse also significantly enhanced LHRH-induced LH surges (P less than 0.05). NPY RIA of plasma confirmed NPY increments after treatments. These results demonstrate that NPY administration can potentiate pituitary responsiveness to LHRH stimulation, and are consistent with the hypothesis that one function of NPY is to operate as a neurohormonal modulator at the level of the gonadotrope during generation of the preovulatory LH surge.     

Role of protein kinase C in facilitation of luteinizing hormone (LH)-releasing hormone-induced LH surges by neuropeptide Y.

In female rats, neuropeptide Y (NPY) facilitates LHRH-induced LH surges without affecting basal LH release. The signal transduction mechanisms mediating this facilitation are unknown. Here, the involvement of PKC in this process was investigated. Anterior pituitaries (APs) were removed from rats at 1400 h proestrus and perifused in vitro with M199 for 5 h. After an equilibration and baseline period, tissue received hourly 5-minute pulses of the PKC inhibitor GF109203X (GFX), 2.5 microM, followed 15 min later by a 5-minute pulse of LHRH (10(-8) M), NPY (10(-6) M), or phorbol 12-myristate 13-acetate (PMA, 50 nM), or some combination. This regimen was repeated hourly for 3 h. As shown previously, NPY had no effect on basal LH release but greatly facilitated LHRH-induced LH release. Treatment with PMA also facilitated LHRH-induced LH release, to approximately the same degree as NPY. Inhibition of PKC activity with GFX completely prevented NPY's and PMA's facilitation of LH release but did not inhibit LH release stimulated by LHRH alone. Because previous work suggested involvement of both NPY and PKC in alterations of LHRH receptor affinity or number, the in vivo effects of NPY on LHRH binding characteristics were also investigated. Although NPY treatment reliably enhanced LHRH-induced LH and FSH surges in proestrous rats, this action was not accompanied by any detectable change in the affinity or concentration of LHRH receptors in anterior pituitary cell membranes. In summary, we have found that NPY's actions are blocked by PKC inhibition, mimicked by PKC stimulation, and not associated with any overt alterations in LHRH receptor affinity or number. We conclude that PKC activation is required for NPY's facilitation of LHRH-induced LH surges, and that this mechanism likely involves PKC targets other than those which may alter LHRH receptor number or affinity.     

Neuropeptide Y gene expression in the arcuate nucleus is increased during preovulatory luteinizing hormone surges.

Recent studies have suggested that neuropeptide Y (NPY) plays an important role in the induction of the preovulatory LH surge. The present study was performed in order to determine if a change in NPY gene expression within arcuate nucleus NPY neurons is associated with the generation of the preovulatory LH surge. In Exp 1, in situ hybridization was used to measure NPY messenger RNA (mRNA) levels in the arcuate nucleus of female rats at 0900 h and every 2 h from 1400-2200 h on the day of proestrus (PRO). Comparisons between groups showed a clear, stepwise increase in NPY gene expression throughout the day of PRO. At 1600 h, when LH values were significantly greater than 0900 h values, NPY mRNA labeling intensities in the arcuate nucleus were significantly greater than 0900 h levels (P < 0.01). By 1800 h, the time at which the LH surge peaked, NPY mRNA levels also peaked and were nearly 3-fold greater than levels observed at 0900 h (P < 0.01). NPY mRNA levels at 2000 h and 2200 h remained elevated above 0900 h levels (P < 0.01) but by 2000 h had decreased significantly from 1800 h levels (P < 0.05). In Exp 2, NPY mRNA levels were measured once again at 0900 h and 1800 h on PRO, and then at 0900 h and 1800 h on metestrus (MET), in order to determine if the change in gene expression seen in Exp 1 was unique to the day of PRO, or if it simply reflected a daily rhythm of gene expression in the nucleus. Analysis of mRNA levels showed no difference in NPY mRNA levels between 0900-1800 h on MET. Also, NPY mRNA levels at 0900 h and 1800 h on MET were significantly less than levels at 1800 h on PRO (P < 0.01). These results are consistent with the hypothesis that NPY neurons participate in the generation of LH surges through increased production of NPY and subsequent potentiation of the release and/or actions of LHRH.     

Inhibition of luteinizing hormone (LH)-releasing hormone-induced secretion of LH in rat anterior pituitary cell culture by testosterone without conversion to 5 alpha-dihydrotestosterone

The role of 5 alpha-reduction of testosterone in the inhibition of LH secretion was investigated in rat anterior pituitary cell cultures. Pituitary cells were preincubated with testosterone or dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for 17 h and then with LHRH for an additional 4 h. Dihydrotestosterone was 6-fold more potent than testosterone in the inhibition of LHRH-induced LH release. Basal LH secretion was not affected by either androgen. The inhibition curves of testosterone and dihydrotestosterone were not shifted by the presence of the 5 alpha-reductase inhibitors 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA) and 17 beta-N,N-diisopropylcarbamoyl-4-aza-androstan-3-one (DIPA). Neither 4-MA nor DIPA alone had an effect on either basal or LHRH-induced LH release. When pituitary cells were incubated with [3H]testosterone for 17 h, the radioactivities were found to be unmetabolized testosterone (66.9 +/- 2.4%), dihydrotestosterone (13.3 +/- 0.5%), androstenedione (15.9 +/- 1.3%), 5 alpha-androstane-3,17-dione (2.8 +/- 0.3%), and 3 alpha (beta), 17 beta-androstanediol (less than 1%). In the presence of 4-MA or DIPA, 5 alpha-reduction of testosterone was completely inhibited; androstenedione was the only metabolite. Androstenedione was only 12% as potent as testosterone in the inhibition of LHRH stimulation of LH release, and conversion of [3H]androstenedione to testosterone and dihydrotestosterone did occur in these cells. When [3H]dihydrotestosterone was incubated with pituitary cells, the radioactivities were dihydrotestosterone (64.4 +/- 0%), 5 alpha-androstanedione (19.3 +/- 1%), 3 alpha (beta), 17 beta-androstanediol (7.7 +/- 1.7%), and unknown polar metabolites. 4-MA and DIPA had no effect on the metabolism of dihydrotestosterone. These results indicate that both testosterone and dihydrotestosterone inhibit LHRH-induced LH release and that this activity of testosterone does not depend on its 5 alpha-reduction.     

Inhibin suppresses luteinizing hormone (LH)-releasing hormone self-priming: direct action on follicle-stimulating hormone secretion and opposition of estradiol-enhanced LH secretion.

Previous studies have suggested that the ovary produces a factor that maintains the pituitary in a state of low LHRH responsiveness that must be overcome by the self-priming action of LHRH. To determine the role of inhibin in maintaining low LHRH responsiveness in pituitaries of diestrous female rats, endogenous inhibin was passively immunoneutralized in vivo, and the pituitaries were removed 18-20 h later and examined for LHRH responsiveness in vitro. Pituitaries from diestrous control rats produced the biphasic pattern of gonadotropin secretion that typifies LHRH self-priming: an initial low secretory response to LHRH (lag phase), followed by a protein synthesis-dependent transition to an enhanced rate of secretion with continued LHRH exposure (primed phase). Immunoneutralization of endogenous inhibin [antiserum (AS) treated] resulted in an increased rate of LH secretion during the lag phase, while no change was observed in the primed phase rate of LH secretion. FSH secretion from pituitaries of AS-treated rats was increased during the lag phase to a rate of secretion similar to that observed during the primed phase of FSH secretion from control pituitaries, and it was increased further during the primed phase of secretion. These results suggest that inhibin is at least partially responsible for the low secretion of LH observed during the lag phase response to LHRH exposure and is totally responsible for the lowered rate of FSH secretion during the lag phase. The observation that the enhanced rate of gonadotropin secretion observed with AS-treated pituitaries during the lag phase was resistant to inhibition of protein synthesis provides further evidence that a partial transition from the lag to the primed phase had already occurred. Pituitaries from ovariectomized rats were also examined in order to place the contribution of inhibin in perspective with the total ovarian influence on pituitary responsiveness to LHRH. Unexpectedly, LH secretion during the lag phase was similar to the low secretion rate of diestrous control pituitaries, and the higher primed rate of secretion failed to fully develop, suggesting that an additional ovarian factor was required to induce and maintain pituitary responsiveness to LHRH in terms of LH secretion. FSH secretion from the ovariectomized rats was similar to that observed from pituitaries of AS-treated rats, thus further supporting the concept that inhibin is fully responsible for the suppression of FSH secretion in response to LHRH. Plasma from the AS-treated rats revealed a 2-fold increase in estradiol levels compared with diestrous control rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuropeptide Y enhances the release of luteinizing hormone (LH) induced by LH-releasing hormone

Depending upon the steroid hormonal milieu, centrally administered neuropeptide Y (NPY) exerts differential effects on the release of LH. Ovarian hormones also effect the concentrations of NPY in hypothalamic nuclei, and some of the changes are similar to those caused by LHRH. The present studies tested whether NPY acts directly on the pituitary gland, either alone or in combination with LHRH, to modify LH secretion. Hemipituitary fragments obtained from ovariectomized rats were incubated in medium 199, and the in vitro effects on LH release of LHRH, NPY, or the two peptides together were assessed. As expected, LHRH (10(-9)-10(-7) M) produced a dose-dependent release of LH, whereas NPY alone had a lesser stimulatory effect at concentrations of 10(-7) or 10(-6) M. On the other hand, 10(-6) M NPY significantly enhanced LH release in response to 10(-9) M LHRH. A potentiation by NPY of the LHRH-induced LH response was observed in an anterior pituitary cell culture system. Cells from the pituitaries of ovariectomized rats were dispersed and cultured for 3 days in medium 199 with BSA, gentamicin, horse serum, and fetal calf serum. During a 3-h incubation, NPY alone (10(-9)-10(-7) M) failed to affect LH release, but significantly potentiated the release induced by 10(-9) or 10(-8) M LHRH. These findings are in accord with the hypothesis that hypothalamic NPY neurons may participate in the regulation of LH secretion in the rat and indicate that one of the mechanisms of its action may be to increase the pituitary LH response to LHRH.     

Effects of luteinizing hormone (LH)-releasing hormone pulse amplitude and frequency on LH secretion by perifused rat anterior pituitary cells

Recent studies have shown that LH secretion in vivo is pulsatile. In the present study, a cell perifusion system was employed to characterize the pituitary response to changes in LHRH pulse amplitude and frequency. Increases in pulse amplitude consistently elevated both mean LH levels and the amount of LH released in response to individual LHRH pulses. The EC50 for LHRH was approximately 3 nM. Increases in pulse frequency also increased mean LH levels, but frequencies of three or more pulses per h were associated with a decrease in the amount of LH released per pulse. Alterations in LHRH pulse characteristics changed qualitative as well as quantitative aspects of LH secretion, with high frequency, high amplitude pulses producing a biphasic response to LHRH. Initially a self-priming response was seen during the second and third hours of stimulation; this was followed by increasing desensitization of the cultures to LHRH. These results, by defining the pituitary response to specific conditions of stimulation, will help to clarify the relationship of LHRH stimulation to LH secretion in vivo.     

Neuropeptide Y affects secretion of luteinizing hormone and growth hormone in ovariectomized rats.

Neuropeptide Y (NPY) has recently been localized in the rat hypothalamus. We have evaluated the effects of NPY on hypothalamic and pituitary function by injecting NPY into the third ventricle in vivo and by examining its action on perifused pituitary cells in vitro. Injections of NPY into the third ventricle of conscious ovariectomized rats led to a dramatic and highly significant reduction in plasma luteinizing hormone (LH) relative to pretreatment levels in these animals or to those of controls injected with physiological saline. Significant inhibition was obtained with doses ranging from 0.02 to 5.0 micrograms (4.7-1175 pmol) of NPY. These inhibitory effects on LH release were dose dependent and lasted for at least 120 min after injection of 5.0 micrograms of NPY. Intraventricular injection of NPY also significantly decreased plasma growth hormone; however, the threshold dose was 2.0 micrograms (470 pmol), a dose 100-fold greater than the lowest dose that inhibited LH release. Plasma follicle-stimulating hormone was unaffected by injection of NPY. NPY (10(-6) and 10(-7) M) stimulated secretion of LH, growth hormone, and follicle-stimulating hormone from perifused anterior pituitary cells loaded in a Bio-Gel P-2 column. These results indicate that NPY acts on structures adjacent to the third ventricle to inhibit the secretion of LH and growth hormone but not follicle-stimulating hormone, whereas it can directly stimulate the secretion of all three hormones from the cells of the anterior pituitary in vitro. Since NPY has been found in the hypothalamus and median eminence, it is quite likely that it plays a physiologically significant role at both hypothalamic and pituitary sites: influencing secretion of pituitary hormones.     

An opioid-neuropeptide-Y transmission line to luteinizing hormone (LH)-releasing hormone neurons: a role in the induction of LH surge

We tested the hypothesis that a decrease in hypothalamic inhibitory opioid tone produced by naloxone (NAL) will activate neuropeptide-Y (NPY) neurosecretion in 17 beta-estradiol (E2)-primed ovariectomized (ovx) rats. NPY neurosecretion was assessed in two ways. First, we studied the effects of iv saline (controls) or NAL infusion (2 mg/h) between 1100-1400 h on NPY concentrations in seven microdissected sites in the medial basal hypothalamus (MBH) and preoptic area in association with the increase in the rate of LH secretion, and then we examined the effects of NAL on the in vitro release of NPY and LHRH from the MBH of E2-primed ovx rats. We observed that in control rats, NPY concentrations in selected hypothalmic sites (median eminence, medial preoptic area, and arcuate nucleus) increased either just before LH rise at 1400 h or in association with the moderate LH surge in the afternoon. NAL infusion advanced the onset and amplified the magnitude of LH surge in the afternoon. In association with this augmentation of LH response, NAL infusion significantly increased NPY concentrations selectively in the median eminence, medial preoptic area, and arcuate nucleus. During NAL infusion at 1300 h and at the end of infusion at 1400 h, NPY concentrations in these sites increased compared to preinfusion levels at 1100 h and corresponding control levels at 1300 h. During the post-NAL infusion period until 1800 h, NPY levels remained elevated in these sites, but were not significantly different from those in control rats, which also displayed increments at this time. Further, NAL increased the in vitro efflux of both NPY and LHRH from the MBH; the increased release of two neuropeptides was dose related between 0.01-0.5 mg/ml, with the maximal increase occurring at 1.0 mg/ml NAL. Cumulatively, these studies show that 1) in association with the spontaneous LH surge in E2-primed rats, NPY concentration increased only in sites confined to the preoptic-tuberal pathway, which previously has been shown to mediate the induction of the LH surge; and 2) a decrease in the inhibitory opioid tone imposed by NAL readily augmented the hypothalamic NPY neurosecretion concomitant with an increase in duration and magnitude of the LH surge. These findings are in accord with the thesis that a decrease in the inhibitory opioid tone by the neural clock, postulated to occur before the LH surge, initiates a chain of neurosecretory events that may include a site-specific activation of NPYergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Simultaneous measurement of luteinizing hormone (LH)-releasing hormone, LH, and follicle-stimulating hormone release in intact and short-term castrate rats

The temporal relationship between LHRH release and gonadotropin secretion as well as the effects of castration on LHRH release were investigated in conscious, freely moving male rats. LHRH release was measured in hypothalamic/median eminence perfusates, while levels of pituitary gonadotropins (LH, FSH) were determined in sequential blood samples obtained via atrial catheters. Twenty-four to 26 h before experiments, rats underwent sham surgery or castration. LHRH release in push-pull perfusates from both groups was pulsatile, and nearly all identified LH pulses (83.3%) were temporally associated with LHRH pulses. Of the fewer irregular FSH pulses that were observed, only 43.7% were temporally associated with LHRH pulses. Mean LHRH pulse amplitude and mean LHRH levels were not different in intact and castrate animals. The frequency of LHRH pulses was moderately increased in castrate rats (1.30 pulses/h) compared to that in intact animals (0.83 pulses/h), and this acceleration was accompanied by a significant increase in LH pulse frequency, pulse amplitude, and mean level. It was also noted that the number of silent LHRH pulses (those not associated with LH pulses) was dramatically reduced in castrate animals. Characteristics of gonadotropin release (pulse frequency, pulse amplitude, and mean level) were not significantly different in animals undergoing push-pull perfusion/bleeding procedures from those in rats not receiving push-pull cannula implants. We conclude from these studies that 1) LH pulses show a high concordance with LHRH pulses, providing evidence that the LHRH pulse generator operates as the neural determinant of LH pulses in male rats, 2) FSH secretion is not associated with LHRH release in an obvious and consistent manner, suggesting that LHRH/FSH relationships are not easily discerned in these animals or that a FSH-releasing factor distinct from the LHRH decapeptide may regulate FSH secretion, 3) a modest increase in LHRH pulse frequency occurs 24-30 h after castration, and 4) silent LHRH pulses occur with much greater regularity in intact than in castrate rats. The latter two observations suggest that both hypothalamic and intrapituitary sequelae of castration may be critically important in the development of postcastration increases in LH secretion and the negative feedback of gonadal steroids.     

Tunicamycin and neuraminidase effects on luteinizing hormone (LH)-releasing hormone binding and LH release from rat pituitary cells in culture

We have studied the effects of tunicamycin (TM) and neuraminidase on the binding of 125I-labeled Buserelin, a GnRH agonist, and on GnRH-stimulated LH release in cultured rat pituitary cells. Treatment with TM, an antibiotic which inhibits protein glycosylation, abolished the development of elongated cell processes without any effect on cell viability. Concomitantly, TM caused a time- and dose-dependent inhibition of specific binding of Buserelin and of GnRH-stimulated LH release. The inhibition of binding was due to a decrease in the number of GnRH receptors without any significant effect on binding affinity. Protein synthesis was not affected under these experimental conditions, suggesting that the aglycosylated GnRH receptors are probably intracellularly accumulated and are not expressed on the cell surface. Treatment with neuraminidase inhibited only 50% of GnRH agonist binding and did not affect GnRH-stimulated LH release. These results indicate that the oligosaccharide portion is essential for the functional properties of the GnRH receptor.     

Endogenous opioid peptides mediate the interleukin-1-induced inhibition of the release of luteinizing hormone (LH)-releasing hormone and LH

We have reported recently that central administration of both the alpha- and beta-subtypes of the cytokine interleukin-1 (IL-1) inhibited the estrogen-progesterone-induced LH surge in ovariectomized (ovx) rats. This inhibition was probably due to a central effect, since IL-1 alpha and IL-1 beta also suppressed the in vitro LHRH output from the hypothalami of steroid-primed ovx rats. Whether IL-1 inhibits LHRH release by a direct action or via some other neuronal system is not known. Since IL-1 reportedly stimulates the release of POMC peptides, which are known to be inhibitory to the LHRH-LH axis, we have tested the hypothesis that the inhibitory influence of IL-1 may be mediated via activation of hypothalamic opioid peptides. Ovx rats, preimplanted with cannulae in the third ventricle of the brain, were injected with 30 micrograms estradiol benzoate, followed by 2 mg progesterone 48 h later. Three hours after P injection, IL-1 alpha, IL-1 beta, or saline (SAL) was injected intracerebroventricularly (30 ng/3 microliters) at 1300 h, followed immediately by iv infusion of SAL or the opiate antagonist naloxone hydrochloride (NAL; 2 mg/0.6 ml.h) for 2 h. Plasma LH levels were measured in blood samples withdrawn hourly until 1800 h. Both IL-1 alpha and IL-1 beta blocked the afternoon LH surge. NAL infusion into control SAL-injected rats did not alter the LH surge; however, it reversed the IL-1 alpha- and IL-1 beta-induced suppression of the LH surge. To determine whether this reversal of IL-1 suppression of the LH surge was due to NAL action at the hypothalamic level, the preoptic area-medial basal hypothalamus of similarly primed ovx rats was obtained at 1300 h and incubated in vitro in the presence of 10 nM IL-1 alpha or IL-1 beta with or without 100 micrograms/ml NAL. Both subtypes of IL-1 suppressed LHRH output significantly. NAL alone did not affect LHRH release, but it completely reversed the inhibitory effects of the cytokine on LHRH release. These results suggest that IL-1 alpha and IL-1 beta inhibit LHRH-LH release by stimulating the activity of hypothalamic endogenous opioid peptide systems.     

Luteinizing hormone (LH)-releasing hormone: chronic effects on LH and follicle-stimulating hormone cells and secretion in adult male rats

We investigated whether chronic administration of LHRH to normal adult rats could increase the percentages of anterior pituitary gland (APG) cells that contain immunoreactive LH and/or FSH and gonadotropin secretion. Vehicle or 1 microgram LHRH was injected sc twice daily for 6 days, and rats were decapitated 16 h after the last injection. Treatment with LHRH caused nearly a doubling in the numerical density of LH and FSH cells and in the percentage of APG cells that contained LH or FSH. It also caused a shift in the gonadotroph population from LH and LH/FSH cells to LH/FSH cells. It did not change the mean size of gonadotrophs or APG weight. These changes at the light microscopic level were not accompanied by any apparent changes in LH cells at the ultrastructural level. However, they were accompanied by an approximate doubling of the basal serum LH and FSH concentrations, an increase in the APG FSH concentration, and an increase in the basal FSH release rate (measured in vitro). The results indicate that exogenous LHRH can be administered to increase numbers of gonadotrophs in the APG, synthesis of FSH in gonadotrophs, and basal serum LH and FSH concentrations.     

Involvement of nitric oxide in growth hormone (GH)-releasing hormone-induced GH secretion in rat pituitary cells.

The involvement of nitric oxide (NO) in human GH-releasing hormone (hGHRH)-induced GH secretion was studied with freshly dissociated male rat pituitary cells. The cells were packed in a column of Bio-Gel-P2 and continuously perifused at 37 C. Hemoglobin (Hb; 10 microM), which is known to strongly bind NO, potentiated 0.01, 0.1, and 1 nM hGHRH-induced GH secretion by 73%, 52%, and 39%, respectively, without affecting the basal secretion of GH. As reported previously, 1-nM or higher concentrations of hGHRH elicit an increase in GH secretion during the application of hGHRH (on-response) and also a transient increase after the cessation of hGHRH application (off-response). It was found that Hb potentiated only the off-response in 1 nM hGHRH-induced GH secretion, and the same concentration of Hb had no effect on 10 nM hGHRH-induced GH secretion. N-Methyl-L-arginine (MeArg; 500 microM), a competitive inhibitor of NO synthase, also potentiated both the on- and off-responses of 1 nM hGHRH-induced GH secretion by 39% without affecting basal GH secretion. Since cAMP is thought to be an intracellular messenger of hGHRH action, the effects of Hb and MeArg on 1 mM (Bu)2AMP-induced GH secretion were examined. Their actions were found to be greater than those in hGHRH-induced GH secretion. Excess K+ (15 and 50 mM)-induced GH secretion, which does not involve cAMP, however, was not affected by either Hb or MeArg. In contrast, 3 mM sodium nitroprusside, which releases NO, suppressed the 1 nM hGHRH-induced off-response by 18%. The same concentration of sodium nitroprusside had no effect on excess K(+)-induced GH secretion. The effect of 8-bromo-cGMP on hGHRH-induced GH secretion was also examined, since NO is thought to exert its action through cGMP by activating guanylate cyclase in neural tissue. The application of 8-bromo-cGMP, however, did not affect 1 nM hGHRH-induced GH secretion. These observations suggest that hGHRH stimulates the synthesis of NO at least partly through cAMP, thereby partially inhibiting hGHRH-induced GH secretion.     

Median eminence dopamine and serotonin neuronal activity. Temporal relationship to preovulatory prolactin and luteinizing hormone surges

Using a high-performance liquid chromatography (HPLC) system coupled with an electrochemical detector, the concentrations of dopamine (DA) and 5-hydroxytryptamine (5-HT) and their major specific metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole-3-acetic acid (5-HIAA), respectively, were measured in the median eminence (ME) throughout the rat estrous cycle. The ME DA content remained fairly constant throughout the estrous cycle except on estrus when 17.00 h values were significantly lower than 10.00 h values (40% decrease, p less than 0.05). The ME 5-HT content determined at 10.00 h was higher on proestrus than on any other day of the cycle. The ME DOPAC concentrations did not differ between 10.00 and 17.00 h on diestrus I, diestrus II or estrus. On the contrary, there was an almost linear decline between 10.00 and 17.00 h on proestrus (36% decrease, p less than 0.05). The ME 5-HIAA content did not differ between 10.00 and 17.00 h on any day of the estrous cycle. Significant changes were recorded for the DOPAC/DA and 5-HIAA/5-HT ratio in the ME on proestrus. There was a progressive decrease, starting from 10.00 h in the DOPAC/DA ratio with minimal values (42% decrease, p less than 0.05) at 16.00 h followed by an increase from 16.00 to 19.00 h. On the other hand, the 5-HIAA/5-HT ratio increased between 10.00 and 17.00 h (97% increase, p less than 0.05) and subsequently declined until 19.00 h (67% decrease vs. 17.00 h, p less than 0.05).2+hese data show that a concomitant     

Evidence that a decrease in opioid tone on proestrus changes the episodic pattern of luteinizing hormone (LH) secretion: implications in the preovulatory LH hypersecretion

We have studied the LH secretion pattern evoked by diminution in the opioid tone produced by iv naloxone (NAL) infusion between 1100-1400 h on proestrus, the LH secretion pattern occurring spontaneously between 1430-1730 h on proestrus and the LH secretion pattern produced by exogenous LHRH administered either as a 10 ng/pulse at 20-, 30-, or 60-min intervals or infused continuously at a rate of 30 ng/h between 1200-1700 h in rats given pentobarbital at 1100 h on proestrus. Infusion of 0.5 ng NAL/h raised plasma NAL levels to 200-300 ng/ml and augmented LH secretion, as evident by increments in pulse amplitude and frequency discharge to one every 37.5 min from an average of one every 75 min in saline-infused control rats. A 4-fold increase in circulating NAL levels, produced by 2 mg/h NAL infusion, further augmented the frequency of LH episodes to 30-33 min and induced a surge-like LH secretion pattern which resembled that seen on the afternoon of proestrus. Further analysis of the secretory pattern of the preovulatory LH surge (n = 7) showed LH pulses of increased amplitude during the basal phase (n = 4), ascending phase (n = 2), and plateau and descending phases (n = 3); in two rats the LH rise was steep, and no LH pulses were identified. A LHRH pulse (10 ng/pulse) delivered at 20- or 30-min intervals or continuous infusion of LHRH at a rate of 30 ng/h produced LH surges, with peak levels reaching the range seen on the afternoon of proestrus. Further, despite the fact that 10 ng LHRH/pulse at 20-min intervals reproduced a proestrous-type LH surge, only 40% of the LHRH pulses were followed by identifiable LH pulses. Surprisingly, despite the observations that NAL evoked robust LH episodes, the basal pattern of FSH secretion in these rats was not altered. These findings show that a decrease in opioid tone on proestrus accelerates episodic LH discharge to the range that occurs after gonadectomy. A quantitative relationship between the degree of restraint on the opioid tone imposed by NAL and the magnitude of the LH response can be demonstrated. The evidence suggests that the preovulatory LH surge may occur in an episodic fashion and that it can be reproduced by LHRH delivered at a frequency rate of LH pulses seen in ovariectomized rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation effect of galanin-like peptide (GALP) on luteinizing hormone-releasing hormone-mediated luteinizing hormone (LH) secretion in male rats.

Galanin-like peptide (GALP) is a recently isolated hypothalamic peptide which has sequence homology to galanin and binds to galanin receptors with high affinity. It has been shown that GALP neurons are localized in the arcuate nucleus and that GALP-immunoreactive fibers are in close apposition with LHRH neurons in the medial preoptic area (MPA). In the present study, we found that intracerebroventricular (icv) administration of GALP increased the plasma LH level but did not change the levels of other hormones. Concomitantly, accumulation of c-Fos protein was dramatically increased in the nuclei of LHRH-positive cells in the MPA by icv GALP administration. Furthermore, the GALP-induced plasma LH response was completely abolished by pretreatment with Cetrorelix, a LHRH receptor antagonist. On the other hand, GALP did not affect the release of LH, FSH, TSH, ACTH, GH or PRL directly from dispersed rat pituitary cells in vitro. These results strongly suggest a role for GALP in the control of gonadotropin secretion through a hypothalamic mechanism involving the release of LHRH.