Stimulation of luteinizing hormone release from chicken pituitary cells by analogues of luteinizing hormone-releasing hormone.

The luteinizing hormone (LH) releasing activities of luteinizing hormone-releasing hormone (LH-RH) and four related analogues were compared using isolated chicken anterior pituitary cells. The analogues, des-Gly¹⁰-LH-RH and Phe⁵-LH-RH, exhibited a greater potency than LH-RH (150 and 237%, respectively), whereas LH-RH(OH) was much less active (1.1%). The potency of Phe⁵-LH-RH was reduced to 0.9% by the insertion of a tyrosine molecule at position 11, indicating that chain length is a significant feature of the biological activity of the molecule. des-Gly¹⁰-LH-RH and Phe⁵-LH-RH were more active in the present system, than is indicated by available information for the rat.     

Morphological evidence that luteinizing hormone-releasing hormone neurons participate in the suppression by estradiol of pituitary luteinizing hormone secretion in ovariectomized rats

Morphological characteristics of LHRH neurons identified by immunocytochemistry were studied using light and electron microscopy in female rats in which estradiol was replaced at the time of ovariectomy ('pseudo-intact' rats) or 3 weeks after ovariectomy (long-term ovariectomized, estradiol-treated). While estradiol levels were equivalent in these two groups, the rise in LH after ovariectomy was prevented by the immediate administration in the pseudo-intact rats, while the augmented plasma LH levels present three weeks following ovariectomy were only reduced by 50% as a result of delayed estradiol treatment. The LHRH content of the medial basal hypothalamus (MBH) including the median eminence (ME) was greater in pseudo-intact females than in untreated long-term ovariectomized control females or long-term ovariectomized, estradiol-treated females, both 1 and 14 days after estradiol exposure. Immunocytochemistry revealed fewer LHRH-immunopositive neuronal processes coursing throughout the MBH and terminating in the ME of long-term ovariectomized, estradiol-treated rats compared to those in pseudo-intact rats. However, within individual neurovascular terminals in the ME, image analysis revealed that the area of reaction product was greater in long-term ovariectomized, estradiol-treated animals. Equivalent amounts of LHRH were assayed in the MBH within each group of animals by several LHRH antisera regardless of their different binding requirements (R42, IJ29 and A-R743), suggesting that the predominant moiety present in neuronal terminals is the fully mature decapeptide. In contrast, in the preoptic area-anterior hypothalamus (POA-AH) these antisera assayed amounts of LHRH that varied as a function of binding characteristics, although the quantities did not vary with the estradiol treatment schedule. Immunocytochemical results paralleled these assay data; antisera requiring an interior sequence of amino acids (A-R743 and A-R419) detected approximately 3 times as many immunoreactive perikarya in the POA-AH as did an antiserum requiring the free amidated C terminal (IJ29). The estradiol treatment schedules had no effect on the total number of LHRH-immunopositive neurons detected by each antiserum or the distribution of LHRH-immunopositive neuronal perikarya. These data support the hypothesis that the predominant moieties present in neuronal cell bodies are precursor forms. The fine-structural characteristics of LHRH-immunopositive neuronal cell bodies are consistent with greater secretory and biosynthetic activity in LHRH neurons of long-term ovariectomized, estradiol-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of prostaglandin accumulation by luteinizing hormone-releasing hormone (LHRH) and LHRH analogs in rat granulosa cells in vitro

LHRH and LHRH agonists have been reported to exhibit somewhat paradoxical antireproductive effects. The current studies were undertaken to examine the actions of these substances on one parameter of ovarian function which is required for ovulation, i.e. prostaglandin production. Granulosa cells were obtained from immature rats 48 h after injection of 20 IU PMSG and incubated for up to 5 h in vitro. LHRH, [D-Ala6, des-Gly-NH2(10)]LHRH-ethylamide (Analog I) and [D-Leu6, des-Gly-NH2(10)]LHRH-ethylamide (Analog II) were observed to stimulate prostaglandin accumulation by granulosa cells. Analog I, for example, at 100 ng/ml increased PGE from 0.19 +/- 0.06 in the control to 7.7 +/- 2.01 ng/2 x 10(6) cells (n = 7; P less than 0.01) after 5 h. TRH, on the other hand, had no effect on prostaglandin accumulation. When added with LH or FSH, the stimulation by maximal concentrations of Analog I and the gonadotropins appeared additive. Although the stimulation of prostaglandin accumulation by LH appears to be mediated by cAMP, no effect of Analog I on the amount of cAMP could be detected. cAMP was determined in cells plus medium at times of 1.5 min to 5 h and at concentrations of 10-2000 ng/ml Analog I, but did not change from the control. LHRH and LHRH agonists, therefore, stimulate the accumulation of ovarian granulosa cell prostaglandins and do so in a manner apparently distinct from that of LH or FSH.     

Specific release of a novel pituitary polypeptide, 7B2, from rat anterior pituitary cells in vitro by luteinizing hormone-releasing hormone

We have recently purified a novel pituitary polypeptide, designated 7B2. Subsequently, we developed a sensitive and specific radioimmunoassay (RIA) for this novel polypeptide. Our aim in the present study was to investigate the release of 7B2 from rat pituitary induced by various hypothalamic factors [luteinizing hormone-releasing factor (LH-RH), corticotropin-releasing factor (CRF), and growth hormone-releasing factor (GRF)]. The anterior pituitaries were removed from rats and immediately dispersed enzymatically (a mixture of collagenase/dispase/deoxyribonuclease/chicken serum) and plated on collagen-coated multiwell plates in culture medium containing 10% fetal bovine serum. After 2 days of attachment period, the medium was replaced with fresh medium every 24 h. The primary cell culture was incubated with various concentrations of LH-RH, CRF or GRF. Subsequently, the concentrations of IR-7B2, IR-LH, IR-FSH, and IR-ACTH released into the medium were quantified by specific RIA. LH-RH, at a concentration as low as 7.5 ng/ml (6 X 10(9) M: dose range 7.5-60 ng/ml) stimulated the release of IR-7B2, IR-LH, and IR-FSH, by 2- to 3-fold, 17- to 18-fold, and 3-fold, respectively, over basal levels. No significant increase of IR-7B2 was observed when stimulated by CRF or GRF at doses as high as 100 ng/ml. In addition, K+ (50 mM) stimulated the release of all the peptides measured. In conclusion, our studies suggest that the novel peptide 7B2 is under LH-RH control and indirectly confirm the immunohistochemical results of its cellular co-localization in FSH and LH cells.     

Processing of angiotensin II (A-II) and (Sar1,Ala8)A-II by cultured bovine adrenocortical cells

Bovine adrenocortical cells, cultured in a chemically defined medium, were used to study the fate of [125I] iodoangiotensin II ([125I]iodo-A-II) and its antagonist (Sar1,Ala8)A-II ([125I]iodo-Saralasin). The binding of both ligands was time and temperature dependent. The maximum specific binding at 37 C, which was reached within 1 h, was followed by a decline with a half-life of about 2 h and 8 h for [125I]iodo-A-II and [125I]iodo-Saralasin, respectively. The decrease of the specific binding was parallel to the appearance in the medium of degraded ligand. At 4 C, the binding of [125I]iodo-A-II was stable for 12 h and no degradation of ligand occurred. Under several experimental conditions, about 70% of the total [125I]iodo-A-II bound was internalized, whereas, in the case of [125I]iodo-Saralasin, less than 25% of the total bound ligand was internalized. These differences in the binding kinetics between A-II and its antagonist were mainly the differences in the rate of internalization of the bound ligands, more rapid for [125I]iodo-A-II (t1/2 approximately equal to 10 min) than for [125I]iodo-Saralasin (t1/2 = 90 min). On the other hand, the rate of degradation of internalized ligand was similar for both ligands (t1/2 = 15 min). Ionophore monensin enhanced the total cellular uptake of both ligands by increasing the amount of internalized ligands. Monensin did not modify the rate of internalization of the two ligands but markedly decreased their rate of degradation (t1/2 approximately equal to 60 min). These results indicate that both A-II and its antagonist are internalized and degraded by adrenocortical cells, but the rate of internalization of the antagonist is lower than that of the agonist. They also show that receptor-mediated endocytosis is the main pathway by which A-II is rapidly degraded by adrenocortical cells. Since A-II receptors are present in many tissues, the receptor-mediated degradation could explain the very short half-life in plasma of this hormone.     

Effects of gamma-aminobutyric acid receptor agonists on the secretion of growth hormone, luteinizing hormone, adrenocorticotrophic hormone and thyroid-stimulating hormone from the rat pituitary gland in vitro

The effects of the gamma-aminobutyric acid receptor agonists muscimol and baclofen were investigated on the secretion of GH, LH, ACTH and TSH from the anterior pituitary in vitro using a rapid superfusion system. A bicuculline-sensitive stimulatory effect of muscimol was demonstrated on the secretion of GH, LH and ACTH but not TSH. Baclofen had no effect on the basal secretion of any of the hormones, but inhibited LH-releasing hormone-stimulated release of LH and K+- and Ba2+-stimulated release of ACTH. The benzodiazepine Roll-6896 and the barbiturate secobarbital were found to potentiate the effect of muscimol on GH secretion. These results demonstrate the presence of GABAA receptors on somatotrophs, gonadotrophs and corticotrophs, and the presence of GABAB receptors on gonadotrophs and corticotrophs. Thyrotrophs appear devoid of GABA receptors.     

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.     

Stimulation by alpha-adrenergic mechanisms of the secretion of growth hormone-releasing factor (GRF) from perifused rat hypothalamus

The possible involvement of adrenergic mechanisms in regulating the secretion of growth hormone (GH)-releasing factor (GRF) from the rat hypothalamus was examined in vitro with a perifusion system. A high potassium concentration (56 mM) stimulated GRF release from the hypothalamus. The infusion of clonidine (10(-4) M), an alpha 2-adrenergic stimulant, resulted in an increase in the spontaneous release of GRF. In the presence of propranolol (10(-5) M), a beta-adrenergic blocking agent, clonidine (10(-5) and 10(-4) M) stimulated GRF release more prominently in a dose-related manner, whereas propranolol (10(-5) and 10(-4) M) by itself did not affect the spontaneous GRF release. The stimulatory effect of clonidine (10(-4) M) on GRF release in the presence of propranolol was inhibited by yohimbine (10(-4) M), an alpha 2-adrenergic blocking agent. These findings suggest that alpha 2-adrenergic mechanisms play a role in stimulating GRF release from the hypothalamus in rats.     

Basal and hypothalamic extract-induced luteinizing hormone and prolactin secretion by cultured anterior pituitary cells from female turkeys in various stages of the reproductive cycle

The wet weight of the anterior pituitary gland of the domestic turkey increases as the hen progresses through the reproductive cycle. The greatest wet weight was observed with anterior pituitary glands from incubating hens, whose weight was twice that of anterior pituitary glands from nonphotostimulated hens. Anterior pituitary glands from hens in each of the various stages of the reproductive cycle were dissociated and cultured, and subsequently basal and hypothalamic extract (HE)-induced releases of prolactin (PRL) and luteinizing hormone (LH) were compared between cultures containing a defined number of anterior pituitary cells. Basal and HE-induced releases of PRL were greatest (P less than 0.05) in the cultures of anterior pituitary cells derived from incubating hens, with those of laying, photorefractory, and nonphotostimulated hens being successively less (P less than 0.05), respectively. HE-induced release of LH was greatest (P less than 0.05) in cell cultures derived from nonphotostimulated hens, with cultures of cells derived from laying, photorefractory, and incubating hens exhibiting successively smaller releases of LH (P less than 0.05), respectively. The concentration of HE that induced the first significant increase in the release of PRL or LH above that of basal levels also varied with the reproductive stage of the donor hens. The changes in circulating PRL levels during the various stages of the reproductive cycle reflect changes in anterior pituitary sensitivity to hypothalamic releasing activity and/or changes in the PRL releasing capacity of the anterior pituitary. In contrast, there does not appear to be a strong correlation between anterior pituitary LH releasing capacity in vitro and circulating LH levels in the domestic turkey hen.     

Possible delta receptor mediation of the effect of beta-endorphin on luteinizing hormone (LH) release, but not on prolactin (PRL) release, in the ovariectomized rat

Administration of opioid receptor antagonists was utilized to determine the opioid receptor type involved in the suppression of LH release by beta-endorphin (beta-END). Long-term (three to four weeks) ovariectomized rats with chronic third ventricular cannulae were fitted with jugular catheters and received treatment with vehicle or one of three opioid antagonists. The delta antagonist ICI 154, 129, but not the mu1 or mu antagonists naloxazone or beta-funaltrexamine, respectively, blocked the suppressive effect of beta-END on plasma LH levels and transiently but significantly increased LH levels above preinfusion value. None of the antagonists significantly reduced the beta-END-induced release of PRL. These results provide evidence that the inhibitory effect of beta-END on LH release may be mediated by delta receptors.     

Effects of a novel gonadotropin-releasing hormone antagonist (ORG 30850) on gonadotropin and prolactin secretion by rat pituitary cells in culture

The effect of a new GnRH antagonist (ORG 30850 ANT) on FSH, LH, and PRL secretion was studied using male rat pituitary cells in monolayer cell culture. In the absence of GnRH, ORG 30850 ANT did not alter spontaneous FSH and LH secretion into culture medium or the cell content of these hormones. In the presence of GnRH (10(-8) mol/l), ORG 30850 ANT significantly and dose-dependently inhibited FSH and LH secretion into culture medium while increasing their cell content. Conversely, in the presence of a single dose of ORG 30850 ANT, FSH and LH secretion rose significantly when subjected to increasing amounts of GnRH, whereas the hormonal cell content diminished. Furthermore, inhibition of GnRH-induced FSH and LH release by ORG 30850 ANT was not changed by pre-incubation with the GnRH antagonist regardless of the pre-incubation time. The inhibitory effect of the GnRH antagonist was observed early, with its peak occurring within 6 h of culture. These short-term studies indicate that ORG 30850 ANT specifically inhibits GnRH-induced gonadotropin release into culture medium, exerts no effect on the rate of gonadotropin production in the presence or absence of GnRH, competitively and reversibly inhibits the binding of natural GnRH to its receptors, and does not lead to any modifications in PRL secretion.     

Dissociated changes of pituitary luteinizing hormone-releasing hormone (LHRH) receptors and responsiveness to the neurohormone induced by 17 beta-estradiol and LHRH in vivo in the rat

A single injection of 17 beta-estradiol into castrated male or female rats results in an initial decrease in plasma concentrations of LH and pituitary responsiveness to LHRH, followed by a rapid return to normal or slightly elevated values. Under such experimental conditions, no acute change of binding of [125I-labeled D-Ser(TBU)6]LHRH ethylamide to anterior pituitary homogenate could be observed. Moreover, the self-priming effect of LHRH, as illustrated by a 10-fold increase in the LH response to a second injection of LHRH in the afternoon of proestrus, is accompanied by a 40% loss of pituitary LHRH receptors. During the estrous cycle, a 100% increase in pituitary LHRH receptors is already found on diestrus II, while the maximal LH responsiveness to LHRH occurs later, namely on the afternoon of proestrus. The present findings of a dissociation between changes in LHRH receptor levels and LH responsiveness to the neurohormone suggest that postreceptor events play a predominant role in the control of gonadotropin secretion by sex steroids and LHRH itself. Moreover, LHRH can cause an acute down-regulation of its own receptor in the anterior pituitary gland.     

Stimulation of growth hormone and prolactin release from rat pituitary cell aggregates by bombesin- and ranatensin-like peptides is potentiated by estradiol, 5 alpha-dihydrotestosterone, and dexamethasone

The effect of the bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-C (NMC), and the ranatensin-like peptides, neuromedin-B (NMB), neuromedin-B30 (NMB30), and neuromedin-B32 (NMB32), on pituitary GH and PRL release was studied in perifused anterior pituitary aggregate cell cultures from 9- to 12-week-old male rats cultured in serum-free defined medium supplemented with 0.05 nM T3 and 4 nM dexamethasone (DEX). All peptides stimulated PRL and GH release. GRP and NMC stimulated hormone release in a concentration-dependent manner between 0.1-10 nM. NMB was slightly more potent than NMB30 and NMB32, but was significantly less potent than GRP and NMC. The magnitude of the PRL response to GRP and NMC inversely correlated with that of the GH response. Cultures with relatively low PRL response levels displayed high GH responses, whereas the opposite was found in cultures with high PRL response levels. The stimulatory actions of GRP, NMC, and NMB were blocked by the bombesin receptor antagonist Leu13 psi (CH2NH) Leu14-bombesin, supporting the specificity of the findings. Addition of 1 nM estradiol (E2) to the culture medium provoked an impressive (4- to 10-fold) increase in the magnitude of the GH response to NMC without changing the EC50 value (0.5 nM). In contrast, E2 significantly decreased the stimulation of GH release by rat GH-releasing factor. In the E2-treated aggregates 3 nM NMC stimulated GH release to a comparable extent as 0.1 nM GRF. 5 alpha-Dihydrotesterone (10 and 100 nM) and DEX (80 nM) also enhanced the GH response to NMC, but to a much smaller extent than E2. E2 had also a stimulatory effect on the PRL response to NMC, particularly in cultures with a low intrinsic PRL response. The PRL response to NMC was decreased by DEX and slightly augmented by 5 alpha-dihydrotestosterone. It is concluded that bombesin- and ranatensin-like peptides have a stimulatory effect on GH and PRL release at the pituitary level. Since their action on GH release is strongly potentiated by E2 and much less so by glucocorticoids, these peptides clearly distinguish their activity and specificity from that of the protagonist releasing factor GH-releasing factor, suggesting a role in sex-related differences in GH release or in the control of GH secretion during sexual maturation.     

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.     

Stimulation of luteinizing hormone (LH) release and phospholipid breakdown by guanosine triphosphate in permeabilized pituitary gonadotropes: antagonist action suggests association of a G protein and gonadotropin-releasing hormone receptor

The stimulation of gonadotropin release from pituitary cell cultures by GnRH has been linked to inositol phospholipid breakdown to diacylglycerols and subsequent activation of protein kinase C as well as Ca2+ mobilization. In order to examine the means of receptor coupling to a phospholipase C-type reaction, we evaluated the role of guanine nucleotides in inositol phospholipid breakdown. In these studies ATP (50 microM) was used for cell permeabilization to allow guanine nucleotides access to the intracellular compartment. Under these conditions GTP and the GTP analog, guanylylimidodiphosphate (GMP-PNP), stimulated a time- and dose-dependent increase in LH release and inositol phosphate accumulation. These actions of GTP and GMP-PNP were not observed unless ATP was included in the treatment media. Other closely related nucleotides and nucleosides alone, or in the presence of ATP, did not elevate LH release above basal levels. We also evaluated the actions of pertussis toxin and cholera toxin on mediating the effect of GTP, GMP-PNP, and GnRH on LH release and inositol phosphate accumulation. After treatment with these agents, no changes were observed in the ability of GnRH, GTP, or GMP-PNP to stimulate either LH release or inositol phosphate accumulation. The additional observation that GnRH-, GTP-, or GMP-PNP-stimulated LH release and inositol phosphate accumulation were blocked by a potent GnRH antagonist suggests that a G protein is functionally associated with the GnRH receptor recognition site.