The effect of valproate (VPA) treatment on inositol phosphates (IPs) accumulation in non-stimulated and GnRH-treated female rat anterior pituitary cells in vitro.

OBJECTIVE: Mechanism(s) responsible for VPA-induced effects on reproductive axis activity are not fully recognized. Previously we reported that VPA suppressed only GnRH-stimulated but not the basal LH release from rat anterior pituitary (AP) cells in vitro. Since the inhibitory effect of VPA was exerted only in GnRH-activated cells, potential VPA impact on GnRH-R-coupled IP3/PKC signaling could not be excluded. In this study the effect of VPA on IPs synthesis in non-stimulated and GnRH-treated rat AP cells was examined. MATERIAL AND METHODS: In the first experiment 5 × 105 cells/ml were incubated for 3h with VPA (10 nM-10 µM), PMA (100 nM), GnRH (100 nM), PMA (100 nM) + VPA (10 nM-10 µM), GnRH (100 nM) + VPA (10 nM-10 µM). In the second experiment cells were preincubated for 24h with 1µCi myo-[23 H]-inositol, then for 30 min with 10 mM LiCl and finally for 3hr with GnRH (100 nM) VPA (1 µM, 10 µM), GnRH (100 nM) + VPA (1 µM, 10 µM). LH concentration was measured by RIA and intracellular IPs accumulation by ion-exchange chromatography analysis. RESULTS: VPA diminished GnRH-stimulated LH release without affecting PMA-induced LH release at any dose tested. Moreover, VPA-induced increase of IPs accumulation occurred in both non-stimulated and GnRH-treated cells and intensity of cellular response was similar in both groups. CONCLUSION: VPA affects IP3/PKC pathway activity through its up-regulatory effect on IPs synthesis in AP cells. VPA-induced inhibition of GnRH-stimulated LH release from gonadotrope cells appears to be the result of still unrecognized cellular mechanism.

The evaluation of estradiol and leptin action on the activity of the somatotropic and gonadotropic axes in peripubertal female rats.

OBJECTIVE: Available data suggest that estrogens and leptin play a role in the control of the pubertal process. In humans and some mammal species the increase of the activity of gonadotropic axis accompanies the decrease in the rate of growth at puberty. The effect of 17ß-estradiol and/or leptin administration on the somatotropic and gonadotropic axes was studied using prepubertal female rats as an animal model. MATERIAL AND METHODS: Prepubertal female rats received estradiol/saline, estradiol/leptin, oil/leptin or oil/saline (vehicles) respectively. The changes of growth rate, and serum 17ß-estradiol, leptin, GH, IGF-I and gonadotropins levels as well as LHRH and estrogen receptor (ER) concentrations in the medial basal hypothalamus (MBH) and the pituitary were determined. All hormones concentrations were measured by radioimmunoassay and ER by radioligand methods . RESULTS: In estradiol and/or leptin treated animals noticeable reduction of rate of growth was found. The decrease of growth in response to estradiol treatment accompanied the increase GH level and the decrease of IGF-I concentration in the circulation. Both hormones operating together activated reproductive axis, what was manifested by a significant increase of LHRH abundant in the hypothalamus as well as elevated LH and FSH levels in the circulation. In these rats a significant decrease of the estrogen receptor concentrations in the pituitary was observed. CONCLUSION: The role of estradiol and leptin in the control of growth and reproduction seems to overlap only partially. Estradiol plays a significant role in the activation of the reproductive axis, and leptin takes part as a permissive factor in pubertal process.

Valproate inhibits GnRH-induced gonadotropin release from anterior pituitary cells of male rat in vitro.

OBJECTIVE: Valproate (VPA) a potent antiepileptic drug has been claimed to induce reproductive disturbances in men. Long-term VPA treatment can affect sperm morphology and induce testicular atrophy in non-epileptic rats. It has been reported that VPA reduced testosterone secretion stimulated by hCG in isolated rat Leydig cells. These results suggest direct effect of VPA on testes in rats. However centrally mediated effects at hypothalamo-pituitary level can therefore not be excluded. This study focused on the dose and time-dependent effects of VPA on basal and GnRH-induced LH and FSH release from the primary anterior pituitary cells culture of male rats. MATERIAL AND METHODS: The dose-dependent effect of 10 nM-100 mM of VPA on basal LH release from anterior pituitary cells after 3h of incubation was examined. To determine the time-dependent effects on LH, FSH, TSH and PRL release short (3 h) and long-term (24 h) incubations in the presence of 10 nM, 100 nM and 1 µM of VPA were maintained.To assess whether VPA can affect GnRH-induced LH and FSH release, cells were incubated for 3 h with 10 nM, 100 nM and 1 µM of VPA in the presence of GnRH. The concentration of rLH, rFSH, rPRL and rTSH in incubation medium was determined by RIA method. RESULTS: VPA did not affect the basal LH, FSH, PRL and TSH release from the primary anterior pituitary cells culture of male rats. VPA in concentration 1µM significantly suppressed GnRH-induced LH secretion. However VPA at all tested doses diminished GnRH-induced FSH release. CONCLUSIONS: VPA may diminish gonadotropin release in vitro but this effect can only be achieved after GnRH-dependent specific receptor activation. Both gonadotropins differ in their pattern of response for increasing doses of VPA.

Orexin A and its role in the regulation of the hypothalamo-pituitary axes in the rat.

Orexin A (OxA), a recently discovered neuropeptide, is synthesized mainly by neurons located in the posterolateral hypothalamus and is a 33 amino acid peptide with N-terminal pyroglutamyl residue and two inter-chain disulfide bonds. It is a potent agonist for both the orexin-1 (OxR1) and orexin-2 (OxR2) receptors. Orexin A and its receptors are widely distributed in the central nervous system (CNS) and peripheral organs suggesting the pleiotropic functions of this peptide. Orexin A is involved in food intake and energy expenditure in many species, but also plays an important role in the regulation of the hypothalamo-pituitary axes. The role of orexin A in the regulation of the hypothalamo-pituitary-adrenal, -thyroid, -somatotropic, and -gonadal axes has been inadequately investigated. Orexinergic fibres project to the septal-preoptic and arcuate nucleus-median eminence regions--two areas of the brain directly involved in the synthesis and release of gonadotropin-releasing hormone (GnRH). Contentious opinions concerning the influence of orexin A over the hypothalamo-gonadotropic axis have been reported in both in vivo and in vitro studies. Further studies are necessary to clarify relationships between orexin A and the hypothalamo-pituitary hormones involved in reproduction.

The role of neuropeptides in the disturbed control of appetite and hormone secretion in eating disorders.

OBJECTIVE: It has been reported that neuropeptides may play a role in the control of appetite and in the mechanism of hormone release. Neuropeptides such as beta-endorphin, neuropeptide Y (NPY), galanin and leptin may affect hormones release, on the other hand the hormonal status may modulate neuropeptide activity. METHODS: The material consisted of 90 obese women, 30 women with Anorexia Nervosa, and 30 healthy, lean women of control group. Plasma beta-endorphin, NPY, leptin, somatostatin and serum pituitary and gonadal hormones concentrations were measured with RIA methods. RESULTS: We observed the highest plasma NPY levels in obese hypertensive and diabetic patients. After carbohydrate administration (OGTT) a marked increase of insulin, beta-endorphin and NPY was found. The blunted response of GH to GH-RH may be connected with increased somatostatin activity and hyperinsulinemia. The abnormal response of LH to opioid blockade may be a result of disturbed opioid and NPY activities in obese patients. However in patients with anorexia nervosa, plasma leptin and NPY concentrations were low. The disturbances in beta-endorphin release are also observed. CONCLUSIONS: The neuroendocrine disturbances in obesity and in anorexia nervosa are opposite. The feedback mechanism between leptin and NPY is disturbed in both in obesity and in anorexia nervosa. An abnormal activity of neuropeptides may lead to disturbed control of appetite and hormonal dysregulation in eating disorders.

Effects of leptin and neuropeptide Y (NPY) on hormones release in female rats.

OBJECTIVES: The aim of this study was to investigate the influence of NMMA on the mechanism of leptin and NPY action in in vivo and in vitro on pituitary hormone release by female rats in diestrus. METHODS: In vivo experiments based on four-day ip injections of: 1) murine recombinant leptin (mrLep); 2) human NPY; 3) saline; 4) NMMA (50 microg) was applied prior to administration of mrLep; 5) NMMA (50 microg) was applied prior to administration of NPY; 6) NMMA (50 microg); 7) NMMA (350 microg) was applied prior to administration of mrLep; 8) NMMA (350 microg). The blood samples for LH and FSH were collected after decapitation of animals on the fifth day of study. In vitro experiments studied the effects of these peptides on LH release from pituitary cell culture. RESULTS: In vivo: Leptin+ NMMA (50 microg) stimulated LH. NPY+ NMMA (50 microg) increased LH. NMMA (350 microg) stimulated FSH release. In vitro: The stimulatory effect of leptin on LH release in vitro was observed after 120 min of incubation, but after 60 mins of incubation the inhibitory effect of leptin on LH release was increased by NMMA. Leptin +NMMA (vs NMMA) decreased LH release with highest statistical significance. NPY 10 nM 60 min after incubation decreased LH release. There was no effect of NMMA on LH release. CONCLUSION: In vivo results show, that NO probably mediates in leptin action on LH release. Moreover, effects of leptin on LH release from cultured pituitary cells are dependent on time of incubation. NPY decreased LH release in vitro.

Alfa 1 adrenergic potentiation of progesterone accumulation stimulated by vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in cultured rat granulosa cells.

OBJECTIVE: VIP and PACAP, two structurally related peptides, stimulate c AMP, steroidogenesis and progesterone (PROG) release from cultured rat granulosa cells. VIP and PACAP(38) are known to mimic the effects of beta-adrenergic receptor stimulation in the rat pinealocytes causing an increased melatonin synthesis. These effects were markedly potentiated by alpha(1) adrenergic receptor stimulation. METHODS: We examined the influence of phenylonephrine (PHEN) (10(-4) M and 10(-5) M)-alpha(1) adrenergic receptor agonist and fenoterol (FEN) (10(-5) M)-beta(2) adrenergic receptor agonist on PROG accumulation stimulated by VIP (10(-6) M) and PACAP (10(-7) M) in cultured ovarian granulosa cells of cyclic rats (diestrus) after 2 h and 24 h incubation. The PROG concentrations in supernatants were measured with RIA tests. RESULTS: VIP, PACAP(38), PHEN and FEN stimulated PROG accumulation after 2 h incubation. The PROG accumulation stimulated both by FEN and VIP, and by FEN and PACAP(38) was not additive. PROG accumulation stimulated by VIP and PACAP(38) was strongly potentiated by PHEN-alpha(1) adrenergic agonist. CONCLUSION: The alpha(1) adrenergic potentiation of VIP and PACAP(38) stimulatory effects on PROG release from granulosa cells culture was found. VIP, PACAP(38) and beta(2) adrenergic receptors activation may share the same postreceptor mechanism. There exists simultaneous activation of different receptors -- peptidergic and adrenergic ones in cultured granulosa cells of adult cyclic rat.

Effects of growth hormone (GH) and growth hormone releasing hormone (GHRH) on progesterone and estradiol release from cultured rat granulosa cells.

OBJECTIVES: It has been reported that GHRH-GH-IGF-1 system plays an important role in the regulation of ovarian follicular development and maturation. METHODS: In order to evaluate the direct effects of growth hormone releasing hormone (GHRH) and growth hormone (GH) on steroidogenesis, the effects of GHRH and GH on progesterone and estradiol release from cultured rat granulosa cells were examined. The progesterone and estradiol in supernatants were measured with RIA methods. RESULTS: Our results demonstrated that the addition of GH to the culture medium produced a marked stimulation of progesterone and estradiol. The stimulating effects were observed after administration of GH in all concentrations: 1, 10, 100 nM during 60 and 120 mins of incubation. During 240 mins of incubation the minimal stimulation of progesterone and estradiol was found. However, GHRH administered in 1, 10 and 100 nM did not change progesterone and estradiol release from cultured granulosa cells. CONCLUSION: Growth hormone (GH) but not GHRH has direct stimulating effects on progesterone release from cultured rat granulosa cells.

Effects of neuropeptide Y (NPY), galanin and vasoactive intestinal peptide (VIP) on pituitary hormone release and on ovarian steroidogenesis.

Ovarian folliculogenesis is regulated by the gonadotrophins, but in recent years other peptides have been found to serve as local regulators of ovarian function. The aim of this study is to evaluate the effects of NPY, galanin and VIP on pituitary and gonadal hormone release. Effects of NPY, galanin and VIP on progesterone, estradiol production by cultured rat granulosa and effects of these peptides on pituitary hormone release by cultured pituitary cells were examined according to methods previously described. Maximal effects of NPY, galanin and VIP on pituitary hormone release and on gonadal steroids were observed after administration of 10 nM of these peptides during 60 mins incubation. VIP and NPY, but not galanin, stimulated PRL release from cultured pituitary cells. VIP increased also LH release whenever NPY and galanin did not change LH release from pituitary cells. Galanin, but not NPY and VIP, leads to an increase of GH production. VIP, NPY and galanin did not change TSH and FSH release. NPY, galanin and VIP markedly stimulated progesterone release from cultured granulosa cells. NPY, galanin and VIP did not change estradiol and testosterone release. Conclusions. Direct effects of NPY, galanin and VIP on pituitary hormone release may indicate their role in the mechanism of pituitary hormone release. A marked increase of progesterone from cultured granulosa cells after VIP, NPY and galanin suggests that these peptides may be involved in the local ovarian steroidogenesis.

New analogs of somatostatin: inhibiting effectively GH, glucagon and insulin levels.

The in vivo effects of three new analogs of somatostatin (ASS-51, ASS-52 and ASS-53 analogs) on GH, insulin and glucagon were studied in WKY rats. The solid phase method was used for the synthesis of ASS. Octreotide and ASS were given iv. in a dose of 0.05 &mgr;g/kg per animal in a time-dependent manner. ASS-52 and ASS-53 were longer acting and more potent somatostatin analogs when compared to octreotide in producing the inhibition of GH. ASS-51 was found to be the most potent and selective inhibitor of insulin and glucagon release. Our results show that the increased inhibitory effect and the higher selectivity of the new somatostatin analogs may result from the differences in their chemical structure. ASS-52 is most active in inhibiting GH release. The mechanism by which ASS-52 inhibits preferentially GH release may involve the opioid system and the activation of GABA-ergic receptors. In studies in vitro ASS-52 inhibited GH release from pituitary cells" culture.