Effect of ethanol on follicle stimulating hormone-induced steroidogenic acute regulatory protein (StAR) in cultured rat granulosa cells.

Steroidogenic acute regulatory protein (StAR) plays a critical role in trophic hormone-stimulated steroid biosynthesis by facilitating the transfer of cholesterol across the mitochondrial membrane, where the cytochrome P450scc enzyme resides to initiate steroid hormone biosynthesis. Because follicle stimulating hormone (FSH) is a critically important regulator of estradiol (E2) synthesis in granulosa cells and because ethanol is known to suppress gonadotropin-stimulated ovarian steroidogenesis, we evaluated the effects of ethanol on FSH-stimulated StAR in ovarian granulosa cells. Granulosa cells from immature rats pretreated with pregnant mare serum gonadotropin were cultured for 24 h in serum-free medium, either alone (medium only) or with FSH (25 ng/ml) in the presence or absence of ethanol (50 mM). Real-time polymerase chain reaction (PCR) analysis showed increased (p < 0.01) expression of the StAR transcript in FSH-treated cells, when compared with cells that received medium only. The FSH stimulation of StAR transcript was blocked (p < 0.01) by the presence of ethanol. This effect coincided with a decrease in E2 secretion into the culture medium. We also examined whether ethanol could affect the production of cyclic AMP (cAMP), the main second messenger that mediates gonadotropin action within the ovary. FSH treatment of granulosa cells markedly increased (p < 0.001) cAMP levels, an effect that was not altered by ethanol. Importantly, FSH induced an increase (p < 0.01) in the release of prostaglandin E2 (PGE2), an effect that was blocked by ethanol. Real-time PCR analysis showed that ethanol had no effect on the expression of cyclooxygenase-1 (COX-1), but blocked (p < 0.01) FSH-stimulated expression of COX-2. These results demonstrate that ethanol is capable of inhibiting FSH-induced ovarian StAR and thus, contributing to suppressed E2 secretion, at least in part, through an inhibitory action on the COX-2-PGE2 pathway.

Physiological significance of neurotensin in pituitary glycoprotein hormone release as revealed by in vivo and in vitro studies with neurotensin antiserum.

The neuropeptide, neurotensin, is localized to neurons within the hypothalamus which project to the median eminence. It is released from the terminals in the median eminence into the hypophyseal portal vessels and is carried to the gland. The content in the pituitary gland cells may be partly related to the delivery of the peptide via the portal vessels, but it also appears to be produced directly in pituitary cells. The peptide has actions on the release of glycoprotein hormones from the pituitary and in the present experiments, we attempted to determine whether these actions of the peptide were physiologically significant by microinjecting purified antiserum directed against neurotensin into the third cerebral ventricle or intravenously into conscious freely moving rats. Blood samples were withdrawn from an indwelling intra-right atrial catheter. In ovariectomized rats with high levels of plasma gonadotropins because of removal of ovarian steroid negative feedback, the intraventricular injection of the higher (3 microliters) dose of neurotensin antiserum (NT-AS) induced a more than 2-fold increase in plasma LH within 2 h which was maintained until 3 h after the injection and returned to basal values in the 4th and 5th hour. The lower 1-microliter dose was ineffective and there was no response to the control normal rabbit serum (NRS) injections into the third ventricle in this and the other experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute and short-term effects of intraventricular injection of somatostatin and LHRH on glutamate and GABA levels in rat brain.

Glutamate and GABA content in different regions of adult female rat brain were determined at 10 and 30 min following intraventricular injection of LHRH or somatostatin. Cerebral cortex, cerebellar and hypothalamic glutamate levels were significantly elevated at 30 min following injection of 1 micrograms somatostatin, whereas hypothalamic glutamate levels were elevated at 10 min following a 0.5 micrograms dose. LHRH at a dose of 1 micrograms elevated cerebellar and brain stem glutamate levels at 10 and 30 min, whereas a 0.5 micrograms dose significantly elevated cerebral cortex, cerebellar and hypothalamic glutamate levels at 30 min. Third ventricular injection of 1 micrograms somatostatin produced a significant decrease in hypothalamic GABA levels at 10 and 30 min, whereas a 0.5-microgram dose decreased brain stem GABA levels at 10 min. LHRH at a dose of 0.1 microgram significantly increased cerebral cortex and cerebellar GABA levels at 10 min and brain stem GABA levels at 10 and 30 min following injection. Intraventricular injection of LHRH at a dose of 0.5 microgram significantly elevated cerebral cortex, cerebellar and brain stem GABA levels at 30 min. Hypothalamic GABA levels were elevated at 10 and 30 min following 0.5 and 1 microgram intraventricular LHRH injection. The implications of these results are discussed in relation to probable interaction between these neuroactive amino acids and neuropeptides in the rat brain.

Control of anterior pituitary hormone secretion by neurotensin.

Neurotensin is localized in discrete populations of neuronal cell bodies with terminals in the hypothalamus and median eminence. High-affinity binding sites for neurotensin have been demonstrated not only in the hypothalamus but also in the pituitary gland. These studies suggest a role for neurotensin in control of hypothalamic-pituitary function. We initially demonstrated that neurotensin could block the release of prolactin in conscious, ovariectomized and male rats after its injection into the third ventricle, whereas intravenous injection of the peptide significantly elevated plasma prolactin and increased prolactin release by pituitaries incubated in vitro. These results suggested that neurotensin had opposite actions on prolactin release, an inhibitory effect at a hypothalamic site and an excitatory one at the pituitary. Further studies employing dopamine receptor blockers and inhibitors of catecholamine synthesis indicated that the action of the peptide to block prolactin release was probably mediated by release of dopamine, which then inhibited prolactin release by the pituitary gland directly. We have evaluated the physiological significance of the peptide in control of prolactin release by intraventricular injection of highly specific antiserum against neurotensin. The antiserum evoked dose-related elevations in plasma prolactin in intact males that were significant but smaller in magnitude than those seen in females, actions opposite to those of the peptide itself, which indicates that the inhibitory action of the peptide within the brain is physiologically significant. Intravenous injection of this antiserum produced a significant suppression of plasma prolactin in females but not males, which indicates that the previously demonstrated stimulatory effect of the peptide on prolactin release by the gland is also physiologically significant because immunoneutralization of the peptide resulted in a decline in plasma prolactin. Our earlier experiments revealed that neurotensin had a dose-related ability to inhibit LH release in ovariectomized and ovariectomized, estrogen progesterone-treated rats. Since it had no effect on the release of LH in vitro, we assigned a hypothalamic site for this action. It appears that this inhibitory effect of the peptide to suppress LH release is also physiologically significant since the intraventricular injection of the antiserum against the peptide produced a dose-related stimulation of LH release in ovariectomized and ovariectomized, estrogen progesterone-blocked rats. The mechanism by which endogenous neurotensin inhibits the release of LHRH has yet to be evaluated.(ABSTRACT TRUNCATED AT 400 WORDS)

Glutathione and gamma-glutamyl transpeptidase in the adult female rat brain after intraventricular injection of LHRH and somatostatin.

Glutathione content and glutamyl transpeptidase activity in different regions of adult female rat brain were determined at 10 and 30 min following intraventricular injection of LHRH and somatostatin. Hypothalamic glutathione levels were significantly elevated at 10 and 30 min after a single injection of a 0.1 micrograms dose of LHRH. On the contrary, glutathione levels significantly decreased in the hypothalamus, cerebral cortex and cerebellum at 10 and 30 min after 0.5 or 1 microgram dose. However, significant decrease in brain stem glutathione was evident at 30 min after 0.5 microgram and 10 min after the 1 microgram dose. Somatostatin at doses of 0.5 microgram and 1 microgram significantly decreased glutathione levels in all four brain regions both at 10 and 30 min following injection into the 3rd ventricle. Gamma-glutamyl transpeptidase activity in the hypothalamus and cerebral cortex was significantly elevated after intraventricular injection of LHRH. However, a significant increase in gamma-glutamyl transpeptidase activity in cerebellum and brain stem was seen only with 0.5 and 1 micrograms doses of LHRH. Somatostatin also significantly increased gamma-glutamyl transpeptidase activity in hypothalamus, cerebral cortex, brain stem and cerebellum. The decrease in glutathione levels with corresponding increase in gamma-glutamyl transpeptidase activity after intraventricular administration of LHRH and somatostatin suggests a possible interaction between glutathione and hypothalamic peptides.

Glutathione distribution in rat brain at different ages and the effect of intraventricular glutathione on gonadotropin levels in ovariectomized steroid-primed rats.

Glutathione levels were estimated in different regions of the brain of 21-, 30-, 40-, 42-, 45-day-old and adult female rats. Glutathione content in the cerebral cortex, cerebellum and the brain stem remained almost the same beginning from day 21 to sexually mature adult rats. There is a significant increase in hypothalamic glutathione content reaching a peak at puberty (42 days) and thereafter decreasing to the adult levels. Plasma gonadotropin levels were evaluated at 5 and 15 min after third ventricular injection of 15 and 30 microgram doses of glutathione in ovariectomized steroid-primed rats. Intraventricular injection of either 15 or 30 micrograms dose of glutathione significantly elevated plasma FSH levels. The 15 micrograms dose of glutathione significantly decreased plasma LH levels whereas 30 micrograms dose had no effect. Lower dose of glutathione inhibits LH release and stimulates FSH release whereas the higher dose of glutathione specifically elevates FSH levels without any change in LH levels suggesting a selective FSH releasing action of glutathione.

Differential responses of hypothalamic cAMP and cGMP to substance P and neurotensin in ovariectomized rats.

Hypothalamic cAMP and cGMP levels in ovariectomized rats were evaluated after intravenous (IV) pulse injection and/or intraventricular (IVT) injection of substance P and/or neurotensin. Intravenous substance P lowered hypothalamic cAMP concentration whereas IVT injection of 2.5 micrograms substance P produced significant increase in cAMP levels. On the other hand, IV administration of neurotensin failed to alter hypothalamic cAMP levels while IVT injection induced significant decrease in cAMP. Intravenous pulse injection of substance P elevated hypothalamic cGMP levels while IVT injection decreased cGMP concentration. Hypothalamic cGMP concentration was not modified by IV administration of neurotensin. However, IVT injection of neurotensin significantly elevated cGMP levels. Since a number of neurotransmitters/neuropeptides exert their action through cyclic nucleotides the present results indicate differential responses of cAMP and cGMP to substance P and neurotensin and implicate a mediatory role for cAMP and cGMP in the neuroendocrine action of substance P and neurotensin.

Recent developments in female contraception: LHRH.

During the last few years new approaches to female contraception based on LHRH and its analogs have been developed. The physiological significance of pulsatile LHRH release and its stimulation of the pituitary has been elucidated by recent studies in rhesus monkey. Immunization against LHRH results in complete inhibition of reproductive function in animals and may find as a useful method of long-term fertility control in domestic animals. Clinical studies have utilized this knowledge to treat infertile hypogonadal women with chronic intermittent low-dose of LHRH. The superactive stimulatory LHRH analogs, used to treat infertility, paradoxically proved to have antifertility effects. They induce desensitization of the processes responsible for gonadotropic and gonadal hormone secretion, mediated by specific LHRH receptors in the pituitary and gonad. While contraceptive effectiveness of luteolytic approach remains to be proven, inhibition of ovulation by intranasal LHRH analog administration or continuous LHRH infusion by programmed minipumps seem to provide safe and effective contraception in women.

Neurotensin enhances estradiol induced DNA synthesis in immature rat uterus.

Systemic administration of Neurotensin, a tridecapeptide, in immature rats treated with estradiol benzoate significantly enhances uterine DNA synthesis as reflected by the incorporation of 3H-thymidine. The peptide may have a direct action on the uterus. Substance P, a related peptide, had no effect on uterine DNA synthesis.

Hypothalamic tyrosine hydroxylase activity and plasma gonadotropin and prolactin levels in ovariectomized-steroid treated rats.

Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase (TH) activity were evaluated in ovariectomized (OVX) estradiol benzoate (EB) or progesterone (P) treated rats. Single injection of 10 micrograms or daily injection of 5 micrograms EB/rat for 7 days significantly lowered gonadotropin levels in OVX animals and elevated PRL levels. Single injection of 2 mg or daily injection of 200 micrograms P/rat for 7 days increased gonadotropin and PRL levels. Hypothalamic TH activity was significantly elevated by estradiol. Single injection of 2 mg P suppressed TH activity in contrast to the elevation in enzyme activity following chronic treatment. These results indicate that hypothalamic noradrenergic as well as dopaminergic neurons participate in the stimulatory or inhibitory feedback effects of ovarian hormones on gonadotropin and PRL secretion.

Gamma aminobutyric acid (GABA), a modulator of anterior pituitary hormone secretion by hypothalamic and pituitary action.

We have evaluated the role of GABA in the control of anterior pituitary (AP) hormone secretion by injecting it into the third ventricle of ovariectomized, ovariectomized-steroid primed and male rats. Specificity of the effects was determined by injecting the GABA blocker, bicuculline. The action of GABA directly on the pituitary was evaluated in vitro. The results indicate that intraventricular GABA can stimulate LH, growth hormone (GH) and, at high doses, prolactin (Prl) release, whereas low doses inhibit Prl and al doses inhibit TSH release. All of these actions are blocked by bicuculline. Intraventricular GABA administration is followed by an elevation of hypothalamic norepinephrine (NE) and median eminence dopamine (DA) levels and AP DA levels, which indicates that the compound stimulates both NE and DA release. The actions on GH and LH appear to proceed independently of DA, since the DA receptor blocker, pimozide, did not interfere with these effects, whereas the action to elevate Prl and to lower TSH was blocked by DA receptor blockade. Anterior pituitary hormone release by AP's incubated with GABA in vitro was unaltered except for an inhibition of Prl release by very high GABA doses, which could be blocked by bicuculline. Intravenous injection of bicuculline to assess the physiological significance of GABA in control of AP hormone secretion revealed no effect on FSH but a delayed rise in LH, an initial rise in Prl, followed by a fall, a tendency for GH values to rise and dramatic fall in TSH levels. These results suggest the possibility that GABA plays a physiological role in the control of AP hormone secretion, mainly via a hypothalamic action.

Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase activity following intraventricular bombesin and secretin in ovariectomized conscious rats.

Plasma gonadotropins, prolactin and hypothalamic tyrosine hydroxylase (TH) activity were evaluated at 15 and 30 min after third ventricular injection of bombesin at doses of 100 or 1000 ng and secretin at doses of 1000 and 5000 ng in ovariectomized (OVX) unanesthetized rats. Bombesin had no effect on plasma gonadotropin levels. Intraventricular injection of either 100 or 1000 ng dose of bombesin significantly suppressed prolactin levels with parallel elevation in hypothalamic TH activity and there appears to be no dose response relationship. Secretin at 1000 ng dose, significantly lowered plasma LH and PRL levels and elevated hypothalamic TH activity whereas a 5000 ng dose increased PRL concentrations but had no effect on gonadotropin levels and hypothalamic TH activity. Bombesin appears to be a potent inhibitor of PRL release in OVX, conscious rats and this effect may be mediated via hypothalamic dopamine. Lower dose of secretin appears to inhibit PRL release by possibly activating the hypothalamic dopaminergic system, while at higher dose peripheral activation results in enhanced prolactin release.

Gastrointestinal hormones: central nervous system localization and sites of neuroendocrine actions.

It was established that VIP (vasoactive intestinal peptide), secretin, CCK (cholecystokinin), gastrin and motilin can be localized to the CNS by immunologic means. Whether or not these immuno-crossreactivities represent peptides identical to those in the g.i. tracts remains to be established. The neuronal localization of these five peptides in the gut predicted, however, their presence in neurons of the CNS. Furthermore, their presence within the hypothalamus and pituitary suggested physiological roles for these hormones in anterior pituitary function. We have now demonstrated the direct actions of VIP, secretin, gastrin and motilin on pituitary hormone release in vitro. Perhaps more importantly, we have described a hypothalamic site of action of VIP, secretin, CCK and gastrin to alter hormone release in vitro. Our data, taken in concert with those of other groups, suggest a modulatory role for the g.i. hormones and indicate the possible symphonic control by many hormones and transmitter candidates of distinct secretory events in the pituitary. Indeed, these data indicate the complexity underlying the finally tuned hypothalamus-pituitary-target tissue axis.

Demonstration of tyrosinase in the vitiligo skin of human beings by a sensitive fluorometric method as well as by 14C(U)-L-tyrosine incorporation into melanin.

Tyrosinase activity (Monophenol, dihydroxyphenylalanine: oxygen oxidoreductase EC 1.14.18.1) in vitiligo and normal epidermal homogenates of skin from human beings was measured by estimating beta 3,4-dihydroxyphenylalanine (dopa) by a highly sensitive fluorometric method described in this paper. The tyrosine activity in the vitiligo skin was about 4 to 37% of corresponding normal skin. The activity of tyrosinase in normal human skin from different individuals and from different regions of the body was in the range of 4 to 140 picomoles of beta 3,4-dihydroxyphenylalanine formed per min/mg protein of epidermal homogenate. The enzyme from vitiligo and normal skin was severely inhibited by substance(s) of low molecular weight. The enzyme exhibits a lag of about 4 hr in the absence of added beta 3,4-dihydroxyphenylalanine and 1 hr in presence of 5 microM dopa. Tyrosinase from the normal and vitiligo skin was inhibited by excess concentration of tyrosine. The homogenates from vitiligo skin could synthesize melanin from C14(U)-L-Tyrosine. The rate of tyrosine incorporation into melanin by the epidermal homogenates is increased by 3,4-dihydroxyphenylalanine (dopa) disproportionate to its effect on tyrosinase activity. Based on the data presented in this paper it is concluded that melanocytes are present in the vitiligo skin. A tentative hypothesis is put forward to explain the lack of melanin synthesis by the vitiligo skin under in vivo conditions, although melanocytes are present.

The effects of neurotensin on anterior pituitary hormone secretion.

The present experiments were conducted to determine the effects of neurotensin on secretion of a variety of anterior pituitary hormones. Conscious rats with indwelling cannulae in the third ventricle and external jugular vein were used and the effects on plasma hormone levels measured by radioimmunoassay. Neurotensin was found to decrease plasma prolactin levels in ovariectomized females, normal males, and males in which prolactin levels had been elevated by ether or by a combination of fluoxetine and 5-hydroxytryptaphane. The prolactin-lowering effect was blocked by alpha-methyl-tyrosine to inhibit catecholamine synthesis and by the specific dopamine receptor blocker, spiroperidol. In ovariectomized females, neurotensin was also capable of suppressing LH and elevating growth hormone following its intraventricular injection. Intravenous injection of the peptide elevated prolactin but had no effect on the release of the other pituitary hormones. When hemipituitaries of ovariectomized rats were incubated in vitro, neurotensin elevated prolactin and TSH release into the medium. The minimal effective dose to elevate prolactin and TSH release was 50 ng/ml. Release of gonadotropins and growth hormone was unaffected. It is concluded that neurotensin inhibits prolactin release by a CNS, presumably hypothalamic action, to stimulate the tuberoinfundibular dopaminergic neurons. The dopamine released then inhibits prolactin release either by a direct action on the pituitary or by release of another prolactin-inhibiting factor. In addition, the peptide has a direct prolactin-releasing action on the pituitary. Neurotensin can inhibit LH and stimulate growth hormone presumably by a hypothalamic action since there was no effect on the release of these pituitary hormones by glands incubated in vitro. Although the peptide had no effect on TSH release following its intraventricular injection, it stimulated prolactin release by pituitaries incubated in vitro. The physiological significance of these results is not yet established; however, the presence of the peptide in regions concerned with pituitary control suggests that it may play a physiological role.

Neurotensin and substance P: differential effects on plasma cholesterol levels in conscious ovariectomized rats.

Circulating plasma cholesterol levels were measured in conscious ovariectomized rats, bearing an indwelling silastic catheter in the external jugular vein, after intravenous (i.v.) pulse injection of 100 microliter 0.9% NaCl containing varying doses of neurotensin and/or substance P. Control injections of saline or decapeptide LH-RH or phosphate buffer did not modify plasma cholesterol levels. 10 or 20 micrograms doses of neurotensin produced a significant and dose-related increase in plasma cholesterol levels while similar doses of substance P had an opposite effect and induced a significant decline in plasma cholesterol levels in ovariectomized rats. 4-APP, a drug which selectively inhibits hepatic secretion of lipoproteins, significantly lowers plasma cholesterol to levels comparable to those produced by substance P. 4-APP and substance P induced hypocholesterolemia was readily reversed by a single dose of neurotensin. These findings indicate that neurotensin acts to increase circulating cholesterol levels and substance P antagonizes this hypercholesterolemic effect of neurotensin presumably by acting at some step in cholesterol transport. Reversal of the inhibitory effects of 4-APP and substance P on blood cholesterol by neurotensin may be through its action on hepatic secretion of lipoproteins, since 4-APP is known to lower circulating cholesterol by its specific action on hepatic secretion of lipoproteins.