Phorbol esters stimulate in vitro GnRH release from the hypothalamus of the estrogen-primed, ovariectomized rat: inhibitory effects of cocaine.

The purpose of this study was (1) to characterize more fully the effects of phorbol esters to stimulate GnRH release in vitro and (2) to determine whether cocaine (which disrupts estrous cyclicity in rats) affected phorbol ester stimulation of GnRH release in vitro. Hypothalami were collected from ovariectomized rats injected subcutaneously with 50 micrograms/kg of 17 beta-estradiol benzoate the two previous mornings. Sagittal sections of this block of CNS tissue comprising the preoptic area/anterior hypothalamus and mediobasal hypothalamus/median eminence were perfused at a rate of 6.2 ml/h with a modified Krebs-Ringer buffer (pH 7.4) using a programmable perfusion system. Perfusion results showed that 10-min pulses of phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate (PDBu) increased GnRH release in dose-dependent fashion (10(-10) to 10(-6) M) but had no effect on aminergic transmitter release. The biologically inactive alpha-phorbol was without effect on GnRH release. PDBu-stimulated GnRH release was blocked by both tetrodotoxin and cocaine, known inhibitors of Na+ influx. These results suggest a role for protein kinase C in regulating the release of GnRH. Our results that cocaine and tetrodotoxin attenuated phorbol ester stimulation of GnRH, presumably through inhibition of Na+ influx, suggest a direct biochemical mechanism for cocaine disruption of hypothalamic GnRH secretion and, consequently, cyclic reproductive function.

Cocaine disrupts estrous cyclicity and alters the reproductive neuroendocrine axis in the rat.

Although a common drug of abuse, cocaine's effects on cyclic reproductive functions and the neuroendocrine systems regulating these functions have not been studied. Here, we report the effects of cocaine on (1) estrous cyclicity and ovulation rates and (2) the stimulated in vitro release of hypothalamic GnRH and aminergic neurotransmitters directly involved in regulating or modulating GnRH release. Within 7 days of treatment with 10 mg kg-1 day-1 of cocaine HCl subcutaneously, rats demonstrated significant estrous cycle irregularity including repetitive days of estrus and prolonged periods of diestrus. After 6 weeks of treatment, cocaine-treated rats exhibited a 44.3% decrease in ovulation rates. For the in vitro studies, bilaterally ovariectomized rats were injected with cocaine (10 mg kg-1 day-1) or with saline for 2 weeks. Each rat received estradiol benzoate (50 mg kg-1 day-1 s.c.) for 2 days before sacrifice. Hypothalamic slices were prepared, placed in 0.1 ml microchambers and perfused with modified Krebs buffer (pH 7.4) using a programmable perfusion system. Basal release of norepinephrine (NE) and serotonin (5HT) was significantly increased in the cocaine-treated group versus controls. Ten-minute pulses of 10(-7)M progesterone (P4) increase NE and 5HT, but not dopamine (DA), release in the saline-treated group. In contrast, pulses of P4 increased NE, but not 5HT or DA, in the cocaine-treated rats. Ten-minute pulses of 0.1 microM NE increased GnRH release in both saline- and cocaine-treated rats. However, the response to pulsed NE was significantly attenuated in the cocaine-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced aminergic synthesis in the hypothalamus of the infertile, genetically diabetic (C57BL/KsJ-db/db) male mouse.

Diabetes mellitus is commonly associated with reproductive neuroendocrinopathy in both humans and animal models for the disease. Diabetes-associated reproductive failure in the male is a result of multilevel dysfunction within the hypothalamo-pituitary-testicular axis. In view of the known effects of diabetes on hypothalamic gonadotropin-releasing hormone (GnRH) and gonadotropins in chemically-induced animal models for diabetes, we examined hypothalamic aminergic activities (important to the regulation of GnRH release), circulating gonadotropin levels and testicular morphology in the infertile, genetically diabetic (C57BL/KsJ-db/db) male mouse. Groups of 2-5 month old (average age: 3.4 months) and 6-11 month old (average age: 8.8 months) diabetic mice were compared with age-matched non-diabetic (C57BL/KsL(-)+/?) male mice. Diabetic mice in both age groups were markedly obese and hyperglycemic. Hypothalamic serotonin synthesis was inhibited in the preoptic area-anterior hypothalamus (POA-AH) in both 2-5 month old and 6-11 month old diabetic mice as well as in the mediobasal hypothalamus-median eminence (MBH-ME) of 6-11 month old diabetic mice. Catecholamine synthesis (norepinephrine and dopamine) was reduced in the POA-AH of 2-5 month old diabetic mice and in the MBH-ME of 6-11 month old mice. These aminergic changes were associated in 2-5 month old diabetic mice with reduced circulating levels of LH and in 6-11 month old diabetic mice, of both LH and FSH. In 6-11 month old diabetic mice, testes were characterized by a thickened tunica albuginea, numerous Sertoli cells and the near absence of any spermatogenic cells. The epididymis from these diabetic mice was devoid of spermatozoa.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoamine biosynthesis in hypothalamic regions of dwarf mice: effect of replacement of deficient anterior pituitary hormones.

Female Ames dwarf and phenotypically normal female mice were killed 30 min after treatment with NSD-1015, an aromatic L-amino acid decarboxylase inhibitor. The accumulation of dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan were measured by high-performance liquid chromatography with electrochemical detection and provided estimates of the endogenous biosynthesis of dopamine (DA) in the median eminence (ME) and serotonin biosynthesis (5-HT) in all brain regions which were examined. Dopamine synthesis was markedly suppressed in the ME while 5-HT synthesis was enhanced in both the ME and mediobasal hypothalamus (MBH) of dwarfs as compared to phenotypically normal mice. Overall, catecholamine biosynthesis (DOPA accumulation) was suppressed in the MBH of the dwarf mice but was not different from that observed in normal mice in the preoptic area anterior hypothalamus (POA-AH). The biosynthesis of 5-HT was not different in the POA-AH of dwarf mice as compared to normal mice. In the second experiment dwarf mice received saline vehicle, ovine prolactin (PRL), growth hormone (GH) or thyroxin (T4) daily for 14 days. Normal mice received saline only. Replacement with PRL significantly enhanced DA synthesis in the ME and was the only hormone to suppress significantly the elevation of 5-HT synthesis normally observed in the ME and the MBH of the dwarfs. Both GH and T4 only partially reduced 5-HT synthesis in the ME and MBH so that this parameter was no longer statistically different from either saline-treated dwarfs or normal mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ovarian steroids to stimulate region-specific hypothalamic 5-hydroxytryptamine synthesis in ovariectomized rats.

Accumulations of 5-hydroxytryptophan (5HTP) and L-dihydroxyphenylalanine (L-DOPA) following decarboxylase inhibition as indices for 5-hydroxytryptamine (5HT) and catecholamine (dopamine and/or norepinephrine) synthesis, respectively, were both increased in the preoptic area-anterior hypothalamus (POA-AH) of ovariectomized rats treated with a combination of 17 beta-estradiol benzoate (E2) and progesterone (P). Similarly, an increased accumulation of L-DOPA was seen in the mediobasal hypothalamus (MBH) and median eminence (ME) of these animals although no change was observed in 5HTP accumulation in the MBH or ME of these rats. Hypophysectomy negated these steroid-induced effects on L-DOPA accumulation. However, hypophysectomy had no apparent effect on steroid-stimulated 5HTP accumulation in the POA-AH of these rats. Under the experimental conditions of our study, the results suggest that the stimulatory effect of ovarian steroids on hypothalamic catecholamine synthesis is dependent on an intact pituitary gland, that the stimulatory effect of ovarian steroids on 5HT synthesis in the POA-AH is not dependent on an intact pituitary gland, and (3) that ovarian steroids do not seem to influence 5HT synthesis in the ME or MBH. The significance of these results may lie in the function of these hypothalamic monoaminergic neurotransmitter systems to regulate gonadotrophin release and subsequent steroid feedback modulation of such central regulatory mechanisms.

Hyperprolactinemia attenuates ovarian steroid stimulation of region-specific hypothalamic serotonin synthesis and luteinizing hormone release in ovariectomized rats.

Hyperprolactinemia adversely affects reproductive functions, presumably through an effect at the hypothalamic level. Given the numerous published reports linking hypothalamic serotonin (5-hydroxytryptamine, 5HT) mechanisms to the regulation of gonadotrophin secretion, we sought to determine the effects of experimentally induced hyperprolactinemia on ovarian steroid-induced increases in serum LH levels and region-specific hypothalamic 5HT synthesis in ovariectomized rats. In the first study, bilaterally ovariectomized Sprague-Dawley rats either received two pituitary homografts implanted beneath the left kidney capsule or were sham-grafted. Both groups of rats were injected subcutaneously with 5 micrograms/100 g of estradiol benzoate (E2) in corn oil vehicle at 08.00 h, 1 and 2 days before serum collection and 5 mg/100 g of progesterone (P) in corn oil vehicle at 07.00 h on the day of serum collection. Blood samples were collected via chronic indwelling jugular cannulae from each rat at 10.00, 12.00, 13.00, 14.00, 16.00 and 18.00 h. A statistically significant elevation in serum LH levels was detected at 13.00, 14.00 and 16.00 h. This increase in serum LH levels was significantly attenuated in rats bearing pituitary homografts, an effect attributed to the high serum PRL levels measured in these animals. In the second study, bilaterally ovariectomized Sprague-Dawley rats were divided into three experimental groups: (1) rats bearing two pituitary homografts and injected with E2 and P on the schedule and at the dosages previously described, (2) sham-grafted rats injected with E2 and P on the schedule and at dosages previously described and (3) sham-grafted rats injected with corn oil vehicle only.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hypophysectomy and subsequent prolactin administration on hypothalamic 5-hydroxytryptamine synthesis in ovariectomized rats.

Increased levels of PRL whether of natural or experimentally induced cause result in an increased turnover of the putative PRL-inhibiting factor dopamine (DA) within tuberoinfundibular neurons, suggesting that PRL can regulate its own release via a short loop feedback mechanism. Although it is known that activation of the hypothalamic 5-hydroxytryptamine (5HT) system stimulates PRL release, the possibility that PRL could alter metabolism or release of this amine within the hypothalamus has not been examined. In the first experiment, bilaterally ovariectomized adult Sprague-Dawley rats were divided into three groups: hypophysectomized rats (HYPOX), HYPOX rats injected sc with 0.5 mg kg-1 ovine PRL (oPRL) at 1000 h and 1800 h on the day before killing the rats (HYPOX + PRL), and rats without further surgical manipulation (controls: NON-HYPOX). Three weeks after surgical manipulation, the rats were injected iv with 100 mg kg-1 NSD-1015 and killed 15 or 30 min later. 5-Hydroxytryptophan accumulation was measured by HPLC with electrochemical detection as an index for the rate of 5HT synthesis. The rate of 5HT synthesis was increased in the median eminence (ME) and mediobasal hypothalamus (MBH) but not anterior hypothalamus of HYPOX rats in comparison to the rate of synthesis of this amine in these areas of control rats. This effect was reversed in the ME and MBH of HYPOX + PRL rats. If 5HT stimulation of PRL release were achieved by 5HT inhibition of the tuberoinfundibular DA system as has been proposed previously, then it is also conceivable that the PRL short loop feedback on hypothalamic 5HT synthesis as suggested by the results of the first experiment is mediated via this DA system. To test this hypothesis in the second experiment, adult Sprague-Dawley rats were injected at 1000 h and 1800 h on the day before their killing with 0.5 mg kg-1 oPRL; 0.5 mg kg-1 oPRL, and 10 mg kg-1 pimozide (PIM) a DA receptor antagonist; 10 mg kg-1 PIM; or, on the day of their killing, with 1 mg kg-1 apomorphine (APO), a DA receptor agonist. An additional group of ovariectomized-HYPOX as well as a group of ovariectomized-NON-HYPOX rats were also included in this experiment. On the day of the experiment the rats were injected iv with 100 mg kg-1 NSD-1015 and killed 30 min later. 5-Hydroxytryptophan accumulation was measured by HPLC with electrochemical detection as an index of the rate of 5HT synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)