Differential in vitro secretion of gonadotropin-releasing hormone (GnRH) and [hydroxyproline]GnRH from the rat hypothalamus during postnatal development.

The differential secretion of gonadotropin-releasing hormone (GnRH) and [hydroxyproline9]GnRH (HypGnRH) has been recently reported from the adult rat hypothalamus. We report here in vitro cosecretion of HypGnRH and GnRH by the hypothalamus of 2-45 day-old-rats and provide evidence that they are differentially regulated throughout development. The secretion of both forms of GnRH was increased in a dependent manner during depolarization by high K+ solutions, and was stimulated by forskolin and 12-O-tetradecanoylphorbol-13-acetate (TPA), activators of adenylate cyclase and protein kinase C pathways, respectively. The proportion of HypGnRH in the release of GnRH-like peptides remained stable and high (33-40%) under basal and K+-induced conditions until days 13 and 21, respectively. By contrast, the proportion of HypGnRH in the total GnRH-like content of the developing hypothalamus continuously decreased (from 37% to 14%). Similarly, the proportion of HypGnRH: total GnRH-like material released remained stable in TPA- (30%) and forskolin- (50%) induced secretion until postnatal day 8. Evaluation of release over tissue store ratios revealed a 1.3-to 2.8-fold higher release of HypGnRH compared to GnRH according to the different secretions and postnatal periods examined. The preferential recruitment of HypGnRH was maintained under basal and K+ conditions during postnatal development, but it disappeared under TPA stimulation from day 13 onwards. After forskolin stimulation, the preferential mobilization of HypGnRH was markedly reduced from day 2 to day 13 but recovered its high perinatal level during puberty. Taken together, our results support the hypothesis that HypGnRH may play a specific role in development. In addition, a specific function of this peptide taking place during puberty through the activation of the adenylate cyclase pathway is suggested.

In vitro secretion of gonadotropin-releasing hormone (GnRH) and [hydroxyproline9]GnRH from the rat hypothalamus exhibits a differential sensitivity to castration and second messengers.

The decapeptide [hydroxyproline9]GnRH (HypGnRH) has been characterized as an endogenous posttranslational product of the gonadotropin-releasing hormone (GnRH) precursor in a wide range of mammalian brains. Despite consistent biological effects, its secretion by the hypothalamus remains hypothetical. We report here in vitro secretion of HypGnRH and GnRH by the hypothalamus from intact and castrated male rats and provide evidence that they are differentially regulated. Both peptides were identified by two anti-GnRH antibodies of different specificities after separation under two high-performance liquid chromatography conditions. Calcium dependency of HypGnRH release was demonstrated under stimulation with KCl in the absence or presence of Ca2+, as well as with Bay K 8644, veratridine, methoxyverapamil, or tetrodotoxin. Activation of signaling pathways involving adenylate cyclase and protein kinases A and C (PKC) induced HypGnRH release. Expression of data as percentage of release over tissue stores revealed a two- to threefold higher release of HypGnRH than of GnRH under the different modes of stimulation used, except under PKC activation which triggered a comparable recruitment of both peptides. Castration selectively affected PKC-coupled GnRH secretion which showed a twofold lesser release than in intact rats, while the HypGnRH release was unaffected. We conclude that HypGnRH and GnRH are not secreted from the hypothalamus according to the same mechanisms.

Effect of an essential fatty acid deficiency on the phospholipid composition in anterior pituitary membranes.

The effects of an essential fatty acid deficient diet were investigated on the phospholipid fatty acids of several membrane fractions of the rat anterior pituitary, the secretion of which is known to be partly dependent on the membrane phospholipidic constituents. In standard dietary conditions, arachidonic acid (20:4n-6) and its elongation product, adrenic acid (22:4n-6), were the two main polyunsaturated fatty acids in all fractions studied. In rats deprived of EFA for 6 weeks after weaning, the levels of both 20:4n-6 and 22:4n-6 were not changed in microsomal + plasma membrane and nuclear fractions, whereas they were decreased in heavy mitochondrial and light mitochondrial fractions. The present data suggest a mechanism of compensation between membrane fractions which may preferentially preserve 20:4n-6 and 22:4n-6 in discrete membrane fractions.

Evidence that clomethiazole interacts with the macromolecular GABA A-receptor complex in the central nervous system and in the anterior pituitary gland.

Clomethiazole (CLOM) is known to be an anticonvulsant drug and has been also reported to decrease serum prolactin (PRL) in humans. Both effects may be mediated by an enhancement of gabaergic transmission. In order to determine if (CLOM) interacts with GABA metabolism and/or at the GABA receptor level, we studied its effect on PRL release and on the binding of various compounds that interact with the GABAA-benzodiazepine-receptor complex. Intraperitoneal (IP) administration of CLOM to rats significantly decreased PRL levels, and this effect was antagonized by IP administration of bicuculline, an antagonist of the GABAA receptor. In vitro, the inhibitory effect of muscimol on PRL release from rat hemiadenohypophysis was potentiated in a dose-dependent manner by preincubation with CLOM. This effect was antagonized by picrotoxin (10(-6) M). On the other hand, CLOM had no effect on GABA metabolism and did not compete with GABAA, GABAB or benzodiazepine binding sites in cortical membranes. CLOM competed, however, with the picrotoxin binding site labelled with [35S]-butylbicyclophosphorothionate (TBPS), at an IC50 value of 1.2 x 10(-4) M, which is in the same range as some barbiturates. These results concerning PRL release and binding experiments with cortical membranes suggest that CLOM interacts with the picrotoxin/barbiturate site of the GABAA-receptor-chloride channel complex.

Independent inhibition of prolactin secretion by dopamine and gamma-aminobutyric acid in vitro.

gamma-Aminobutyric acid (GABA) inhibits PRL release from incubated hemipituitaries in a dose-dependent manner. The maximum inhibition obtained with GABA is less than that obtained with dopamine. Its affinity is 100 times lower. The effect is blocked by picrotoxin but not by a dopamine inhibitor; alpha-flupentixol but not picrotoxin antagonizes dopamine inhibition. This indicates that dopamine and GABA inhibit PRL release through independent receptors. The hypothalamic extract contains sufficient GABA to inhibit PRL release in our in vitro conditions. Picrotoxin, however, does not significantly inhibit the nondopaminergic PRL-inhibiting activity of mediobasal hypothalamic extracts. Another nondopaminergic PRL-inhibiting factor, therefore, seems to be present in the hypothalamus.

Subcellular distribution of corticotropin-releasing factor in the medio-basal hypothalamus of the rat.

Subcellular fractionation of the mediobasal hypothalamus (MBH) and frontal cerebral cortex was performed by differential and discontinuous sucrose gradient centrifugation. Corticotropin-releasing factor (CRF) activity of the different fractions was evaluated by bioassay. Significant CRF activity was found in acidic extracts of the MBH but not of the cerebral cortex. About 80% of the MBH effect on adrenocorticotropic hormone release was recovered in the crude mitochondrial pellet (P2) which contains synaptosomes. After further fractionation, distribution of CRF activity paralleled that of lactate dehydrogenase activity, a marker of the soluble cytoplasm. It is concluded that most CRF in the MBH is located in nerve endings as already shown for several other neurohormones.

Midbrain raphe lesion in the newborn rat: II. Biochemical alterations in serotoninergic innervation.

Electrolytic raphe lesion was performed in 4-6-day-old rats and the resulting changes of 5HT metabolism within the central nervous system were analyzed up to 9 months later. As soon as the 2nd day following the selective destruction of B7 and B8 nuclei, forebrain 5HT levels were decreased by more than 75%. This reduction persisted for at least 9 months with no sign of recovery. The time course of 5-HIAA decrease was parallel to that of the indoleamine so that the ratio of 5-HIAA over 5-HT levels in the forebrain of lesioned rats was similar to that estimated in controls, whatever their age. This result would suggest that the remaining serotoninergic neurons in the lesioned rats did not develop a compensatory hyperactivity. The raphe lesion induced no change in MAO activity and synaptosomal tryptophan uptake but a pronounce decrease in the Vmax of synaptosomal KHT uptake process in various forebrain areas occurred. The serotonin sensitive adenylate cyclase activity in colliculi homogenate was not altered by the lesion suggesting that this enzyme was probably located in postsynaptic membranes. In addition, this observation would indicate that 5-HT receptors which are linked to this adenylate cyclase did not become supersensitive following the selective degeneration of serotoninergic neurons. Animals without forebrain serotoninergic innervation might be of great interest to analyse the role of serotoninergic neurons in various functions (sleep, analgesia, thermoregulation).

Prolactin inhibiting activity of dopamine-free subcellular fractions from rat mediobasal hypothalamus.

In order to check the hypothesis of an identity of dopamine (DA) and prolactin inhibiting activity (PIF), their subcellular distribution was studied in the mediobasal hypothalamus (MBH) and the striatum, which served as a control structure. PIF was tested both in vivo and on pituitary incubates. Fractions were also assayed after adsorption of their catecholamine content on alumina, as well as in presence of haloperidol or alpha-flupentixol, potent DA receptor inhibitors. In the MBH, PIF was evenly distributed in the 17,000 g supernatant (S2) and in the crude mitochondrial fraction (P2) which contains synaptosomes. PIF activity was completely removed by alumina adsorption of S2, but not of P2 in spite of an over 99.9% elimination of DA. In contrast, striatal PIF activity was detected only in P2, and disappeared completely upon alumina adsorption, thus indicating that, in this structure, it is entirely due to DA. Addition of haloperidol (10--5M) or alpha-flupentixol (10--6M) reduced PIF activity of crude MBH homogenates, but no longer affected it after alumina adsorption. Quantitative studies suggest that only half of the total MBH PIF activity is accounted for by DA. It is concluded that the MBH contains dopamine-free PIF, which, as already shown for several other neurohormones, is exclusively distributed in nerve-endings.

Effect of suckling on plasma prolactin and hypothalamic monoamine levels in the rat.

In lactating rats that were separated from their pups for 8 hours, suckling induced a rapid rise in plasma prolactin levels. Correlatively, it induced a depletion in dopamine (DA) and serotonin (5-HT) Concentrations within the hypothalamus as early as 5 min after the onset of suckling. This depletion lasted as long as the stimulus was maintained. A parallel increase in the level of the metabolite of 5-HT, 5-hydroxyindolacetic acid (5-HIAA), was observed. Under these conditons, the ratio of 5-HIAA/5-HT is augmented. This ratio is a good index of turnover of the transmitter. Suckling affected neither the content of noradrenaline (NA) in the hypothalamus nor the levels of any amine tested in the cerebral cortex. When pups were separated from their mother for 24 hours, suckling no longer resulted in changes in either plasma prolactin levels or hypothalamic DA, 5-HT, or 5-HIAA concentrations. We have concluded that the activation of a discrete system of 5-HT containing neurons is associated with the triggering effect of suckling on prolactin release.