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The vasopressin V3 receptor (V3) is specifically expressed in pituitary corticotropes and mediates the stimulatory effect of vasopressin on adrenocorticotropic hormone (ACTH) release. The V3 gene is overexpressed in corticotrope pituitary tumours compared to normal pituitaries. We hypothesized that V3 overexpression might induce changes in corticotrope function and alter the regulation of the hypothalamic-pituitary-adrenal axis. Thus, we generated transgenic mice (POMV3) expressing the human V3 receptor in the pituitary under the control of rat pro-opiomelanocortin (POMC) promoter sequences. The transgene was efficiently transcribed and vasopressin binding was increased in both corticotropes and melanotropes. In-vitro ACTH release and inositol phosphate formation were unchanged in POMV3 pituitaries, but the responses to vasopressin were significatively increased. In vivo, basal circulating concentrations of ACTH in POMV3 mice were similar to those of controls but corticosterone concentrations were moderately increased. In addition, the levels of POMC mRNA in the transgenic pituitaries were comparable to those of control mice. Finally, POMV3 mice responded with a similar maximal increase of ACTH and corticosterone to a 20-min acute restraint stress. Together, these results show that hypophyseal V3 overexpression led to increased basal concentrations of corticosterone and suggest that the negative glucocorticoid feedback may be altered at the pituitary level.

The effects of ionotropic excitatory amino acids agonists on the release of vasopressin from rat hypothalamic slices were studied. Incubation with increasing doses of NMDA, kainate or AMPA decreased the release of vasopressin in a dose-dependent manner. The values of the IC50 were 1.0, 9.6, or 3.7 x 10-8 M, respectively. The inhibitory effect of the various excitatory amino acids tested was blocked by coincubation with their respective antagonists. Vasopressin secretion was stimulated to 140.3 +/- 7.6% of controls when the slices were obtained from chronically (7 days) salt-loaded rats. Addition of 1 x 10-7 M NMDA or 1 x 10-6 M kainate to the incubation medium antagonized the salt loading-induced increase in vasopressin release. Incubation with 1 x 10-4 M tetrodotoxin did not change basal vasopressin release, but it blocked the decrease in vasopressin secretion induced by 1 x 10-7 M NMDA or 1 x 10-6 M kainate or 1 x 10-6 M AMPA. Incubation with 1 x 10-5 M phaclophen (a GABAB antagonist) and 1 x 10-5 M bicuculline (a GABAA antagonist) was without effect on basal vasopressin secretion while it reversed the inhibition of vasopressin release induced by 1 x 10-7 M NMDA. Incubation with 1 x 10-6 M GABA alone decreased vasopressin secretion to 64.6 +/- 6.9% of control values. The inhibitory effect of GABA did not change when 1 x 10-7 M NMDA was added to the incubation medium. These findings demonstrate that ionotropic excitatory amino acids agonists inhibit vasopressin secretion from hypothalamic slices. They strongly suggest that this inhibitory effect is mediated through local GABAergic interneurones.

Neonatal rats were daily injected with 100 microg/kg T4 and killed at 4, 8 or 15 days. Circulating corticosterone and corticosteroid binding globulin concentrations increased in 8- and 15-day-old rats after T4 treatment. Plasma adrenocorticotropic hormone (ACTH) concentrations, pituitary ACTH content and pro-opiomelanocortin mRNA expression were unaffected in T4-treated rats. T4 treatment induced an increase in corticotropin-releasing factor (CRF) mRNA expression in the whole population of CRF synthesizing cells of the paraventricular nucleus (PVN) that became significant at day 8 and disappeared at day 15. Double labelling in situ hybridization revealed that CRF gene expression in the CRF+/arginine vasopressin (AVP)+ subpopulation was increased at days 4 and 8 and decreased at day 15. CRF immunoreactivity in the zona externa of the median eminence increased with age but was not affected by the experimental hyperthyroidism. The degree of CRF and AVP colocalization, the concentration of AVP mRNA in the parvo and magnocellular cell bodies of the PVN and the density of immunoreactive AVP in the zona interna or zona externa of the median eminence did not change after T4 treatment. Our data demonstrate that experimental hyperthyroidism accelerates the maturation of hypothalamic CRF gene expression, including in particular in the CRF+/AVP+ subpopulation, during the stress hyporesponsive period. These observations suggest that the physiological peak of plasma thyroxine that occurs between days 8-12 may participate in the maturation of hypothalamic CRF cells.

We examined the expression of functional growth hormone secretagogue receptors (GHS-R) in a series of 30 human pituitary adenomas-six secreting GH, three GH-PRL, six prolactin (PRL), five adrenocorticotrophic hormone (ACTH), one thyroid stimulating hormone (TSH), four gonadotroph and five non-secreting adenomas. By reverse transcriptase polymerase chain reaction (RT-PCR), the coexpression of the two GHS-R isoforms (Ia and Ib) was found in all the GH-, GH-PRL- and PRL-secreting adenomas, and only in two out of three corticotroph, two out of four gonadotroph and one out of five non-secreting tumours. They were absent in the TSH-secreting adenoma. The PCR products of GHS-R Ia and Ib were identical in size to those from two normal pituitaries. PCR cloning and sequencing of isoforms performed in two somatotroph adenomas revealed only two single, silent base mutations. Triple in-situ hybridization showed colocalization of GHS-R mRNA with messengers of GH and PRL, conjointly or separately, in individual cells of somatotroph, mammosomatotroph, and lactotroph adenomas. The presence of GHS-R mRNA in cells expressing PRL mRNA is emphasized. In cultured cells from six somatotroph and two mammosomatotroph adenomas, the powerful GHS MK-0677 stimulated GH release in a dose-dependent manner, with maximal effect at 6 h. Contrarily, when GHRH was applied, only three somatotrophs and two mamosomatotrophs were stimulated. In the two mammosomatotrophs, the PRL response to MK-0677 and to GHRH was similar to the GH response. An homologous desensitization of the GHS-R and the GHRH receptor was observed 24 h after a first stimulation by a single dose of the corresponding agonist. Heterologous desensitization was not observed. Interestingly, MK-0677 also stimulated, in a dose-dependent way, the hormone release of cells from all tested lactotroph and corticotroph adenomas. The existence of a functional expression of GHS-R in somatotroph, mammosomatotroph, lactotroph and corticotroph adenomas rises the question of the role played by GHS-R in pituitary adenomas, particularly those not engaged in GH secretion.

The hypothalamo-pituitary-adrenal axis is already functional in rat fetuses in late gestation. We have reported previously that prenatal morphine exposure induced a severe atrophy of the adrenals and a decrease of corticosterone release in newborn rats at birth and during the early postnatal period. The first aim of the present study was to determine the effects of prenatal morphine exposure (1) on corticotrophin releasing factor (CRF) content of the hypothalamus, CRF immunofluorescence in the median eminence, CRF mRNA in the paraventricular nucleus (PVN) and pro-opiomelanocortin (POMC) mRNA in the anterior pituitary gland; (2) on CRF-induced ACTH release from the anterior pituitary gland in vitro; and (3) on ACTH-induced corticosterone release by the adrenals in vitro. Moreover, as morphine is a hepatotoxic factor, we determined the effects of prenatal morphine on liver weight and plasma corticosteroid binding globulin (CBG) binding capacity in newborn rats. Since acute administration of morphine stimulates corticosterone secretion in adult rats and since maternal corticosterone can cross the placental barrier, we also measured both adrenal weight and glucocorticoid activity in newborns from adrenalectomized mothers treated with morphine. The present results show that prenatal morphine given to intact mothers induced adrenal atrophy and hypoactivity in newborns but did not affect the responsiveness of the anterior pituitary gland to CRF or that of the adrenal gland to ACTH. Prenatal morphine reduced both CRF content in the newborn hypothalamus and CRF immunofluorescence in the median eminence without a significant effect on CRF mRNA expression in the PVN. Moreover, morphine induced a significant decrease of POMC mRNA in the anterior pituitary gland. However, morphine did not significantly affect the weight of the liver, or the plasma CBG binding capacity for corticosterone, in rat pups. In contrast, morphine treatment of the adrenalectomized mothers did not induce adrenal atrophy in newborns and did not impair adrenal activation during the early postnatal period. Maternal adrenalectomy also prevented the effects of prenatal morphine on hypothalamic content of CRF, CRF immunofluorescence in the median eminence, and POMC mRNA in the anterior pituitary gland. However, adrenal atrophy was observed at term in newborns of adrenalectomized mothers treated with both morphine and corticosterone or only corticosterone. In conclusion, morphine given to pregnant rats induced inhibition of the hypothalamo-pituitary-adrenal axis in pups at term. As maternal adrenalectomy prevented these effects, we speculate that an adrenal factor of maternal origin, probably corticosterone, mediated these drug effects on newborns.

It is known that in vivo excitatory amino acids (EAA) stimulate the hypothalamo-pituitary-adrenal axis. However their site of action is not fully understood. We investigated the possibility of a direct action of EAA on the secretion of the major adrenocorticotropin hormone (ACTH) secretagogue: corticotropin-releasing factor (CRF) from incubated rat hypothalamic slices. N-methyl-D-aspartic acid (NMDA) or L-glutamate (1 x 10(-7) to 1 x 10(-3) M) stimulated in a dose-dependent fashion CRF release. The maximal effect was obtained at a concentration of 1 x 10(-4) M for both drugs. The IC50 was 1.3 x 10(-5) M and 3.3 x 10(-5) M for NMDA and L-glutamate, respectively. Incubation with 2.5 x 10(-4) M D-2-amino-5-phosphonovalerate (a NMDA receptor antagonist) or 2-amino-4-phosphonobutyrate (a metabotropic receptor antagonist) was without significant effect on basal CRF secretion and completely blocked the increase in CRF release induced by 5 x 10(-5) M NMDA or L-glutamate, respectively. Incubation with 1 x 10(-4) M kainate or 0.5 x 10(-4) M AMPA did not change basal CRF secretion. Incubation with 2 x 10(-4) M gamma-D-glutamylglycine (a specific antagonist of kainate and AMPA receptor) had no effect under basal conditions or during exposure to kainate or AMPA. Our data demonstrate that EAA could stimulate directly CRF secretion, by an action through NMDA and metabotropic receptors, but not kainate or AMPA receptors. These findings may be relevant to the regulation of the hypothalamo-pituitary adrenal axis, both under basal conditions and during exposure to stress.

In vitro, estrogens stimulate corticotropin-releasing factor (CRF) gene transcription. In ovariectomized (OVX) rats, estrogens have a negative effect on CRF mRNA levels in the hypothalamic paraventricular nucleus. This suggests that the stimulatory influence of estrogens found in vitro may be masked in vivo by an inhibitory effect mediated through neural and/or humoral factors. Glucocorticoids may be involved in this phenomenon since estrogens increase circulating corticosterone levels in OVX rats. We studied the effect of 7-day 17 beta-estradiol (E2) treatment on CRF gene expression in the paraventricular nucleus of 2-week OVX mature rats after sham operation or adrenalectomy (ADX) with or without corticosterone replacement. In sham ADX animals, E2 administration increased plasma corticosterone concentrations, did not change the binding capacity or the affinity of circulating corticosteroid binding globulin, and decreased hypothalamic CRF gene expression. Following ADX, CRF mRNA levels increased and were normalized by corticosterone treatment. Estrogens induced a significant build up in CRF mRNA concentrations in both ADX or ADX corticosterone-replaced animals. Our data demonstrate that the positive effect of estrogens on CRF gene expression found in vitro or in vivo after ADX is antagonized, in vivo when the adrenal glands are intact, by a negative influence. They strongly suggest that the increased circulating corticosterone levels induced by E2 administration mediate the inhibitory effect of estrogens on CRF mRNA levels. These observations may explain the gender related differences in the basal and stress-induced hypothalamo-pituitary-adrenal activity.

Corticotropin-releasing factor and arginine vasopressin (AVP) are the two major hypothalamic factors that regulate anterior pituitary adrenocorticotropin secretion during stress. We have previously reported that the expression of the corticotröpin-releasing factor gene in the hypothalamus and its regulation by glucocorticoids were not mature during the first week of life in the rat, i.e. during the stress non-responsive period. In this report, we studied the ontogeny of expression of the AVP gene in the hypothalamic paraventricular nucleus in rats using in situ hybridization. AVP mRNA was detected as early as day 20 of gestation (E20) both in the parvocellular and the magnocellular portion of the paraventricular nucleus. The levels of expression of the AVP gene increased steadily from E20 to the third day after birth (P3) and remained stable from P3 to P14. Bilateral surgical adrenalectomy induced an increase in AVP mRNA levels in the parvocellular portion of the paraventricular nucleus, but not in the magnocellular portion, in both 7-day-old and 14-day-old rats, suggesting that the glucocorticoid regulation of the AVP synthesizing neurons of the paraventricular nucleus is mature in the developing postnatal rat.