Pituitary adenylate cyclase-activating peptide (PACAP) is a 38-amino-acid polypeptide, first isolated from hypothalamus, which directly stimulates in vitro the production of cAMP as well as the release of several pituitary hormones, such as growth hormone and luteinizing hormone. In vivo, PACAP has been shown to stimulate ACTH release. The presence of PACAP receptors in several brain areas, including the hypothalamus, suggests that this peptide might play a role as a neurotransmitter/neuromodulator and might be involved in the regulation of hypophysiotropic neurohormones. In order to study the role of PACAP on corticotropin-releasing hormone (CRH) neuron, we have investigated the effects of intracerebroventricular (i.c.v.) and intravenous (i.v.) injections of PACAP and the potent PACAP antagonist PACAP(6-38) on CRH gene expression in the hypothalamic paraventricular nucleus (PVN) in the male rat. The levels of CRH mRNA were evaluated by quantitative in situ hybridization. The i.c.v. injection of PACAP (4 microg/kg b.wt.) produced a 22% increase in the hybridization signal, an effect which was completely prevented by the concomitant injection of the PACAP antagonist (4 microg/kg b.wt.). On the other hand, the administration of the PACAP antagonist induced by itself a 40% decrease in the amounts of CRH mRNA. The i.v. injection of the same peptides (100 microg/kg. b.wt.) produced very similar results. These data strongly suggest that PACAP is involved in the positive regulation of CRH gene expression via specific central receptors and then can play a role as a neurotransmitter/neuromodulator. The effect observed after i.v. injection of PACAP also suggests that the circulating levels of PACAP can play a role in the modulation of CRH gene expression. PACAP might then be involved in the regulation of the HPA axis by a double mechanism: stimulation of CRH gene expression at the central level and direct effect on pituitary corticotrophs.

Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are pivotal mediators of the hormonal response to stressors and are found within neurons of the paraventricular nucleus of the hypothalamus (PVN) and several extrahypothalamic sites where expression is activity-dependent. Previous work has shown increased CRH immunoreactivity in extrahypothalamic sites after kainic-acid (KA)-induced seizures in male rats. This study examined the induction of CRH heterogeneous nuclear RNA (hnRNA), AVP hnRNA and c-fos as a measure of gene transcription and cell activation following kainic-acid (KA)-induced seizures. KA or saline was administered to intact male rats, ovariectomized (OVX) females and OVX females treated with 17beta-estradiol (E2). Animals were sacrificed 0, 15, 60 or 120 min following KA treatment. In the PVN, CRH hnRNA levels were increased by KA treatment at 15, 60, and 120 min. AVP hnRNA and c-fos mRNA in the PVN were also significantly elevated above controls at all time points. Elevations in CRH hnRNA were also identified in hippocampus, the lateral bed nucleus of the stria terminalis (BNST) and globus pallidus at 60 and 120 min following KA and in the piriform cortex, and central nucleus of the amygdala at 120 min after KA. CRH hnRNA levels at 120 min in the PVN, amygdala, cingulate cortex, hippocampus (CA1), piriform cortex, and BNST were lower in OVX+E2 females compared to females without E2. To determine if the increases in CRH hnRNA translated to increased CRH peptide, immunocytochemistry was performed. CRH immunoreactivity was increased in the amygdala, BNST, cingulate cortex, PVN and globus pallidus within 3 h after KA treatment and in the piriform cortex and hippocampus by 6 h after KA. These results suggest a time-dependent activation of the CRH system following activation of kainate receptors, which may result in long-term changes in the expression of extrahypothalamic CRH.

Sustained pretreatment with angiotensin II AT(1) receptor antagonists prevents the sympathoadrenal and hormonal responses to 24 h isolation stress. To elucidate the mechanism of the anti-stress effects of AT(1) receptor antagonism, we examined the effect of subcutaneous infusion of candesartan, a non-competitive AT(1) receptor antagonist, 0.5 mg/kg/day for 14 days, to Wistar rats on the hypothalamic pituitary adrenal (HPA) axis after 24 h isolation stress. In the morning of day 15, we measured AT(1) receptors corticotropin-releasing factor (CRF) mRNA and immunoreactive CRF in the paraventricular nucleus (PVN), the pituitary adrenocorticotropin hormone (ACTH) and adrenal corticosterone content, and the urinary corticosterone excretion. In rats not treated with candesartan, 24 h isolation stress increased pituitary ACTH, adrenal corticosterone content and AT(1) receptor binding in the PVN but decreased CRF mRNA and CRF content in the PVN. This indicates enhanced CRF utilization not compensated by CRF gene transcription and effective glucocorticoid feedback inhibition in spite of the increase in AT(1) receptor expression. The effects of stress on HPA axis activation and CRF mRNA and content in the PVN were prevented by candesartan pretreatment, suggesting that activation of AT(1) receptors is required for the HPA axis response to isolation. Our results support the hypothesis that the activity of PVN AT(1) receptors is part of the mechanism necessary for development of a full stress-induced HPA axis activation. Inhibition of central AT(1) receptors limits the CRF response to stress and should be considered as a therapeutic tool to preserve homeostasis under chronic stress conditions.

Intermedin/Adrenomedullin-2 (IMD), a newly described peptide with structural homology to adrenomedullin (AM), is present in brain and pituitary gland and binds to the same receptors as AM and calcitonin gene-related peptide (CGRP). We hypothesized that IMD would exert actions similar to AM and CGRP and previously have demonstrated that indeed IMD, like AM and CGRP, increases sympathetic tone and inhibits feeding and drinking when administered centrally. Here, we extend those observations by demonstrating that like AM, IMD acts in brain to stimulate the secretions of prolactin (PRL) and adrenocorticotropin (ACTH) and to inhibit the secretion of growth hormone (GH) in conscious rats. In addition, in conscious rats, central administration of IMD results in increased plasma levels of oxytocin (OT) and vasopressin (AVP). The ability of IMD to activate the hypothalamo-pituitary-adrenal (HPA) axis can be blocked by intravenous pretreatment with the corticotropin releasing factor (CRF) antagonist, astressin. These results suggest that multiple members of the AM family of peptides may be involved in the cardiovascular, behavioral and neuroendocrine responses to stress.

Several brain structures responsible for controlling stress responses reach maturity during adolescence. Therefore, acute or chronic stress in prepuberty may negatively affect stress responses as well as behavior in adulthood. The hypothalamus-pituitary-adrenal axis (HPA) is part of the stress system whose inhibitory control is regulated by glucocorticoids through mineralocorticoid (MR) and glucocorticoid (GR) receptors. In this study, we aimed to determine whether MR or GR blockade after stress in adolescence prevents changes in exploratory behavior and HPA axis control in adult female rats. Adolescent female rats (26 days old) were submitted to one or seven daily restraint sessions followed by administration of MR (spironolactone) or GR (RU-486) antagonists. At 60 days old, animals were evaluated in the elevated plus maze and at 61 days old rats were subjected to acute stress to evaluate the HPA response. The chronic restraint in the adolescence induced an anxiogenic effect in the adult animals that was reverted by either MR or GR antagonist. In the same way chronic stress reduced the HPA axis activity by blunted corticosterone (CORT) secretion and decreased the activation of Corticotropin Releasing Hormone (CRH) neurons in the paraventricular nucleus. The post-stress blocking of GR independently restored the CORT secretion without effect on central activation. The acute stress in the adolescence had minor enduring effects. We concluded that the use of RU-486 and spironolactone after stress in the early adolescence can improve behavioral changes induced by stress whereas RU-486 only showed effect on the HPA axis response in adulthood.