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Corticotropin-releasing hormone (CRH) has been shown capable of inhibiting hypothalamic gonadotropin-releasing hormone (GnRH) release through activation of an endogenous opioid peptide (EOP)-dependent mechanism. Recently, we have shown that prolactin (PRL) stimulates CRH release and inhibits GnRH release by hypothalami explanted from male rats. Thus, the present study was undertaken to investigate whether the inhibitory effect of PRL on GnRH release in vitro is mediated by CRH and/or EOP. To this aim, the release of GnRH in response to PRL was evaluated in presence of CRH9-41 alpha-helical (CRH-9-41), a CRH receptor antagonist, and/or naloxone (NAL), a nonselective opioid receptor antagonist, using a static hypothalamic organ culture system which enabled us to evaluate immunoreactive GnRH (iGnRH) release from individually incubated longitudinally halved hypothalamic. As previously shown, PRL at the concentration of 100 nmol/l inhibited basal iGnRH release by about 35%. CRH9-41 or NAL overcame the inhibitory effect of PRL on iGnRH release in a concentration-dependent fashion. The simultaneous co-incubation with both antagonists was not more effective than each single antagonist. CRH9-41 did not have any effect on basal iGnRH release whereas NAL, as previously reported, increased it. In addition, PRL at the concentration of 100 nmol/l stimulated basal hypothalamic beta-endorphin (beta-EP) release. In conclusion, these data show that antagonism to CRH receptors counteracts the suppressive effects of PRL upon GnRH release and that PRL is able to stimulate hypothalamic beta-EP release in vitro.

To study the role of corticotropin-releasing hormone (CRH) in the circadian rhythm of circulating corticotropin (ACTH), beta-endorphin (beta-END), corticosterone, and prolactin (PRL), we measured the effects of CRH immunoneutralization over a 24-hour period in chronically cannulated, conscious, freely moving, male Sprague-Dawley rats, maintained at a constant light-dark cycle. Blood samples were collected in the morning (08.00 h), at noon (12.00 h), and in the evening (18.00 h) on the day of treatment, and in the morning (08.00 h) of the next day. Hyperimmune rabbit serum raised against rat CRH (1.0 ml/rat, i.v.) or normal rabbit serum (NRS, 1.0 ml/rat, i.v.) was administered at 08.00 h, immediately after the first blood sample had been collected. CRH immunoneutralization caused no significant decreases in circulating immunoreactive ACTH, beta-END and corticosterone plasma levels at noon, but abolished the evening rises of these hormones. PRL levels were not significantly different between the groups at any time point measured. To compare the effects of CRH immunoneutralization to those of glucocorticoid negative feedback, we measured the effects of dexamethasone (0.5 mg/kg, i.v. at 08.00 h) on the above parameters. ACTH and beta-END concentrations were significantly decreased, and corticosterone and PRL levels were markedly suppressed after glucocorticoid administration both at 12.00 and 18.00 h. However, 24 h after the administration of dexamethasone, PRL concentrations were elevated despite persistently low corticosterone levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholecystokinin (CCK), a gastrointestinal (GI) hormone, is also present in structures of the central nervous system such as cortex, hippocampus, amygdala, olfactory tubercle and in regions involved in the regulation of the pituitary function. Although a number of studies have evaluated the effects of CCK on hypothalamic-pituitary-adrenal (HPA) axis function and on arginine vasopressin (AVP), prolactin (PRL) and growth hormone (GH) plasma levels in the laboratory animal, its role in humans has not been explored. Hence, we examined the effects of the exogenous administration of this GI hormone on corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), cortisol, AVP, PRL and GH plasma levels in humans. To accomplish this, graded doses (0, 50, 140 and 420 ng/kg) of sulfated CCK octapeptide (CCK-8), the full biologically active peptide, were infused intravenously to healthy men in 30 min. Blood samples were collected 30 min and immediately before the infusion was started (baseline) and 15, 30, 45, 60 and 90 min thereafter. CRH, ACTH, and AVP were extracted from plasma proteins using cartridges of SepPak C18. These hormones and cortisol were measured by radioimmunoassay whereas PRL and GH were measured by immunoradiometric assay. CCK-8 increased plasma ACTH and cortisol levels only at the dose of 420 ng/kg, whereas it had no detectable effect on plasma CRH levels. It increased also plasma AVP levels at the doses of 140 and 420 ng/kg. However, this effect reached the statistical significance only at the highest dose tested. CCK-8 stimulated PRL and GH release in a dose-dependent fashion. The lowest stimulatory dose was 140 ng/kg for both hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

The susceptibility of female Lewis (LEW/N) rats to the development of streptococcal cell wall (SCW)-induced arthritis and other autoimmune phenomena is associated with the inability of their hypothalamic-pituitary-adrenal (HPA) axis to adequately respond to inflammatory stimuli. In contrast, resistance to the development of SCW-induced arthritis and other inflammatory autoimmune manifestations in histocompatible female Fischer rats (F344/N) is related to their intact HPA axis response to inflammatory mediators. To evaluate the mechanism and the specificity of the HPA axis defect in LEW/N rats, we examined the ability of three major excitatory neurotransmitter systems to activate the HPA axis in both Lewis and Fisher rats. The responsiveness of plasma ACTH and corticosterone to the cholinergic muscarinic receptor agonist arecoline, the alpha 1-adrenergic receptor agonist methoxamine and the serotonin (5-HT) type 2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane were significantly blunted and/or abolished in LEW/N compared to F344/N rats. To localize the HPA axis defect to the hypothalamic CRH neuron, we evaluated the ability of explanted hypothalami from the two strains to secrete immunoreactive CRH in vitro, in response to acetylcholine (ACh), norepinephrine (NE), 5-HT and the 5-HT agonist quipazine. LEW/N hypothalami released less immunoreactive CRH (iCRH) in response to ACh, NE, 5-HT and quipazine than F344/N hypothalami. The dose-response curves of these compounds in the former were shifted to the right and/or abolished, suggesting decreased sensitivity of LEW/N hypothalami to these neurotransmitters.(ABSTRACT TRUNCATED AT 250 WORDS)

In rats hyperprolactinemia increases corticotropin-releasing hormone (CRH) concentration and secretion in hypophysial portal blood and the serum concentration of adrenocorticotropic hormone (ACTH). To determine whether the stimulatory effect of prolactin (PRL) on CRH and ACTH in vivo is exerted directly on the hypothalamus, hypothalamic explants and primary anterior pituitary cell cultures from adult male and female rats were used. Hypothalami explanted from male and female rats were preincubated during 90 min and treated for 30 min with rat PRL (rPRL) at concentrations of 10–8, 10–7, and 10–6M (about 200, 2,000, and 20,000 ng/ml, respectively), corticosterone at concentrations of 10–7, 10–6, and 10–5M (about 35, 350 and 3,500 ng/ml, respectively), ACTH at concentrations ranging from 10–10 to 10–7M (0.46, 4.6, 46, and 460 ng/ml, respectively), and graded concentrations of testosterone or estradiol. Concentrations of immunoreactive CRH (iCRH) were measured by radioimmunoassay. rPRL at 10–6M stimulated iCRH secretion by 360 and 400% of the basal iCRH output (about 14 pg/hypothalamus), respectively, from hypothalami explanted from male and female rats. ACTH and corticosterone did not suppress rPRL (10–6M) induced iCRH secretion. Corticosterone at the concentration of 10–6M potentiated rPRL (10–6M) induced iCRH secretion in hypothalami explanted from male, but not female rats. Gonadal steroids had no effect either on the basal or rPRL (10–6M) stimulated iCRH secretion, with the exception of estradiol which augmented the response to 10–6M rPRL by about fivefold, but only at the concentration of 10–8M (about 2.7 ng/ml). Pituitary cells were treated with rPRL, corticosterone, gonadal steroids, or CRH for 4 h. The concentrations of ACTH in the culture medium were measured by radioimmunoassay. rPRL at the concentration of 10–7M caused an ACTH release of 169 ± 8 and 185 ± 14% of basal values (about 2,000 pg/105 cells), respectively, in pituitary cells explanted from male and female rats, whereas at the concentration of 10–6M rPRL caused a release of 265 ± 15 (male) and 336 ± 62% (female). Corticosterone suppressed PRL (10–6M) induced ACTH release in a dose-dependent fashion, but the ACTH secretion did not reach basal levels. Inhibition of CRH-induced ACTH release by corticosterone was not reversed by the addition of 10-6M rPRL. Gonadal steroids had no effect. Rat growth hormone did not influence iCRH or ACTH secretion in these systems. We conclude that the activation of the hypothalamic-pituitary-adrenal axis observed during the course of hyperprolactinemia may be explained by a direct stimulatory effect of PRL on both hypothalamic CRH and pituitary ACTH secretions.

The effects of chronic cortisol treatment on neuroendocrine and behavioral responses to serotonim (5-HT1) receptor agonists were studied in conscious, freely moving rats. Seven-day cortisol treatment (25 mg/kg/day with osmotic minipumps) markedly suppressed basal plasma corticotropin (ACTH) and corticosterone concentrations, indicating a suppression of the hypothalamo-pituitary-adrenocortical axis. Cortisol also decreased body weight, food intake, plasma norepinephrine (NE), and epinephrine (E) levels. In the drug challenge studies, we used two 5-HTι agonists, the 5-HT1B and 5-HT1C agonist, m-chlorophenylpiperazine (m-CPP), and the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT), to examine the effect of cortisol on their behavioral and neuroendocrine effects. After 7-day cortisol treatment, plasma prolactin responses to both m-CPP and 8-OHDPAT were significantly decreased. While the plasma NE, E, and food intake responses to m-CPP were also significantly reduced by cortisol treatment, these same responses to 8-OHDPATwere unchanged. The effect of m-CPP on locomotor activity was also decreased. Since only the responses to m-CPP and 8-OHDPAT previously shown to be antagonized by pretreatment with the 5-HT1/5-HT2 antagonist, metergoline, were significantly attenuated after cortisol treatment, these changes may be specific to 5-FΓT receptors. These data indicate that chronic exposure to high glucocorticoid levels alters 5-HT1 receptor-mediated functions and provides additional evidence relevant to the contribution of glucocorticoid elevation to the symptoms of depression.

Several lines of experimental evidence suggest that acetylcholine (ACh) is excitatory to the hypothalamic-pituitary-adrenal (HPA) axis. Since previous experiments have shown that ACh does not affect pituitary adrenocorticotropin secretion in vitro, we hypothesized that ACh stimulates the HPA axis by causing hypothalamic corticotropin-releasing hormone (CRH) secretion. We examined this hypothesis using an organ culture system that measures the ability of single rat hypothalami to secrete immunoreactive CRH (IR-rCRH) in vitro. ACh stimulated hypothalamic IR-rCRH secretion in a dose-dependent fashion, at concentrations ranging from 3.3 x 10(-10) to 10(-5) M. This effect was antagonized by the simultaneous presence of atropine and hexamethonium, a muscarinic and a nicotinic receptor antagonist, respectively (p less than 0.05). Further evidence for the cholinergic regulation of the CRH neuron was provided by the findings that both carbachol, a muscarinic receptor agonist, and nicotine, a nicotinic receptor agonist, stimulated IR-rCRH secretion in a dose-dependent fashion. These effects were antagonized by atropine and hexamethonium, respectively, suggesting that both muscarinic and nicotinic receptors are involved in the process. ACh stimulated hypothalamic IR-rCRH secretion in the presence of phentolamine, an alpha-adrenergic antagonist, and ritanserin, a serotonin2 receptor antagonist, suggesting that the cholinergic stimulation of CRH secretion is not mediated by alpha-adrenergic or serotonergic interneurons. We conclude that ACh stimulates hypothalamic CRH secretion via both muscarinic and nicotinic receptor mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)