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Diagnostic tests of hypothalamic-pituitary-adrenocortical function in psychiatric illness largely report the interaction of hypothalamic secretagogues with glucocorticoids at the pituitary level. This study investigated whether the efficiency of glucocorticoid inhibition is subject to modulation by intracellular processes that enhance cAMP accumulation and/or facilitate membrane depolarization. The secretion of ACTH induced by corticotropin-releasing factor (CRF; 0.1 nM) in primary cultures of rat anterior pituitary cells was markedly inhibited upon a 2-h exposure to 100 nM corticosterone. Arginine vasopressin (2 nM) enhanced the cAMP as well as the ACTH responses to CRF and reduced the efficiency of glucocorticoid inhibition of ACTH release. The action of arginine vasopressin was mimicked by rolipram, an inhibitor of cyclic nucleotide phosphodiesterase type 4. Application of the broad specificity K(+) channel blockers clofilium and astemizole produced minor or no significant enhancement of CRF-induced ACTH release, respectively, but opposed the inhibitory effect of corticosterone. Specific blockers of HERG, KCNQ, and Isk channels had no effect on ACTH release under any condition examined. In summary, these data reveal multiple sites of posttranslational modulation of adrenal corticosteroid action at the level of the pituitary gland, which appear important for the outcome of diagnostic tests of hypothalamic-pituitary- adrenocortical function.

The utility of the established ACTH secreting mouse pituitary tumor cell line AtT20 for investigating early glucocorticoid inhibition was examined. Three different strains of the cell line D1, D16v, and D16:16, respectively, were analyzed. In initial studies CRF and phorbol esters were used as secretagogues to examine the properties of hormone secretion. In a perifusion system (cells in suspension) D1 cells failed to respond to the secretagogues, whereas both D16v and D16:16 cells were responsive. However, hormone release declined upon repeated exposure to secretagogue in both D16v and D16:16 cells and similar data were obtained when cells adhering to cover slips were perifused. In static incubation D16:16 cells gave more consistent results especially with respect to inhibition by glucocorticoids and were used in all subsequent studies. Synthetic glucocorticoids acting through the type II receptor inhibited CRF-induced ACTH release within 45 min; at 120 min, stimulated release was strongly (80-90%) suppressed. In contrast, no consistent inhibition by corticosterone could be found. In the presence of glycyrrhetinic acid, an inhibitor of 11 beta-hydroxysteroid dehydrogenase, a high concentration of corticosterone (10 microM) did produce a slight inhibition of ACTH release. Dexamethasone also inhibited ACTH release induced by the calcium channel activator compound (+)202-791. The accumulation of cAMP in response to CRF was not altered by dexamethasone. The inhibitory effect of synthetic glucocorticoids on ACTH release was prevented by blockers of messenger RNA (actinomycin D, dichlorobenzimidazole ribofuranoside) or protein (puromycin) biosynthesis, indicating the induction of new proteins. Immunoblotting for lipocortin I (annexin I) and chromogranin A revealed no induction by dexamethasone of any of these proteins in D16:16 cells. Messenger RNA encoding lipocortin I was not detectable and was not induced by treatment with dexamethasone in D16:16 cells. These data show that the AtT20 D16:16 strain is a useful model for early glucocorticoid action, which is mediated by type II receptors and involves the induction of new protein(s). Notably, induction of lipocortin I messenger RNA or protein could not be detected at a time when the inhibitory effect of glucocorticoids on stimulated hormone secretion was maximal.

The aim of this study was to resolve previous controversies regarding the effect of atriopeptin on the secretion of ACTH in vivo. Male Wistar rats were used throughout. The animals were subjected to lesioning of the hypothalamic paraventricular nucleus (PVN) or sham operation and implanted with indwelling jugular cannulae 5 days later for blood sampling and drug infusion. Two days after the insertion of the cannulae the animals were treated with saline or 103-126 amino acid residue atriopeptin iv: a bolus injection was given (200 or 40 pmol/rat) followed by an infusion (40 or 8 pmol/min) which was maintained for the entire duration of the experiment (70 min). Ten minutes after the bolus of atriopeptin the animals received iv a combination of 1 pmol 41-residue CRF and 10 pmol arginine vasopressin (CRF/AVP) to stimulate ACTH secretion. Serial blood samples (0.1 ml) were obtained at -10 min and immediately before the injection of CRF/AVP and at 5, 10, 20, 30, and 60 min afterwards. Plasma ACTH concentration was measured by RIA. In sham-operated rats CRF/AVP caused a 4-fold increase in plasma ACTH which peaked at 5 min and returned to baseline by 60 min. In sham-operated rats the higher dose of atriopeptin (200 pmol bolus, 40 pmol/min infusion) did not alter the effect of the stimulus between 5 and 30 min, and augmented plasma ACTH at 60 min. The smaller dose of atriopeptin reduced plasma ACTH at 10 and 20 min by 54% and 48%, respectively, and also decreased by 48% the net amount of ACTH released over 30 min in response to CRF/AVP. When given alone, the higher dose of atriopeptin caused a persistent (60 min) 10-13% reduction of mean arterial blood pressure, while the lower dose decreased blood pressure by about 9% for less than 10 min. In parallel, the higher dose of atriopeptin increased plasma ACTH concentration while the lower dose produced no change. In PVN-lesioned rats the CRF/AVP induced ACTH response was similar to that seen in sham-operated controls. Only the higher dose of atriopeptin was tested, and this markedly reduced CRF/AVP stimulated ACTH secretion at 5-60 min after CRF/AVP. Given alone, atriopeptin had no marked effect on plasma ACTH in PVN-lesioned rats, while its hypotensive action was similar to that in sham-operated animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Previous work has shown that corticosterone, cell-membrane permeant analogs of cGMP, as well as activators of guanylyl cyclase inhibit secretagogue-stimulated ACTH release. In the present study we have examined whether cGMP mediates the inhibitory effect of corticosterone in perifused isolated rat anterior pituitary cells. A brief 22.5-min exposure to corticosterone strongly inhibited ACTH secretion evoked by arginine vasopressin (AVP), 48 mM KC1, and two types of combined stimuli, i.e. 41-residue CRF and AVP (0.05 and 0.5 nM, respectively; CRF/AVP), or ionomycin and phorbol-dibutyrate (200 and 10 nM, respectively; PdBu/IM). The time course of inhibition by corticosterone was similar in all cases; a rapid approximately 30% reduction in ACTH was evident within 25 min, which increased to 60% by 50-70 min and will be referred to as the delayed effect. The corticosteroid inhibition of PdBu/IM-induced ACTH release was fully antagonized by the glucocorticoid/progestin antagonist RU 38486, indicating that it is exerted through type II glucocorticoid receptors. In contrast to corticosterone, the cGMP derivative 8-bromo-cGMP failed to suppress ACTH release evoked by PdBu/IM, whereas it effectively inhibited the action of CRF/AVP. Furthermore, ionomycin reversed the reduction of CRF/AVP-stimulated ACTH release by 8-bromo-cGMP, but had no effect on the delayed inhibition caused by corticosterone. These data indicate that there are two distinct cellular pathways of inhibiting stimulus-evoked ACTH secretion in vitro. One of these is activated by corticosterone, whereas the other involves cGMP as a cellular messenger.

Activation of the hypothalamic-pituitary-adrenocortical axis is a major component of the body's response to stress. Current theories on the pathophysiology of disorders associated with hyperfunction of the axis, such as depression and Cushing's stress, are based on the concept that anterior pituitary adrenocorticotropin (ACTH) secretion is stimulated by hypothalamic corticotropin-releasing hormones and inhibited by adrenal corticosteroids. Hypothalamic inhibitory control of pituitary ACTH secretion has been also postulated, but has not gained general acceptance because of the lack of definitive evidence for a corticotropin-release inhibiting hormone. It is shown here that in conscious rats stress-induced secretion of ACTH and corticosterone is markedly enhanced by the immunoneutralisation of atriopeptin. Therefore, we propose that atriopeptin is a physiologically relevant corticotropin-release inhibiting hormone.

Glucocorticoid hormones suppress the release of ACTH by the anterior pituitary gland: rapid feedback inhibits hormone secretion within 30 min of steroid application, delayed feedback is most effective at 1-2 h, and slow feedback becomes manifest in several hours. The aim of the present study was to determine the type of glucocorticoid receptor that mediates the rapid and delayed feedback actions of glucocorticoids and whether genomic activation occurs during the rapid and delayed time domains. Rat anterior pituitary cell columns were perfused with Dulbecco's minimum essential medium, 41-residue CRF (10(-9) M) was used as the secretagogue, which stimulated ACTH secretion to a peak of about 8- to 10-fold of basal release. The amount of ACTH released upon repeated 5 or 10 min stimulation with CRF was constant. Treatment with 10(-7) M corticosterone for 20 min immediately before and for 10 min during stimulation with CRF reduced ACTH release by about 50% (rapid feedback), while at 1 h and 2 h after the initial exposure to corticosterone the secretory response was 33% and 15% of control, respectively. The effect of corticosterone was prevented by the type II glucocorticoid/progesterone antagonist RU 38486 (10(-6) M). The selective type II receptor agonist RU 28362 (10(-7) M) was even more potent than corticosterone in inhibiting ACTH release; the time course of action was similar. When actinomycin D (10(-4) M) was applied in conjunction with RU28362 or corticosterone, no inhibitory effects appeared up to 2 h after the exposure to steroid. Puromycin (10(-4) M), given during and for 1 h after the administration of the steroid prevented the rapid as well as the delayed (1 h) inhibitory action of RU28362. When puromycin was removed from the system, a 75% inhibition of stimulated ACTH release developed at 2 h after the application of the steroid, indicating that translatable messenger RNA (mRNA) was still present in the cells. Cycloheximide (10(-4) M) was only partially effective at inhibiting rapid or delayed feedback, and increasing its concentration impaired the ACTH response to CRF-41. In summary, at the pituitary level the rapid as well as the delayed feedback inhibition of ACTH secretion by adrenal corticoids is exerted via type II glucocorticoid receptors. Furthermore, both rapid and delayed feedback require the synthesis of new mRNA and protein.

In the present study we report the properties of vasopressin (VP) receptors in the anterior pituitary gland and show that the number of these receptors is markedly affected by adrenalectomy and hypothalamic lesions. VP-binding activity was assayed in particulate fractions of rat anterior pituitary glands using tritium-labeled arginine VP ([3H] AVP) as tracer. In the presence of Mg2+ the radioligand interacted with a single class of high affinity, low capacity binding sites. Magnesium ions modulated the affinity of the receptors but had no effect on binding capacity. Guanine nucleotides decreased the amount of tracer bound in a dose-dependent manner by increasing the dissociation constant (Kd) of the binding reaction by approximately 2-fold. Increasing the concentration of Mg2+ did not prevent this effect. Bilateral adrenalectomy (ADX) decreased pituitary AVP-binding activity: binding fell by 30% 4 h after surgery and declined further to 10% or less of control at 4 days. The decrease in binding was primarily due to a reduction in the number of receptors. Daily administration of corticosterone inhibited the reduction of binding activity at 4 days in a dose-dependent manner. Destruction of hypophyseotropic VP neurons by means of surgical lesioning of the hypothalamic paraventricular nucleus or the medial basal hypothalamus abolished the effect of ADX on pituitary AVP binding at 24 h but only attenuated the degree of receptor loss at 4 days. Furthermore, the lesions themselves caused a significant (approximately 30%) reduction in receptor number 4-7 days after hypothalamic surgery. Adrenalectomy reduced pituitary AVP-binding activity in homozygous (di/di) Brattleboro rats. The extent as well as the time course of the loss of receptor activity resembled that in normal rats. Rat anterior pituitary segments were exposed to synthetic CRF, AVP, or oxytocin (all 10(-7) M) for 4 h in vitro, and [3H] AVP-binding activity was subsequently determined. Both AVP and oxytocin reduced the amount of radioligand bound, while CRF had no effect. These observations allow the following conclusions: Magnesium ions and guanine nucleotides modulate the affinity of pituitary AVP receptors by different mechanisms and have no effect on binding capacity; Pituitary receptors for AVP are regulated by the amount of AVP released by paraventricular nucleus neurons as well as through a mechanism that requires the presence of corticosterone; Homozygous Brattleboro rats may respond to ADX by increased hypothalamic release of an endogenous ligand for pituitary AVP receptors.

Specific, high affinity sites that bound tritium-labeled arginine-vasopressin (3H-AVP) were detected in a crude membrane fraction of rat adrenal capsules (chiefly zona glomerulosa). Binding displacement experiments with peptide analogs of AVP suggested that the binding site is a pressor (V1) type receptor for AVP. When added to dispersed rat adrenal glomerulosa cells, vasopressin (10(-8)-10(-6)M) stimulated the incorporation of 32P-phosphate into phosphatidylinositol, and the effect was blocked by the AVP receptor antagonist peptide d(CH2)5Tyr(Me)AVP. Vasopressin also increased the breakdown of phosphatidylinositol-4,5-bisphosphate within 1 min after its addition to the incubation medium. Superfused zona glomerulosa cells responded to AVP (10(-8)-(-6)M) by increasing their aldosterone production. The response could be blocked by the antagonist peptide. These data show that functionally active V1 receptors are present in rat glomerulosa cells, and suggest that vasopressin may regulate the function of the adrenal glomerulosa.

The nature of purported receptors for oxytocin (OT) in the rat adenohypophysis was investigated by means of radioligand binding. Tritium-labeled oxytocin (3H-OT) was used as the radioligand. Particulate fractions prepared from the adenohypophysis of estrogen-treated female rats bound 3H-OT reversibly and with high affinity (Kd = 1.5 nM). Scatchard plots indicated the presence of a single, low-capacity binding-site (Bmax = 33 fmol/mg protein). However, ligand displacement experiments suggested that 3H-OT labels OT- as well as vasopressin-preferring sites in adenohypophysial membrane-suspensions. When the number of pituitary vasopressin receptors was reduced by adrenalectomy, it was revealed that the ligand-specificity of the high-affinity OT binding site is similar to that of classical OT receptors. The results show the presence of separate receptors for OT and vasopressin in the rat adenohypophysis, and hence reinforce the notion that OT may function as a hypophysiotrophic hormone.