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The effect of vasoactive intestinal peptide (VIP) on cortisol production was studied in a primary culture enriched with guinea pig Zona Fasciculata (ZF) cells. In ZF cells, VIP stimulates cortisol secretion and enhances the steroidogenic action of ACTH. Compared to ACTH on an equal molar basis, the cortisol-stimulatory effect of VIP is at least 10-fold less potent. As VIP exhibits a wide range of biological actions with widespread distribution in the body, the steroidogenic action of VIP on the adrenal glands is not tissue-specific. There are VIP receptors in ZF cells. With the aid of a VIP receptor antagonist, we found that ACTH and VIP mutually bind each other's receptors with an affinity-ranking order of ACTH > VIP receptor antagonist > VIP. VIP stimulates cortisol production most likely through the cyclic AMP (cAMP) signaling pathway. Both ACTH receptors and the VIP receptors bind VIP receptor antagonist more avidly than VIP, but the bindings do not lead to a consequential effect on cAMP production and cortisol secretion. However, the VIP receptor antagonist counteracted ACTH and VIP to lower both cAMP and cortisol production. In addition, ASIF and BNP-32, which are the proven ACTH receptor antagonists, reduced the cortisol-stimulatory effect of ACTH and VIP. These results suggest that besides ACTH, VIP be an important factor in regulating the cortisol secretion from the adrenal cortex at the site of ACTH receptors. In cases with hypercortisolemia being detected concomitantly with normal or low ACTH levels, we may need to investigate the influential role of VIP.

The suppressive effect of aldosterone secretion-inhibitory factor (ASIF) and brain natriuretic peptide (BNP-32) on the basal and ACTH-stimulated cortisol production in a primary culture enriched with guinea pig Zona Fasciculata (ZF) cells was further studied. The binding of 125I-labeled ACTH(1-24) and ASIF to ZF cells was found to be displaced by ACTH(1-24), [Phe2, Nle4 and Ala24]-ACTH(1-24), ASIF, and BNP in a concentration-dependent manner. The binding of 125I-labeled [Phe2, Nle4 and Ala24]-ACTH(1-24) to two transformed clones of mammalian cells expressing the guinea pig ACTH receptor was also competitively inhibited by ASIF and BNP. ASIF and BNP significantly suppressed ACTH-stimulated cAMP production in ZF cells. The 10- and 30-min cellular changes in cAMP induced by ASIF and BNP did not correlate in the rank order with the ultimate magnitude of cortisol suppression observed in ZF cells after a 24-hour treatment with these peptides. Nevertheless, the results did conform to the signaling mechanism of their action. Overall, the findings clearly demonstrated that ASIF and BNP suppressed the adrenocortical function and inhibited ACTH for their antagonistic action against ACTH primarily at the ACTH receptor site. These results support the notion that a physiological role of adrenal medulla in regulating the adrenocortical function may be mediated by the neuropeptides through a paracrine pathway.

The effects of thyroxine (T4) on the secretion of corticosterone both in vivo and in vitro in male rats were studied. Rats were thyroidectomized (Tx) or sham Tx. The Tx rats were subcutaneously with T4 (20 micrograms/kg) or saline once daily for two weeks. In an in vitro experiment, adrenal glands were incubated with ACTH, T4, or ACTH plus T4 in the presence or absence of 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) at 37 degrees C for 60 min. Medium and ether-extracted plasma samples were analyzed for corticosterone by radioimmunoassay (RIA). The accumulation of cyclic adenosine monophosphate (cAMP) in adrenal tissues after incubation with IBMX was measured by RIA. The levels of plasma corticosterone in Tx rats were significantly increased as compared with euthyroid rats. T4 replacement in Tx rats restored plasma corticosterone to euthyroid level. Administration of T4 in vitro resulted in an inhibition of both basal and ACTH-stimulated release of corticosterone. Both basal and ACTH-stimulated generations of cAMP in adrenal tissues were decreased by T4. These results suggest that T4 inhibits the spontaneous and ACTH-stimulated secretion of corticosterone by acting directly at adrenal glands via a decrease in cAMP production.

Circumstantial evidence has indicated that the adrenal gland may produce certain factors regulating its own corticoid production through a paracrine pathway. The putative factors play only a subtle role modulating adrenocortical function in response to ACTH. Aldosterone secretion-inhibitory factor (ASIF) and brain natriuretic peptides (BNP) are among the attractive candidates. ASIF was tested in a primary culture system of guinea-pig adrenal zona fasciculata (ZF) cells and found to be effective in suppressing both the basal and ACTH-stimulated cortisol production in a dose-dependent manner. At the effective doses, ASIF was not toxic to the cells in culture morphologically but attenuated the growth-stimulating action of ACTH. Tested with three types of natriuretic peptides in the same cell culture system, we found that BNP was as effective as ASIF. Studies on the relationship between peptide primary structures and activities revealed that the inhibitory activity of ASIF on cortisol production required an intact Cys-Cys loop with both N- and C-terminal extensions from Cys residues of not less than 2 amino acids.

Although organization and hormonal regulation of the hypothalamic-pituitary-adrenocortical (HPA) and the hypothalamic-pituitary-thyroid (HPT) axes share a remarkable degree of similarities, distinctive patterns of their plasma hormone secretions are observed. We measured plasma levels of ACTH-cortisol (PA) and hTSH-T3 (PT) pairs of hormones in 24-hour sequential blood specimens sampled at 30-minute intervals from 3 patients with suspected adrenal disorders and 4 normal volunteers and found the same percentage of discordant secretions of the PA and PT hormones but significantly greater coefficients of variations of the PA values than the PT values (p < 0.002). This confirms that plasma PT hormones are more tightly self-regulated between themselves than plasma PA hormones. Moreover, cluster analysis of the 24-hour plasma hormonal fluctuations revealed one or more ACTH-cortisol hyposecretory clusters only in subjects with a normal status of adrenal function. There was no similar hTSH-T3 hyposecretory cluster detected in any one of the 7 subjects. Based on these results, we postulate that there is some adrenal factor normally exerting a subtle antagonistic action against ACTH and causes the incidence of such ACTH-cortisol hyposecretory cluster.

To test the long-standing concept that secretion of adrenal glucocorticoids is solely dependent upon plasma level of adrenocorticotropic hormone (ACTH), we collected sequential blood samples from normal subjects and patients with pituitary-adrenal axis disorders at 30-min intervals for 24 hours and analyzed for both the plasma ACTH and cortisol profiles. In a majority of time points, the changes of cortisol levels coincided with those of ACTH. However, in all individuals tested so far, on more than 24 per cent of occasions, plasma cortisol rose together with a declining or fell with an elevating plasma ACTH level, that indicates a secretory discordance between these two hormones. These discordant patterns cannot be explained simply by the ACTH-stimulatory and cortisol-feedback inhibitory mechanisms. Since a generalized quantitative relationship between the overall profiles of plasma ACTH and cortisol did exist and the mutually regulatory pathways between pituitary and adrenal functions were mostly observable, there were no other known reasons explaining why these frequent and unsynchronized secretory activities should happen. Our observation reveals that cortisol secretion is probably under some other minor but normal influences in addition to the major regulatory action of ACTH.

We used guinea-pig adrenal tissue to develop a primary culture, enriched with zona fasciculata (ZF) cells. In a continuous culture up to 2 weeks, the cells maintained the characteristic of glucocortical function by producing cortisol as the final steroidogenic product and secreting it into culture medium. When culture medium, which was replaced at 24-hr intervals, was assayed for cortisol, the basal steroidogenic function peaked on day 5 and then declined. In response to 24-hr treatment with the bioactive adrenocorticotropic hormones (ACTH) on day 4, production of cortisol was stimulated and prolonged, and also cells were morphologically hypertrophic. This in vitro system provides a convenient laboratory method which can be used for studying adrenocortical function under a possibly physiological condition.