Hypothalami, anterior pituitary gland segments and adrenal glands were removed from female Wistar-derived rats decapitated at various times of the day. Blood and tissue hormone concentrations were measured and the tissues challenged with appropriate stimuli in vitro. Both bioactive and immunoreactive corticotrophin-releasing factor (CRF) content of the hypothalami were significantly higher in the evening than in the morning, as was the basal release of bioactive CRF in vitro. The response of the hypothalami to serotonin or acetylcholine added in vitro did not change with time of day. Basal bioactive and immunoreactive adrenocorticotrophin (ACTH) release from the anterior pituitary gland was significantly increased in the evening, as was the response to synthetic ovine CRF in vitro. Plasma ACTH concentrations in intact rats given crude CRF (hypothalamic extract) in vivo were higher in the evening at all times after injection tested, but this difference was markedly reduced in animals with mediobasal hypothalamic lesions. Corticosterone released basally from adrenal glands in vitro was significantly increased in the evening and the response to added ACTH 1–24 was slightly enhanced. For adrenal glands removed from lesioned rats, the pattern was reversed, corticosterone release in vitro being lower in the evening for all doses of ACTH added. Similarly in vivo, in intact rats given ACTH 1–24, plasma corticosterone concentrations and corticosterone release in vitro from adrenal glands removed after the injection were higher in the evening. After the placement of basal hypothalamic lesions, the situation was reversed, the response to ACTH administration in vivo being greater in the morning. The peaks in plasma concentrations of ACTH and corticosterone occurred 1 h later than the peak in hypothalamic CRF release. Therefore, it appears that the timing of the diurnal variation in plasma levels is dictated by the hypothalamus, but circadian changes in the output and responsiveness of the pituitary gland to CRF and the adrenal glands to ACTH play a major role in determining the amplitude of the rhythm.

The occurrence and nature of corticosteroid inhibition of ACTH secretion at the rat anterior pituitary gland was investigated using three experimental models: animals bearing lesions of the basal hypothalamus, and two preparations of the gland incubated in vitro; these were tissue segments and collagenase-dispersed cells. Release of ACTH in the experiments was provoked using one of three distinct stimuli: acid extracts of whole hypothalami, corticotrophin releasing activity released by serotonin from hypothalami incubated in vitro and synthetic ovine corticotrophin releasing factor. Irrespective of whether ACTH was measured directly by radioimmunoassay (in the experiments in vitro) or indirectly in terms of corticosterone production (in the lesioned animals), its stimulated release from the anterior pituitary gland was inhibited by corticosterone. Two phases of inhibition were observed; these had some of the characteristics inferred previously from experiments with intact animals and designated fast feedback and delayed feedback. However, the fast feedback demonstrable in lesioned animals did not show the rate-sensitivity shown previously in intact animals. 11-Deoxycortisol (or 11-deoxycorticosterone) and prednisolone proved to be agonists of corticosterone in provoking fast feedback in lesioned animals, whereas they had been shown respectively to act as an antagonist or to have no effect in intact rats. Several steroids were able to cause delayed feedback in lesioned rats, but beclomethasone dipropionate (shown to be an agonist of corticosterone in intact rats) proved to have no inhibitory effect at the anterior pituitary gland of lesioned animals. It is concluded that the dynamics of corticosteroid feedback mechanisms at the anterior pituitary gland, as indicated by experiments in lesioned animals, differ from those operative in the intact animals. Other work suggests that a more important site for such inhibitory mechanisms in vivo is the hypothalamus.

Subcutaneous injection of 400 µg/100 g body weight of corticosterone (B) 2 h previously in male rats prevented the stress response, as assessed by the ability of adrenal glands removed from these animals to produce endogenous B. Two injections of a combination of 17α-hydroxyprogesterone and 11-epicortisol, the first given 30 min before and the second with the B, were able to block this inhibitory effect on the stress response. Neither of the steroids alone was effective in this regard. The combination was also effective against the early delayed feedback effects of 400 µg/100 g body weight cortisol, prednisolone or beclomethasone dipropionate in the same system. The minimum effective dose for reversal of feedback by B or beclomethasone dipropionate (2 mg/100 g body weight of each antagonist) was lower than that required for the same effect against prednisolone or cortisol (5 mg/100 g body weight). Previous injection of B also abolished the ability of anterior pituitary gland fragments to respond to corticotropin-releasing factors (CRFs) added in vitro, an effect which was not abolished by the injection of the combination of putative antagonistic steroids. From experiments designed to measure the ability of 17α-hydroxyprogesterone and 11-epicortisol to compete with ³H-corticosterone in binding to macromolecular components in hypothalamic, hippocampal and pituitary cytosolic preparations, it was deduced that the competition seen in the hypothalamic and hippocampal, rather than the pituitary, preparations was in better accord with the effect seen on the stress response. These results, taken together with our studies of the effects of the two steroids on CRF release from the hypothalamus in vitro, suggest that the site of action of 17α-OH-progesterone and 11-epicortisol is at the hypothalamus (and/or higher centres) and not at the pituitary gland.