Adrenocortical cell apoptosis was studied by using an established in vivo model, the hypophysectomized rat, and an in vitro model, viz., rat adrenal glands in short-term organ culture. In vivo, apoptosis (biochemical autoradiographic analysis of internucleosomal DNA cleavage) was weak and not apparent until 12-24 h after hypophysectomy. In situ histochemical localization of 3'-end DNA strand breaks revealed that apoptosis in vivo occurred nearly exclusively in subpopulations of zona reticularis cells. Adrenocorticotropic hormone (ACTH) maintenance completely blocked these indices of apoptosis. By contrast, apoptosis (DNA fragmentation) in cultured rat adrenal glands without ACTH was extensive and relatively rapid, being apparent after 1 h and increasing with the duration of incubation. ACTH attenuated (by 44%) but did not completely block apoptosis in vitro. Thus, ACTH appears to be the sole pituitary hormone that forestalls apoptosis of terminally differentiated adrenocortical (zona reticularis) cells. However, the discrepancy between in vitro and in vivo models in terms of the magnitude and rate of DNA fragmentation suggests that, in vivo, other factors finely regulate the magnitude of adrenocortical apoptotic cell death.

The relation between steroidogenesis induced by ACTH and that induced by exogenous concentrations of glucocorticoids was studied in isolated adrenocortical cells. Exogenous corticosterone and cortisol, in concentrations within the production capacity of the adrenal gland, suppressed steroidogenesis induced by ACTH in rat and beef cells, respectively. The precursors pregnenolone and progesterone enhanced steroidogenesis in both rat and beef cells. Aldosterone in rat cells and 17 beta-estradiol in rat and beef cells had little if any effect on steroidogenesis. Either suppression or stimulation by exogenous steroids was acute, that is, after 2-h incubation for rat cells and 1-h incubation for beef cells. A direct suppressive action of end product glucocorticoids is indicated. This observed self-suppression of adrenocortical cells suggests the existence of a mechanism for the find adjustment of steroidogenesis that operates in addition to the classical control exerted by the anterior pituitary.

Effects of handling, administration of a local anesthetic, and electrical dehorning on plasma cortisol were measured in Holstein heifer calves. Jugular blood was sampled from 24 calves (age, 7 to 16 wk) within 1 to 2 min after entering their pen (baseline, 0800 h). All calves were then haltered, placed in a restraint chute, an unheated electrical dehorner applied to each horn stump, and a second jugular blood sample obtained 30 min later (handling). Calves were then divided into control and test groups (12 calves). For the next 2 consecutive d the sequence of sampling blood and handling were the same except that 1) for control calves a heated dehorner was used on d 2 (dehorning), and 2) for test calves the cornual nerve of each horn stump was injected with 5 ml lidocaine prior to applying an unheated dehorner on d 2 (administration of anesthetic) and a heated dehorner on d 3 (combination of anesthetic and dehorning). Handling, injection of an anesthetic, and dehorning were stressful and increased plasma cortisol of calves 5.4, 16.8, and 28.3 ng/ml above baseline, respectively. Dehorning and the combination of injecting lidocaine and dehorning resulted in similar increases in plasma cortisol. Different responses in plasma cortisol in individual calves exposed to similar stimuli suggest that synthesis and release of cortisol can be modified.

The effect of synthetic ovine CRF on ACTH secretion of dispersed, domestic fowl pituitary cells was investigated. Cells preincubated for 2 h, 16 h, or after 48-h culture were incubated briefly with CRF (up to 4 h). ACTH was bioassayed using isolated rat adrenocortical cells; ACTH-(1-24) served as the standard for expressing the data. Results with 16-h preincubated cells were as follows: CRF induced ACTH secretion in a concentration-dependent manner: ED50 and maximal stimulatory concentrations were 1.0 nM and 5.0 nM, respectively. CRF (10 nM) induced significant ACTH secretion within 10 min of incubation; maximal secretion (370% over basal value) was attained at 2 h. Dexamethasone (DEX) inhibited basal and CRF-induced ACTH secretion in a concentration-dependent manner; half-maximal inhibitory and maximal inhibitory concentrations were approximately 10 nM and 1 microM, respectively. In addition, DEX (10 microM) acutely (within 2 h) inhibited maximal CRF-induced ACTH secretion by 46%. 8-Bromo-cAMP (1 mM) also induced ACTH secretion, and DEX inhibited this secretion with a potency equivalent to that for CRF-induced ACTH secretion. In contrast to the effect of CRF, high concentrations (100 nM) of ovine LHRH, TRH, and synthetic human pancreatic GH-releasing factor (1-32) failed to induce significant ACTH secretion, thus suggesting that the effect of CRF was peptide specific. Domestic fowl pituitary cells cultured for 48 h before treatment also responded to CRF but not to any greater extent than that of 16-h preincubated cells. In contrast to 16-h preincubated cells or 48-h cultured cells, 2-h preincubated cells had high basal values of ACTH secretion that may have partially diminished or masked the actions of CRF. These data suggest that 1) CRF is a potent and specific stimulator of ACTH secretion by domestic fowl pituitary cells and 2) 16-h preincubated cells or 48-h cultured cells are amenable for other in vitro investigations on the regulation of avian ACTH secretion.

Adrenocortical function was investigated in immature, dietary protein-restricted domestic fowl (Gallus gallus domesticus). White Leghorn cockerels (2 weeks old) were fed isocaloric semipurified diets containing either 8% [low (L)] or 20% [normal (N)] soy protein for 4 weeks ad libitum. Cockerels were quickly killed by decapitation and exsanguination. Trunk plasma corticosterone (B) and ACTH levels were measured by RIA. Maximal B-binding capacity (CBC) of plasma was also measured. In addition, in randomly selected cockerels, a rough index of the B clearance rate was determined. Finally, to determine the influence of protein malnutrition on adrenocortical cell function per se, we measured the acute (2-h) B responses of highly enriched adrenocortical cell populations to various ACTH analogs, 8-bromo-cAMP (8-Br-cAMP), and cellular B production maximally supported with 25-hydroxycholesterol. Plasma B and ACTH concentrations of L cockerels were, respectively, 160% greater and 32% less than those of N cockerels. In addition, plasma B clearance rate of L birds was 85% greater than that of N birds, thus suggesting a greater B secretion rate in L birds. However, maximal plasma CBC of L cockerels was 59% less than that of N cockerels. Thus, the free plasma B concentration of L birds was greater than that of N birds. The increase in the plasma B concentration of L cockerels is explained in part by the relative adrenal weight of these birds which was 88% greater than that of N cockerels. In addition, there were differences at the adrenocortical cell level. On an equal cell concentration basis, basal and maximal B production values (stimulated by ACTH analogs and 8-Br-cAMP, and supported by 25-hydroxycholesterol) of L cockerel adrenocortical cells were, respectively, 73% and 139% greater than those of N cockerel adrenocortical cells. In addition, maximal ACTH-induced aldosterone production of L bird cells was 104% greater than that of N bird cells, such that the ratio of aldosterone production to B production was not altered by protein deprivation. The data suggest that the greater steroidogenic capacity of L cockerel cells was due to an increase in intracellular steroidogenic enzyme content and/or activity and not to an alteration in the composition of adrenocortical cell types within the populations of isolated cells. Furthermore, ACTH analog ED50 values for B, aldosterone, and cAMP production by L bird cells were about one third to one fifth the values for N bird cells, thus indicating that L bird cells were about 3-5 times more sensitive to ACTH than were N bird cells.(ABSTRACT TRUNCATED AT 400 WORDS)