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In mammalian zona glomerulosa cells, angiotensin II (AII)-induced increases in intracellular Ca2+ ([Ca2+]i) and AII-induced aldosterone production seem to be inextricably linked. However, in avian adrenal steroidogenic (adrenocortical) cells studied thus far, inducible aldosterone production seems to be insensitive to alterations in the mobilization of cellular Ca2+. This raises the hypothesis that alternative signal transduction pathways are implemented to induce aldosterone production in avian adrenocortical cells. In the present study, this hypothesis was investigated by using isolated turkey (Meleagris gallopavo) adrenocortical cells that are known to be three times more sensitive to AII than to ACTH for aldosterone production. In isolated turkey adrenocortical cells, the mammalian AII receptor antagonist, [Sar1,Ile8]AII, was as efficacious as [Ile5]AII in stimulating aldosterone production, albeit it had about 1/150 the potency of [Ile5]AII. The actions of both analogs required extracellular K+, suggesting a voltage-sensitive event. However, a maximal aldosteronogenic concentration of [Sar1,Ile8]AII not only failed to increase [Ca2+]i but also completely blocked maximal (10(-8) M)[Ile5]AII-induced increases in [Ca2+]i when added before [Ile5]AII and partially dampened (approximately 50%) maximal [Ile5]AII-induced increases in [Ca2+]i when added after (3 min) [Ile5]AII. This blockade in [Ca2+]i elevation was surmounted by high concentrations of [Ile5]AII (> 10(-6) M). By contrast, [Sar1,Ile8]AII did not alter maximal aldosterone production induced by [Ile5]AII and vice versa, thus suggesting that the action of both analogs converged on the same aldosteronogenic pathway, and that AII-induced aldosterone production was not coupled to elevations in [Ca2+]i. Detailed homologous-heterologous ligand-binding analyses supported the presence of two AII-binding sites that were discriminated by [Sar1,Ile8]AII (dissociation constants, 4.2 +/- 1.4 and 21.9 +/- 2.2 nM; concentration distribution, approximately 40% and approximately 60%, respectively; mean +/- SE, n = 4) but not by [Ile5]AII (dissociation constant, 2.1 +/- 0.1 nM for both sites). In addition, [Sar1,Ile8]AII- and [Ile5]AII-binding sites exhibited different physicochemical and pharmacological properties. The sensitivity of [Sar1,Ile8]AII-binding sites was about twice that of [Ile5]AII-binding sites to dithiothreitol. In addition, whereas both the high- and low-affinity sites detected by [Sar1,Ile8] AII exhibited equivalent competitive sensitivities to the type-1 receptor, the nonpeptidic antagonist, losartan (DuP 753), the sensitivity of the low-affinity site was 2.7 times that of the high-affinity site to the type-2 receptor, nonpeptidic antagonist, PD123319. Taken collectively, the data suggest that in turkey adrenocortical cells, elevations in [Ca2+]i and aldosterone production are dissociable events regulated by distinct AII receptor subtypes or isomorphs.

Previous work has demonstrated that 4-week protein restriction of the domestic fowl (Gallus gallus domesticus) increases both adrenocortical cell sensitivity to ACTH and corticosteroidogenic capacity. In the present study we examined the transience (study 1) and the persistence (study 2) of this effect of protein restriction. In study 1, two strains of domestic fowl were used: a slower-growing White Leghorn strain and a faster-growing Cobb broiler strain. Cockerels (2 weeks old) were fed isocaloric diets containing either low (L; 5% or 8%) or control (C; 20%) levels of soy protein for 2 weeks, and then were either switched to the alternate diet (C-L, L-C) or maintained on the initial diet (C-C, L-L) for an additional 2 weeks. Cockerels were killed at 6 weeks of age. In study 2, White Leghorn cockerels (2 weeks old) were fed either diet for 4 weeks and then switched to or maintained on the control diet for an additional 4 weeks (i.e. C vs. restriction followed by repletion). In this study cockerels were killed at 10 weeks of age. In both studies highly enriched populations of adrenocortical cells were isolated from cockerel adrenal glands, and their steroidogenic properties (basal and maximally induced corticosterone and cAMP production; steroidogenic agent ED50 values) were evaluated in 2-h suspension incubations. In study 1, regardless of strain, the greater the length of the restriction period, the greater the magnitude of maximal cellular corticosterone production induced by ACTH; the average value for 4-week restriction (L-L) was 39.5% greater than that for 2-week restriction (L-C, C-L) and 117.5% greater than that for control (C-C). In addition, the value for restriction from 4-6 weeks of age (C-L) was 34% greater than that for restriction from 2-4 weeks of age (L-C), suggesting that the enhancement of maximal ACTH-induced corticosterone production after a 2-week restriction interval might be transient. Although there were no strain differences in the effect of protein restriction on maximal ACTH-induced corticosterone production, there were strain differences in its effect on cellular sensitivity to ACTH, as indicated by the ACTH ED50 values (the lower the ED50 value, the greater tha cellular sensitivity). With the White Leghorn strain, the greater the duration of protein restriction, the greater the adrenocortical cell sensitivity to ACTH; the sensitivity of L-L cells was 3.0 times the sensitivities of L-C and C-L cells and 4.1 times the sensitivity of C-C cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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)

To identify the possible role of calmodulin in ACTH function, we tested the ability of chlorpromazine (CP) and other calmodulin antagonists to inhibit steroidogenesis of isolated adrenocortical cells of the rat. CP reversibly inhibited maximal ACTH-induced corticosterone (B) production. The presence of the drug did not alter the ED50 of ACTH stimulation (3.2 X 10(3) pg/ml), suggesting that it inhibited ACTH-induced steroidogenesis in a noncompetitive manner. The CP concentration required for half-maximal inhibition was 8.2 microM, a value close to the dissociation constant of the CP-calmodulin complex (5.3 microM). Concentrations greater than 40 microM resulted in complete inhibition. Similar concentrations of CP inhibited ACTH-induced cAMP accumulation in a dose-dependent manner, indicating an effect of the drug on early events in ACTH action. In addition, CP also apparently acted at a site distal to the point of cAMP formation, as shown by the finding that it inhibited cAMP-induced B production. CP inhibition of ACTH-induced B production was independent of the Ca2+ concentration, suggesting that the drug did not compete with Ca2+ directly. Concentrations of CP greater than 20 microM inhibited protein synthesis as measured by leucine incorporation into cellular proteins. Thus, although the inhibitory effect of high concentrations of CP on steroidogenesis might be explained by an effect on protein synthesis, the inhibition seen at 10 microM appeared to be independent of protein synthesis. Other antagonists of calmodulin action inhibited maximal ACTH-induced B production with the following relative potencies: trifluoperazine greater than CP greater than haloperidol greater than chlordiazepoxide. This order is similar to that reported for inhibition of calmodulin-activated phosphodiesterase and for binding to calmodulin. These findings suggest that calmodulin may modulate the effect of ACTH on steroidogenesis at multiple sites.

Exogenous corticosterone (B), the natural glucocorticoid product of rats, suppressed endogenous B production of isolated rat adrenocortical cells induced by alpha ACTH-(1-24), [9-tryptophan (O-nitrophenylsulfenyl)]ACTH-(1-24) [( Trp (Nps)9]ACTH-(1-24], and cAMP as well as pregnenolone supported-steroidogenesis. This self-suppression occurred within 2 h. It was dependent on the concentration of exogenous B. However, self-suppression did not alter the half-maximal steroidogenic concentration (ED50) of each steroidogenic agent. In addition, exogenous B did not suppress ACTH-induced cAMP production or gross protein synthesis, as measured by leucine incorporation into bulk cellular proteins. These results with isolated cells suggested at least two mechanisms for self-suppression: 1) exogenous B inhibited steroidogenic steps in a noncompetitive manner, and/or 2) exogenous B induced B degradation. In this study we examined the effect of exogenous B on the degradation of B. Accordingly, we measured the adrenal 5 alpha-reductase activity (5 alpha RA) of cell homogenates prepared from treated cells. Isolated adrenocortical cells were incubated for 2 h with alpha ACTH-(1-24), ovine PRL (oPRL), and B. They were then homogenized and assayed for 5 alpha RA, as indicated by the disappearance of exogenous B, as shown by RIA. In addition, the percentage of exogenous tritium-labeled B [( 3H]B) converted to 5 alpha-dihydrocorticosterone (DHB), the principal reduced metabolite of B, was determined by TLC. Isolated adrenocortical cells from intact rats showed insignificant 5 alpha RA and DHB formation when incubated with or without alpha ACTH-(1-24) and with or without oPRL. However, with exogenous B, there was significant 5 alpha RA and DHB formation. oPRL plus B decreased DHB formation. The effects of B and oPRL were more demonstrable with cells from hypophysectomized rats. These cells exhibited high 5 alpha RA and DHB formation; exogenous B increased these values, whereas oPRL acutely reversed the effects of hypophysectomy and exogenous B. In other work avoiding cell homogenization, exogenous B suppressed ACTH-induced B accumulation and increased DHB formation in intact cell suspensions from intact rats and intact male domestic fowl. Furthermore, exogenous B increased the conversion of [3H] pregnenolone to DHB in intact cell suspensions from intact rats, showing that B synthesized de novo as well as exogenous B can be degraded during self-suppression. These data indicate that acute self-suppression of corticosteroidogenesis is at least partly mediated by an increase in 5 alpha RA.

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.

The control of adrenocortical function in the domestic fowl (Gallus domesticus) was investigated using adrenocortical cells isolated from hypophysectomized (hypox) and intact cockerels and from hypox cockerels injected with T3, purified chicken GH (cGH), and T3 plus cGH. Corticosterone in plasma and cell incubations was measured by RIA. Cellular responses to varying concentrations of steroidogenic agents were fitted and statistically analyzed by computer. Hypophysectomy reduced the basal plasma corticosterone (B) concentration to 53% of the value in intact birds and eliminated a stress response. Although hypophysectomy reduced adrenal weight by 20%, it did not change relative adrenal weight (milligrams per 100 g BW). However, replacement with cGH increased relative adrenal weight by 24%, whereas replacement with T3 or T3 plus cGH reduced relative adrenal weight by 16%. In the absence of steroidogenic agents, there were no detectable differences in B production by adrenocortical cells isolated from various experimental groups. However, with a maximal steroidogenic concentration of ACTH, B production by isolated adrenocortical cells from hypox birds was 61% of that by cells from intact birds. The ED50 of ACTH for cells from hypox cockerels was 2.7 times greater than that of cells from intact cockerels, thus indicating a loss of cell sensitivity to ACTH after hypophysectomy (the greater the ED50, the lesser the cell sensitivity). Although cGH replacement increased maximal B production (Bmax) induced by ACTH to 329% that by cells from hypox cockerels, it increased the ACTH ED50 3.6 times, thus decreasing cell sensitivity more than hypophysectomy alone. In contrast to cGH, T3 replacement maintained the cell sensitivity to ACTH at the level of cells from intact birds, but lowered Bmax to 54% that of cells from hypox cockerels. The combination of cGH and T3 administered to hypox cockerels both maintained cell sensitivity to ACTH and raised the Bmax to 358% that of cells from hypox animals. These treatments also affected 8-bromo-cAMP-induced Bmax and pregnenolone-supported Bmax, but did not significantly alter the ED50 of these agents.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Our previous work suggests that persistent protein malnutrition in immature domestic fowl (Gallus gallus domesticus) alters ACTH-adrenocortical cell interaction, possibly including ACTH receptors. To investigate this possibility, we measured some ACTH receptor parameters in isolated adrenocortical cells from normal and dietary protein-restricted domestic fowl. 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, and adrenal glands were removed and prepared for cell isolation. Highly enriched (greater than 80% pure) adrenocortical cells (collagenase isolated, followed by separation on a Percoll continuous density gradient) were evaluated for ACTH receptors using pharmacological and radioligand approaches. In a pharmacological approach, we measured the influence of the complete, competitive antagonist, human (h) ACTH-(7-38) on hACTH-(7-39)-induced corticosterone production by adrenocortical cells from L and N cockerels. Inhibitor constants of hACTH-(7-38), calculated from Schild plots, were 3.16 X 10(-7) and 9.82 X 10(-7) M for L and N cockerel cells, respectively, thus suggesting differences in ACTH receptor function between the two treatment groups. To characterize ACTH receptors directly, we measured the binding of a monoiodinated ACTH analog [125I-Tyr23]hACTH-(1-39) to domestic fowl adrenocortical cells. Binding was linear with cell concentration, highly specific (only ACTH peptides caused significant displacement), rapid (maximal binding by 1 h), reversible (half-time of dissociation, approximately 40 min), and saturable. Curvilinear Scatchard plots were obtained, and vectorial analysis resolved both high and low affinity sites. The concentrations (femtomoles per 50 micrograms DNA) and dissociation constants (Kd) of both classes of sites were different between N and L bird cells. Values of these receptor parameters for N and L cockerel cells were, respectively, as follows: concentrations of low affinity sites, 7.45 and 11.60; concentrations of high affinity sites, 3.16 and 5.50; Kd of low affinity sites, 2.05 X 10(-8) and 2.58 X 10(-9) M; Kd of high affinity sites, 1.01 X 10(-9) and 1.27 X 10(-10) M. Thus, the overall binding capacity of L bird cells was 65% greater than that of N bird cells. In addition, the overall affinity (1/Kd) of sites of L bird cells was 9 times that of sites of N bird cells. These data indicate that persistent protein malnutrition in the domestic fowl increased both the number and affinity of adrenocortical cell ACTH receptors.