DISSOCIATION OF INCREASES IN INTRACELLULAR CALCIUM AND ALDOSTERONE PRODUCTION INDUCED BY ANGIOTENSIN-II (AII) - EVIDENCE FOR REGULATION BY DISTINCT AII RECEPTOR SUBTYPES OR ISOMORPHS

In mammalian zona glomerulosa cells, angiotensin II (AII)-induced incr eases in intracellular Ca2+ ([Ca2+](i)) and AII-induced aldosterone pr oduction seem to be inextricably linked. However, in avian adrenal ste roidogenic (adrenocortical) cells studied thus far, inducible aldoster one production seems to be insensitive to alterations in the mobilizat ion of cellular Ca2+. This raises the hypothesis that alternative sign al transduction pathways are implemented to induce aldosterone product ion in avian adrenocortical cells. In the present study, this hypothes is was investigated by using isolated turkey (Meleagris gallopavo) adr enocortical cells that are known to be three times more sensitive to A II than to ACTH for aldosterone production. In isolated turkey adrenoc ortical cells, the mammalian AII receptor antagonist, [Sar(1),Ile(8)]A II, was as efficacious as [Ile(5)]AII in stimulating aldosterone produ ction, albeit it had about 1/150 the potency of [Ile(5)]AII. The actio ns of both analogs required extracellular K+, suggesting a voltage-sen sitive event. However, a maximal aldosteronogenic concentration of [Sa r(1),Ile(8)]AII not only failed to increase [Ca2+](i) but also complet ely blocked maximal(10(-8) M) [Ile(5)]AII-induced increases in [Ca2+]( i) when added before [Ile(5)]AII and partially dampened (similar to 50 %) maximal [Ile(5)]AII-induced increases in [Ca2+](i) when added after (3 min) [Ile(5)]AII. This blockade in [Ca2+](i) elevation was surmoun ted by high concentrations of[Ile(5)]AII(>10(-6) M). By contrast, [Sar (1),Ile(8)]AII did not alter maximal aldosterone production induced by [Ile(5)]AII and vice versa, thus suggesting that the action of both a nalogs converged on the same aldosteronogenic pathway, and that AII-in duced 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 [Sar( 1),Ile(8)]AII (dissociation constants, 4.2 +/- 1.4 and 21.9 +/- 2.2 nM ; concentration distribution, similar to 40% and similar to 60%, respe ctively; mean +/- SE, n = 4) but not by [Ile(5)]AII (dissociation cons tant, 2.1 +/- 0.1 nM for both sites). In addition, [Sar(1),Ile(8)]AII- and [Ile(5)]AII-binding sites exhibited different physicochemical and pharmacological properties. The sensitivity of [Sar(1),Ile(8)]AII-bin ding sites was about twice that of [Ile(5)]AII-binding sites to dithio threitol. In addition, whereas both the high- and low-affinity sites d etected by [Sar(1), Ile(8)] AII exhibited equivalent competitive sensi tivities 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 antagon ist, PD123319. Taken collectively, the data suggest that in turkey adr enocortical cells, elevations in [Ca2+](i) and aldosterone production are dissociable events regulated by distinct Ail receptor subtypes or isomorphs.