Regulation of guanine nucleotide binding regulatory proteins in cultured adrenal cells by adrenocorticotropin and angiotensin-II

In addition to their steroidogenic effect on cultured bovine adrenal fasciculate cells ACTH and angiotensin-II (A-II) have a long term effect on the ability of these cells to respond to subsequent hormonal stimulation. The present work explores the effects of a 72-h pretreatment of adrenal cells with both hormones on the first steps of the mechanism of action of ACTH and A-II and on the amounts of the α-subunits of guanine nucleotide binding proteins Gs and Gi. ACTH but not A-II increased acute ACTH or cholera toxin-induced cAMP production. Moreover, ACTH but not A-II enhanced the amount of α_s protein evaluated by cholera toxin ADP ribosylation, whereas both hormones elevated immunoblotted α_s. Both hormones increased A-II-induced phosphoinositide breakdown and Ca²⁺ uptake without modification of the A-II-potentiating effect on ACTH-induced cAMP production. Treatment of cells with pertussis toxin (PT, 0.5 μg/ml) for the last 24 h reduced by 27% the A-II-induced phosphoinositide breakdown in A-II-pretreated cells but had no significant effect in ACTH-pretreated cells. No effect of PT was observed on A-II-induced Ca²⁺ uptake or on its potentiating action on ACTH-induced cAMP production in ACTH as well as A-II-pretreated cells. Moreover, both hormones increased Gi proteins (40-41 kDa) evaluated by PT ADP ribosylation. Immunoblot analysis revealed that ACTH preferentially enhanced α_i3, whereas the stimulatory effect of A-II was more marked on α_i1 and α_i2. These results indicate that in bovine adrenal fasciculata cells, peptide hormones settle target cell responsiveness not only by regulating the membrane-bound receptors, but also by modulating the level of G proteins coupling these receptors to the intracellular signals.