The receptors and intracellular signaling mechanisms activated by angiotensin II (AII) and ACTH were studied in bovine adrenal glomerulosa cells and Xenopus laevis oocytes. The pathways of phosphoinositide metabolism in glomerulosa cells were markedly shifted by ACTH stimulation, with increased production of higher inositol phosphates. This change was mediated by cyclic AMP, and resulted from increased phosphorylation of Ins(1,3,4)P3 to Ins(1,3,4,6)P4, and decreased Ins(1,4,5)-5-phosphatase activity. Such interactions between the cAMP and inositol phosphate pathways cause enhanced production of InsP4 isomers and InsP5, which may be involved in long-term cell responses. Analysis of the role of receptor internalization in AII-induced signaling revealed that blockade of endocytosis of AII-receptor complexes markedly reduced the sustained phases of Ins(1,4,5)P3 generation and elevation of cytoplasmic calcium concentration. These effects were similar to those of extracellular calcium deficiency, and suggest that sustained generation of intracellular signals depends on endocytosis of the hormone-receptor complex. A receptor-operated vesicular calcium uptake process could also contribute to the maintenance of the prolonged phase of the cytoplasmic calcium response to AII. About 85% of the sites in the zona glomerulosa were of the AT1 subtype whereas the AT2 subtype was predominant in the medulla. In contrast, the bovine adrenal cortex and medulla contained only AT1 sites. In both species AII-stimulated aldosterone production was mediated by AT1 receptors, as shown by the inhibitory effects of the AT1 antagonist, Dup 753, on binding to AT1 sites and the lack of effect of selective AT2 antagonists. The inhibition of adenylate cyclase by AII in rat glomerulosa cells was also blocked by Dup 753, indicating that the AT1 receptor subtype is coupled to adenylate cyclase via Gi as well as to phospholipase C via Gq. In follicular Xenopus oocytes, in which endogenous AII receptors in the follicle cells are coupled through gap junctions to calcium mobilization in the oocyte, AII activation of calcium responses was blocked by peptide AII antagonists but not by the non-peptide antagonists. Likewise, AII receptors of the Xenopus myocardium bound AII and its peptide analogs with high affinity but did not recognize the non-peptide antagonists. Thus, the amphibian receptor is functionally of the AT1 subtype, but is pharmacologically unique in distinguishing with high selectivity between peptide and non-peptide AII antagonists. The mammalian ACTH receptor was expressed from adrenal mRNA in Xenopus oocytes and shown to be fully functional in terms of ligand recognition and signal generation.
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