Characterization and coupling of angiotensin-II receptor subtypes in cultured bovine adrenal fasciculata cells

Abstract

Angiotensin-II (A-II) receptor subtypes and their potential coupling mechanisms were investigated in bovine adrenal fasciculata cells (BAC) in culture, by the use of selective antagonists for AT1 (DUP 753 or Losartan) and AT2 (PD 123177 and CGP 42112A) sites. Competition for [ 125I]A-II specific binding with AT1 or AT2 selective ligands produced a biphasic displacement curve, suggesting two distinct A-II binding sites. In the presence of PD 123177 (10 −5 M), a concentration at which most of the AT2 sites were saturated, DUP 753 displaced [ 125I]A-II specific binding in a monophasic manner with an IC 50 of 6.2 ± 1.4 × 10 −7 M. In the presence of DUP 753 (10 −5 M), the displacement produced by CGP 42112A and PD 123177 was also monophasic, with IC 50s of 8 ± 3 × 10 −10 and 4.6 ± 2.1 × 10 −7 M, respectively. The reducing agent dithio-1,4-erythritol inhibited the binding of [ 125I]A-II to AT1 (DUP 753 sensitive) sites, but increased its binding to AT2 sites 2-fold. The IC 50 values for these two effects were about 0.5 and 3mM, respectively. The biological effects of A-II in BAC, phosphoinositide hydrolysis and cortisol production, were inhibited in a dose-dependent manner by DUP 753, but not by AT2 antagonists. Similarly, the potentiating action of A-II on corticotropin-induced cAMP production was blocked by DUP 753, but not by AT2 antagonists. These data indicate that BAC contain both receptor subtypes, but that all the known effects of A-II in BAC were induced via the AT1 receptor subtype.