ANGIOTENSIN-IV RECEPTORS AND SIGNALING IN OPOSSUM KIDNEY-CELLS

The pharmacological properties and signaling of angiotensin TV (ANG TV) receptors were studied in opossum kidney cell line OK7A. Saturation binding experiments with I-125-labeled ANG IV demonstrated the presence of high-affinity ANG IV binding sites in OK7A cell membranes with a dissociation constant (K-d) Of 0.40 +/- 0.08 nM and a maximal amount of binding sites (B-max) of 180 +/- 50 fmol/mg protein. In competition experiments, unlabeled ANG TV inhibited I-125-ANG IV binding biphasically: 20% of binding sites had high affinity [inhibition constant (K-i) = 0.44 +/- 0.04 nhl] and 80% had low affinity (K-i = 130 +/- 10 nM). ANG III displaced I-125-ANG IV from binding sites with low affinity (K-i = 205 +/- 10 nM), and ANG II did not compete with I-125-ANG IV at concentrations up to 10 mu M. The binding of ANG IV to OK7A cell membranes was significantly enhanced in the presence of 5 mM EDTA and completely blocked by 5 mM dithiothreitol. Guanosine 5'-O-(3-thiotriphosphate) inhibited the binding of I-125-ANG IV, indicating the G protein coupling of ANG IV receptors in OK7A cells. In signaling studies, ANG IV induced transient increase in intracellular calcium concentration ([Ca2+](i)) from 49 +/- 3 to 280 +/- 45 nM. ANG IV failed to influence phosphoinositol metabolism, indicating that Ca2+ mobilization is not linked to ANG IV signaling. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid completely abolished ANG IV-induced increase in [Ca2+](i), consistent with Ca2+ influx. The voltage-sensitive Ca2+ channel blocking agents verapamil and nifedipine attenuated the effect of ANG IV on [Ca2+](i) to 133 +/- 33 and 174 +/- 32 nM, respectively. These data suggest that ANG TV induces Ca2+ influx in OK7A cells, at least partially, through the voltage-sensitive Ca2+ channels.