Angiotensin IV receptors and signaling in opossum kidney cells

The pharmacological properties and signaling of angiotensin IV (ANG IV) receptors were studied in opossum kidney cell line OK7A. Saturation binding experiments with 125I-labeled ANG IV demonstrated the presence of high-affinity ANG IV binding sites in OK7A cell membranes with a dissociation constant (Kd) of 0.40 +/- 0.08 nM and a maximal amount of binding sites (Bmax) of 180 +/- 50 fmol/mg protein. In competition experiments, unlabeled ANG IV inhibited 125I-ANG IV binding biphasically: 20% of binding sites had high affinity inhibition constant (Ki) = 0.44 +/- 0.04 nM and 80% had low affinity (Ki = 130 +/- 10 nM). ANG III displaced 125I-ANG IV from binding sites with low affinity (Ki = 205 +/- 10 nM), and ANG II did not compete with 125I-ANG IV at concentrations up to 10 microM. 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 125I-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-aminoethylether)-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 IV induces Ca2+ influx in OK7A cells, at least partially, through the voltage-sensitive Ca2+ channels.