AJP - Renal Physiology, Vol 269, Issue 5 644-F652, Copyright © 1995 by American Physiological Society

ARTICLES

Angiotensin IV receptors and signaling in opossum kidney cells

N. Dulin, Z. T. Madhun, C. H. Chang, L. Berti-Mattera, D. Dickens and J. G. Douglas
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4982, USA.

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.

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