title: Angiotensin IV receptors and signaling in opossum kidney cells.
abstract: 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.
authors.affiliation: Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4982, USA.
authors.forename: N
authors.initials: N
authors.lastname: Dulin
authors.forename: Z T
authors.initials: ZT
authors.lastname: Madhun
authors.forename: C H
authors.initials: CH
authors.lastname: Chang
authors.forename: L
authors.initials: L
authors.lastname: Berti-Mattera
authors.forename: D
authors.initials: D
authors.lastname: Dickens
authors.forename: J G
authors.initials: JG
authors.lastname: Douglas
mesh_terms: D000804:Angiotensin II / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000378:metabolism; D009893:Opossums; D010716:Phosphatidylinositols / Q000378:metabolism; D011945:Receptors, Angiotensin / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: C090830:AT4 receptor; D009712:Nucleotides, Cyclic; D010716:Phosphatidylinositols; D011945:Receptors, Angiotensin; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D010738:Type C Phospholipases; D002118:Calcium