Identification and characterization of functional angiotensin II receptors on cultured heart myocytes.
Cultured neonatal rat myocytes have been investigated as a potential system to study the molecular mechanism of angiotensin II (All) stimulation of heart contractile behavior. Intact cultured cells and the membranes from these cells bind [125I] All in a biphasic manner. The data were analyzed as two classes of binding sites on membrane preparations: Kd1 = 0.65 nM; maximum binding (Bmax1) = 245 fmol/mg of protein and Kd2 = 5.57 nM, Bmax2 = 720 fmol/mg of protein. The receptor on intact cells is specific as All peptide analogs were potent at inhibiting the binding of [125I]All. An All antagonist, [125I]Sar1,Leu8-All, recognized a single class of high affinity receptors on intact cells: Kd = 0.63 nM, Bmax = 318 fmol/mg of protein, or 45,300 sites per cell. Guanine nucleotides regulated the binding of All to membranes by shifting the receptor from a high affinity to a low affinity form without changing Kd2. Conversely, these nucleotides altered the high affinity binding of [125I]Sar1,Leu8-All by increasing Kd without changing Bmax. All was found to stimulate the contractile frequency of spontaneously beating myocytes with maximal effects (a 50% stimulation) observed at 5 nM hormone. The responses were reversible with half-maximal effects observed at doses around 1 nM. The antagonist, Sar1,Ala8-All, could inhibit the chronotropic stimulatory responses. It is concluded that these cultured myocytes express an All receptor system with properties consistent with a true hormone receptor system. Furthermore, studies on the pharmacology of the chronotropic responses of these cells to All demonstrate that these receptors are functional.
D015059:1-Sarcosine-8-Isoleucine Angiotensin II / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008274:Magnesium / Q000494:pharmacology; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D011945:Receptors, Angiotensin / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000032:analysis*; D012964:Sodium / Q000494:pharmacology
D007457:Iodine Radioisotopes; D011945:Receptors, Angiotensin; D011956:Receptors, Cell Surface; D000804:Angiotensin II; D006165:Guanylyl Imidodiphosphate; D015059:1-Sarcosine-8-Isoleucine Angiotensin II; D012964:Sodium; D008274:Magnesium; D002118:Calcium