Solubilization and characterization of endothelin-1 receptors in rat cardiac tissue

Abstract

Adult rat cardiac endothelin-1 (ET-1) receptors were solubilized with 0.5% digitonin and then characterized. The receptors retained binding activity after solubilization. Binding was saturable (K 0 of 0.065±0.004 nM, Bmax of 94.6±4.5 fmol/mg protein; Hill coefficient of 0.987±0.017 n=6) and pH dependent, with the binding increasing as the pH was decreased from 10 to 4, but decreasing dramatically as pH dropped to 2. Specifically bound [ 125I]-ET-1 was not dissociated by 2×10 −7M unlabelled ET-1, but was dissociated by pH 10 and 2. Returning the pH to 7.4 restored the binding activity of the receptors. Unlabelled ET-1 (10 −12–10 −7M) and sarafotoxin S6b(10 −12–10 −7M) competed with [ 125I]-ET-1 for binding to the receptors.