Identification of a 65 kDa endothelin receptor in bovine cardiac sarcolemmal vesicles

Endothelin-1, an endothelial cell-derived vasoconstrictor peptide, also exerts a potent positive inotropic effect on cardiac tissue. Characterization of specific binding of endothelin-1 to bovine cardiac sarcolemmal vesicles is reported. In the presence of 1 mM CaCl2, the observed binding for 125I-endothelin-1 had a Kd of 6.2 nM with an observed Bmax of 14 pmol/mg sarcolemmal protein. In the presence of 1 mM EDTA (and no added Ca2+) Bmax was reduced to 9 pmol/mg sarcolemmal protein while the Kd remained unchanged. Binding affinity for sarafotoxin S6b was at least one order of magnitude less than for endothelin-1. 125I-Endothelin-1 covalently cross-linked to a sarcolemmal protein with an apparent molecular weight of 65 kDa. Site-directed polyclonal antibodies to a sequence located on the third extramembranal segment of a previously cloned endothelin ETA receptor from bovine lung were produced. Using Western blot analysis, the site-directed polyclonal antibody recognized a sarcolemmal protein at 65 kDa. We conclude that sarcolemmal membranes from bovine ventricular myocardium contain an endothelin binding site and that it is a protein with an apparent molecular weight of 65 kDa.