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.
Shannon
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Dalton Cardiovascular Research Center, University of Missouri, Columbia 65211.
Hale
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D003432:Cross-Linking Reagents; D016232:Endothelins / Q000493:pharmacokinetics; D006352:Heart Ventricles / Q000378:metabolism; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009206:Myocardium / Q000276:immunology / Q000378:metabolism*; D011863:Radioimmunoassay; D017466:Receptors, Endothelin / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012508:Sarcolemma / Q000276:immunology / Q000378:metabolism*
D003432:Cross-Linking Reagents; D016232:Endothelins; D007457:Iodine Radioisotopes; D017466:Receptors, Endothelin