An impurity in xanthine oxidase increases endothelin binding to pig coronary artery smooth muscle

Since endothelin (ET) is a potent vasoconstrictor and the free radicals are produced in ischemia-reperfusion, we determined the effect of superoxide on ET-1 binding to its receptors. Microsomes from pig coronary artery smooth muscle contained mostly ETa- and some ETb -receptors. Initially, we used xanthine oxidase (XO) purified from bovine milk + 0.3 mM xanthine (X) to generate superoxide. We pretreated the microsomes with X, XO, or X + XO to use them for binding experiments. The treatment with X + XO increased the ET-1 binding to ETa and ETb sites. The binding increase was depended on the [XO] used. However, XO alone also caused the binding increase. We repeated this experiment using a bacterial XO (Sigma). In the later experiment X, XO, or X + XO did not alter the ET-1 binding. We conclude that superoxide does not affect ET-1 binding to ETa or ETb receptors and that the increase observed with the bovine milk preparation is an artefact unrelated to superoxide generation.