Ebselen as a peroxynitrite scavenger in vitro and ex vivo

We have previously shown that peroxynitrite (PN) selectively impaired prost acyclin (PGI,)dependent vasorelaxation by tyrosine nitration of PGI, syntha se in an in situ model (Zou MH, Jendral M and Ullrich V, Br] Pharmacol 126: 1283-1292, 1999). By using this established model, we tested whether or no t ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one which reacts rapidly wit h the anionic form of PN, affected PN inhibition of PGI(2) synthase. Admini stration of ebselen (1 to 50 mu M) to bovine coronary strips 5 min Frier to PN (1 mu M) treatment neither prevented PN-triggered vasoconstriction nor the inhibition of PGI(2) release. In line with these results, ebselen affec ted neither PN inhibition of the conversion of [C-14]-PGH(2) into 6 keto PG F(1 alpha) nor the nitration of PGI(2) synthase in bovine aortic microsomes . Following the hypothesis that a reaction of ebselen with cellular thiols could have caused the inefficiency of ebselen, we observed that free ebsele n quickly reacted with thiols in both coronary strips and in aortic microso mes to form two metabolites, one of which was identified as the ebselen-glu tathione adduct, whereas the other had a similar retention time to that of the ebselen-cysteine adduct. The nitration of phenol by PN in a metal-free solution could be blocked more efficiently in the presence of ebselen or gl utathione alone than in the presence of both, indicating that like selenome thionine and other selenocompounds, ebselen-thiol adducts were less reactiv e towards PN than ebselen itself. Further evidence came from the results th at ebselen became effective in preventing the inhibition and nitration of P GI(2) synthase after thiol groups of microsomal proteins were previously ox idized with Ellman's reagent. We conclude that in cellular systems ebselen is present as thiol adducts and thus loses its high reactivity towards PN, which is required to compete with the nitration of PGI, synthase. BIOCHEM P HARMACOL 59;2: 153-160, 2000. (C) 1999 Elsevier Science Inc.