ROLE OF PEROXYNITRITE AND ACTIVATION OF POLY (ADP-RIBOSE) SYNTHETASE IN THE VASCULAR FAILURE INDUCED BY ZYMOSAN-ACTIVATED PLASMA

1 Zymosan is a wall component of the yeast Saccharomyces Cerevisiae. I njection of zymosan into experimental animals is known to produce an i ntense inflammatory response. Recent studies demonstrated that the zym osan-induced inflammatory response in vivo can be ameliorated by inhib itors of nitric oxide (NO) biosynthesis. The cytotoxic effects of NO a re, in part, mediated by the oxidant preoxynitrite and subsequent acti vation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). In t he present in vitro study, we have investigated the cellular mechanism s of vascular failure elicited by zymosan-activated plasma and the con tribution of peroxynitrite production and activation of PARS to the ch anges. 2 Incubation of rat aortic smooth muscle cells with zymosan-act ivated plasma (ZAP) induced the production of nitrite, the breakdown p roduct of NO, due to the expression of the inducible isoform of NO syn thase (iNOS) over 6-24 h. In addition, ZAP triggered the production of peroxynitrite in these cells, as measured by the oxidation of the flu orescent dye dihydrorhodamine 123 and by nitrotyrosine Western blottin g. 3 Incubation of the smooth muscle cells with ZAP induced DNA single strand breakage and PARS activation. These effects were reduced by in hibition of NOS with N-G-methyl-L-arginine (L-NMA, 3 mM), and by gluta thione (3 mM), a scavenger of peroxynitrite. The PARS inhibitor 3-amin obenzamide (1 mM) inhibited the ZAP-induced activation of PARS. 4 Incu bation of thoracic aortae with ZAP in vitro caused a reduction of the contractions of the blood vessels to noradrenaline (vascular hyporeact ivity) and elicited a reduced responsiveness to the endothelium-depend ent vasodilator acetylcholine (endothelial dysfunction). 5 Preincubati on of the thoracic aortae with L-NMA (1 mM), glutathione (3 mM) or by the PARS inhibitor 3-aminobenzamide (1 mM) prevented the development o f vascular hyporeactivity in response to ZAP. Moreover, glutathione an d 3-aminobenzamide treatment protected against the ZAP-induced develop ment of endothelial dysfunction. The PARS-related loss of the vascular contractility was evident at 30 min after incubation in endothelium-i ntact, but not in endothelium-denuded vessels and also manifested at 6 h after incubation with ZAP in endothelium-denuded rings. The acute r esponse is probably related, therefore, to peroxynitrite formation (in volving the endothelial NO synthase), whereas the delayed response may be related to the expression of iNOS in the smooth muscle. 6 The data obtained suggest that zymosan-activated plasma causes vascular dysfun ction by inducing the simultaneous formation of superoxide and NO. The se radicals combine to form peroxynitrite, which, in turn causes DNA i njury and PARS activation. The protective effect of 3-aminobenzamide d emonstrates that PARS activation contributes both to the development o f vascular hyporeactivity and endothelial dysfunction during the vascu lar failure induced by ZAP.