Peroxynitrite-induced nitrotyrosination of proteins is blocked by direct 5-lipoxygenase inhibitor zileuton

We have previously shown that the ability of overnight pretreatment with li popolysaccharide (LPS) to suppress alveolar macrophage (AM) leukotrienes (L T) synthesis is explained by induction of nitric oxide (NO), and reactive o xygen intermediates (ROI). More recently we have demonstrated that the gene ration of peroxynitrite (ONOO-) from the combination of NO and ROI directly nitrotyrosinates the 5-lipoxygenase (5-LO) enzyme and reduces cell-free an d intact AM 5-LO metabolism. This effect of ONOO- was associated with nitro tyrosination of the 5-LO enzyme in intact cells and after treatment of reco mbinant enzyme. We postulated that LPS treatment of cells resulted in activ ation of 5-LO with the generation of ROI, which in turn led to autoinactiva tion of the enzyme. In an effort to suppress ROI generated from activation of 5-LO we examined the effect of a direct 5-LO inhibitor on LPS-induced su ppression of LT synthesis. Coincubation with the reversible 5-LO inhibitor zileuton during the LPS pretreatment of intact cells dose dependently block ed the inhibition of 5-LO metabolism by LPS. The effect of zileuton on LPS- induced suppression of LT synthesis was similar to that of N-monomethyl-L-a rginine. Zileuton had no effect on inducible nitric-oxide synthase inductio n. Interestingly, zileuton blocked ONOO--induced nitrotyrosination of recom binant 5-LO in a cell-free system as well as of native enzyme in intact cel ls. Moreover, zileuton blocked the nitrotyrosination of other proteins. We conclude that the suppression of 5-LO activity occurring with LPS treatment can be blocked by zileuton. The mechanism by which zileuton is effective i s in part explained by blocking nitrotyrosination of 5-LO.