Inhibition of vascular NADH/NADPH oxidase activity by thiol reagents: Lackof correlation with cellular glutathione redox status

Vascular NAD(P)H oxidase activity contributes to oxidative stress. Thiol ox idants inhibit leukocyte NADPH oxidase. To assess the role of reactive thio ls on vascular oxidase, rabbit iliac/carotid artery homogenates were incuba ted with distinct thiol reagents. NAD(P)H-driven enzyme activity, assessed by lucigenin (5 or 250 muM) luminescence, was nearly completely (> 97%) inh ibited by the oxidant diamide (1mM) or the alkylator p-chloromercuryphenyls ulfonate (pCMPS, 0.5mM). Analogous inhibition was also shown with EPR spect roscopy using DMPO as a spin trap. The oxidant dithionitrobenzoic acid (0.5 mM) inhibited NADPH-driven signals by 92% but had no effect on NADH-driven signals. In contrast, the vicinal dithiol ligand phenylarsine oxide (PAO, 1 muM) induced minor nonsignificant inhibition of NAC)PH-driven activity, bu t significant stimulation of NADH-triggered signals. The alkylator N-ethyl maleimide (NEM, 0.5mM) or glutathione disulfide (GSSG, 3mM) had no effect w ith each substrate. Coincubation of N-acetyl-cysteine (NAC, 3mM) with diami de or pCMPS reversed their inhibitory effects by 30-60%, whereas NAC alone inhibited the oxidase by 52%. Incubation of intact arterial rings with the above reagents disclosed similar results, except that PAO became inhibitor and NAC stimulator of NADH-driven signals. Notably, the cell-impermeant rea gent pCMPS was also inhibitory in whole rings, suggesting that reactive thi ol(s) affecting oxidase activity are highly accessible. Since lack of oxida se inhibition by NEM or GSSG occurred despite significant cellular glutathi one depletion, change in intracellular redox status is not sufficient to ac count for oxidase inhibition. Moreover, the observed differences between NA DPH and NADH-driven oxidase activity point to complex or multiple enzyme fo rms. (C) 2000 Elsevier Science Inc.