M. Janiszewski et al., Inhibition of vascular NADH/NADPH oxidase activity by thiol reagents: Lackof correlation with cellular glutathione redox status, FREE RAD B, 29(9), 2000, pp. 889-899
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.