Inhibition of vascular NADH/NADPH oxidase activity by thiol reagents: lack of correlation with cellular glutathione redox status.
Vascular NAD(P)H oxidase activity contributes to oxidative stress. Thiol oxidants inhibit leukocyte NADPH oxidase. To assess the role of reactive thiols on vascular oxidase, rabbit iliac/carotid artery homogenates were incubated with distinct thiol reagents. NAD(P)H-driven enzyme activity, assessed by lucigenin (5 or 250 microM) luminescence, was nearly completely (> 97%) inhibited by the oxidant diamide (1mM) or the alkylator p-chloromercuryphenylsulfonate (pCMPS, 0.5mM). Analogous inhibition was also shown with EPR spectroscopy using DMPO as a spin trap. The oxidant dithionitrobenzoic acid (0.5mM) inhibited NADPH-driven signals by 92% but had no effect on NADH-driven signals. In contrast, the vicinal dithiol ligand phenylarsine oxide (PAO, 1 microM) induced minor nonsignificant inhibition of NADPH-driven activity, but significant stimulation of NADH-triggered signals. The alkylator N-ethyl maleimide (NEM, 0.5mM) or glutathione disulfide (GSSG, 3mM) had no effect with each substrate. Coincubation of N-acetylcysteine (NAC, 3mM) with diamide 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 reagent pCMPS was also inhibitory in whole rings, suggesting that reactive thiol(s) affecting oxidase activity are highly accessible. Since lack of oxidase inhibition by NEM or GSSG occurred despite significant cellular glutathione depletion, change in intracellular redox status is not sufficient to account for oxidase inhibition. Moreover, the observed differences between NADPH and NADH-driven oxidase activity point to complex or multiple enzyme forms.
Janiszewski
M
M
Emergency Medicine Department, University of São Paulo Medical School;, São Paulo, Brazil.
Pedro
M A
MA
Scheffer
R C
RC
van Asseldonk
J H
JH
Souza
L C
LC
da Luz
P L
PL
Augusto
O
O
Laurindo
F R
FR
D000166:Acridines; D000818:Animals; D001808:Blood Vessels / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D002339:Carotid Arteries / Q000187:drug effects / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005978:Glutathione / Q000378:metabolism*; D007083:Iliac Artery / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011817:Rabbits; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D000166:Acridines; D013439:Sulfhydryl Reagents; C033472:10,10'-dimethyl-9,9'-biacridinium; D009247:NADH, NADPH Oxidoreductases; D005978:Glutathione