According to current views, oxidation of aldehyde dehydrogenase-2\n(ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially\ninvolved in vascular nitrate tolerance and explains the dependence\nof this reaction on added thiols. Using a novel fluorescent\nintracellular nitric oxide (NO) probe expressed in vascular smooth\nmuscle cells (VSMCs), we observed ALDH2-catalyzed formation of\nNO from GTN in the presence of exogenously added dithiothreitol\n(DTT), whereas only a short burst of NO, corresponding to a single\nturnover of ALDH2, occurred in the absence of DTT. This short burst\nof NO associated with oxidation of the reactive C302 residue in the\nactive site was followed by formation of low-nanomolar NO, even\nwithout added DTT, indicating slow recovery of ALDH2 activity by an\nendogenous reductant. In addition to the thiol-reversible oxidation of\nALDH2, thiol-refractive inactivation was observed, particularly under\nhigh-turnover conditions. Organ bath experiments with rat aortas\nshowed that relaxation by GTNlasted longer than that caused by the\nNO donor diethylamine/NONOate, in line with the long-lasting\nnanomolar NO generation from GTN observed in VSMCs. Our\nresults suggest that an endogenous reductant with low efficiency\nallows sustained generation of GTN-derived NO in the lownanomolar\nrange that is sufficient for vascular relaxation. On a\nlonger time scale, mechanism-based, thiol-refractive irreversible\ninactivation of ALDH2, and possibly depletion of the endogenous\nreductant, will render blood vessels tolerant to GTN. Accordingly, full\nreactivation of oxidizedALDH2may not occur in vivo and may not be\nnecessary to explain GTN-induced vasodilation.
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