Sustained Formation of Nitroglycerin-Derived Nitric Oxide by Aldehyde Dehydrogenase-2 in Vascular Smooth Muscle without Added Reductants: Implications for the Development of Nitrate Tolerance.

Marissa Opelt,Wolfgang F Graier,John T Fassett,Bernd Mayer,Gerald Wölkart,Markus Waldeck-Weiermair,Emrah Eroglu,Roland Malli,Astrid Schrammel,Antonius C F Gorren,Alexander Kollau

doi:10.1124/mol.117.110783

Abstract

According to current views, oxidation of aldehyde dehydrogenase-2 (ALDH2) during glyceryltrinitrate (GTN) biotransformation is essentially involved in vascular nitrate tolerance and explains the dependence of this reaction on added thiols. Using a novel fluorescent intracellular nitric oxide (NO) probe expressed in vascular smooth muscle cells (VSMCs), we observed ALDH2-catalyzed formation of NO from GTN in the presence of exogenously added dithiothreitol (DTT), whereas only a short burst of NO, corresponding to a single turnover of ALDH2, occurred in the absence of DTT. This short burst of NO associated with oxidation of the reactive C302 residue in the active site was followed by formation of low-nanomolar NO, even without added DTT, indicating slow recovery of ALDH2 activity by an endogenous reductant. In addition to the thiol-reversible oxidation of ALDH2, thiol-refractive inactivation was observed, particularly under high-turnover conditions. Organ bath experiments with rat aortas showed that relaxation by GTN lasted longer than that caused by the NO donor diethylamine/NONOate, in line with the long-lasting nanomolar NO generation from GTN observed in VSMCs. Our results suggest that an endogenous reductant with low efficiency allows sustained generation of GTN-derived NO in the low-nanomolar range that is sufficient for vascular relaxation. On a longer time scale, mechanism-based, thiol-refractive irreversible inactivation of ALDH2, and possibly depletion of the endogenous reductant, will render blood vessels tolerant to GTN. Accordingly, full reactivation of oxidized ALDH2 may not occur in vivo and may not be necessary to explain GTN-induced vasodilation.

Highlights

Aldehyde dehydrogenase-2 (ALDH2) catalyzes formation of nitric oxide (NO) from the antianginal drug nitroglycerin (GTN), resulting in vasodilation mediated by cGMP (Mayer and Beretta, 2008)
Using a novel fluorescent probe expressed in vascular smooth muscle cells (VSMCs), we have recently demonstrated that NO formation is necessary and sufficient to explain GTN bioactivity (Opelt et al, 2016)
Highly selective fluorescent sensors allowing quantification of intracellular NO have become available (Eroglu et al, 2016). Using one of these sensors [cyan genetically encoded NO probe (C-geNOp)], we demonstrated that aldehyde dehydrogenase-2 (ALDH2) catalyzes a burst of GTN-derived NO in VSMCs, followed by a rapid decrease in the signal in the absence of exogenously added thiols (Opelt et al, 2016)

Summary

Introduction

Aldehyde dehydrogenase-2 (ALDH2) catalyzes formation of nitric oxide (NO) from the antianginal drug nitroglycerin (GTN), resulting in vasodilation mediated by cGMP (Mayer and Beretta, 2008). Using a novel fluorescent probe expressed in vascular smooth muscle cells (VSMCs), we have recently demonstrated that NO formation is necessary and sufficient to explain GTN bioactivity (Opelt et al, 2016). Since the reaction of GTN with ALDH2 results. In oxidation of the reactive cysteine residue C302 in the catalytic site, reactivation of the enzyme by a reductant is required for sustained turnover (Chen et al, 2002; Beretta et al, 2008; Wenzl et al, 2011). Dithiothreitol (DTT) has proven most efficient in vitro, but the identity of the putative endogenous reductant remains elusive. Dihydrolipoic acid (LPA-H2) has been suggested as a candidate (Wenzel et al, 2007), its efficiency appears rather low

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