Vascular Bioactivation of Nitroglycerin Is Catalyzed by Cytosolic Aldehyde Dehydrogenase-2

Abstract

Rationale: According to general view, aldehyde dehydrogenase-2 (ALDH2) catalyzes the high-affinity pathway of vascular nitroglycerin (GTN) bioactivation in smooth muscle mitochondria. Despite having wide implications to GTN pharmacology and raising many questions that are still unresolved, mitochondrial bioactivation of GTN in blood vessels is still lacking experimental support.Objective: In the present study, we investigated whether bioactivation of GTN is affected by the subcellular localization of ALDH2 using immortalized ALDH2-deficient aortic smooth muscle cells and mouse aortas with selective overexpression of the enzyme in either cytosol or mitochondria. Methods and Results:Quantitative Western blotting revealed that ALDH2 is mainly cytosolic in mouse aorta and human coronary arteries, with only approximately 15% (mouse) and approximately 5% (human) of the enzyme being localized in mitochondria. Infection of ALDH2-deficient aortic smooth muscle cells or isolated aortas with adenovirus containing ALDH2 cDNA with or without the mitochondrial signal peptide sequence led to selective expression of the protein in mitochondria and cytosol, respectively. Cytosolic overexpression of ALDH2 restored GTN-induced relaxation and GTN denitration to wild-type levels, whereas overexpression in mitochondria (6-fold vs wild-type) had no effect on relaxation. Overexpression of ALDH2 in the cytosol of ALDH2-deficient aortic smooth muscle cells led to a significant increase in GTN denitration and cyclic GMP accumulation, whereas mitochondrial overexpression had no effect. Key Words: adenovirus Ⅲ aldehyde dehydrogenase-2 Ⅲ mitochondria Ⅲ nitroglycerin Ⅲ vasodilation A ldehyde dehydrogenase-2 (ALDH2) has a wellestablished function in the detoxification of reactive aldehydes, in particular ethanol-derived acetaldehyde, in the liver. Because the liver enzyme is almost exclusively located in the mitochondrial matrix space, it is commonly designated as mitochondrial aldehyde dehydrogenase to differentiate it from the cytosolic isoform (ALDH1). 1 In 2002, Stamler et al 2 discovered that vascular ALDH2 catalyzes bioconversion of nitroglycerin to yield 1,2-glycerol dinitrate (GTN) and inorganic nitrite. This reaction appears to be associated with formation of a disulfide in the catalytic site, leading to mechanism-based enzyme inactivation in the absence of an appropriate reductant, like dithiothreitol, 2 dihydrolipoic acid, 3 or the thioredoxin/thioredoxin reductase system. 4 Based on the mitochondrial localization of liver ALDH2, it has been proposed that GTN bioactivation takes place in mitochondria of vascular smooth muscle cells. Reduction of GTN-derived nitrite to nitric oxide (NO) by components of the respiratory chain would then couple ALDH2-catalyzed GTN metabolism to activation of soluble guanylate cyclase (sGC) and vascular relaxation. 2 Mitochondrial GTN bioactivation, supported by the observation that isolated mitochondria generate NO bioactivity from added GTN, 5,6 has been generally accepted in the field and has fo...

Effects of Cyanamide, an Aldehyde Dehydrogenase Type 2 Inhibitor, on Glyceryl Trinitrate- and Isosorbide Dinitrate-Induced Vasodilation in Rabbit Excised Aorta and in Anesthetized Whole Animal

“…Isosorbide dinitrate may be bioactivated by ALDH1 (cytosolic isoform), ALDH2 (mitochondrial isoform) 15,25) and ALDH3, including ALDH3D. 23) These evidences indicate that possible different sensitivities of these enzymes to vasodilators 23,25) and inhibitors, 19) and possible different tissue contribution, 26,27) including intracellular compartments, 24) may account for our present results. Indeed, different sensitivities to different ALDH2 inhibitors have been reported: GTN bioactivation in rat liver was sensitive to chloral hydrate but not to daidzin.…”

“…23) Aldehyde dehydrogenase 2, ALDH3 23) and cytosolic ALDH2 (alternative splicing variant of mitochondrial ALDH2), 24) but not mitochondrial ALDH2, may be responsible for bioactivation of GTN. Isosorbide dinitrate may be bioactivated by ALDH1 (cytosolic isoform), ALDH2 (mitochondrial isoform) 15,25) and ALDH3, including ALDH3D.…”

Endothelial Nitric Oxide Synthase-Independent Release of Nitric Oxide in the Aorta of the Spontaneously Hypertensive Rat

“…In addition, inhibition of Complex I only partly reduces NO release, whereas inhibition of Complex III completely abolishes its release (Kozlov et al, 1999). An inhibitor of another enzyme that is a source of NO, aldehyde dehydrogenase 2 (ALDH-2) (Chen et al, 2002;Beretta et al, 2012), was also examined. Daidzin, used at a concentration reported to inhibit ALDH-2 (Keung et al, 1997), did not affect the inhibition by the endothelium of the contraction to phenylephrine.…”

P300/CBP Associated Factor Regulates Nitroglycerin-Dependent Arterial Relaxation by N ε -Lysine Acetylation of Contractile Proteins

“…Remarkably, ANAC pretreatment did not limit response to nifedipine or sodium nitroprusside (not shown) suggesting specificity for GTN which, opposite from the other 2 inducers of rapid arterial relaxation, requires bioactivation through cytosolic aldehyde dehydrogenase-2. 24 Further experiments are required to elucidate this important point. However, the compound SPV106, a selective GNC5/ PCAF activator, 14 administered daily with GTN prevented the development of tolerance (EC 50T /EC 50C =0.9; Figure 3D or in the absence (E-F) of endothelium.…”