Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Beretta, Matteo; Gruber, Karl; Kollau, Alexander; Russwurm, Michael; Koesling, Doris; Goessler, Walter; Keung, Wing Ming; Schmidt, Kurt; Mayer, Bernd

doi:10.1074/jbc.m801182200

Cited by 69 publications

(84 citation statements)

References 38 publications

(45 reference statements)

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“…The inactivation of ALDH2 after each turnover was shown to be DTT-reversible, suggesting that it contributes to the development of nitrate tolerance. Although this mechanism may account for the main route of ALDH2-catalyzed GTN denitration, our recent work demonstrating an additional pathway yielding NO instead of nitrite (Beretta et al, 2008a) and partially DTT-irreversible inactivation of the enzyme by GTN (Beretta et al, 2008b) indicates that the reaction between ALDH2 and GTN may be more complex than originally assumed. In the present study, we mutated the cysteine residues adjacent to the catalytic nucleophile Cys302 and the putative general base Glu268 in human ALDH2 to clarify their function in GTN metabolism and GTN-induced enzyme inactivation.…”

Section: Discussionmentioning

confidence: 76%

“…In 2002, Stamler and coworkers (Chen et al, 2002) showed that ALDH2 denitrates GTN to 1,2-glyceryl dinitrate (1,2-GDN) and nitrite and proposed that reduction of nitrite by components of the mitochondrial electron transfer chain could provide the link between ALDH2-catalyzed GTN metabolism and vasorelaxation. However, we found that 1) GTN activates sGC in the presence of purified ALDH2 , 2) ALDH2 catalyzes the reduction of GTN to NO, accounting for 5 to 10% of total GTN turnover (Beretta et al, 2008a;Wenzl et al, 2009), and 3) mitochondrial biotransformation of nitroglycerin is not affected by respiratory substrates or inhibitors (Kollau et al, 2009), suggesting that ALDH2 catalysis is sufficient for GTN bioactivation in blood vessels.…”

Section: Introductionmentioning

confidence: 92%

“…Bovine lung sGC was purified as described previously . Human ALDH2 was expressed in Escherichia coli BL21(DE3) and purified as described previously (Zheng et al, 1993;Beretta et al, 2008a). Concentrations are expressed per monomer, assuming a molecular mass of 54 kDa.…”

Section: Methodsmentioning

confidence: 99%

“…To confirm the desired mutations, sequencing of cDNAs was performed with an ABI 373A automated DNA sequencer (Applied Biosystems, Carlsbad, CA). All mutants were expressed in E. coli BL21(DE3) and purified as described previously (Zheng et al, 1993;Beretta et al, 2008a).…”

Section: Methodsmentioning

confidence: 99%

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Site-Directed Mutagenesis of Aldehyde Dehydrogenase-2 Suggests Three Distinct Pathways of Nitroglycerin Biotransformation

Wenzl

Beretta²,

Griesberger³

et al. 2011

Mol Pharmacol

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To elucidate the mechanism underlying reduction of nitroglycerin (GTN) to nitric oxide (NO) by mitochondrial aldehyde dehydrogenase (ALDH2), we generated mutants of the enzyme lacking the cysteines adjacent to reactive Cys302 (C301S and C303S), the glutamate that participates as a general base in aldehyde oxidation (E268Q) or combinations of these residues. The mutants were characterized regarding acetaldehyde dehydrogenation, GTN-triggered enzyme inactivation, GTN denitration, NO formation, and soluble guanylate cyclase activation. Lack of the cysteines did not affect dehydrogenase activity but impeded GTN denitration, aggravated GTN-induced enzyme inactivation, and increased NO formation. A triple mutant lacking the cysteines and Glu268 catalyzed sustained formation of superstoichiometric amounts of NO and exhibited slower rates of inactivation. These results suggest three alternative pathways for the reaction of ALDH2 with GTN, all involving formation of a thionitrate/sulfenyl nitrite intermediate at Cys302 as the initial step. In the first pathway, which predominates in the wild-type enzyme and reflects clearance-based GTN denitration, the thionitrate apparently reacts with one of the adjacent cysteine residues to yield nitrite and a protein disulfide. The predominant reaction catalyzed by the single and double cysteine mutants requires Glu268 and results in irreversible enzyme inactivation. Finally, combined lack of the cysteines and Glu268 shifts the reaction toward formation of the free NO radical, presumably through homolytic cleavage of the sulfenyl nitrite intermediate. Although the latter reaction accounts for less than 10% of total turnover of GTN metabolism catalyzed by wild-type ALDH2, it is most likely essential for vascular GTN bioactivation.

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Section: Discussionmentioning

confidence: 76%

Section: Introductionmentioning

confidence: 92%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Site-Directed Mutagenesis of Aldehyde Dehydrogenase-2 Suggests Three Distinct Pathways of Nitroglycerin Biotransformation

Wenzl

Beretta²,

Griesberger³

et al. 2011

Mol Pharmacol

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“…This enzyme catalyzes the formation of 1,2-glyceryl dinitrate (1,2-GDN) and inorganic nitrite from the metabolism of GTN, leading to the production of cGMP and vasorelaxation (3,10). Nitrite anion can be reduced to NO by enzymatic and nonenzymatic mechanisms, and it has been reported that vasodilator responses to GTN in the coronary and systemic vascular beds can be attenuated by cyanamide, an inhibitor of ALDH2 (3,8,45). However, the effects of cyanamide and allopurinol on responses to GTN have not been determined in the pulmonary vascular bed.…”

mentioning

confidence: 99%

Mitochondrial aldehyde dehydrogenase mediates vasodilator responses of glyceryl trinitrate and sodium nitrite in the pulmonary vascular bed of the rat

Badejo

Hodnette

Dhaliwal

et al. 2010

American Journal of Physiology-Heart and Circulatory Physiology

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PJ. Mitochondrial aldehyde dehydrogenase mediates vasodilator responses of glyceryl trinitrate and sodium nitrite in the pulmonary vascular bed of the rat. Am J Physiol Heart Circ Physiol 299: H819 -H826, 2010. First published June 11, 2010; doi:10.1152/ajpheart.00959.2009.-It has been reported that mitochondrial aldehyde dehydrogenase (ALDH2) catalyzes the formation of glyceryl dinitrate and inorganic nitrite from glyceryl trinitrate (GTN), leading to an increase in cGMP and vasodilation in the coronary and systemic vascular beds. However, the role of nitric oxide (NO) formed from nitrite in mediating the response to GTN in the pulmonary vascular bed is uncertain. The purpose of the present study was to determine if nitrite plays a role in mediating vasodilator responses to GTN. In this study, intravenous injections of GTN and sodium nitrite decreased pulmonary and systemic arterial pressures and increased cardiac output. The decreases in pulmonary arterial pressure under baseline and elevated tone conditions and decreases in systemic arterial pressure in response to GTN and sodium nitrite were attenuated by cyanamide, an ALDH2 inhibitor, whereas responses to the NO donor, sodium nitroprusside (SNP), were not altered. The decreases in pulmonary and systemic arterial pressure in response to GTN and SNP were not altered by allopurinol, an inhibitor of xanthine oxidoreductase, whereas responses to sodium nitrite were attenuated. GTN was ϳ1,000-fold more potent than sodium nitrite in decreasing pulmonary and systemic arterial pressures. These results suggest that ALDH2 plays an important role in the bioactivation of GTN and nitrite in the pulmonary and systemic vascular beds and that the reduction of nitrite to vasoactive NO does not play an important role in mediating vasodilator responses to GTN in the intact chest rat. mitochondrial aldehyde dehydrogenase; xanthine oxidoreductase; nitric oxide; glyceryl trinitrate; sodium nitrite; sodium nitroprusside; allopurinol; cyanamide; U-46619; N G -nitro-L-arginine methyl ester GLYCERYL TRINITRATE (GTN) and, to a lesser extent, amyl nitrite have been used in the treatment of angina and heart failure for more than a century (4, 5, 38). However, the molecular mechanism by which GTN relaxes vascular smooth muscle is still the subject of current investigation and remains unknown. Although it is well established that nitric oxide (NO) activates soluble guanylyl cyclase, increases cGMP formation, and relaxes vascular smooth muscle, the role of NO release in mediating the vasorelaxant response to GTN is uncertain (11,17,21,26,30,37,39). Although studies in the literature provide evidence that NO is released from GTN, other studies show that NO is not released and suggest that the activation of guanylyl cyclase is mediated by a closely related but not currently identified chemical species with NO-guanylyl cyclase stimulating properties (2, 11, 16, 23, 25, 29 -31, 36, 39). There is substantial evidence in the literature that mitochondrial aldehyde dehydrogenase (ALDH2) plays an i...

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Neither nitrite nor nitric oxide mediate toxic effects of nitroglycerin on mitochondria

Dungel¹,

Haindl²,

Behling³

et al. 2011

J Biochem & Molecular Tox

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It is commonly accepted that the major effect of nitroglycerin (NG) is realized through the release of nitric oxide (NO) catalyzed by aldehyde dehydrogenase-2 (ALDH2). In addition, it has been shown that NG inhibits mitochondrial respiration. The aim of this study was to clarify whether NG-mediated inhibition of mitochondrial respiration is mediated by NO. In rat liver mitochondria, NG inhibited complex-I-dependent respiration and induced reactive oxygen species (ROS) production, preferentially at complex I. Both effects were insensitive to chloral hydrate, an ALDH2 inhibitor. Nitrite, an NG intermediate, had no influence on either mitochondrial respiration or the production of ROS. NO inhibited preferentially complex I but did not elevate ROS production. Hemoglobin, an NO scavenger, and blue light had contrary effects on mitochondria inhibited by NO or NG. In summary, our data suggest that although NG induces vasodilatation via NO release, it causes mitochondrial dysfunction via an NO-independent pathway.

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Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Cited by 69 publications

References 38 publications

Site-Directed Mutagenesis of Aldehyde Dehydrogenase-2 Suggests Three Distinct Pathways of Nitroglycerin Biotransformation

Site-Directed Mutagenesis of Aldehyde Dehydrogenase-2 Suggests Three Distinct Pathways of Nitroglycerin Biotransformation

Mitochondrial aldehyde dehydrogenase mediates vasodilator responses of glyceryl trinitrate and sodium nitrite in the pulmonary vascular bed of the rat

Neither nitrite nor nitric oxide mediate toxic effects of nitroglycerin on mitochondria

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