Role of Reduced Lipoic Acid in the Redox Regulation of Mitochondrial Aldehyde Dehydrogenase (ALDH-2) Activity

Chronic therapy with nitroglycerin results in a rapid development of nitrate tolerance, which is associated with an increased production of reactive oxygen species. We have recently shown that mitochondria are an important source of nitroglycerin-induced oxidants and that the nitroglycerin-bioactivating mitochondrial aldehyde dehydrogenase is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on aldehyde dehydrogenase activity and its restoration by dihydrolipoic acid. In vivo tolerance in Wistar rats was induced by infusion of nitroglycerin (6.6 μg/kg/min, 4 days). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to nitroglycerin. Chronic nitroglycerin infusion lead to impaired vascular responses to nitroglycerin and decreased dehydrogenase activity, which was corrected by dihydrolipoic acid co-incubation. Superoxide, peroxynitrite, and nitroglycerin itself were highly efficient in inhibiting mitochondrial and yeast aldehyde dehydrogenase activity, which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate aldehyde dehydrogenase thereby leading to nitrate tolerance. Glutathionylation obviously amplifies oxidative inactivation of the enzyme providing another regulatory pathway. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores mitochondrial aldehyde dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance. Chronic therapy with nitroglycerin results in a rapid development of nitrate tolerance, which is associated with an increased production of reactive oxygen species. We have recently shown that mitochondria are an important source of nitroglycerin-induced oxidants and that the nitroglycerin-bioactivating mitochondrial aldehyde dehydrogenase is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on aldehyde dehydrogenase activity and its restoration by dihydrolipoic acid. In vivo tolerance in Wistar rats was induced by infusion of nitroglycerin (6.6 μg/kg/min, 4 days). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to nitroglycerin. Chronic nitroglycerin infusion lead to impaired vascular responses to nitroglycerin and decreased dehydrogenase activity, which was corrected by dihydrolipoic acid co-incubation. Superoxide, peroxynitrite, and nitroglycerin itself were highly efficient in inhibiting mitochondrial and yeast aldehyde dehydrogenase activity, which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate aldehyde dehydrogenase thereby leading to nitrate tolerance. Glutathionylation obviously amplifies oxidative inactivation of the enzyme providing another regulatory pathway. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores mitochondrial aldehyde dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance. Organic nitrates such as nitroglycerin (glyceryl trinitrate, GTN) 3The abbreviations used are: GTN, glyceryl trinitrate (nitroglycerin); ALDH-2, mitochondrial aldehyde dehydrogenase; DTT, dithiothreitol; HX, hypoxanthine; Sin-1, 3-morpholinosydnonimine hydrochloride; Cu, Zn-SOD, copper and zinc-superoxide dismutase (the cytosolic and extracellular isoform); SPE/NO, spermine NONOate; XO, xanthine oxidase; HPLC, high-performance liquid chromatography. have been used for over a century in the therapy of cardiovascular diseases like myocardial infarction, unstable angina, and arterial hypertension (1Abrams J. Arch. Intern. Med. 1995; 155: 357-364Crossref PubMed Scopus (60) Google Scholar). However, the usefulness of organic nitrates is limited by tolerance, which develops shortly after onset of treatment. The mechanisms underlying nitrate tolerance remain only in part defined and are most likely multifactorial (2Munzel T. Daiber A. Mulsch A. Circ. Res. 2005; 97: 618-628Crossref PubMed Scopus (361) Google Scholar). Previously, we found that 3 days of nitrate treatment doubled vascular superoxide ( O2·¯) production (3Munzel T. Sayegh H. Freeman B.A. Tarpey M.M. Harrison D.G. J. Clin. Invest. 1995; 95: 187-194Crossref PubMed Scopus (611) Google Scholar), which was also found in human bypass material from GTN-treated patients (4Schulz E. Tsilimingas N. Rinze R. Reiter B. Wendt M. Oelze M. Woelken-Weckmuller S. Walter U. Reichenspurner H. Meinertz T. Munzel T. Circulation. 2002; 105: 1170-1175Crossref PubMed Scopus (153) Google Scholar). Chen et al. (5Chen Z. Zhang J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8306-8311Crossref PubMed Scopus (496) Google Scholar) identified the mitochondrial aldehyde dehydrogenase (ALDH-2) as a GTN-metabolizing enzyme and a possible important component in the processes leading to tolerance. This concept was supported by recent studies in ALDH-2-deficient mice (ALDH-2-/-) (6Chen Z. Foster M.W. Zhang J. Mao L. Rockman H.A. Kawamoto T. Kitagawa K. Nakayama K.I. Hess D.T. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12159-12164Crossref PubMed Scopus (192) Google Scholar). Our laboratory further substantiated this concept in an animal model of in vivo tolerance and extended previous observations by demonstrating that mitochondria are a major source of reactive oxygen species formation in response to acute and chronic GTN challenges (7Sydow K. Daiber A. Oelze M. Chen Z. August M. Wendt M. Ullrich V. Mulsch A. Schulz E. Keaney Jr., J.F. Stamler J.S. Munzel T. J. Clin. Invest. 2004; 113: 482-489Crossref PubMed Scopus (283) Google Scholar, 8Daiber A. Oelze M. Coldewey M. Bachschmid M. Wenzel P. Sydow K. Wendt M. Kleschyov A.L. Stalleicken D. Ullrich V. Mulsch A. Munzel T. Mol. Pharmacol. 2004; 66: 1372-1382Crossref PubMed Scopus (168) Google Scholar). The importance of the ALDH-2 concept for clinical nitrate tolerance was proven by two independent clinical studies in Asian subjects with a point-mutated, dysfunctional ALDH-2 (9Mackenzie I.S. Maki-Petaja K.M. McEniery C.M. Bao Y.P. Wallace S.M. Cheriyan J. Monteith S. Brown M.J. Wilkinson I.B. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1891-1895Crossref PubMed Scopus (79) Google Scholar, 10Li Y. Zhang D. Jin W. Shao C. Yan P. Xu C. Sheng H. Liu Y. Yu J. Xie Y. Zhao Y. Lu D. Nebert D.W. Harrison D.C. Huang W. Jin L. J. Clin. Invest. 2006; 116: 506-511Crossref PubMed Scopus (138) Google Scholar). Because mitochondrial reactive oxygen species formation seems to play a major role for development of tolerance and cross-tolerance, we verified this hypothesis in mice with heterozygous deficiency in mitochondrial superoxide dismutase (Mn-SOD+/-) that were more susceptible for the development of in vitro nitrate and cross-tolerance (tachyphylaxis) (11Daiber A. Oelze M. Sulyok S. Coldewey M. Schulz E. Treiber N. Hink U. Mulsch A. Scharffetter-Kochanek K. Munzel T. Mol. Pharmacol. 2005; 68: 579-588Crossref PubMed Scopus (88) Google Scholar). Previous studies implicated that ALDH-2 is oxidatively inhibited by organic nitrates (5Chen Z. Zhang J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8306-8311Crossref PubMed Scopus (496) Google Scholar, 8Daiber A. Oelze M. Coldewey M. Bachschmid M. Wenzel P. Sydow K. Wendt M. Kleschyov A.L. Stalleicken D. Ullrich V. Mulsch A. Munzel T. Mol. Pharmacol. 2004; 66: 1372-1382Crossref PubMed Scopus (168) Google Scholar, 11Daiber A. Oelze M. Sulyok S. Coldewey M. Schulz E. Treiber N. Hink U. Mulsch A. Scharffetter-Kochanek K. Munzel T. Mol. Pharmacol. 2005; 68: 579-588Crossref PubMed Scopus (88) Google Scholar, 12Daiber A. Oelze M. Coldewey M. Kaiser K. Huth C. Schildknecht S. Bachschmid M. Nazirisadeh Y. Ullrich V. Mulsch A. Munzel T. Tsilimingas N. Biochem. Biophys. Res. 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Despite this knowledge, very little is known about the nature of ALDH-2-inhibiting oxidants, especially about restoration of enzyme activity. Because chronic GTN treatment increases vascular superoxide and peroxynitrite formation (3Munzel T. Sayegh H. Freeman B.A. Tarpey M.M. Harrison D.G. J. Clin. Invest. 1995; 95: 187-194Crossref PubMed Scopus (611) Google Scholar, 19Abou-Mohamed G. Johnson J.A. Jin L. El-Remessy A.B. Do K. Kaesemeyer W.H. Caldwell R.B. Caldwell R.W. J. Pharmacol. Exp. Ther. 2004; 308: 289-299Crossref PubMed Scopus (48) Google Scholar, 20Hink U. Oelze M. Kolb P. Bachschmid M. Zou M.H. Daiber A. Mollnau H. August M. Baldus S. Tsilimingas N. Walter U. Ullrich V. Munzel T. J. Am. Coll. Cardiol. 2003; 42: 1826-1834Crossref PubMed Scopus (103) Google Scholar, 21Warnholtz A. Mollnau H. Heitzer T. Kontush A. Moller-Bertram T. Lavall D. Giaid A. Beisiegel U. Marklund S.L. Walter U. Meinertz T. Munzel T. J. Am. Coll. Cardiol. 2002; 40: 1356-1363Crossref PubMed Scopus (66) Google Scholar) and because reduced α-lipoic acid/lipoamide represent potent dithiol reductants with their own reductase system in mitochondria (22Moini H. Packer L. Saris N.E. Toxicol Appl. Pharmacol. 2002; 182: 84-90Crossref PubMed Scopus (455) Google Scholar, 23Lynch M.A. Nutr. Neurosci. 2001; 4: 419-438Crossref PubMed Scopus (34) Google Scholar, 24Wollin S.D. Jones P.J. J. Nutr. 2003; 133: 3327-3330Crossref PubMed Scopus (172) Google Scholar) the present study was focused on these compounds. We here sought to determine the role of reduced α-lipoic acid/lipoamide for maintenance and restoration of ALDH-2 activity and to gain in the oxidative inactivation of ALDH-2 by various isometric tension GTN was used from a infusion from G. of in vivo tolerance, GTN was used from a in which was from and were from acid and were from and yeast aldehyde dehydrogenase was from were of and were from and in animal treatment was in with the of and with the for the and of as and by the of and was by the of the We used Wistar rats from In vivo tolerance was induced by chronic infusion of rats with GTN in for 4 by model for from of the as a The was recently (7Sydow K. Daiber A. Oelze M. Chen Z. August M. Wendt M. Ullrich V. Mulsch A. Schulz E. Keaney Jr., J.F. Stamler J.S. Munzel T. J. Clin. Invest. 2004; 113: 482-489Crossref PubMed Scopus (283) Google Scholar, 8Daiber A. Oelze M. Coldewey M. Bachschmid M. Wenzel P. Sydow K. Wendt M. Kleschyov A.L. Stalleicken D. Ullrich V. Mulsch A. Munzel T. Mol. Pharmacol. 2004; 66: 1372-1382Crossref PubMed Scopus (168) Google Scholar). that the was which is in animal studies on of responses to GTN were with isolated aortic rings for isometric tension in as (3Munzel T. Sayegh H. Freeman B.A. Tarpey M.M. Harrison D.G. J. Clin. Invest. 1995; 95: 187-194Crossref PubMed Scopus (611) Google Scholar, T. Giaid A. S. D.J. Harrison D.G. Proc. Natl. Acad. Sci. 1995; PubMed Scopus Google Scholar). In vitro tolerance (tachyphylaxis) was induced by two with GTN in the acid and were to the after with that to its reduced in aortic rings to efficient restoration of ALDH-2 activity. of ALDH-2 in activity of ALDH-2 in isolated mitochondria was by the of to acid a A. Oelze M. Coldewey M. Bachschmid M. Wenzel P. Sydow K. Wendt M. Kleschyov A.L. Stalleicken D. Ullrich V. Mulsch A. Munzel T. Mol. Pharmacol. 2004; 66: 1372-1382Crossref PubMed Scopus (168) Google Scholar). mitochondria were to a S. Free Radic. Biol. Med. PubMed Scopus Google Scholar), which was A. Oelze M. August M. Wendt M. Sydow K. H. Kleschyov A.L. Munzel T. Free Radic. Res. 2004; PubMed Scopus Google Scholar). The mitochondrial in were for at with dihydrolipoic DTT, of ALDH-2 dehydrogenase activity, was and the were for another at of was to The system of a and and a 4 from The in acid The and its were at a of at and and acid and The were and In the mitochondria in were and with GTN for at peroxynitrite for at this with were and for another at The activity of in isolated mitochondria was by the of to acid. The and its were at a of at and and acid and The were and The effect of on ALDH-2 activity was also in mitochondria in for at we used two in the was with peroxynitrite at in the was peroxynitrite of the the ALDH-2 acid was to the with the ALDH-2 for at The were at and measured as of in in the of and and with the as used for of ALDH-2 activity was as recently M. Daiber A. M. Wenzel P. Hink U. Schulz E. Mollnau H. A. Kleschyov A.L. Mulsch A. H. U. Munzel T. 2006; PubMed Scopus Google Scholar, H. Oelze M. August M. Wendt M. Daiber A. Schulz E. Baldus S. Kleschyov A.L. A. Wenzel P. Hink U. G. Munzel T. Arterioscler. Thromb. Vasc. Biol. 2005; 25: PubMed Scopus Google Scholar). the were with with and to and of we the by the and a J. Biochem. J. 2004; PubMed Google Scholar). The was a from at a of was by with of ALDH-2 of of was with of M. E. H. Clin. Exp. Res. PubMed Scopus Google Scholar) on in at 4 and of the at for the ALDH-2 were to and with as of in to with and in was with of peroxynitrite at Sin-1, SPE/NO, GTN, at In such as Cu, thiol compounds such as DTT, and dihydrolipoic acid to were with the of and the dehydrogenase activity was by the formation of by the at for at are as of for of was used for of and ALDH-2, and yeast activity. The for was by were are in the of ALDH-2 in dehydrogenase activity in isolated mitochondria was inhibited in a by peroxynitrite as as by in peroxynitrite from The on a was for of these GTN was more in inhibiting the enzyme activity of study the of various reactive oxygen and species in more we used highly yeast with an activity the yeast and the mitochondrial ALDH-2 in is the yeast enzyme may as a model to study oxidative because its thiol active site is also highly to oxidative we found that yeast was and inactivated by peroxynitrite, Sin-1, GTN, and system which in the The rather on because which were decreased in the of the system on This increased in the of and superoxide source is to peroxynitrite formation and by in the of was a potent of yeast and a of more for The for with of are in the of oxidants that of yeast yeast in at Sin-1, SPE/NO, SPE/NO, GTN at for on previous yeast in at Sin-1, SPE/NO, SPE/NO, GTN at for on previous A. D. D. Ullrich V. J. Biol. 2002; PubMed Scopus Google Scholar). in a We also the effect of thiol compounds on activity treatment with peroxynitrite, Sin-1, and GTN of peroxynitrite and decreased rather increased the activity to an effect of of GTN and also an of and in of activity. In the dithiol compounds and dihydrolipoic acid a more of activity. that this was to restoration of dehydrogenase activity in rather of oxidants and we the after of the oxidative inactivation after and GTN The effect was of ALDH-2 in effect of dihydrolipoic acid on ALDH-2 activity was in isolated mitochondria from in vivo GTN-treated The dehydrogenase activity in mitochondria was increased by by dihydrolipoic acid the activity was further increased in response to dihydrolipoic acid. ALDH-2 activity was decreased in mitochondria from in vivo GTN-treated rats and was restored in the of dihydrolipoic acid the dihydrolipoic acid to further of ALDH-2 activity in mitochondria from rats to the of the mitochondria from rats were with the of mitochondrial ALDH-2 activity was decreased by the of dihydrolipoic acid to this activity, dihydrolipoic acid further dihydrolipoic acid effect observations were with another dihydrolipoic acid the to impaired ALDH-2 activity In ALDH-2 activity was inhibited by the the ALDH-2 dihydrolipoic acid to effect on ALDH-2 activity This of dihydrolipoic acid with the dehydrogenase activity of in in the of and ALDH-2 activity was by GTN of isolated with in an in activity that was associated with and and peroxynitrite induced was after of peroxynitrite and and However, with peroxynitrite, ALDH-2 inactivation by of peroxynitrite and further ALDH-2 is the we of ALDH-2 by shown in by ALDH-2 was with peroxynitrite by of However, we were to in the of that these is of importance and we used a ALDH-2 of in to in GTN and in to in GTN by as as of GTN in from and rats was by isometric tension studies of isolated in In vivo treatment of rats with GTN to the development of nitrate tolerance as by a of the to the GTN as with aortic rings from rats This was by a decreased of the to GTN a decreased of this in from GTN-treated rats In the of dihydrolipoic acid in the the impaired of GTN in was and the was and was aortic rings of were to two GTN The GTN the treatment induced in vitro tolerance (tachyphylaxis) as by a of the This a in the of GTN in the which was in the of in the to the of the and The of is by and mitochondrial dehydrogenase has a for this compound as with the acid. may also by such as and (22Moini H. Packer L. Saris N.E. Toxicol Appl. Pharmacol. 2002; 182: 84-90Crossref PubMed Scopus (455) Google Scholar, 23Lynch M.A. Nutr. Neurosci. 2001; 4: 419-438Crossref PubMed Scopus (34) Google Scholar, 24Wollin S.D. Jones P.J. J. Nutr. 2003; 133: 3327-3330Crossref PubMed Scopus (172) Google Scholar). ALDH-2 is the mitochondrial of aldehyde and is known to The the dehydrogenase activity, which the and the activity to thiol C.S. Senior D.J. Biochem. Cell Biol. 1991; 69: 193-197Crossref PubMed Scopus (9) Google Scholar, D.J. C.S. Biochem. Cell Biol. 68: PubMed Scopus Google Scholar) and are inhibited by oxidants and compounds such as A. Oelze M. Coldewey M. Bachschmid M. Wenzel P. Sydow K. Wendt M. Kleschyov A.L. Stalleicken D. Ullrich V. Mulsch A. Munzel T. Mol. Pharmacol. 2004; 66: 1372-1382Crossref PubMed Scopus (168) Google Scholar). In an was to this enzyme Chen et al. (5Chen Z. Zhang J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8306-8311Crossref PubMed Scopus (496) Google Scholar) identified ALDH-2 as a of GTN and nitrates is a reduction and from active thiol groups, which are to a disulfide the The activity of ALDH-2 is restored by and reduced acid as by the present the we an extended in the to the stress concept in nitrate vascular and mitochondrial oxidants to play a major role for the development of nitrate and cross-tolerance in response to chronic organic nitrate treatment (2Munzel T. Daiber A. Mulsch A. Circ. Res. 2005; 97: 618-628Crossref PubMed Scopus (361) Google Scholar, T. Sayegh H. Freeman B.A. Tarpey M.M. Harrison D.G. J. Clin. Invest. 1995; 95: 187-194Crossref PubMed Scopus (611) Google Scholar, E. Tsilimingas N. Rinze R. Reiter B. Wendt M. Oelze M. Woelken-Weckmuller S. Walter U. Reichenspurner H. Meinertz T. Munzel T. Circulation. 2002; 105: 1170-1175Crossref PubMed Scopus (153) Google Scholar, 11Daiber A. Oelze M. Sulyok S. Coldewey M. Schulz E. Treiber N. Hink U. Mulsch A. 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Google Scholar). peroxynitrite, in peroxynitrite and GTN in inhibiting the enzyme in mitochondrial We further substantiated this by yeast as a model of oxidative ALDH-2 inactivation and The results to an formation of peroxynitrite from the of with which has been shown for the inactivation of yeast dehydrogenase by formation of superoxide and A. D. D. Ullrich V. J. Biol. 2002; PubMed Scopus Google Scholar). In with the we a in ALDH-2 activity in mitochondria from GTN in rats also in response to acute with the and the mitochondrial and thereby to oxygen in increased superoxide which ALDH-2 activity. has been shown that may the activity of ALDH-2 (5Chen Z. Zhang J. Stamler J.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8306-8311Crossref PubMed Scopus (496) Google Scholar). The that is a compound and ALDH-2 implicated the of another dithiol acid and its at mitochondria and are to the reduced species by such as the mitochondrial dehydrogenase as as cytosolic reductase (22Moini H. Packer L. 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Med. 2004; PubMed Scopus Google Scholar). possible for only restoration of dehydrogenase activity by in the of GTN in vitro that the organic nitrate was and at of The restoration of responses by reduced acid the setting of in vivo and the setting of in vitro tolerance, may also of the reduced and of these compounds activity restoration M. R. Arch. Biochem. Biophys. 2002; PubMed Scopus Google Scholar). this in more the of thiol compounds were yeast only and acid activity The of of is on a ALDH-2 yeast a disulfide a acid that two for the and the two in a Because the active site of aldehyde is rather it is that only the active Because reduction of two this with the disulfide and reduced acid in a also a disulfide with in to the reduction via thiol et al. (14Moon K.H. Kim B.J. Song B.J. FEBS Lett. 2005; 579: 6115-6120Crossref PubMed Scopus (71) Google Scholar) found that is highly in inhibiting ALDH-2 activity, by of the active site of the we an in the these of that reduced acid/lipoamide restores activity of ALDH-2 and yeast and that this dithiol is the for this which is highly susceptible for oxidative inactivation by peroxynitrite, and Our data a role of dihydrolipoic acid and in the restoration of impaired ALDH-2 reductase activity, which is for organic nitrate and thereby may nitrate tolerance in response to chronic GTN treatment. However, acid formation for ALDH-2 of the The of is shown in the in may have important implications for the clinical of organic nitrates that are by ALDH-2 also for ALDH-2 is of the major that are for of especially of which is at The oxidative of ALDH-2 by alcohol-induced reactive oxygen and species lead to of these which in have been to thereby a that in cell damage of the