The long-term objective is to gain a better understanding of the mechanisms of action of organic nitrates. These important cardiovascular drugs, including nitroglycerin (NTG), are used for the acute relief of coronary ischemia and for prophylaxis against angina pectoris. A """"""""mystery"""""""" of organic nitrate action, lasting now about 130 years since its discovery, is how continuous organic nitrate use rapidly produces therapeutic tolerance (loss of effect). Although several hypotheses have been tested, a consensus opinion has not been reached regarding how this phenomenon is initiated, and how the myriad of events associated with nitrate tolerance can be reconciled with the initiating mechanism(s). Intriguingly, long-term organic nitrate use did not produce significant benefits in patient outcomes, and, according to some reports, it may even engender increased cardiac risk. The potential mechanism for this phenomenon is also not understood. We propose that these wide-ranging action of organic nitrates are underpined by their metabolic pathways of bioactivation. Our preliminary data indicate that NTG mediates S-oxidation, including S-glutathionylation, of cellular proteins. This reaction can lead to many of the events observed in nitrate tolerance, and in fact, is likely to be the initiating step. We also showed, for the first time, that NTG activates the cysteine switch of the crucial peptide sequence in pro-forms of matrix metalloproteinases (MMPs) through S-oxidation. This latter reaction may explain the potential long-term deleterious effects of organic nitrates in some patients.
Specific aim 1 of this proposal will define the relative contributions of candidate enzymes that have been shown to metabolize various organic nitrates, using LLC-PK1 cells, rabbit aorta and/or human vascular smooth muscle cells. Different to other studies which employ classical inhibitors (whose specificity in nitrate metabolism has not been established), we shall utilize specific antibodies to inhibit enzyme activity.
Aim 2 will test the relative roles of S-oxidation vs. superoxide generation in initiating nitrate tolerance, including the use of mice deficient in NADPH oxidase activity.
Aim 3 will test the effects of organic nitrate on the post-translational and mRNA/protein expression effects on MMP-2 and MMP-9, using both in vitro and in vivo techniques. We believe that our studies can provide new and meaningful data that will contribute to the establishment of a unifying mechanism for organic nitrate action and deleterious effects, and lead to their better therapeutic use

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Myocardial Ischemia and Metabolism Study Section (MIM)
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Liang, Isabella Y
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State University of New York at Buffalo
Schools of Pharmacy
United States
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Shin, Beom Soo; Fung, Ho-Leung; Upadhyay, Mahesh et al. (2015) Estimation of nitric oxide synthase activity via liquid chromatography/tandem mass spectrometric assay determination of 15N3 -citrulline in biological samples. Rapid Commun Mass Spectrom 29:447-55
Page, Nathaniel A; Fung, Ho-Leung (2013) Organic nitrate metabolism and action: toward a unifying hypothesis and the future-a dedication to Professor Leslie Z. Benet. J Pharm Sci 102:3070-81
Lin, Shunxin; Page, Nathaniel A; Fung, Sun Mi et al. (2013) In vitro organic nitrate bioactivation to nitric oxide by recombinant aldehyde dehydrogenase 3A1. Nitric Oxide 35:137-43
Mohan, Srinidi; Fung, Ho-Leung (2012) Mechanism of cellular oxidation stress induced by asymmetric dimethylarginine. Int J Mol Sci 13:7521-31
Mohan, Srinidi; Wu, Chia-Ching; Shin, Soyoung et al. (2012) Continuous exposure to L-arginine induces oxidative stress and physiological tolerance in cultured human endothelial cells. Amino Acids 43:1179-88
Shin, Soyoung; Fung, Ho-Leung (2011) Evaluation of an LC-MS/MS assay for 15N-nitrite for cellular studies of L-arginine action. J Pharm Biomed Anal 56:1127-31
Krishnatry, Anu Shilpa; Kamei, Tomonori; Wang, Hao et al. (2011) Identification of nitroglycerin-induced cysteine modifications of pro-matrix metalloproteinase-9. Rapid Commun Mass Spectrom 25:2291-8
Shin, Soyoung; Fung, Sun-Mi; Mohan, Srinidi et al. (2011) Simultaneous bioanalysis of L-arginine, L-citrulline, and dimethylarginines by LC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 879:467-74
Tsou, Pei-Suen; Page, Nathaniel A; Lee, Sean G et al. (2011) Differential metabolism of organic nitrates by aldehyde dehydrogenase 1a1 and 2: substrate selectivity, enzyme inactivation, and active cysteine sites. AAPS J 13:548-55
Shin, Soyoung; Mohan, Srinidi; Fung, Ho-Leung (2011) Intracellular L-arginine concentration does not determine NO production in endothelial cells: implications on the ""L-arginine paradox"". Biochem Biophys Res Commun 414:660-3

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