Biotransformation of Glyceryl Trinitrate by Rat Hepatic Microsomal GlutathioneS-Transferase 1

Abstract

Although the biotransformation of organic nitrates by the cytosolic glutathione S-transferases (GSTs) is well known, the relative contribution of the microsomal GST (MGST1) to nitrate biotransformation has not been described. We therefore compared the denitration of glyceryl trinitrate (GTN) by purified rat liver MGST1 and cytosolic GSTs. Both MGST1 and cytosolic GSTs catalyzed the denitration of GTN, but the activity of MGST1 toward GTN was 2-to 3-fold higher. To mimic oxidative/nitrosative stress in vitro, we treated enzyme preparations with hydrogen peroxide, S-nitrosoglutathione, and peroxynitrite. Both oxidants and nitrating reagents increased the activity of MGST1 toward the GST substrate, 1-chloro-2,4-dinitrobenzene (CDNB) whereas these treatments inhibited GTN denitration by MGST1. Alkylation of the sole cysteine residue of MGST1 by N-ethylmaleimide markedly increased enzyme activity with CDNB as substrate but decreased the rate of GTN denitration. In aortic microsomes from GTN-tolerant animals, there was a decreased abundance of MGST1 dimers and trimers. In hepatic microsomes from GTN-tolerant animals, GTN biotransformation was unaltered whereas the rate of CDNB conjugation was doubled, suggesting that chronic GTN exposure causes structural modifications to the enzyme, resulting in increased activity to certain substrates. Collectively, these data indicate that MGST1 contributes significantly to the biotransformation of GTN and that chemical modification of the microsomal enzyme has differential effects on the catalytic activity toward different substrates.Organic nitrates such as glyceryl trinitrate (GTN) have been used clinically for more than a century in the treatment of angina pectoris and congestive heart failure. It is generally accepted that GTN is a prodrug that requires bioactivation to form nitric oxide (NO), or a closely related species, before activation of guanylyl cyclase, increased cyclic GMP, and vascular smooth muscle relaxation. In addition to this mechanism-based biotransformation of GTN to activators of guanylyl cyclase, the denitration of GTN in vascular and nonvascular tissues to inorganic nitrite anion (NO 2 Ϫ ) and its two dinitrate metabolites, glyceryl-1,2-dinitrate (1,2-GDN) and glyceryl-1,3-dinitrate (1,3-GDN) also occurs. This is catalyzed by a number of proteins and enzymes, including hemoglobin and myoglobin (Bennett et al

Constitutive nitric oxide synthase activation is a significant route for nitroglycerin-mediated vasodilation

“…2 and 3). These findings suggest that nitroglycerin is bioactivated and exerts physiological effects through its metabolism at higher doses, which is consistent with findings of multiple enzyme-dependent ways to activate nitroglycerin (6,8,9). Nevertheless, taken together, our experiments demonstrated that such pathways dependent on metabolism are not likely to be the major routes operating in vivo at therapeutic nitroglycerin doses (1-10 nM).…”

“…Since then, intense research has led to extensive and well documented literature regarding nitroglycerin's physiological effects (2)(3)(4) and the identity of its derived metabolites in cells, tissues, laboratory animals, and humans (5,6). Although much of nitroglycerin's pharmacology is known, the mechanisms through which nitroglycerin acts on the endothelium and the heart as well as the enzymatic pathways leading to its bioactivation are still controversial and under intense investigation.…”

“…Xanthine oxidase and mitochondrial aldehyde dehydrogenase (which are closely related oxidoreductases) have been found to mediate nitroglycerin reduction to nitrite (11) and NO itself (8,10). Several intermediate compounds, such as partially nitrated glycerin, nitrite (11), and nitrosothiols (6,7), have been indicated as precursors of nitroglycerin-derived NO, which is ultimately responsible for the observed effects on the vasculature. Collectively, these studies contributed to establishing nitroglycerin as a metabolismdependent NO donor.…”

“…Although much of nitroglycerin's pharmacology is known, the mechanisms through which nitroglycerin acts on the endothelium and the heart as well as the enzymatic pathways leading to its bioactivation are still controversial and under intense investigation. A number of hypotheses for nitroglycerin bioconversion in vivo have been formulated, implicating a multitude of enzymes such as GST (6,7), oxidoreductases (8), and mitochondrial aldehyde dehydrogenase (9,10) in the bioconversion of nitroglycerin to NO and/or other vasoactive compounds. For example, GST has been shown to catalyze the transnitration of lower thiols in the presence of nitroglycerin (6,7).…”

“…A number of hypotheses for nitroglycerin bioconversion in vivo have been formulated, implicating a multitude of enzymes such as GST (6,7), oxidoreductases (8), and mitochondrial aldehyde dehydrogenase (9,10) in the bioconversion of nitroglycerin to NO and/or other vasoactive compounds. For example, GST has been shown to catalyze the transnitration of lower thiols in the presence of nitroglycerin (6,7). Xanthine oxidase and mitochondrial aldehyde dehydrogenase (which are closely related oxidoreductases) have been found to mediate nitroglycerin reduction to nitrite (11) and NO itself (8,10).…”