Combating nitrate tolerance: a novel endogenous mechanism.

With glycerol trinitrate (GTN) as the prototype, nitrates represent one of the safest and most rapidly effective pharmacological means to reduce acute symptoms of myocardial ischemia attributable to obstructive coronary disease. This has led, over the years, to the development of long-acting oral and topical preparations. However, efficacy with chronic administration is more difficult to achieve because of the development of therapeutic resistance, generally occurring a few days after initiating treatment. This phenomenon known as nitrate tolerance has been the stimulus for intense investigation of the metabolic fate of nitroglycerin with the idea that modulation of its biotransformation could improve efficacy of chronic treatment. The mechanism of GTN-induced dilation is complex and was not identified until more than 100 years after its discovery. GTN is not a direct vasodilator, rather it must be converted to dinitrate products for vasoactivity. Biotransformation to the active metabolite nitric oxide (NO) occurs in parallel with the formation of glycerol-1,2-dinitrate and involves a dithiol-dependent process.1 It was not until recently that the principal enzyme responsible for biotransformation of GTN was identified. Chen et al1 showed that mitochondrial aldehyde dehydrogenase (ALDH-2) metabolizes GTN to glycerol-1,2-dinitrate and nitrite. This was confirmed by Sydow et al2 using mitochondrial-deficient cultured endothelial cells, although a cytosolic source of ALDH-2 has also been suggested.3 The mitochondrial enzyme converts nanomolar concentrations of GTN to active nitrodilator metabolites in vivo and in vitro, as shown by direct measurements coupled with the use of selective inhibitors and competing substrates.1,2 Whether the metabolic product NO or a related compound is responsible for the subsequent activation of guanylate cyclase remains in question.4 Other enzymatic pathways of GTN metabolism include glutathione-S-transferase and cytochrome P450 reductase, but neither leads to the formation of glycerol-1,2-dinitrate. Glutathione-S-transferase metabolizes GTN to the inactive product glycerol-1,3-dinitrite and is thought to represent a pathway for metabolic inactivation of GTN.5,6 Cytochrome P450 enzymes are capable of active biotransformation of nitrates, but the Km for the enzyme is such that generation of NO occurs only at high (micromolar) GTN concentrations.7–9 The differential vascular distribution of cytochrome P450 enyzmes may explain spatial regional variability in nitrodilator effects as well as the greater response in venous than arterial segments.8 Which of these metabolic pathways are activated depends on both the concentration of the nitrate compound used as well as the number of nitrate groups present on the parent compound.10 Thus ISDN and ISMN are not bioactivated by ALDH-2, but GTN and PETN are.10 The cell type responsible for nitroglycerin biotransformation is not well-defined because the enzymes involved are expressed throughout the vascular wall. Some evidence indicates that vascular smooth muscle is responsible for conversion to active metabolites.11 However, recent data suggest that the endothelium is important because dilation to GTN is reduced substantially by endothelial removal.12 Nitrate tolerance was demonstrated more than 100 years ago, but the mechanism appears complex and is only now being unraveled (nicely reviewed in13,14). There are 2 processes involved which result in tolerance to chronic nitrate therapy, cross-tolerance to other NO donors, and endothelial dysfunction. First, chronic nitrate therapy increases vascular oxidative stress and reduces bioavailability of NO. The resulting reactive oxygen and nitrogen species inhibit bioactivation of the administered nitrate. Tolerance occurs via GTN-induced production of superoxide, likely from mitochondrial sources because mitochondrially targeted antioxidants prevent it15 and heterozygous MnSOD-deficient mice show heightened sensitivity.11 However, other sources exist including NADPH oxidase16 and uncoupled NOS,17 either through BH4 oxidation or depletion of intracellular arginine.18 The culprit superoxide is derived both from vascular smooth muscle19 and endothelium, as denudation reduces both tolerance to NTG and cross tolerance to other NO donors.12 An increase in PKC activity also occurs with nitrate tolerance, contributing to enhanced constriction, reduced dilation,20 and NOS uncoupling.21 Second, in addition to quenching NO that is derived from GTN, ROS, including NO, can block bioactivation of GTN by disulfide modification and inhibition of ALDH-2.1,2,13 Thus GTN induces tolerance both by reducing GTN biotransformation to dilator metabolites (inhibition of ALDH-2) and From the Departments of Medicine and Pharmacology, Medical College of Wisconsin and Zablocki VA Medical Center, Milwaukee. Correspondence to David D. Gutterman, MD, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail [email protected] (Arterioscler Thromb Vasc Biol. 2007;27:1673-1676.) © 2007 American Heart Association, Inc.