Organic Nitrate Metabolism, Tolerance and Action

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