EVIDENCE FOR ENHANCED VASCULAR SUPEROXIDE ANION PRODUCTION IN NITRATE TOLERANCE - A NOVEL MECHANISM UNDERLYING TOLERANCE AND CROSS-TOLERANCE

We sought to examine mechanisms underlying nitroglycerin (NTG) tolerance and ''cross-tolerance'' to other nitrovasodilators. Rabbits were treated for 3 d with NTG patches (0.4 mg/h) and their aortic segments studied in organ chambers. Relaxations were examined after preconstriction with phenylephrine. In NTG tolerant rabbit aorta, relaxations to cGMP-dependent vasodilators such as NTG (45+/-6%), SIN-1 (69+/-7%), and acetylcholine (ACh, 64+/-5%) were attenuated vs. controls, (90+/-2, 94+/-3, and 89+/-2% respectively, P < 0.05 for all), while responses to the cAMP-dependent vasodilator forskolin remained unchanged. In tolerant aorta, endothelial removal markedly enhanced relaxations to NTG and SIN-1 (82+/-4 and 95+/-3%, respectively). Other studies were performed to determine how the endothelium enhances tolerance. Vascular steady state O-2-radical-anion levels (assessed by lucigenin chemiluminescence) was increased twofold in tolerant vs. control vessels with endothelium (0.31+/-0.01 vs. 0.61+/-0.01 nmol/mg per minute). This difference was less in vessels after denudation of the endothelium. Diphenylene iodonium, an inhibitor of flavoprotein containing oxidases, and Tiron a direct O-2-radical anion scavenger normalized O-2-radical anions levels. In contrast, oxypurinol (1 mM) an inhibitor of xanthine oxidase, rotenone (50 mu M) an inhibitor of mitochondrial electron transport and N-G-nitro-L-arginine (100 mu M) an inhibitor of nitric oxide synthase did not affect the chemiluminescence signals from NTG-tolerant aortas. Pretreatment of tolerant aorta with liposome-entrapped, pH sensitive superoxide dismutase (600 U/ml) significantly enhanced maximal relaxation in response to NTG, SIN-1, and ACh, and effectively reduced chemiluminescence signals. These studies show that continuous NTG treatment is associated with increased vascular O-2-radical-anion-production and consequent inhibition of NO. mediated vasorelaxation produced by both exogenous and endogenous nitrovasodilators.