Effects of the flavoprotein inhibitor, diphenyleneiodonium sulfate, on ex vivo organic nitrate tolerance in the rat.

The flavoprotein inhibitor, diphenyleneiodonium (DPI), inhibits the action of glyceryl trinitrate (GTN) and the D-enantiomer of isoidide dinitrate (IIDN), but not the L-enantiomer (L-IIDN), in isolated rat aorta via inhibition of the bioactivation of these prodrugs. Paradoxically, a vascular NAD(P)H oxidase, which also is inhibited by DPI, has been proposed to generate superoxide that quenches nitric oxide (NO) produced during GTN biotransformation, and increased oxidase levels are proposed to contribute to the phenomenon of organic nitrate tolerance. We examined the effect of DPI on isolated rat aorta using an in vivo model of organic nitrate tolerance. The EC(50) values for GTN-, D-IIDN-, and L-IIDN-induced relaxation of aorta from GTN-tolerant rats were increased 4.5- to 7.5-fold. Treatment of blood vessels with DPI (0.3 microM) increased the EC(50) values for GTN and D-IIDN by the same magnitude in control and tolerant aortae, a result that would not be predicted if DPI and GTN tolerance affected common targets. The expression of NADPH-cytochrome P450 reductase (CPR) during in vivo tolerance was assessed by NADPH-dependent cytochrome c reductase activity of aortic microsomes, immunoblotting, and Northern analysis. By all three determinants, CPR expression was unchanged in aorta from GTN-tolerant rats. Superoxide dismutase-inhibitable NADPH-dependent cytochrome c reductase activity (a measure of superoxide generation) of tolerant rat aortic microsomes was not different from that of controls. Superoxide dismutase-inhibitable NADH-dependent cytochrome c reductase activity was detected only in microsomes from tolerant animals. DPI caused a modest increase in the sensitivity for relaxation by the NO donor DEA NONOate to an equal extent in tolerant and nontolerant tissues, whereas the superoxide scavenger, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), had no effect on the sensitivity for relaxation by GTN. These results would not be expected if tolerance-induced increases in superoxide were a causative factor for the reduced relaxation response in tolerance. We conclude that neither reduced flavoprotein-dependent metabolic activation of organic nitrates, such as that mediated by CPR, nor increased superoxide due to increased NAD(P)H oxidase activity can account for the development of in vivo tolerance to GTN.