cyclic-gmp and resorufin

1. The effects of 7-ethoxyresorufin (7-ER), which is a substrate for and competitive inhibitor of cytochrome P450, were studied on responses to nitric oxide (NO), the NO donors sodium nitroprusside (SNP) and glyceryl trinitrate (GTN), acetylcholine-induced endothelium-dependent relaxations of rat and rabbit aortic rings and nitrergic nerve stimulation-induced relaxations of rat anococcygeus muscles. 2. In rat and rabbit aortic rings, 7-ER (2 microM) inhibited the relaxations to acetylcholine in endothelium-intact preparations and the relaxant action of NO in endothelium-denuded preparations. Relaxant responses to SNP and GTN were inhibited by 7-ER in the rat but not rabbit aortic rings. However, the relaxant actions of papaverine and 8-bromo-cyclic GMP were not affected by 7-ER. 3. In rat anococcygeus muscles, 7ER (2 microM) inhibited the relaxant action of NO, but relaxations elicited by nitrergic nerve stimulation were only partly inhibited by a higher concentration of 7-ER (10 microM). 4. After inhibition by 7-ER, superoxide dismutase (100 u ml-1) restored NO-induced relaxations of the rat aortic rings, but not acetylcholine-, SNP or GTN-induced relaxations, and restored NO- and nitrergic nerve stimulation-induced relaxations of anococcygeus muscles. 5. Another cytochrome P450 inhibitor, troleandomycin (10-30 microM), had no effect on NO- or acetylcholine-induced relaxations of rat aortic rings and NO- or nitrergic nerve stimulation-induced relaxations of anococcygeus muscles. However, resorufin, an analogue of 7-ER, inhibited responses to acetylcholine, NO and GTN in rat aortic rings. 6. The results suggest that 7-ER inhibited responses to NO and nitrergic nerve stimulation through generation of superoxide radicals. However, an additional mechanism may be involved in the reduction in acetylcholine-induced response in aortic rings. 7. A 7-ER sensitive P450 system may be involved in the bioactivation of GTN and SNP in rat aortic rings, but not in rabbit aorta or rat anococcygeus muscles.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Arginine; Binding, Competitive; Catalase; Cyclic GMP; Cytochrome P-450 Enzyme Inhibitors; Enzyme Inhibitors; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Oxazines; Rabbits; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Troleandomycin; Vasodilator Agents

We examined the effect of the cytochrome P-450 substrate, 7-ethoxyresorufin (7-ER), and its corresponding product, resorufin, on nitrovasodilator- and endothelium-dependent relaxation of isolated rat aorta. The EC50 value for glyceryl trinitrate (GTN) induced relaxation was increased over 100-fold by 7-ER and less than 3-fold by resorufin. The EC50 value for sodium nitroprusside (SNP) induced relaxation was increased approximately 12-fold by 7-ER, acetylcholine (ACh) induced relaxation was abolished, and relaxation induced by isopropylnorepinephrine was not significantly affected. GTN-, SNP-, and ACh-induced increases in cyclic GMP accumulation were inhibited by 7-ER, as were basal cyclic GMP levels in endothelium-intact, but not endothelium-denuded tissues. 7-ER decreased GTN biotransformation in intact aorta and decreased the regioselective formation of glyceryl-1,2-dinitrate. The activation by GTN and SNP of aortic guanylyl cyclase in broken cell preparations was not affected by 7-ER, indicating that the inhibitory effect of 7-ER is probably not due to a direct interaction with guanylyl cyclase. The inhibitory effect of 7-ER on GTN-induced relaxation was not altered by the addition of superoxide dismutase, suggesting that 7-ER does not act by increasing superoxide anion concentration (which would serve to increase the degradation of nitric oxide (NO) formed during vascular GTN biotransformation). Our data provide further evidence for the role of the cytochrome P-450--cytochrome P-450 reductase system in the biotransformation of GTN to an activator (presumably nitric oxide) of guanylyl cyclase. The data are consistent with a mode of action of 7-ER involving either competitive inhibition of vascular cytochrome P-450 or uncoupling of vascular cytochrome P-450 reductase from cytochrome P-450. The data also suggest that the cytochrome P-450 system facilitates NO release from SNP and that 7-ER has an inhibitory effect on endothelial nitric oxide synthase.

Topics: Acetylcholine; Adrenergic beta-Agonists; Animals; Aorta; Biotransformation; Cyclic GMP; Cytochrome P-450 Enzyme System; Drug Interactions; Endothelium, Vascular; Enzyme Activation; Guanylate Cyclase; In Vitro Techniques; Isoproterenol; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitroglycerin; Nitroprusside; Oxazines; Rats; Rats, Sprague-Dawley; Vasodilator Agents