EFFECT OF YC-1, AN NO-INDEPENDENT, SUPEROXIDE-SENSITIVE STIMULATOR OFSOLUBLE GUANYLYL CYCLASE, ON SMOOTH-MUSCLE RESPONSIVENESS TO NITROVASODILATORS

1 We studied the effects of 3-(5'-hydroxymethyl-2'furyl)-1-benzyl inda zole (YC-1) on the activity of purified soluble guanylyl cyclase (sGC) , the formation of guanosine-3':5' cyclic monophosphate (cyclic GMP) i n vascular smooth muscle cells (VSMC), and on the tone of rabbit isola ted aortic rings preconstricted by phenylephrine (PE). In addition, we assessed the combined effect of YC-1, and either NO donors, or supero xide anions on these parameters. 2 YC-1 elicited a direct concentratio n-dependent activation of sGC (EC(50) 18.6 +/- 2.0 mu M), which was ra pid in onset and quickly reversible upon dilution. YC-1 altered the en zyme kinetics with respect to GTP by decreasing K-M and increasing V-m ax. Activation of sGC by a combination of sodium nitroprusside (SNP) a nd YC-1 was superadditive at low and less than additive at high concen trations, indicating a synergistic activation of the enzyme by both ag ents. A specific inhibitor of sGC, -(1,2,4)oxdiazolo-(4,3-a)-6-bromo-q uinoxazin-1-one (NS 2028), abolished activation of the enzyme by eithe r compound. 3 YC-1 induced a concentration-dependent increase in intra cellular cyclic GMP levels in rat cultured aortic VSMC, which was comp letely inhibited by NS 2028. YC-1 applied at the same concentration as SNP elicited 2.5 fold higher cyclic GMP formation. Cyclic GMP-increas es in response to SNP and YC-1 were additive. 4 YC-1 relaxed preconstr icted endothelium-denuded rabbit aortic rings in a concentration-depen dent manner (50% at 20 mu M) and markedly increased cyclic GMP levels. Relaxations were inhibited by NS 2028. A concentration of YC-1 (3 mu M), which elicited only minor effects on relaxation and cyclic GMP, in creased the vasodilator potency of SNP and nitroglycerin (NTG) by 10 f old and markedly enhanced SNP- and NTG-induced cyclic GMP formation. 5 Basal and YC-1-stimulated sGC activity was sensitive to inhibition by superoxide (O-2(-)) generated by xanthine/xanthine oxidase, and was p rotected from this inhibition by superoxide dismutase (SOD). YC-1 1-st imulated sGC was also sensitive to inhibition by endogenously generate d (O-2(-) in rat preconstricted endothelium-denuded aortic rings. Rela xation to YC-1 was significantly attenuated in aortae from spontaneous ly hypertensive rats (SHR), which generated O-2(-) at a higher rate th an aortae from normotensive Wistar Kyoto rats (WKY). SOD restored the vasodilator responsiveness of SHR rings to YC-1. 6 In conclusion, thes e results indicate that YC-1 is an NO-independent, O-2(-)-sensitive, d irect activator of sGC in VSMC and exerts vasorelaxation by increasing intracellular cyclic GMP levels. The additive or even synergistic res ponses to NO-donors and YC-1 in cultured VSMC and isolated aortic ring s apparently reflect the direct synergistic action of YC-1 and NO on t he sGC. The synergism revealed in this in vitro study suggests that lo w doses of YC-1 may be of therapeutic value by permitting the reductio n of nitrovasodilator dosage.