Use of a nitronyl nitroxide to discriminate the contribution of nitric oxide radical in endothelium-dependent relaxation of control and diabetic blood vessels.

Nitronyl nitroxides react with nitric oxide radical (.NO) to form imino nitroxides. We used a nitronyl nitroxide, [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3 oxide] (CPTIO) to evaluate the contribution of .NO to basal tone and acetylcholine-induced endothelium-dependent relaxation in control vs. diabetic rat aortic rings. In rings precontracted with phenylephrine, CPTIO produced an additional increment in tension that was greater in control vs. diabetic rings. Tension after CPTIO was similar to that observed in rings pretreated with the NO synthase inhibitor, L-nitroarginine or in rings without endothelium. This increment was insensitive to indomethacin, cysteine, tetraethylammonium or catalase, but was sensitive to inhibition by the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxaline-1-one. L-Nitroarginine blocked relaxation to ACH by 100 and 90% in control and diabetic rings, respectively. In contrast, CPTIO produced a concentration-dependent inhibition of ACH-induced relaxation that was greater in control rings. The residual CPTIO-resistant component of relaxation was equivalent to 26 and 43% of initial precontraction in control vs. diabetic rings, respectively, and was not altered by indomethacin, catalase, cysteine or tetraethylammonium but was significantly inhibited by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxaline-1-one. These data suggest the release of additional unknown factor(s) that cannot be discerned using NO synthase inhibitors only. This CPTIO-resistant dilator is likely not a cyclooxygenase product or a hyperpolarizing factor but a factor that acts, in part, by activation of guanylate cyclase. This substance is possibly .NO that is not available for reaction with CPTIO either by its diffusibility and sequestration or molecular rearrangement to a redox active form (i.e., not free .NO) or is a completely different vasodilator. The use of a more lipid soluble nitronyl nitroxide derivative suggests a portion of the CPTIO-resistant relaxation in diabetic (but not control) rings could be explained by .NO sequestered in the lipid phase.