Differential mechanisms of urethral smooth muscle relaxation by several NOdonors and nitric oxide

We have examined the mechanisms of action of a broad spectrum of nitric oxi de (NO) donors, including several S-nitrosothiols, sodium nitroprusside (SN P) and nitroglycerine (GTN), in relation to their relaxant activity of uret hral smooth muscle. For all the compounds examined, NO release tin solution and in the presence of urethral tissue), relaxation responses, elevations in cGMP levels and the effect of thiol modulators were evaluated and compar ed with the effect of NO itself. Whilst all NO donors, except GTN, released NO in solution due to photolysis or chemical catalysis, this release was not correlated with their relaxant activity in sheep urethral preparations, which were furthermore not affect ed by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline- 1-oxyl 3-oxide (cPTIO; 0.3 mM). A substantial NO-generating activity was fo und for S-nitroso -L-cysteine (CysNO) and S-nitroso-N-acetyl-D,L-penicillam ine (SNAP) in the presence of urethral cytosolic fractions, suggesting meta bolic activation to NO in the cytosol of the target tissue. In contrast, NO generation from S-nitroso-N-acetyl-L-cysteine (N-ac-CysNO), S-nitrosogluta thione (GSNO) and SNP were reduced by the presence of urethral homogenate a nd/or subcellular fractions, suggesting direct NO transfer to tissue consti tuents. NO donors and NO gas induced dissimilar degrees of cGMP accumulatio n in urethral tissue, while they were essentially equipotent as urethral re laxants. Furthermore, 1H-[1,2,4]-oxadiazole-[4,3-a] -quinoxalin-1-one (ODQ; 10 mu M) inhibited both relaxation and cGMP accumulations, but with differ ent potency for the different compounds. Oxidation of sarcolemmal thiol gro ups with 5-5'-dithio-bis[2-nitrobenzoic acid] (DTNB; 0.5 mM) enhanced relax ations to GSNO, an effect that was reversed by dithiotreitol (DTT; 1 mM), s uggesting a direct effect through nitrosylation/oxidation reactions at the cell membrane, while relaxations to NO and to all the other compounds were not affected by these treatments. Finally, photodegradation of SNP induced the formation of a stable intermediate that still evoked NO-cCMP-mediated r elaxations. This indicates that the assumption that SNP is fully depleted o f NO by exposure to light should be revised. It can be concluded that important differences exist in the mechanisms by w hich distinct NO donors relax urethral smooth muscle and they cannot be reg arded simply as NO-releasing prodrugs.