García-Pascual A, Costa G, Labadía A, Jimenez E, Triguero D
Departamento de Fisiología, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
Naunyn Schmiedebergs Arch Pharmacol. 1999 Jul;360(1):80-91. doi: 10.1007/s002109900038.
We have examined the mechanisms of action of a broad spectrum of nitric oxide (NO) donors, including several S-nitrosothiols, sodium nitroprusside (SNP) and nitroglycerine (GTN), in relation to their relaxant activity of urethral smooth muscle. For all the compounds examined, NO release (in solution and in the presence of urethral tissue), relaxation responses, elevations in cGMP levels and the effect of thiol modulators were evaluated and compared 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 affected 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 found for S-nitroso-L-cysteine (CysNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in the presence of urethral cytosolic fractions, suggesting metabolic 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-nitrosoglutathione (GSNO) and SNP were reduced by the presence of urethral homogenate and/or subcellular fractions, suggesting direct NO transfer to tissue constituents. NO donors and NO gas induced dissimilar degrees of cGMP accumulation in urethral tissue, while they were essentially equipotent as urethral relaxants. Furthermore, 1H-[1,2,4] -oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ; 10 microM) inhibited both relaxation and cGMP accumulations, but with different potency for the different compounds. Oxidation of sarcolemmal thiol groups with 5-5'-dithio-bis[2-nitrobenzoic acid] (DTNB; 0.5 mM) enhanced relaxations to GSNO, an effect that was reversed by dithiotreitol (DTT; 1 mM), suggesting 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-cGMP-mediated relaxations. This indicates that the assumption that SNP is fully depleted of NO by exposure to light should be revised. It can be concluded that important differences exist in the mechanisms by which distinct NO donors relax urethral smooth muscle and they cannot be regarded simply as NO-releasing prodrugs.
我们研究了多种一氧化氮（NO）供体的作用机制，包括几种S-亚硝基硫醇、硝普钠（SNP）和硝酸甘油（GTN），及其对尿道平滑肌的舒张活性。对于所有检测的化合物，评估了其NO释放（在溶液中以及存在尿道组织的情况下）、舒张反应、cGMP水平升高以及硫醇调节剂的作用，并与NO本身的作用进行了比较。虽然除GTN外的所有NO供体由于光解或化学催化在溶液中释放NO，但这种释放与它们在绵羊尿道制剂中的舒张活性无关，而且它们不受NO清除剂2-(4-羧基苯基)-4,4,5,5-四甲基咪唑啉-1-氧基3-氧化物（cPTIO；0.3 mM）的影响。在存在尿道胞质组分的情况下，发现S-亚硝基-L-半胱氨酸（CysNO）和S-亚硝基-N-乙酰-D,L-青霉胺（SNAP）具有显著的NO生成活性，表明在靶组织的胞质溶胶中代谢活化为NO。相比之下，尿道匀浆和/或亚细胞组分的存在会降低S-亚硝基-N-乙酰-L-半胱氨酸（N-ac-CysNO）、S-亚硝基谷胱甘肽（GSNO）和SNP的NO生成，表明NO直接转移至组织成分。NO供体和NO气体在尿道组织中诱导的cGMP积累程度不同，而它们作为尿道舒张剂的作用基本相当。此外，1H-[1,2,4]-恶二唑-[4,3-a]-喹喔啉-1-酮（ODQ；10 microM）抑制舒张和cGMP积累，但对不同化合物的效力不同。用5,5'-二硫代双[2-硝基苯甲酸]（DTNB；0.5 mM）氧化肌膜硫醇基团可增强对GSNO的舒张作用，二硫苏糖醇（DTT；1 mM）可逆转这种作用，表明通过细胞膜上的亚硝基化/氧化反应产生直接作用，而对NO和所有其他化合物的舒张作用不受这些处理的影响。最后，SNP的光降解诱导形成一种稳定的中间体，该中间体仍能引发NO-cGMP介导的舒张作用。这表明关于SNP通过光照完全耗尽NO的假设应予以修正。可以得出结论，不同的NO供体舒张尿道平滑肌的机制存在重要差异，不能简单地将它们视为释放NO的前体药物。
