Alcón S, Morales S, Camello P J, Hemming J M, Jennings L, Mawe G M, Pozo M J
Department of Physiology, University of Extremadura, 10071 Cáceres, Spain.
J Physiol. 2001 May 1;532(Pt 3):793-810. doi: 10.1111/j.1469-7793.2001.0793e.x.
The purpose of this study was to determine the effects of sodium nitroprusside (SNP), 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NO) and 3-morpholinosydnonimine (SIN-1), NO donors which yield different NO reactive species (NO+, NO* and peroxynitrite, respectively), as well as exogenous peroxynitrite, on gall bladder contractility. Under resting tone conditions, SNP induced a dose-dependent contraction with a maximal effect (10.3 +/- 0.7 mN, S.E.M.) at 1 mM. Consistent with these findings, SNP caused a concentration-dependent depolarization of gall bladder smooth muscle. The excitatory effects of SNP were dependent on extracellular calcium entry through L-type Ca2+ channels. Furthermore, the contraction and depolarization were sensitive to tyrosine kinase blockade, and an associated increase in tyrosine phosphorylation was detected in Western blot studies. DETA/NO induced dose-dependent relaxing effects. These relaxations were sensitive to the guanylyl cyclase inhibitor 1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one (ODQ, 2 microM) but they were not altered by treatment with the potassium channel blockers tetraethylammoniun (TEA, 5 mM) and 4-aminopyridine (4-AP, 5 mM). When tested in a reducing environment (created by 2.5 mM 1,4-dithiothreitol, DTT), SNP caused a relaxation of gall bladder muscle strips. Similarly, the SNP-induced contraction was converted to a relaxation, and associated hyperpolarization, when DTT was added during the steady state of an SNP-induced response. SIN-1 (0.1 mM), which has been shown to release peroxynitrite, induced relaxing effects that were enhanced by superoxide dismutase (SOD, 50 U ml(-1)). The relaxations induced by either SIN-1 alone or SIN-1 in the presence of SOD were strengthened by catalase (1000 U ml(-1)) and abolished by ODQ pretreatment. However, exogenous peroxynitrite induced a concentration-dependent contraction, which was dependent on activation of leukotriene (LT) metabolism and extracellular calcium. The peroxynitrite-induced contraction was abolished in the presence of the peroxynitrite scavenger melatonin. These results suggest that SIN-1 behaves as an NO* rather than a peroxynitrite source. We conclude that, depending on the redox state, NO has opposing effects on the motility of the gall bladder, being a relaxing agent when in NO * form and a contracting agent when in NO+ or peroxynitrite redox species form. Knowledge of the contrasting effects of the different redox forms of NO can clarify our understanding of the effects of NO donors on gall bladder and other smooth muscle cell types.
本研究的目的是确定硝普钠（SNP）、2,2'-（羟基亚硝基肼基）双乙胺（DETA/NO）和3-吗啉代西多非明（SIN-1）这三种分别产生不同NO反应性物种（分别为NO⁺、NO和过氧亚硝酸盐）的NO供体以及外源性过氧亚硝酸盐对胆囊收缩性的影响。在静息张力条件下，SNP诱导剂量依赖性收缩，在1 mM时达到最大效应（10.3±0.7 mN，标准误）。与这些发现一致，SNP引起胆囊平滑肌浓度依赖性去极化。SNP的兴奋作用依赖于通过L型Ca²⁺通道的细胞外钙内流。此外，收缩和去极化对酪氨酸激酶阻断敏感，并且在蛋白质印迹研究中检测到酪氨酸磷酸化相关增加。DETA/NO诱导剂量依赖性舒张作用。这些舒张作用对鸟苷酸环化酶抑制剂1H-[1,2,4]恶二唑并[4,3-a]喹喔啉-1-酮（ODQ，2 μM）敏感，但用钾通道阻滞剂四乙铵（TEA，5 mM）和4-氨基吡啶（4-AP，5 mM）处理后未改变。在还原环境（由2.5 mM 1,4-二硫苏糖醇，DTT产生）中进行测试时，SNP引起胆囊肌条舒张。同样，当在SNP诱导反应的稳态期间加入DTT时，SNP诱导的收缩转变为舒张，并伴有超极化。已证明能释放过氧亚硝酸盐的SIN-1（0.1 mM）诱导的舒张作用被超氧化物歧化酶（SOD，50 U ml⁻¹）增强。单独的SIN-1或存在SOD时SIN-1诱导的舒张作用被过氧化氢酶（1000 U ml⁻¹）增强，并被ODQ预处理消除。然而，外源性过氧亚硝酸盐诱导浓度依赖性收缩，这依赖于白三烯（LT）代谢的激活和细胞外钙。在过氧亚硝酸盐清除剂褪黑素存在下，过氧亚硝酸盐诱导的收缩被消除。这些结果表明SIN-1表现为NO而非过氧亚硝酸盐来源。我们得出结论，根据氧化还原状态，NO对胆囊运动有相反的作用，以NO*形式存在时是舒张剂，以NO⁺或过氧亚硝酸盐氧化还原物种形式存在时是收缩剂。了解NO不同氧化还原形式的对比效应可以阐明我们对NO供体对胆囊和其他平滑肌细胞类型影响的理解。
