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目的研究NCA[硝酰基(HNO,一氧化氮的单电子还原产物,对活体心脏发挥正性肌力作用)供体,1-nitrosocyclohexylacetate]的作用效果及其巯基靶点机制。方法大鼠右心室的完整梳状肌被连接在张力换能器与刺激电极之间,肌小节长度设定在2.2～2.3μm之间,K-H液表面灌流后(pH 7.4,室温),Fura-2经玻璃微电极负载进行离子透入法检测[Ca2+]i同时测定心肌收缩张力的变化。Western blotting方法检测NCA对Tm(肌原纤维蛋白)、MLC(肌凝蛋白轻链)和TnI(肌钙蛋白I)的影响。结果收缩力在NCA作用下呈剂量依赖性增加(20～100μmol.L-1)。不同频率作用下(0.5～3.0 Hz,NCA 20μmol.L-1,Ca2+0.5μmol.L-1)收缩力的增加(P<0.01)和[Ca2+]i瞬变(P>0.05)没有受到明显的影响。同对照组相比,NCA处理组去肌膜心肌收缩力明显增加(P<0.05),且作用效果具有结构唯一性特点。非还原条件下Western blotting可见Tm出现交联。巯基还原剂二硫苏糖醇(DTT,5.0μmol.L-1)能够阻止并逆转NCA的活动。结论 NCA提供的硝酰基是心脏钙离子增敏剂,心肌调节蛋白质巯基翻译后修饰可能是其靶点作用所在。
Objective To investigate the effect and the mercapto-targeting mechanism of NCA [nitrosyl (HNO), a mono electron reduction product of nitric oxide, a donor of 1-nitrosocyclohexylacetate, an inotropic agent on living heart). Methods The intact comb muscles of the right ventricle were connected between the tension transducer and the stimulation electrode. The length of the muscle segment was set between 2.2 and 2.3 μm. After the surface of KH solution was perfused (pH 7.4, room temperature), Fura- 2 [Ca2 +] i was measured by iontophoresis on the glass microelectrode. Simultaneously, the change of myocardial contractile tension was measured. The effects of NCA on Tm (myofibrillar), MLC (myosin light chain) and TnI (troponin I) were examined by Western blotting. Results Contractility increased in a dose-dependent manner under the action of NCA (20-100 μmol·L-1). Contraction force (P <0.01) and [Ca2 +] i transient (P> 0.05) were not significantly affected by different frequencies (0.5-3.0 Hz, NCA 20μmol.L-1, Ca2 + 0.5μmol.L-1) Impact. Compared with the control group, the contractile force of myocardium in NCA treated group was significantly increased (P <0.05), and the effect was unique in structure. Western blotting under non-reducing conditions shows cross-linking of Tm. The thiol reductant dithiothreitol (DTT, 5.0 μmol·L-1) blocked and reversed NCA activity. Conclusions The nitroxyl provided by NCA is a cardiac calcium ion sensitizer, and the post-translational modification of the myocardial regulatory protein mercapto may be the target of its role.