【摘要】 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。更多还原

【Abstract】 OBJECTIVE To test the effect of 1-nitrosocyclohexyl acetate(NCA),a newly developed nitroxyl(HNO) donor,on myofilaments and its action mechanism for the thiol in tropomyosin.METHODS Trabeculae were dissected from the right ventricule of rat heart and mounted between a force transducer and a motor arm.The muscles were superfused with K-H solution(pH 7.4) at room temperature.Fura-2 salt was loaded into the trabeculae via electrophoresis,and the sarcomere length was set at 2.2-2.3 μm.Western blotting tested the change of tropomyosin.RESULTS Twitch force increased in a dose dependent manner in the presence of NCA in the range of 20-100 μmol·L-1.Force increased(P<0.01) and i transients remained unchanged(P>0.05) with different frequencies(0.5-3.0 Hz) with NCA of 20 μmol·L-1 and Ca2+ of 0.5 μmol·L-1.Skinned force increased as compared to the control(P<0.05).The effect of NCA had characteristics of structure uniqueness.A cross-linking band in tropomyosin was seen by Western blotting under non-reducing condition.The thiol reducing agent dithiothreitol(DTT) both prevented and reversed HNO action.CONCLUSION The donor of HNO,NCA,represents a new class of agents capable of directly sensitizing cardiac myofilaments to Ca2+.Post-translational thiol modification of the key regulatory myofilament protein(i.e.tropomyosin) may underlie the effect of NCA.更多还原