束缚应激大鼠血小板及血管内膜的L-精氨酸/一氧化氮合酶/一氧化氮通路下调

Restraint stress down-regulates L-Arg/NOS/NO pathway of platelet and aortic intima in rats

目的:观察束缚应激对大鼠血管内膜及血小板L精氨酸(LArg)/一氧化氮合酶(nitricoxidesynthase,NOS)/一氧化氮(NO)通路的影响及其相互联系,探讨血管应激损伤的发病机制。方法:大鼠束缚-浸水(waterimmersionrestraint,WIR)应激7h,以胃溃疡指数作为机体应激损伤的的标识,采用Greiss法测定离体血管内膜及血小板孵育生成的亚硝酸盐(NO2-)含量;以同位素示踪法检测其NOS活性及LArg转运。结果:WIR应激大鼠呈现典型应激性胃溃疡,主动脉血管内膜及血小板NO2-生成分别较对照组低46%和57%(均P<0.01),且Person相关分析显示两者变化呈现明显的正相关(r2=0.9835,P<0.01)。与对照组比较,WIR大鼠血小板NOS酶催化效率显著降低,Km值增加18%,最大酶促反应速率(Vmax)降低44%(均P<0.01);血管内膜NOS活性亦显著降低(-50%,P<0.01),血小板和血管内膜NOS活性呈正相关变化(r2=0.9726,P<0.01)。WIR组大鼠血小板LArg的跨膜转运能力较对照组明显抑制,转运的Vmax值低32%,Km值高37%(P<0.01)。血管内膜LArg转运较对照组降低27%,两者呈明显正相关(r2=0.9887,P<0.01)。结论:WIR应激下调血管内膜和血小板的LArg/NOS/NO通路,血管内膜和血小板LArg/NOS/NO通路的变化呈显著正相关,提示检测血小板LArg/NOS/NO通路的变化可能反映应激状态下血管内皮功能的损伤。

Objective： To investigate alteration and cross link of the aortic intima and platelet endogenous L-Arg/NOS/NO pathway induced by water immersion restraint （WIR） stress. Methods： After 7 h of WIR stress, the aortic intima was isolated and prepared the platelet, then NO2 - production released from aortic intima and platelet was measured with Greiss regent, NOS activity and L-arginine transport activity were detected by isotope tracer method. Results： After 7 h of WIR stress, the levels of NO2^- from platelet and aortic intima obviously decreased by 57% and 46% respectively as comparied with the control rats （P 〈0.01 ）. The inhibitory effects of NO2^- showed a positive correlation between platelet and aortic intima by person regression analysis （r^2=0.9835, P 〈0.01 ）. The kinetics analysis of NOS activity of platelet showed that the enzymatic catalytic maximum velocity （ Vmax） decreased by 44%, Km values increase by 18% and the enzymatic catalytic efficiency was obviously reduced in the WIR stress rats （P 〈0.01 ）. The aortic intima NOS activity was inhibited by 50% after WIR stress （P 〈0.01 ）. The person analysis showed the obvious correlation between them （r2 =0.9726, P 〈0.01 ）. In the WIR stress platelet, the L-Arg transport Vmax obviously reduced by 32%, Km parameter increased 37%, and the transport efficiency was inhibited by 50% （P 〈0.01 ）, which exhibited obvious positive correlation with the inhibition of L-Arg transport activity in aortic intima （27%） of WIR stress rats （ r^2 = 0.9887, P 〈 0.01）. Conclusion： WIR stress down-regulated endogenous L-Arg/NOS/NO pathway of aortic intima and platelet, whose alteration showed obvious positive correlation. Detection of the alteration of endogenous L-Arg/NOS/NO pathway in platelet might act as an indirect method to assess the endothelial dysfunction involving the pathogensis of stress.