溶血磷脂酸激活大鼠血小板L-精氨酸/一氧化氮通路

Lysophosphatidic acid activates L-arginine/nitric oxide pathway of platelets in rats

目的观察溶血磷脂酸(LPA)对血小板L-精氨酸/一氧化氮合酶/一氧化氮(L-Arg/NOS/NO)通路的影响,并探讨其与血小板功能变化的关系。方法LPA(10-6,10-5和5×10-5mol/L)与大鼠血小板混悬液共孵育,采用Greiss法测定血小板孵育液中亚硝酸盐(NO2-)含量;同位素示踪法检测血小板NOS活性及L-Arg转运;荧光探针法测定血小板内游离钙浓度。结果LPA孵育30和60min,血小板NO释放分别增加了35%和56%(P<0.01),LPA(10-6,10-5和5×10-5mol/L)呈浓度依赖性增加了血小板NO的生成,EC50为17.8μmol/L,95%CI为13.1～24.2μmol/L(P<0.01)。LPA浓度依赖性增加了NOS活性和血小板L-Arg的转运(P<0.01)。LPA(5×10-5mol/L)孵育30和60min,显著升高血小板[Ca2+]i水平(P<0.01)。NOS抑制剂L-NAME预处理的血小板,LPA升高[Ca2+]i的反应分别增强20%和32%(P<0.01);加入NO供体L-Arg预处理,则明显抑制LPA升高[Ca2+]i,分别减少14%和18%(P<0.01)。结论LPA通过激活血小板L-Arg转运和NOS活性,上调L-Arg/NOS/NO通路,增加血小板NO生成。

Objective： To investigate the mechanism of platelet function caused by Lysophosphatidic acid （LPA）, by observing the change of the L-arginine/nitric oxide synthase/nitric oxide （L-Arg/NOS/ NO） pathway of platelet in rats. Methods：LPA （10^-6, 10^-5 and 5 × 10^-5 mol/L） was administrated in rats and incubated for 30 and 60 minutes. The nitrite production was measured by Greiss assay ; NOS activities and L-arginine transportation were detected by isotope tracer method and intracellular [ Ca^2＋ ] i changes by fluorescent probe. Results：LPA increased NO release by 35% and 56%, after incubating for 30 and 60 minutes, respectively. LPA （10^-6, 10^ -5 and 5 × 10 ^-5 mol/L） enhanced the NO productions of platelets in a concentration-dependent manner （P 〈0.01 ）. EC50 was 17.8 μmol/L, and 95% CI was 13.3 -24.2 μmol/L, involved in the physiological concentration of LPA in plasma （P 〈0.01 ）. Simultaneously, different doses of LPA increased NOS activities and L-arginine uptake in a dose-dependent manner （P 〈0.01 ）. In this study, LPA （50 μmol/L） increased the intracellular free calcium ion concentration （ [ Ca^2＋ ] i, P 〈0.01 ）, after incubating for 30 and 60 minutes. Pre-treated with NOS inhibi- tor-L-NAME for 20 minutes, LPA obviously enhanced the effects by 20% and 32% respectively （ P 〈 0.01 ）. On the contrary, pre-treated with L-arginine （200 μmol/L） for the same times obviously reduced the effects by 14% and 18% respectively （P 〈 0.01 ）. Conclusion：LPA increased NO release by enhancing L-arginine uptake and NOS activities, up-regulating L-arginine/NOS/NO pathway in platelets of rats.