氧化低密度脂蛋白及辛伐他汀对平滑肌细胞信号通路蛋白激酶活性和胞浆内游离钙的影响

THE EFFECT OF ox-LDL AND SIMVASTATIN ON PKC ACTIVITY AND CYTOSOLIC FREE Ca^(2+) IN CULTURED RAT AORTIC SMOOTH MUSCLE CELLS

分别应用γ 3 2 P ATP磷酸转移法和Fluo 3/Am荧光负载、流式细胞术检测氧化修饰LDL(oxLDL)及辛伐他汀对大鼠主动脉平滑肌细胞 (ASMC)蛋白激酶C(PKC)和胞浆内游离钙 ([Ca2 + ]i)水平的变化。结果表明 ,oxLDL呈剂量依赖方式促进ASMCPKC活性增加 ,14min时达峰值 ,然后缓慢下降 ,30min仍维持较高水平。胞浆内 [Ca2 + ]i升高分两个时相 ,即快速相和持续相。移去细胞外液钙 ,oxLDL仍引起快速相 ,但持续相消失 ,而辛伐他汀能明显抑制oxLDL引起的ASMCPKC活性增加 ,并显著降低持续相胞浆内钙水平 ,但对快速相无影响。提示oxLDL能引起ASMC内信号通路PKC及 [Ca2 + ]i的动态变化 ,二者密切相关。oxLDL刺激ASMC[Ca2 + ]i升高的快速相是由胞浆钙池释放引起 ,持续相升高是由胞外钙内流引起。辛伐他汀抑制ASMCPKC活性可能是通过胞内 [Ca2 +

The change of PKC activity and cytosolic free Ca 2+ induced by oxLDL and simvastatin in ASMC were measured by its ability to transfer phosphate from 32P-ATP to lysine-rich histone and flow cytometric analysis loading with the Ca 2+ dye fluo-3/Am, respectively. As a result, oxLDL increased total PKC activity in a dose-dependent manner with phase peaking at 14 min, then decreased slowly and maintained for at least 30 min, while oxLDL induced biphasic [Ca 2+]i responses including rapid initial transient phase and sustained phase. Removal of extracellular Ca 2+did not inhibit the rapid phase of oxLDL-induced rise in [Ca 2+]i, but abolished the sustained phase of [Ca 2+]i in response to oxLDL. Activity of PKC was markedly decreased and simvastatin did not impair the initial peak in response to oxLDL but significantly reduced the subsequent plateau phase when simvastatin was added. The results suggested that oxLDL can induce dynamic changes of signal transduction of PKC and cytosolic free Ca 2+in ASMC. The rapid initial transient phase was due to the mobilization of [Ca 2+]i from intracellular Ca 2+ pool and the sustained phase resulted from the influx of extracellular Ca 2+. The inhibition of PKC activity induced by simvastatin may contribute to the changes of intracellular Ca 2+.