青藤碱对家兔主动脉血管平滑肌细胞内游离钙浓度及蛋白激酶C的影响

EFFECTS OF SINOMENINE ON INTRACELLULAR FREE CALCIUM CONCENTRATION AND THE ACTIVITY OF PROTEIN KINASE IN CULTURED RABBIT AORTIC SMOOTH MUSCLE CELLS

目的观测青藤碱对培养家兔血管平滑肌细胞内游离钙浓度及正常和缺血缺氧刺激下蛋白激酶C(PKC)活性的影响。方法Fura-2/AM作Ca2+指示剂,检测青藤碱对培养家兔主动脉血管平滑肌细胞静息Ca2+浓度及去甲肾上腺素,高K+,咖啡因刺激作用下的改变,并与钙拮抗剂维拉帕米进行对照研究;复制血管平滑肌细胞缺血缺氧模型,液闪仪测定PKC活性。结果青藤碱剂量依赖性抑制高K+去极化引起[Ca2+]i升高,青藤碱10×10-6mol·L-1、3×10-5mol·L-1、10-4mol·L-1,对NE通过受体介导引起的[Ca2+]i增高也有明显抑制。但对静息状态下及咖啡因刺激的血管平滑肌细胞[Ca2+]i无明显影响。正常时,青藤碱处理后血管平滑肌细胞胞浆、胞膜PKC活性均升高;缺血缺氧状态下,胞浆PKC活性升高,但胞膜PKC活性降低,青藤碱处理后胞浆PKC活性下降,胞膜PKC活性上升。结论青藤碱可能抑制血管平滑肌细胞电压依赖性钙通道和受体操纵性钙通道,降低细胞内游离钙水平,调节缺血缺氧条件下血管平滑肌细胞PKC活性。

To explore the effects of sinomenine（Sin） on intracellular free calcium（[Ca^2＋]i） and the activity of PKC（ protein kinase C） of the cultured aortic vascular smooth muscle celIs（VSMC） during ischemia and hypoxia. Methods： The effect of Sin on changes in[Ca^2＋]i were determined in cultured rabbit VSMC after exposure to high K^＋ , norepinephrine（NE） and caffeine（Caf）. Fluorescent Ca^2＋ -indicater fura-2/AM was used.The effects of Sin were compared, with that of verapamil（Ver） .The hypoxia model was made, then the activity of PKC was measured by γ scintillation counting instrument. Results： Sin（ 10 × 10^-6mol.L^-1,3× 10^-5mol.L^-1, 10^-4mol.L^-1） inhibited the elevation of [Ca^2＋]i induced by high K＋ -depolarization in a concentration dependent manner. In addi-tion,Sin inhibited the elevation of [Ca^2＋]i induced by NE in the presence of extracellular Ca^2＋ . In the ab^nce of extracetlular Ca^2＋ , Sin（3 × 10^-5mol.L^-1） also had no blocking effect on the NE-induced[Ca^2＋]i increase. It was found that the activity of PKC treated with Sin in VSMC cytoplasm and cell membrane in normal condition increased, the activity of PKC in cytoplasm in ischemia and hy-poxia situation increased, but the activity of PKC in cell membrane decreased. When VSMC was treated with Sin, the activity of PKC in cytoplasm decreased and that of cell membrane increased. Conclusion： The results suggest that Sin might decrease the[Ca^2＋] i of VSMC by blocking hoth VDC and ROC, could retaliate the PKC activities induced by ischemia and hypoxia.