摘要目的 探讨腺苷三磷酸(ATP)对冠状动脉平滑肌细胞大电导钙激活钾通道(BK通道)的激活作用及其机制.方法 采用酶消化法急性分离正常大鼠冠状动脉平滑肌细胞;膜片钳实验技术记录全细胞状态下BK通道电流;钙离子成像技术记录ATP对细胞内钙离子浓度影响.结果 膜片钳实验结果显示,加入1 mmol/L ATP前后,BK通道电流密度分别为(137±13) pA/pF和(179±15)pA/pF(P ＜0.05);钙离子成像结果显示,加入0.5 mmol/L ATP前后,细胞内钙离子浓度荧光信号强度比值分别为2.46±0.08和4.04±0.21 (P＜0.05).分别采用嘌呤(P2Y1)受体阻滞剂MRS2179、磷脂酶C(PLC)受体阻滞剂U73122和三磷酸肌醇(IP3)受体阻滞剂2-APB孵育冠状动脉平滑肌细胞,再测定加入0.5 mmol/L ATP后3组细胞内钙离子浓度荧光信号强度比值,分别是2.70±0.06、2.65±0.12和2.69±0.13,与未加入3种阻滞剂相比,钙离子浓度荧光信号强度比值均明显降低(P＜0.05).结论 ATP可以通过P2Y1-PLC-IP3通路升高细胞内钙离子浓度,从而激活BK通道.

abstractsObjective To explore the effects and activation mechanism of adenosine triphosphate (ATP) on large conductance calcium-activated potassium channel (BK channel) in coronary artery smooth muscle cells.Methods Coronary smooth muscle cells were isolated by enzyme digestion from SpragueDawley rats.And BK currents were recorded by patch clamp technique in whole cell configuration.The effects of ATP on cytosolic calcium concentrations were examined by recording the changes of fluorescence intensity ratios.Results BK current densities were (137 ± 13) pA/pF and (179 ± 15) pA/pF before and after a perfusion of 1 mmol/L ATP (P ＜ 0.05).The fluorescence ratios were 2.46 ± 0.08 and 4.04 ± 0.21 (P ＜ 0.05) before and after 0.5 mmol/L perfusion.After incubating with purine receptor (P2Y1) blocker MRS2179,phospholipase C (PLC) blocker U73122 and inositol triphosphate (IP3) blocker 2-APB,the fluorescence ratios were 2.7 ± 0.06,2.65 ± 0.12 and 2.69 ± 0.13 respectively.Compared with control group,all fluorescence ratios decreased after incubating with three blockers (P ＜ 0.05).Conclusion ATP may elevate intracellular calcium concentration via P2Y1-PLC-IP3 pathway consequently activating BK channel.