二十二碳六烯酸通过磷脂酶C-三磷酸肌醇-钙离子途径激活正常大鼠冠状动脉平滑肌细胞BK通道  

Docosahexaenoic acids activate large conductance calcium-activated potassium channels via phospholipase C- inositol triphosphatecalcium pathway in normal rat coronary smooth muscle cells

作　　者：孙曼青[1] 钱玲玲[1] 党时鹏[1] 吴莹[1] 汤徐 季圆[1] 王湘芸[1] 夏大云[1] 王文[2] 柴强[3] 陆彤 王如兴[1] 

机构地区：[1]南京医科大学附属无锡市人民医院心内科,214023 [2]徐州医学院药理学系 [3]山东省医学科学院基础医学研究所心血管病研究室 [4]美国Mayo临床细胞电生理研究室

出　　处：《中华心血管病杂志》2016年第6期530-535,共6页Chinese Journal of Cardiology

基　　金：国家自然科学基金（81370303,81500249,81400297,81500323）;江苏省自然科学基金（BK2015110）;江苏省人事厅“六大人才高峰”第七批高层次项目（006）;江苏省医学重点人才资助项目（RC2011034）

摘　　要：目的探讨二十二碳六烯酸（DHA）激活正常大鼠冠状动脉平滑肌细胞大电导钙激活钾通道（BK通道）的机制。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；全细胞膜片钳实验技术记录正常大鼠冠状动脉平滑肌细胞BK通道电流；膜内向外型单通道膜片钳实验技术记录不同钙离子浓度条件下BK通道电流，并计算开放概率（NP1）；钙离子成像技术测定不同浓度DHA作用后及分别采用磷脂酶（CPLC）受体抑制剂和三磷酸肌醇（IR）受体抑制剂预孵育冠状动脉平滑肌细胞后细胞内钙离子浓度变化。结果1μmol／LDHA可激活BK通道；在刺激电位60mV，电极外液钙离子浓度为0、0．01、0．1、1、3、10、50和100μmol／L条件下，BK通道NP。分别为0．0027±0．0004、0．0060±0．0014、0．0972±0．0106、0．1379±0．0329、0．4687±0．1637、2．0971±0．3104和3．1204±0．2427（P〈0．05，n=4）。未加DHA时，细胞内钙离子浓度荧光信号比值（R值）为0．51±0．01；加入0．001和0．01μmol／LDHA后，R值分别为0．53±0．02和0．55±0．01，与未加DHA相比差异无统计学意义（P〉0．05，n／〉5）；当加入的DHA浓度为0．1、0．3、1、3、5和10μmol／L时，其R值分别为0．64±0．01、0．65±0．01、0．70±0．01、0．69±0．01、0．68±0．01和0．67±0．02，ECEn为（0．04±0．02）μmol／L，与未加DHA比较，差异有统计学意义（P〈0．05，nI〉4）。分别采用PLC受体抑制剂U73122和IR受体抑制剂2-APB预孵育冠状动脉平滑肌细胞，测定加入0．1μmol／LDHA后R值分别为0．52±0．01和0．49±0．02，低于未加抑制剂的R值（0．64±0．01，P〈0．05，n≥4）。结论DHA可通过PLC—IP，Ca^2＋途径增加正常大鼠冠状动脉平滑肌细胞内钙离子浓度激活BK通道，从而扩张血管，对心血管系统有一定的保护作用。Objective To investigate the mechanisms of docosahexaenoic acids （DHA） on activating large conductance calcium-activated potassium channels （BK channels） in normal rat coronary smooth muscle cells. Methods Normal coronary smooth muscle cells were isolated by enzyme digestion from Sprague-Dawley rats. BK currents were recorded by patch clamp in whole cell and single channel configurations, respectively. The effects of DHA on cytosolic calcium concentrations were examined by recording the changes of fluorescence intensity ratios. Results DHA （ 1 μmol/L ） could activate BK channels. Open probabilities （ NP0 ） of BK channels at test potential 60 mV, and calcium concentrations in external solution at 0,0. 01, 0. 1, 1, 3, 10, 50 and 100 μmol/L were 0. 002 7 ± 0. 000 4, 0. 006 0 ± 0. 001 4, 0.097 2±0.010 6, 0. 137 9 ±0. 032 9, 0.468 7 ±0. 163 7, 2. 097 1 ±0. 310 d and 3. 120 4 ± 0. 242 7, respectively （ P 〈 0. 05, n =4 ）. Before DHA perfusion, the fluorescence intensity ratio was 0. 51 ±0.01, and the ratios were O. 53 ± 0. 02 and 0. 55 ± 0. 01 after 0. 001 and 0.01 μmol/L DHA perfusion, respectively （P 〉0. 05 ,n≥5）. The ratios were 0. 64 ±0. 01, 0. 65 ±0. 01, 0. 70 ±0. 01, 0. 69 ±0.01, 0.68 ± 0.01 and 0.67 ±0.02 after 0.1, 0.3, 1, 3, 5 and 10 tLmol/L DHA perfusion, respectively, and ECs0 was （0. 04 ±0. 02） μmol/L（P 〈0. 05, n≥4）. They were all higher than that before DHA perfusion. After incubating with phospholipase C （PLC） blocker U73122 and inositol triphosphate （IP3 ） blocker 2-APB, the ratios were 0. 52 ± 0. 01 and 0. 49 ± 0. 02 on the setting of 0. 1 μmol/L DHA, respectively. Compared with control group （0. 64 ± 0. 01 ）, the ratios decreased after incubating with blockers （P 〈 0. 05, n≥4）. Conclusions Docosahexaenoic acids can activate large conductance calcium-activated potassium channels by the pathway of PLC-IP3-Ca2± to increase cytosolic calcium concentration in normal coronary smooth muscle ceils, dilate the coronary vessel

关 键 词：冠状血管 肌细胞 平滑肌 二十二碳六烯酸类 大电导钙激活钾通道 

分 类 号：R965[医药卫生—药理学]