尼可地尔降低血管平滑肌细胞内ATP诱导的游离钙浓度

1、    尼可地尔降低血管平滑肌细胞内 ATP诱导的游离钙浓度        摘要目的：探讨尼可地尔对血管平滑肌细胞内游离钙(Ca2+i)的影响及机理。方法：培养的兔主动脉平滑肌细胞加入Fura-2AM 2.5 mol/L，在37下孵育50 min，Ca2+i用荧光分光光度计检测。结果：ATP(0.1 mmol/L)诱导的Ca2+i峰相和持续相增加可被尼可地尔抑制，且呈剂量依赖性，尼可地尔(10 mol/L)的抑制作用可被优降糖(10 mol/L)完全阻断(峰相：53

2、0±31 vs 544±41 nmol/L，持续相：370±19 vs 381±11 nmol/L，P0.05)；在无钙溶液中，先给尼可地尔能显著抑制ATP诱导的Ca2+i峰相增加。结论：尼可地尔抑制ATP诱导的Ca2+i增加，可能与减少细胞外钙内流及细胞内钙释放有关。主题词尼可地尔； ATP； 血管平滑肌；钙；优降糖Nicorandil inhibits ATP-induced cytosolic free calcium increase in cultured vascular smooth muscle cellsPENG Jian, HE Ji

3、an-Xin, XIAO Zhong-Ju1, LIU Yi-LiDepartment of Internal Medicine Cardiology Division of Nanfang Hospital ;1Department of Physiology; The First Military Medical University, Guangzhou 510515) Abstract AIM:To study the effects of nicorandil on cytosolic free calcium(Ca2+i) changes and their possible me

4、chanisms in vascular smooth muscle cells.METHODS：Cultured rabbit aortic smooth muscle cells were treated with Fura-2 AM 2.5 mol.L-1 at 37 for 50 min.Ca2+i level was measured by fluorospectrometer.RESULTS：ATP (0.1 mmol.L-1)-induced peak and sustained phase Ca2+i increase were inhibited by nicorandil

5、in a concentration-dependent manner. The effects of nicorandil(10mol.L-1) was completely canceled (peak phase:530±31 vs 544±41 nmol.L-1; sustained phase: 370±19 vs 381±11 nmol.L-1) by glibenclamide (10 mol.L-1). Pretreated with the nicorandil, the peak Ca2+i elevation induced by

6、ATP was reduced in the Ca2+-free solution (140±22 vs 260±33 nmol.L-1,P0.01).CONCLUSION：Nicorandil inhibits ATP-inducedCa2+i increase, associated with the decreases of both Ca2+ release from intracellular store and Ca2+ influs from extracellular store.MeSHNicorandil; Adenosine triphosphate;

7、 Vascular smooth muscle; Calcium; Glibenclamide尼可地尔有血管扩张作用，在冠状动脉通过激活钾通道，使膜电位超极化，钙内流减少，而扩张血管1，2。细胞内Ca2+i是决定血管平滑肌张力和活动的主要因素3，4，本研究探讨尼可地尔对血管平滑肌细胞内Ca2+i的影响。材料与方法(一)试剂：胰蛋白酶、依地酸、依他酸、小牛血清、ATP、尼可地尔和优降糖均为美国Sigma公司产品，尼可地尔用蒸馏水溶解，优降糖溶于0.001%二甲基亚砜(DMSO)。(二)细胞培养：取8周龄兔的胸主动脉行移植法，即精心剥去内皮和外膜，中层组织切成12 mm2的碎块，在含2 g NaH

8、CO3、 20%小牛血清、 100 kU/L青霉素和100 mg/L链霉素的RPMI 1640 培育液和37条件下，用CO2孵箱培养，3 d更换1次培养基，23周后放入含0.1%的胰蛋白酶和0.02%依地酸的Hanks溶液中，细胞松散，用组织细胞计数器计数，然后放入含RPMI 1640培养液的培养皿中，用电镜进行细胞鉴定5，采用810代细胞进行实验。(三)Ca2+i的测定：培养5 d后，血管平滑肌细胞用Hanks液清洗2遍，用含0.02%依地酸的0.1%胰蛋白酶消化后，再清洗。细胞存放液为(mmol/L)： NaCl 137， KCl 5.37， MgSO4*7H2O 0.57， NaH2PO

9、4 0.42， KH2PO40.44，葡萄糖11， HEPES 109，CaCl21.8，小牛血清0.2% (w/v)。调整细胞数为5×108/L，然后用Fura-2AM(终浓度2.5 mol/L)在37下培养50 min，取出离心4 min，清洗后放入测定液用台盼蓝检测细胞变异情况，用RF-5 000(日本)荧光分光光度计检测加入ATP、尼可地尔、优降糖后Ca2+i6。(四)统计分析：数据用表示，采用t检验进行统计分析。结果(一)ATP诱导的Ca2+i增加：加入ATP(0.1% mmol/L)后Ca2+i的增加有峰相和持续相，在细胞外液含钙时，血管平滑肌细胞内Ca2+i为(170&

10、#177;20) nmol/L (n=20)，如先加入尼可地尔可抑制ATP诱导的Ca2+i的两个增高相(表1)。表1尼可地尔对培养血管平滑肌细胞ATP诱导的Ca2+i增加的影响Tab 1 Effects of nicorandil(Nic) on ATP (0.1 mmol/L)-inducedincreased of cytosolic Ca2+ in Fura-2 AM-loaded cultured rabbitvascular smooth muscle cells in the presence of extracellular Ca2+(ATP added 0.001% DMSO

11、as control, ，n=5)Ca2+i peak phase(nmol/L)Ca2+i sustained phase(nmol/L)Control550±40370±12Nic 1 mol/L508±49*350±17*Nic 10 mol/L410±38*270±16*Nic 100 mol/L280±22*160±19*P0.01， vs control；P0.01， vs Nic 1 mol/L；P0.01， vs Nic 10 mol/L在细胞外液无钙条件下，Ca2+i为(86±9) nm

12、ol/L (n=20),尼可地尔(10 mol/L)可抑制ATP诱导的峰相增加，但对持续相增加无影响(见表2)。表2尼可地尔、优降糖对ATP诱导的血管平滑肌Ca2+i变化的影响Tab 2 Effects of nicorandil 10mol/L)on ATP(0.1 mmol/L)-induced biphasic increase of Ca2+i in Fura-2 AM-loaded culturedrabbit vascular smooth muscle cells in the absence of extracellular Ca2+. (ATP added 0.001% DM

13、SO as control ，n=5)Ca2+i peak phase(nmol/L)Ca2+i sustained phase(nmol/L)Control260±33118±28Nicorandil140±22*110±15*P0.01，vs control,P0.05， vs control(二)优降糖的阻断作用：在细胞外液含钙条件下用优降糖培养时，血管平滑肌细胞内Ca2+i无变化，优降糖也不影响ATP诱导的Ca2+i增高，但尼可地尔对ATP诱导的Ca2+i增加的抑制作用可被优降糖阻断峰相：(530±31) vs (544±41

14、) nmol/L；持续相：(370±19) vs (381±11) nmol/L，P0.05。讨论ATP诱导的Ca2+i增加主要由于三磷酸肌醇敏感的细胞内钙库中Ca2+释放，也可能来源于细胞外Ca2+内流7。本实验结果，当细胞外液无钙时ATP诱导的Ca2+i增加可被尼可地尔抑制，表明尼可地尔的作用与细胞内钙库中Ca2+释放减少有关。其次细胞外液含钙时，ATP诱导的Ca2+i两个增高相均被抑制，说明尼可地尔也可能抑制了细胞外Ca2+内流，其细胞机制目前仍不清楚，可能与钾通道激活，动作电位时限缩短有关8，也有学者认为尼可地尔促使膜超极化，减少了电压依赖性钙通道的开放9。优降糖通

15、过阻断ATP敏感性钾通道，而抑制尼可地尔对心脏和血管平滑肌的作用，本研究结果显示尼可地尔对ATP诱导的Ca2+i增加可被优降糖完全阻断，提示尼可地尔有开放ATP敏感性钾通道的作用。作者单位：彭健何建新刘伊丽第一军医大学附属南方医院心内科肖中举生理教研室(510515)参考文献1Uchida Y, Yoshimoto N, Murao S. Effect of 2-nicotinamidoethyl nitrate(SG-75) on coronary circulation. Jpn Heart J, 1978, 19:112.2Furukawa K, Itoh T, Kajiwara M,

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