尼可地尔降低血管平滑肌细胞内ATP诱导的游离钙浓度
作者：彭 健 何建新 刘伊丽 肖中举
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)
关键词：尼可地尔；ATP；血管平滑肌；钙；优降糖
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 μmol/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 μmol/L)的抑制作用可被优降糖(10 μmol/L)完全阻断(峰相：530±31 vs 544±41 nmol/L，持续相：370±19 vs 381±11 nmol/L，P>0.05)；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。
, 百拇医药
Nicorandil inhibits ATP-induced cytosolic free calcium increase
in cultured vascular smooth muscle cells
PENG Jian, HE Jian-Xin, XIAO Zhong-Ju1, LIU Yi-Li
Department of Internal Medicine Cardiology Division of Nanfang Hospital ;1 Department of Physiology;
The First Military Medical University, Guangzhou 510515)
, 百拇医药
Abstract AIM:To study the effects of nicorandil on cytosolic free calcium([Ca2+]i) changes and their possible mechanisms in vascular smooth muscle cells.METHODS：Cultured rabbit aortic smooth muscle cells were treated with Fura-2 AM 2.5 μmol.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 in a concentration-dependent manner. The effects of nicorandil(10 μmol.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 μmol.L-1). Pretreated with the nicorandil, the peak [Ca2+]i elevation induced by ATP was reduced in the Ca2+-free solution (140±22 vs 260±33 nmol.L-1,P<0.01).CONCLUSION：Nicorandil inhibits ATP-induced[Ca2+]i increase, associated with the decreases of both Ca2+ release from intracellular store and Ca2+ influs from extracellular store.
, http://www.100md.com
MeSH Nicorandil; Adenosine triphosphate; 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1～2 mm2的碎块，在含2 g NaHCO3、 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，2～3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8～10代细胞进行实验。
, 百拇医药
(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， NaH2PO4 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 μmol/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+]i[6]。
(四)统计分析：数据用
表示，采用t检验进行统计分析。
, http://www.100md.com
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±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)-induced
, http://www.100md.com
increased of cytosolic Ca2+ in Fura-2 AM-loaded cultured rabbit
vascular smooth muscle cells in the presence of extracellular Ca2+
(ATP added 0.001% DMSO as control,，n=5)
[Ca2+]i peak phase
(nmol/L)
, 百拇医药
[Ca2+]i sustained phase
(nmol/L)
Control
550±40
370±12
Nic 1 μmol/L
508±49*
350±17*
Nic 10 μmol/L
410±38*△
, 百拇医药
270±16*△
Nic 100 μmol/L
280±22*△☆
160±19*△☆
*P<0.01， vs control；△P<0.01， vs Nic 1 μ mol/L；☆P<0.01， vs Nic 10 μmol/L
在细胞外液无钙条件下，[Ca2+]i为(86±9) nmol/L (n=20),尼可地尔(10 μmol/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
, http://www.100md.com
[Ca2+]i变化的影响
Tab 2 Effects of nicorandil 10 μmol/L) on ATP(0.1 mmol/L)
-induced biphasic increase of [Ca2+]i in Fura-2 AM-loaded cultured
rabbit vascular smooth muscle cells in the absence of extracellular
Ca2+. (ATP added 0.001% DMSO as control，n=5)
, 百拇医药
[Ca2+]i peak phase
(nmol/L)
[Ca2+]i sustained phase
(nmol/L)
Control
260±33
118±28
Nicorandil
140±22*
110±15△
, http://www.100md.com
*P<0.01，vs control, △P>0.05， 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) nmol/L；持续相：(370±19) vs (381±11) nmol/L，P>0.05]。
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。
, 百拇医药
参考文献
1 Uchida Y, Yoshimoto N, Murao S. Effect of 2-nicotinamidoethyl nitrate(SG-75) on coronary circulation. Jpn Heart J, 1978, 19:112.
2 Furukawa K, Itoh T, Kajiwara M, et al. Vasodilating actions of 2-nicotinamidoethyl nitrate on porcine and guinea-pig coronary arteries. J Pharmacol Exp Ther, 1981,218:248.
3 Morgan KG. Calcium and vascular smooth muscle tone. Am J Med, 1987, 82 (suppl 3b):9.
, http://www.100md.com
4 Inoue I, Matsuura H, Shingu T, et al. Role of intracellular cation abnormalities in development of left ventricular hypertrophy. J Cardiovasc Pharmacol, 1991, 17 suppl 2:s107.
5 Southgate KM, Newby AC. Serum-induced prolifereation of rabbit aortic smooth muscle cells from the contractile state is inhibited by 8-Br-cAMP but not 8-Br-cGMP. Atherosclerosis, 1990,80:113.
6 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved flurescence properties. J Biol Chem, 1985, 260:3440.
, 百拇医药
7 Von der Weid PY, Serebryakov VN, Orallo F, et al. Effects of ATP on cultured smooth muscle cells from rat aorta. Br J Pharmacol, 1993, 108:638.
8 Nakayama K, Fan Z, Marumo F, et al. Action of nicorandil on ATP-sensitive K+ channel in guinea-pig ventricular myocytes. Br J Pharmacol, 1991,103:1641.
9 Yanagisawa T, Hashimoto H, Taira N. Interaction of potassium channel openers and blockers in canine atrial muscle. Br J Pharmacol, 1989, 97:753.
(1997年12月18日收稿，1998年7月15日修回), http://www.100md.com
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)
关键词：尼可地尔；ATP；血管平滑肌；钙；优降糖
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 μmol/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 μmol/L)的抑制作用可被优降糖(10 μmol/L)完全阻断(峰相：530±31 vs 544±41 nmol/L，持续相：370±19 vs 381±11 nmol/L，P>0.05)；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。
, 百拇医药
Nicorandil inhibits ATP-induced cytosolic free calcium increase
in cultured vascular smooth muscle cells
PENG Jian, HE Jian-Xin, XIAO Zhong-Ju1, LIU Yi-Li
Department of Internal Medicine Cardiology Division of Nanfang Hospital ;1 Department of Physiology;
The First Military Medical University, Guangzhou 510515)
, 百拇医药
Abstract AIM:To study the effects of nicorandil on cytosolic free calcium([Ca2+]i) changes and their possible mechanisms in vascular smooth muscle cells.METHODS：Cultured rabbit aortic smooth muscle cells were treated with Fura-2 AM 2.5 μmol.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 in a concentration-dependent manner. The effects of nicorandil(10 μmol.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 μmol.L-1). Pretreated with the nicorandil, the peak [Ca2+]i elevation induced by ATP was reduced in the Ca2+-free solution (140±22 vs 260±33 nmol.L-1,P<0.01).CONCLUSION：Nicorandil inhibits ATP-induced[Ca2+]i increase, associated with the decreases of both Ca2+ release from intracellular store and Ca2+ influs from extracellular store.
, http://www.100md.com
MeSH Nicorandil; Adenosine triphosphate; 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1～2 mm2的碎块，在含2 g NaHCO3、 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，2～3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8～10代细胞进行实验。
, 百拇医药
(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， NaH2PO4 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 μmol/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+]i[6]。
(四)统计分析：数据用
表示，采用t检验进行统计分析。, http://www.100md.com
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±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)-induced
, http://www.100md.com
increased of cytosolic Ca2+ in Fura-2 AM-loaded cultured rabbit
vascular smooth muscle cells in the presence of extracellular Ca2+
(ATP added 0.001% DMSO as control,
，n=5)[Ca2+]i peak phase
(nmol/L)
, 百拇医药
[Ca2+]i sustained phase
(nmol/L)
Control
550±40
370±12
Nic 1 μmol/L
508±49*
350±17*
Nic 10 μmol/L
410±38*△
, 百拇医药
270±16*△
Nic 100 μmol/L
280±22*△☆
160±19*△☆
*P<0.01， vs control；△P<0.01， vs Nic 1 μ mol/L；☆P<0.01， vs Nic 10 μmol/L
在细胞外液无钙条件下，[Ca2+]i为(86±9) nmol/L (n=20),尼可地尔(10 μmol/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
, http://www.100md.com
[Ca2+]i变化的影响
Tab 2 Effects of nicorandil 10 μmol/L) on ATP(0.1 mmol/L)
-induced biphasic increase of [Ca2+]i in Fura-2 AM-loaded cultured
rabbit vascular smooth muscle cells in the absence of extracellular
Ca2+. (ATP added 0.001% DMSO as control
，n=5), 百拇医药
[Ca2+]i peak phase
(nmol/L)
[Ca2+]i sustained phase
(nmol/L)
Control
260±33
118±28
Nicorandil
140±22*
110±15△
, http://www.100md.com
*P<0.01，vs control, △P>0.05， 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) nmol/L；持续相：(370±19) vs (381±11) nmol/L，P>0.05]。
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。
, 百拇医药
参考文献
1 Uchida Y, Yoshimoto N, Murao S. Effect of 2-nicotinamidoethyl nitrate(SG-75) on coronary circulation. Jpn Heart J, 1978, 19:112.
2 Furukawa K, Itoh T, Kajiwara M, et al. Vasodilating actions of 2-nicotinamidoethyl nitrate on porcine and guinea-pig coronary arteries. J Pharmacol Exp Ther, 1981,218:248.
3 Morgan KG. Calcium and vascular smooth muscle tone. Am J Med, 1987, 82 (suppl 3b):9.
, http://www.100md.com
4 Inoue I, Matsuura H, Shingu T, et al. Role of intracellular cation abnormalities in development of left ventricular hypertrophy. J Cardiovasc Pharmacol, 1991, 17 suppl 2:s107.
5 Southgate KM, Newby AC. Serum-induced prolifereation of rabbit aortic smooth muscle cells from the contractile state is inhibited by 8-Br-cAMP but not 8-Br-cGMP. Atherosclerosis, 1990,80:113.
6 Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved flurescence properties. J Biol Chem, 1985, 260:3440.
, 百拇医药
7 Von der Weid PY, Serebryakov VN, Orallo F, et al. Effects of ATP on cultured smooth muscle cells from rat aorta. Br J Pharmacol, 1993, 108:638.
8 Nakayama K, Fan Z, Marumo F, et al. Action of nicorandil on ATP-sensitive K+ channel in guinea-pig ventricular myocytes. Br J Pharmacol, 1991,103:1641.
9 Yanagisawa T, Hashimoto H, Taira N. Interaction of potassium channel openers and blockers in canine atrial muscle. Br J Pharmacol, 1989, 97:753.
(1997年12月18日收稿，1998年7月15日修回), http://www.100md.com