尼可地爾降低血管平滑肌細(xì)胞內(nèi)ATP誘導(dǎo)的游離鈣濃度

1、    尼可地爾降低血管平滑肌細(xì)胞內(nèi) ATP誘導(dǎo)的游離鈣濃度        摘要目的：探討尼可地爾對(duì)血管平滑肌細(xì)胞內(nèi)游離鈣(Ca2+i)的影響及機(jī)理。方法：培養(yǎng)的兔主動(dòng)脈平滑肌細(xì)胞加入Fura-2AM 2.5 mol/L，在37下孵育50 min，Ca2+i用熒光分光光度計(jì)檢測。結(jié)果：ATP(0.1 mmol/L)誘導(dǎo)的Ca2+i峰相和持續(xù)相增加可被尼可地爾抑制，且呈劑量依賴性，尼可地爾(10 mol/L)的抑制作用可被優(yōu)降糖(10 mol/L)完全阻斷(峰相：53

2、0±31 vs 544±41 nmol/L，持續(xù)相：370±19 vs 381±11 nmol/L，P0.05)；在無鈣溶液中，先給尼可地爾能顯著抑制ATP誘導(dǎo)的Ca2+i峰相增加。結(jié)論：尼可地爾抑制ATP誘導(dǎo)的Ca2+i增加，可能與減少細(xì)胞外鈣內(nèi)流及細(xì)胞內(nèi)鈣釋放有關(guān)。主題詞尼可地爾； ATP； 血管平滑??；鈣；優(yōu)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尼可地爾有血管擴(kuò)張作用，在冠狀動(dòng)脈通過激活鉀通道，使膜電位超極化，鈣內(nèi)流減少，而擴(kuò)張血管1，2。細(xì)胞內(nèi)Ca2+i是決定血管平滑肌張力和活動(dòng)的主要因素3，4，本研究探討尼可地爾對(duì)血管平滑肌細(xì)胞內(nèi)Ca2+i的影響。材料與方法(一)試劑：胰蛋白酶、依地酸、依他酸、小牛血清、ATP、尼可地爾和優(yōu)降糖均為美國Sigma公司產(chǎn)品，尼可地爾用蒸餾水溶解，優(yōu)降糖溶于0.001%二甲基亞砜(DMSO)。(二)細(xì)胞培養(yǎng)：取8周齡兔的胸主動(dòng)脈行移植法，即精心剝?nèi)?nèi)皮和外膜，中層組織切成12 mm2的碎塊，在含2 g NaH

8、CO3、 20%小牛血清、 100 kU/L青霉素和100 mg/L鏈霉素的RPMI 1640 培育液和37條件下，用CO2孵箱培養(yǎng)，3 d更換1次培養(yǎng)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，細(xì)胞松散，用組織細(xì)胞計(jì)數(shù)器計(jì)數(shù)，然后放入含RPMI 1640培養(yǎng)液的培養(yǎng)皿中，用電鏡進(jìn)行細(xì)胞鑒定5，采用810代細(xì)胞進(jìn)行實(shí)驗(yàn)。(三)Ca2+i的測定：培養(yǎng)5 d后，血管平滑肌細(xì)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。細(xì)胞存放液為(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)。調(diào)整細(xì)胞數(shù)為5×108/L，然后用Fura-2AM(終濃度2.5 mol/L)在37下培養(yǎng)50 min，取出離心4 min，清洗后放入測定液用臺(tái)盼藍(lán)檢測細(xì)胞變異情況，用RF-5 000(日本)熒光分光光度計(jì)檢測加入ATP、尼可地爾、優(yōu)降糖后Ca2+i6。(四)統(tǒng)計(jì)分析：數(shù)據(jù)用表示，采用t檢驗(yàn)進(jìn)行統(tǒng)計(jì)分析。結(jié)果(一)ATP誘導(dǎo)的Ca2+i增加：加入ATP(0.1% mmol/L)后Ca2+i的增加有峰相和持續(xù)相，在細(xì)胞外液含鈣時(shí)，血管平滑肌細(xì)胞內(nèi)Ca2+i為(170&

10、#177;20) nmol/L (n=20)，如先加入尼可地爾可抑制ATP誘導(dǎo)的Ca2+i的兩個(gè)增高相(表1)。表1尼可地爾對(duì)培養(yǎng)血管平滑肌細(xì)胞ATP誘導(dǎo)的Ca2+i增加的影響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在細(xì)胞外液無鈣條件下，Ca2+i為(86±9) nm

12、ol/L (n=20),尼可地爾(10 mol/L)可抑制ATP誘導(dǎo)的峰相增加，但對(duì)持續(xù)相增加無影響(見表2)。表2尼可地爾、優(yōu)降糖對(duì)ATP誘導(dǎo)的血管平滑肌Ca2+i變化的影響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)優(yōu)降糖的阻斷作用：在細(xì)胞外液含鈣條件下用優(yōu)降糖培養(yǎng)時(shí)，血管平滑肌細(xì)胞內(nèi)Ca2+i無變化，優(yōu)降糖也不影響ATP誘導(dǎo)的Ca2+i增高，但尼可地爾對(duì)ATP誘導(dǎo)的Ca2+i增加的抑制作用可被優(yōu)降糖阻斷峰相：(530±31) vs (544±41

14、) nmol/L；持續(xù)相：(370±19) vs (381±11) nmol/L，P0.05。討論ATP誘導(dǎo)的Ca2+i增加主要由于三磷酸肌醇敏感的細(xì)胞內(nèi)鈣庫中Ca2+釋放，也可能來源于細(xì)胞外Ca2+內(nèi)流7。本實(shí)驗(yàn)結(jié)果，當(dāng)細(xì)胞外液無鈣時(shí)ATP誘導(dǎo)的Ca2+i增加可被尼可地爾抑制，表明尼可地爾的作用與細(xì)胞內(nèi)鈣庫中Ca2+釋放減少有關(guān)。其次細(xì)胞外液含鈣時(shí)，ATP誘導(dǎo)的Ca2+i兩個(gè)增高相均被抑制，說明尼可地爾也可能抑制了細(xì)胞外Ca2+內(nèi)流，其細(xì)胞機(jī)制目前仍不清楚，可能與鉀通道激活，動(dòng)作電位時(shí)限縮短有關(guān)8，也有學(xué)者認(rèn)為尼可地爾促使膜超極化，減少了電壓依賴性鈣通道的開放9。優(yōu)降糖通

15、過阻斷ATP敏感性鉀通道，而抑制尼可地爾對(duì)心臟和血管平滑肌的作用，本研究結(jié)果顯示尼可地爾對(duì)ATP誘導(dǎo)的Ca2+i增加可被優(yōu)降糖完全阻斷，提示尼可地爾有開放ATP敏感性鉀通道的作用。作者單位：彭健何建新劉伊麗第一軍醫(yī)大學(xué)附屬南方醫(yī)院心內(nèi)科肖中舉生理教研室(510515)參考文獻(xiàn)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,

16、et al. Vasodilating actions of 2-nicotinamidoethyl nitrate on porcine and guinea-pig coronary arteries. J Pharmacol Exp Ther, 1981,218:248.3Morgan KG. Calcium and vascular smooth muscle tone. Am J Med, 1987, 82 (suppl 3b):9.4Inoue I, Matsuura H, Shingu T, et al. Role of intracellular cation abnormal

17、ities in development of left ventricular hypertrophy. J Cardiovasc Pharmacol, 1991, 17 suppl 2:s107.5Southgate 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, 1990,80:113.6Grynkie

18、wicz G, Poenie M, Tsien RY. A new generation of Ca2 indicators with greatly improved flurescence properties. J Biol Chem, 1985, 260:3440.7Von der Weid PY, Serebryakov VN, Orallo F, et al. Effects of ATP on cultured smooth muscle cells from rat aorta. Br J Pharmacol, 1993, 108:638.8Nakayama K, Fan Z, Marumo F, et al. Ac