乌拉地尔干预急性心肌缺血的细胞机制

1、    乌拉地尔干预急性心肌缺血的细胞机制        【摘要】目的新的肾上腺素受体拮抗剂乌拉地尔（阻断1受体为主），治疗高血压急症、急性心肌缺血和肺水肿有效。本研究试阐述乌拉地尔对缺血心肌内能量代谢状态、SR钙泵活力和钙释放通道的作用，探讨其治疗急性心肌缺血的分子和细胞机制。方法制备急性心肌缺血家兔模型，给予乌拉地尔1.0mgkg-1d-1，3天后测定缺血和非缺血区心肌内ATP、ADP、AMP和乳酸量，计算能荷；测定SR Ca2+ATP酶活力和钙释放通道3HRyanodi

2、ne结合的Bmax与Kd值。测定心肌细胞缺氧培养及药物干预时的SR钙泵活力。结果2+ATP酶活力从（1.19±0.11）mol/g降至0.94mol/g(P<0.05）；钙释放通道数目下降，亲和力不变。治疗组缺血心肌内ATP、ADP、AMP含量明显回升、能荷升高，SR Ca2+2+泵和钙释放通道功能、减轻钙超载是治疗的分子和细胞机制之一。【关键词】乌拉地尔心肌缺血Ca2+转运ATP酶钙通道 Cellular mechanism of urapidil on acuteischemic myocardium in rabbitsZHANG JinanYANG Guo-pingSU

3、 Enbenet alThe First Affiliated Hospital, Nanjing Medical University, Nanjing 210029【Abstract】ObjectiveTo investigate the effects of urapidil, a new drug mainly blocks alpha 1 adrenergic receptor, on energy metabolism, calcium ATPase activity and calcium release channel of sarcoplasmic reticulum in

4、the ischemic myocardium following ligation of the anterior branch of left coronary artery in rabbits.MethodsRabbits (n=24) were randomly divided into three groups: control (n=7), ligation of the anterior branch of left coronary artery and intravenous urapidil (1.0 mgkg-1d-1) following ligation. At t

5、he third day after operation, the hearts were taken out and frozen in liquid nitrogen. The contents of ATP, ADP, AMP and lactate, SR calcium pump activities as well as Bmax and Kd of calcium release channel in the ischemic and non-ischemic myocardium were measured by enzymatic and radiobinding analy

6、ses. Meanwhile SR calcium pump activities in cultured neonatal rat cardiomyocytes under anoxic conditions with and without urapidil or lacidipine, a calcium antagonist, were also evaluated. ResultsLevels of ATP, ADP and AMP in the ischemic myocardium decreased by 31%, 40% and 33% respectively, lacta

7、te increased about 65% than that in the non-ischemic area of the myocardium (P<0.05 or 0.01). Activity of SR calcium pump and number of calcium release channel in the ischemic myocardium reduced sigificantly (P<0.05). In the group treated with urapidil (n=7), contents of ATP, ADP, AMP and ener

8、gy charge changed to higher levels in comparison to the ischeimic myocardium without urapidil treatment (n=10), both the activity of SR calcium pump and number of calcium release channel reached normal range. In cultured cardiomyocytes under anoxic condition, the activity of SR calcium pump reduced

9、by 55% comparing to that in normal condition. The activities of SR calcium pump of cardiomyocytes cultured in anoxic culture medium with urapidil or lacidipine were higher than that in the anoxic cultured cells.ConclusionThe present data firstly demonstrated that urapidil could improve the energy me

10、tabolism, reduce the level of lactate, and adjust the concentration of cellular free calcium through regulating the function of SR calcium pump and calcium release channel in the acute ischemic myocardium following ligation of coronary artery.【Key words】urapidilmyocardial ischemiaCa2+-transporting A

11、TPasecalcium channels近年，肾上腺素能受体阻断剂乌拉地尔被推荐为急诊室治疗高血压危象的首选用药。它还可显著减轻经皮冠状动脉血管成形术后短暂心肌缺血所致的左室功能不全,减弱冠脉阻力和冠脉收缩1,2。国内报告能改善急性左心衰竭肺水肿及慢性心力衰竭患者血液动力学参数，降低外周阻力、肺毛细血管嵌压，增加心排血量及左室作功指数3。本实验观察乌拉地尔对缺血心肌内能量代谢的作用，对肌浆网（SR）钙泵（Ca2+ ATP pump）和钙释放通道（Calcium Release Channel） 的影响，探讨乌拉地尔干预急性心肌缺血的细胞机制。材料与方法1健康青紫兰家兔24只，体重1.92.3

12、kg，雌雄不限，随机分为开胸不结扎（对照组7只）、结扎冠状动脉左前降枝（未治疗组10只）和结扎乌拉地尔（治疗组7只）三组。治疗组结扎前用乌拉地尔（德国BYK药厂生产）1.5mg/次，术后1、2、3天静脉缓慢注射1.0mg/kg。连续采血标本，按本室ELISA法测肌钙蛋白I（cTnI）4，3天后取心脏，分缺血区和非缺血区，液氮速冻后储存-75备用。SD乳鼠心肌细胞培养和缺氧培养，按Hohi等5方法，分缺氧乌拉地尔或加钙通道拮抗剂拉西地平培养，收取细胞，测SR钙泵活力。2心肌ATP、ADP、AMP和乳酸量测定按过氯酸提取酶反应法，SR钙泵即Ca2+ ATP酶、SR钙释放通道按Zhang等6改良法，

13、用3HRyanodine（0.2540nmol/L）结合法测定6,7，获饱和曲线及Scatchard分析。3试剂NADH、NAD、HK、G6PDH、乳酸、ATP等订自Sigma公司，3HRyanodine购自Amershan，其余均为国产分析纯试剂。4资料先以方差分析F检验显著后，再行组间q检验。结果结扎家兔冠状动脉后4小时血清cTnI即明显升高，36、48小时高峰达20g/L，72小时始下降，类似临床急性心肌梗塞患者血清cTnI动态变化4表1乌拉地尔对家兔急性缺血心肌内能量代谢的影响(mol/g湿重，±s)组别只数ATPADPAMPEnergy Charge乳酸对照组72.83&#

14、177;0.822.96±0.661.66±0.220.6317.44±11.02未治疗组非缺血区52.42±0.89*2.94±1.10*1.95±0.65*0.5722.23±12.14缺血区101.67±0.781.76±0.911.31±1.100.5235.58±29.57治疗组非缺血区55.04±1.233.91±0.482.07±1.020.62*16.59±4.20缺血区74.87±2.092.95±1.01*

15、2.27±0.49*0.64*23.5±8.30注：Energy Charge(EC)计算公式：(ATP+0.5ADP)/(ATP+ADP+AMP)。统计学分析：*为P<0.05(未治疗组中：非缺血区与缺血区比较；治疗组中：缺血区与未治疗组缺血区比较，非缺血区与未治疗组非缺血区比较)；为P<0.01(治疗组中：缺血区与未治疗组的缺血区比较，非缺血区与未治疗组的非缺血区比较)；表2同此 3HRyanodine配基结合测定钙释放通道最佳条件为37哺育60120分钟时达饱和，心肌匀浆蛋白量0400mg为直线，3HRyanodine 10nmol/L时即达饱和，冷配基1

16、0-5M Ryanodine抑制配基结合率80，非特异结合低于20。未治疗组缺血区钙释放通道密度较非缺血区下降26（P<0.05）。治疗组缺血区密度回升27分别为（93.39±9.69）fmol/mg蛋白及（118.68±12.29）fmol/mg蛋白，亲和力没有改变。乌拉地尔对缺血心肌SR3HRyanodine结合与亲和力及对Ca2泵活力的作用见表2。急性缺血心肌Ca2泵即Ca2ATP酶活力从（1.19±0.11）molg蛋白-1min-1降至（0.94±0.14）molg蛋白-1min-1(P<0.05)，用药后Ca2ATP酶活力上升1.

17、5倍，超过未治疗组非缺血区1倍（P<0.01）。钙释放通道结合位点下降（P<0.05），亲和力不变。将retenone(16mol/L)加入培养第4天的乳鼠心肌细胞50分钟，在缺氧条件下，钙泵活力下降55(2.75mol/g蛋白和1.25mol/g蛋白)。20mol/L的乌拉地尔保护钙泵活力，接近有氧培养的对照组水平（2.75mol/g蛋白和2.48mol/g蛋白）。20mol/L拉西地平也有保护作用，但低于乌拉地尔（2.75mol/g蛋白和2.0mol/g蛋白）。表2乌拉地尔对家兔急性缺血心肌肌浆网钙泵和钙释放通道的影响(±s)组别只数钙释放通道钙泵活力（molg蛋白-

18、1min-1）最大结合力(fmol/mg蛋白)解离常数(nmol/L)对照组7155.74±23.500.90±0.381.70±0.25未治疗组非缺血区5126.35±9.701.03±0.121.19±0.11缺血区1093.39±9.68*1.10±0.190.94±0.14*治疗组非缺血区5134.50±19.351.18±0.441.34±0.16缺血区7118.68±12.290.92±0.182.30±0.16讨论 哺乳动物心肌内存

19、在受体，生理条件下调节心肌正性肌力作用不如受体显著。急性缺血心肌内受体及信号调节活跃，通过激活细胞内PKC活力，增加二磷酸甘油DAG和磷酸肌醇IP3第二信使，使心肌细胞内钙负荷超载8。1R阻滞剂哌唑嗪似乎不直接影响心肌受体，而减轻细胞内Ca2+超载。Gregorini等报告乌拉地尔明显改善经皮冠状动脉血管成形术后冠脉痉挛，也改善结扎冠脉后总冠脉收缩和流量。有关乌拉地尔治疗心肌缺血的代谢机制尚未见报告。本研究资料明确显示，乌拉地尔使缺血心肌ATP、ADP、AMP极显著升高，比未治疗组升高0.71.9倍，改善心肌能量生成、利用和储存的过程。能荷是组织内能量生成利用和运转的重要标志，乌拉地尔使缺血心

20、肌内能荷值上升，接近和超过对照组。表明能量平衡恢复，这可能与乌拉地尔改善结扎以外的冠脉血管痉挛、恢复冠脉血流有关。心肌能量来源于葡萄糖有氧分解或无氧酵解和脂肪酸氧化，心肌乳酸含量是有氧或无氧代谢、丙酮酸能否顺利进入三羧酸循环的客观证据，乌拉地尔能降低缺血心肌内乳酸量（52），其具体作用环节尚待进一步阐明。多种细胞膜蛋白影响胞内Ca2+i浓度。主要调控蛋白是SR钙泵和钙释放通道9，Ca2+泵将胞浆Ca2+泵入SR内储存，释放通道将SR储存Ca2+放入胞浆。维持心肌细胞Ca2+i平衡。从而调节兴奋收缩偶联。本研究发现，缺血心肌内Ca2+泵活力明显降低，释放通道亲和力无改变。乌拉地尔恢复缺血区Ca2

21、+泵活力和通道数目，从钙调节蛋白水平上与Moraru资料相一致。乌拉地尔恢复缺血、缺氧心肌细胞SR Ca2+泵活力，它对缺血、缺氧心肌细胞内钙稳定调节可能作用在SR钙调节蛋白水平上。乌拉地尔恢复缺血心肌内能量代谢状态、降低乳酸含量、调整SR Ca2+泵和钙释放通道功能，从而降低胞内Ca2+超载，是影响急性缺血的细胞基础之一。作者单位：210029南京医科大学第一附属医院（第八作者系北京友谊医院）参考文献1Hirschl MM, Seidier D, Laggner AN, et al. Effecacy of different antihypertensive drugs in the em

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