烏拉地爾干預(yù)急性心肌缺血的細(xì)胞機(jī)制

1、    烏拉地爾干預(yù)急性心肌缺血的細(xì)胞機(jī)制        【摘要】目的新的腎上腺素受體拮抗劑烏拉地爾（阻斷1受體為主），治療高血壓急癥、急性心肌缺血和肺水腫有效。本研究試闡述烏拉地爾對缺血心肌內(nèi)能量代謝狀態(tài)、SR鈣泵活力和鈣釋放通道的作用，探討其治療急性心肌缺血的分子和細(xì)胞機(jī)制。方法制備急性心肌缺血家兔模型，給予烏拉地爾1.0mgkg-1d-1，3天后測定缺血和非缺血區(qū)心肌內(nèi)ATP、ADP、AMP和乳酸量，計(jì)算能荷；測定SR Ca2+ATP酶活力和鈣釋放通道3HRyanodi

2、ne結(jié)合的Bmax與Kd值。測定心肌細(xì)胞缺氧培養(yǎng)及藥物干預(yù)時的SR鈣泵活力。結(jié)果2+ATP酶活力從（1.19±0.11）mol/g降至0.94mol/g(P<0.05）；鈣釋放通道數(shù)目下降，親和力不變。治療組缺血心肌內(nèi)ATP、ADP、AMP含量明顯回升、能荷升高，SR Ca2+2+泵和鈣釋放通道功能、減輕鈣超載是治療的分子和細(xì)胞機(jī)制之一?！娟P(guān)鍵詞】烏拉地爾心肌缺血Ca2+轉(zhuǎn)運(yùn)ATP酶鈣通道 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，腎上腺素能受體阻斷劑烏拉地爾被推薦為急診室治療高血壓危象的首選用藥。它還可顯著減輕經(jīng)皮冠狀動脈血管成形術(shù)后短暫心肌缺血所致的左室功能不全,減弱冠脈阻力和冠脈收縮1,2。國內(nèi)報告能改善急性左心衰竭肺水腫及慢性心力衰竭患者血液動力學(xué)參數(shù)，降低外周阻力、肺毛細(xì)血管嵌壓，增加心排血量及左室作功指數(shù)3。本實(shí)驗(yàn)觀察烏拉地爾對缺血心肌內(nèi)能量代謝的作用，對肌漿網(wǎng)（SR）鈣泵（Ca2+ ATP pump）和鈣釋放通道（Calcium Release Channel） 的影響，探討烏拉地爾干預(yù)急性心肌缺血的細(xì)胞機(jī)制。材料與方法1健康青紫蘭家兔24只，體重1.92.3

12、kg，雌雄不限，隨機(jī)分為開胸不結(jié)扎（對照組7只）、結(jié)扎冠狀動脈左前降枝（未治療組10只）和結(jié)扎烏拉地爾（治療組7只）三組。治療組結(jié)扎前用烏拉地爾（德國BYK藥廠生產(chǎn)）1.5mg/次，術(shù)后1、2、3天靜脈緩慢注射1.0mg/kg。連續(xù)采血標(biāo)本，按本室ELISA法測肌鈣蛋白I（cTnI）4，3天后取心臟，分缺血區(qū)和非缺血區(qū)，液氮速凍后儲存-75備用。SD乳鼠心肌細(xì)胞培養(yǎng)和缺氧培養(yǎng)，按Hohi等5方法，分缺氧烏拉地爾或加鈣通道拮抗劑拉西地平培養(yǎng)，收取細(xì)胞，測SR鈣泵活力。2心肌ATP、ADP、AMP和乳酸量測定按過氯酸提取酶反應(yīng)法，SR鈣泵即Ca2+ ATP酶、SR鈣釋放通道按Zhang等6改良法，

13、用3HRyanodine（0.2540nmol/L）結(jié)合法測定6,7，獲飽和曲線及Scatchard分析。3試劑NADH、NAD、HK、G6PDH、乳酸、ATP等訂自Sigma公司，3HRyanodine購自Amershan，其余均為國產(chǎn)分析純試劑。4資料先以方差分析F檢驗(yàn)顯著后，再行組間q檢驗(yàn)。結(jié)果結(jié)扎家兔冠狀動脈后4小時血清cTnI即明顯升高，36、48小時高峰達(dá)20g/L，72小時始下降，類似臨床急性心肌梗塞患者血清cTnI動態(tài)變化4表1烏拉地爾對家兔急性缺血心肌內(nèi)能量代謝的影響(mol/g濕重，±s)組別只數(shù)ATPADPAMPEnergy Charge乳酸對照組72.83&#

14、177;0.822.96±0.661.66±0.220.6317.44±11.02未治療組非缺血區(qū)52.42±0.89*2.94±1.10*1.95±0.65*0.5722.23±12.14缺血區(qū)101.67±0.781.76±0.911.31±1.100.5235.58±29.57治療組非缺血區(qū)55.04±1.233.91±0.482.07±1.020.62*16.59±4.20缺血區(qū)74.87±2.092.95±1.01*

15、2.27±0.49*0.64*23.5±8.30注：Energy Charge(EC)計(jì)算公式：(ATP+0.5ADP)/(ATP+ADP+AMP)。統(tǒng)計(jì)學(xué)分析：*為P<0.05(未治療組中：非缺血區(qū)與缺血區(qū)比較；治療組中：缺血區(qū)與未治療組缺血區(qū)比較，非缺血區(qū)與未治療組非缺血區(qū)比較)；為P<0.01(治療組中：缺血區(qū)與未治療組的缺血區(qū)比較，非缺血區(qū)與未治療組的非缺血區(qū)比較)；表2同此 3HRyanodine配基結(jié)合測定鈣釋放通道最佳條件為37哺育60120分鐘時達(dá)飽和，心肌勻漿蛋白量0400mg為直線，3HRyanodine 10nmol/L時即達(dá)飽和，冷配基1

16、0-5M Ryanodine抑制配基結(jié)合率80，非特異結(jié)合低于20。未治療組缺血區(qū)鈣釋放通道密度較非缺血區(qū)下降26（P<0.05）。治療組缺血區(qū)密度回升27分別為（93.39±9.69）fmol/mg蛋白及（118.68±12.29）fmol/mg蛋白，親和力沒有改變。烏拉地爾對缺血心肌SR3HRyanodine結(jié)合與親和力及對Ca2泵活力的作用見表2。急性缺血心肌Ca2泵即Ca2ATP酶活力從（1.19±0.11）molg蛋白-1min-1降至（0.94±0.14）molg蛋白-1min-1(P<0.05)，用藥后Ca2ATP酶活力上升1.

17、5倍，超過未治療組非缺血區(qū)1倍（P<0.01）。鈣釋放通道結(jié)合位點(diǎn)下降（P<0.05），親和力不變。將retenone(16mol/L)加入培養(yǎng)第4天的乳鼠心肌細(xì)胞50分鐘，在缺氧條件下，鈣泵活力下降55(2.75mol/g蛋白和1.25mol/g蛋白)。20mol/L的烏拉地爾保護(hù)鈣泵活力，接近有氧培養(yǎng)的對照組水平（2.75mol/g蛋白和2.48mol/g蛋白）。20mol/L拉西地平也有保護(hù)作用，但低于烏拉地爾（2.75mol/g蛋白和2.0mol/g蛋白）。表2烏拉地爾對家兔急性缺血心肌肌漿網(wǎng)鈣泵和鈣釋放通道的影響(±s)組別只數(shù)鈣釋放通道鈣泵活力（molg蛋白-

18、1min-1）最大結(jié)合力(fmol/mg蛋白)解離常數(shù)(nmol/L)對照組7155.74±23.500.90±0.381.70±0.25未治療組非缺血區(qū)5126.35±9.701.03±0.121.19±0.11缺血區(qū)1093.39±9.68*1.10±0.190.94±0.14*治療組非缺血區(qū)5134.50±19.351.18±0.441.34±0.16缺血區(qū)7118.68±12.290.92±0.182.30±0.16討論 哺乳動物心肌內(nèi)存

19、在受體，生理?xiàng)l件下調(diào)節(jié)心肌正性肌力作用不如受體顯著。急性缺血心肌內(nèi)受體及信號調(diào)節(jié)活躍，通過激活細(xì)胞內(nèi)PKC活力，增加二磷酸甘油DAG和磷酸肌醇IP3第二信使，使心肌細(xì)胞內(nèi)鈣負(fù)荷超載8。1R阻滯劑哌唑嗪似乎不直接影響心肌受體，而減輕細(xì)胞內(nèi)Ca2+超載。Gregorini等報告烏拉地爾明顯改善經(jīng)皮冠狀動脈血管成形術(shù)后冠脈痙攣，也改善結(jié)扎冠脈后總冠脈收縮和流量。有關(guān)烏拉地爾治療心肌缺血的代謝機(jī)制尚未見報告。本研究資料明確顯示，烏拉地爾使缺血心肌ATP、ADP、AMP極顯著升高，比未治療組升高0.71.9倍，改善心肌能量生成、利用和儲存的過程。能荷是組織內(nèi)能量生成利用和運(yùn)轉(zhuǎn)的重要標(biāo)志，烏拉地爾使缺血心

20、肌內(nèi)能荷值上升，接近和超過對照組。表明能量平衡恢復(fù)，這可能與烏拉地爾改善結(jié)扎以外的冠脈血管痙攣、恢復(fù)冠脈血流有關(guān)。心肌能量來源于葡萄糖有氧分解或無氧酵解和脂肪酸氧化，心肌乳酸含量是有氧或無氧代謝、丙酮酸能否順利進(jìn)入三羧酸循環(huán)的客觀證據(jù)，烏拉地爾能降低缺血心肌內(nèi)乳酸量（52），其具體作用環(huán)節(jié)尚待進(jìn)一步闡明。多種細(xì)胞膜蛋白影響胞內(nèi)Ca2+i濃度。主要調(diào)控蛋白是SR鈣泵和鈣釋放通道9，Ca2+泵將胞漿Ca2+泵入SR內(nèi)儲存，釋放通道將SR儲存Ca2+放入胞漿。維持心肌細(xì)胞Ca2+i平衡。從而調(diào)節(jié)興奮收縮偶聯(lián)。本研究發(fā)現(xiàn)，缺血心肌內(nèi)Ca2+泵活力明顯降低，釋放通道親和力無改變。烏拉地爾恢復(fù)缺血區(qū)Ca2

21、+泵活力和通道數(shù)目，從鈣調(diào)節(jié)蛋白水平上與Moraru資料相一致。烏拉地爾恢復(fù)缺血、缺氧心肌細(xì)胞SR Ca2+泵活力，它對缺血、缺氧心肌細(xì)胞內(nèi)鈣穩(wěn)定調(diào)節(jié)可能作用在SR鈣調(diào)節(jié)蛋白水平上。烏拉地爾恢復(fù)缺血心肌內(nèi)能量代謝狀態(tài)、降低乳酸含量、調(diào)整SR Ca2+泵和鈣釋放通道功能，從而降低胞內(nèi)Ca2+超載，是影響急性缺血的細(xì)胞基礎(chǔ)之一。作者單位：210029南京醫(yī)科大學(xué)第一附屬醫(yī)院（第八作者系北京友誼醫(yī)院）參考文獻(xiàn)1Hirschl MM, Seidier D, Laggner AN, et al. Effecacy of different antihypertensive drugs in the em

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