缺血预适应对缺氧—复氧后细胞离子稳态的影？…

研究缺血预适应对大鼠心肌细胞缺氧－复氧后离子稳态的影响及可能机制以及探讨缺血预适应抗心律失常的离子基础。采用Ｌａｎｇｄｏｒｆｆ逆行灌流技术分离ＳＤ大鼠心肌细胞。按①缺血预适应组、②Ｇｌｉｂｅｎｃｌａｍｉｄｅ干预缺血预适应组、③哇巴因（ＯＵＢ）干预缺氧－复氧组、④缺氧－复氧组、⑤Ｃｒｏｍａｋａｌｉｍ（ＣＲＫ）干预缺血预适应组、⑥ＯＵＢ和ＣＲＫ干预缺氧－复氧组、⑦正常对照组，分组孵育细胞，观察缺血预适     

严重烧伤早期心肌细胞动作电位和ATP酶活性的变化

为研究严重烧伤早期心肌细胞动作电位 (AP)和ATP酶 (ATPase)活性的变化 ,用玻璃微电极记录大鼠离体心室乳头肌细胞AP ,用差速离心和蔗糖密度梯度离心分离心肌细胞膜 ,用钼蓝比色定磷法测定总ATPase、Mg2 + ATPase和Na+ K+ ATPase活性。结果 :与对照组相比 ,①烧伤后各组静息电位、AP振幅和 0相最大去极化电位均降低 ,以伤后 8h最显著 ,分别为 - 6 4± 2vs - 87± 2mV ,88.4± 0 .0 5vs 12 3.1± 2 .8mV ,88± 9vs 2 33± 37V/s,P均 <0 .0 1,伤后 2 4h有所恢复。②AP复极化时程 (APD)缩短 ,以APD50 和APD90 缩短明显 ,烧伤后 3h和 8h二者均显著缩短 ,8h尤为明显 ,而APD10 仅伤后 8h稍缩短 ,且APD10 、APD50 和APD90 于烧伤后 2 4h已基本恢复正常 ;③伤后 8h大鼠心肌细胞膜总ATPase、Mg2 + ATPase和Na+ K+ ATPase活性均显著下降。结论 :烧伤早期主要影响Na+ 、K+ 离子流 ,Na+ 、K+ 内流均被抑制 ,K+ 外流增加     

苯那普利对乳鼠缺氧复氧心肌细胞肿瘤坏死因子的影响

目的 观察苯那普利对乳鼠心肌细胞缺氧复氧损伤时肿瘤坏死因子(TNF-α)的影响和可能机制.方法 采用纯化的乳鼠心肌细胞复制缺血再灌注模型.实验分5组正常对照组、单纯缺氧复氧组(HR)、缺氧复氧+苯那普利低剂量组(1×10-7 mol/L)、缺氧复氧+苯那普利中剂量组(1×10-6 mol/L)和缺氧复氧+苯那普利高剂量组(1×10-5 mol/L).细胞缺氧1 h复氧2 h后取细胞培养液测定TNF-α及乳酸脱氢酶(LDH)的活性,取细胞测定超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量.结果 单纯缺氧复氧组与对照组比较,TNF-α(40.32±5.54和5.43±3.32,P＜0.05)、LDH(27.83±2.71和8.0±1.41,P＜0.01)和MDA(0.757±0.036和0.131±0.029,P＜0.01)含量增高,SOD活性明显降低(对照组为36.13±1.43,其他各组为13.20±1.10、8.87±1.43、21.84±1.31、24.34±1.07,分别为P＜0.01和P＜0.05);各组苯那普利均减弱TNF-α的表达,减少MDA的生成和LDH的释放,明显提高SOD活性,并呈剂量依赖趋势.结论 苯那普利可抑制乳鼠缺氧复氧心肌细胞过度分泌TNF-α,具有明显的抗缺氧复氧损伤、保护心肌的作用.     

硫酸镁预处理对人脐静脉内皮细胞缺氧再复氧损伤的保护作用

目的探讨高浓度镁离子对培养人脐静脉内皮细胞(HUVECs)损伤的保护作用。方法对培养的HUVECs传代并分组:对照组常规培养;缺氧再复氧组缺氧1 h,再复氧1 h;镁离子干预组分别将细胞加入镁离子终浓度为2、4、6、8、10 mmol/L的培养液中进行培养,12 h后进行缺氧再复氧,观察三组细胞形态学变化,测定培养液乳酸脱氢酶(LDH)、丙二醛(MDA)、一氧化氮(NO)及内皮素(ET-1)的含量,并测定细胞内谷胱甘肽(GSH)含量并进行统计分析。结果①缺氧再复氧组细胞损伤严重,随镁浓度增加细胞形态结构趋于正常。②缺氧再复氧组培养液LDH、MDA及ET-1含量较对照组明显升高(P<0.001),镁离子组较缺氧复氧组降低(P<0.05);缺氧复氧组NO及细胞GSH含量较对照组降低〔(32.50±8.12)vs(96.46±8.77),P<0.001;(6.37±1.03)vs(16.01±2.41),P<0.001〕,镁离子组较缺氧复氧组升高(P<0.05)。结论高浓度镁离子能预防缺氧复氧损伤引起细胞结构改变,保护细胞膜和内皮功能。     

麦冬皂苷D对大鼠心肌细胞缺氧复氧损伤的保护作用及机制研究

目的:探讨麦冬皂苷D对心肌细胞缺氧复氧损伤的保护作用及机制.方法:体外培养心肌细胞H9c2,实验分为心肌细胞未行任何干预的对照组、缺氧复氧组(H/R组)、缺氧复氧+麦冬皂苷D组(H/R+OpD组)、缺氧复氧+麦冬皂苷D+磷脂酰肌醇-3激酶(PI3K)过表达组(H/R+OpD+PI3K组)、缺氧复氧+麦冬皂苷D+pcDN...     

缺氧预处理对缺氧复氧乳鼠心室肌细胞游离钙的影响

观察缺氧预处理对缺氧复氧乳鼠心室肌细胞游离钙的影响。建立培养乳鼠心肌细胞缺氧/复氧损伤模型。设正常对照组(A组)、缺氧复氧组(B组)、缺氧预适应组(C组)。经Flou-3/AM负载染色后,采用流式细胞分析技术,测定细胞内钙离子浓度;利用膜片钳技术,观察L型钙通道和钠钙交换电流的变化。结果:①与A组比较,B组可显著增加细胞内的游离钙离子浓度(P<0.01);L型钙电流密度明显下降,I-V曲线上移,半数失活电压(V1/2)减小,ICa,L失活曲线明显左移,而Na+/Ca2+交换电流显著增加。②与B组比较,C组可减轻缺氧再灌注时[Ca2+]i增加(P<0.01);可减轻再灌注对L型钙电流的抑制,使I-V曲线下移程度减轻,V1/2增加及稳态失活曲线的右移;减少Na+/Ca2+交换电流的增加;③与A组比较,C组钙内流和Na+/Ca2+交换电流有轻度增加(P<0.05)。结论:缺氧预处理使缺氧/复氧造成的[Ca2+]i增高的程度减轻,是通过抑制Na+/Ca2+交换电流的增加实现的。     

替米沙坦对OK细胞Na+-K+ ATP酶活性的影响

目的探讨血管紧张素Ⅱ受体拮抗剂(ARB)替米沙坦和血管紧张素转换酶抑制剂(ACEI)苯那普利对负鼠近端小管上皮细胞(OK细胞)Na+-K+-ATP酶活性的影响.方法培养的OK细胞采用低渗方法制备细胞膜悬液,使用BCA-100蛋白质定量测定试剂盒测定膜蛋白;Na+-K+ ATP酶活性采用孔雀绿比色分析法测定释放的无机磷(Pi)含量,培养液中分别加入血管紧张素Ⅱ(Ang Ⅱ)、Ang Ⅱ+血管紧张素Ⅱ受体拮抗剂替米沙坦(Telmisartan)、Ang Ⅱ+血管紧张素转换酶抑制剂苯那普利(Benazepril),观察它们对OK细胞Na+-K+-ATP酶活性的影响.结果 (1)培养液中加入10-10 mol/L Ang Ⅱ组与对照组相比,OK细胞Na+-K+-ATP酶活性明显上升.(0.0972±0.0080 vs 0.0896±0.0065 μmol·L-1·mg pro-1·h-1, P<0.05)(2) 当培养液中同时加入10{10 mol/L Ang Ⅱ和10-9mol/L Telmisartan,与单加入10-10mol/L AngⅡ组相比,OK细胞Na+-K+-ATP酶活性明显降低.(0.0623±0.0053 vs 0.0972±0.0080 μmol·L-1·mg pro-1·h-1,P<0.05)(3)当培养液中同时加入10-10 mol/L AngⅡ和10-9 mol/L Benazepril,与单加入10-10 mol/L AngⅡ组相比,OK细胞Na+-K+-ATP酶活性无明显变化.(0.1027±0.0166 vs 0.0972±0.0080 μmol·L-1·mg pro-1·h-1, P>0.05).结论血管紧张素Ⅱ作为一种生长因子,不仅能刺激细胞增殖,又能调节近端小管的离子转运,增加Na+-K+-ATP酶活性;替米沙坦能抑制血管紧张素Ⅱ引起的OK细胞Na+-K+-ATP酶活性增加,而苯那普利则无此作用.     

卡托普利晚期预处理对缺氧复氧心肌细胞游离钙的影响

目的观察卡托普利晚期预处理对缺氧复氧心肌细胞游离钙的影响,评价其延迟心肌的保护作用。方法建立培养乳鼠心肌细胞缺氧复氧损伤模型。设正常对照组、缺氧复氧组、缺氧预适应组、卡托普利预处理组、蛋白激酶C阻断剂+卡托普利组、一氧化氮合酶阻断剂+卡托普利组和核因子κB阻断剂+卡托普利组。利用分光光度计测定丙二醛和超氧化物岐化酶含量;全自动生物化学分析仪测定乳酸脱氢酶含量。Flou3AM负载染色和流式细胞分析技术测定细胞内钙离子浓度。结果卡托普利预处理和缺氧预处理均可减轻缺氧再灌注时心肌细胞内钙离子浓度增加(594±5nmolL、507±32nmolL比789±9nmolL,P<0.01),抑制乳酸脱氢酶和丙二醛的增加和超氧化物岐化酶含量的降低;但与对照组(414±37nmolL)比较钙内流仍有轻度增加(P<0.05);预先分别加入蛋白激酶C阻断剂、一氧化氮合酶阻断剂、核因子κB阻断剂与卡托普利共同孵育细胞,行缺氧复氧损伤所测得细胞内钙离子浓度分别为676±32nmolL、700±37nmolL和689±11nmolL,与卡托普利预处理组比较有所增加(P<0.05);但与缺氧复氧组比较仍有降低(P<0.05)。结论以上提示,卡托普利预处理可抑制缺氧复氧时的钙超载及其脂质过氧化损伤。其机制可能是通过轻度增加钙内流、启动心肌延迟保护作用;其过程可能涉及蛋白激酶C、一氧化氮合酶和核因子κB信号转导通路中的多个环节。     

线粒体ATP敏感钾通道介导缺氧预处理期细胞外信号调节蛋白激酶活化的机制

目的探讨线粒体ATP敏感钾通道(mKATP)与细胞外信号调节蛋白激酶(ERK1/2)在缺氧预处理延迟保护机制中的相互关系. 方法采用SD大鼠心肌细胞培养复制、缺氧/复氧损伤模型及缺氧预处理模型,建立对照组、缺氧/复氧组、缺氧预处理组、二氮嗪组、5-羟基癸酸+二氮嗪组、5-羟基癸酸+缺氧预处理组、二氮嗪+MGP组、缺氧预处理+2-巯基丙酰基甘氨酸(MGP)组、二氮嗪+PD98059组,以细胞存活率等作为反映心肌细胞损伤的指标,并于预处理后不同时间点分别测定细胞内活性氧含量及ERK1/2的活性. 结果缺氧预处理组及二氮嗪组细胞存活率和细胞内超氧化物歧化酶活性[(81.9±11.4)%、(13.6 ± 3.7)U/L,(79.2±12.4)%、(16.5±4.6)U/L]均显著高于缺氧/复氧组[(42.2±7.3)%、(8.8±2.8)U/L],缺氧预处理组及二氮嗪组乳酸脱氢酶释放[(101.9±18.9)U/L、(97.5±17.7)U/L]均显著低于缺氧/复氧组[(250.5±43.6)U/L,均为P＜0.01].缺氧预处理及二氮嗪均可快速诱导细胞内大量的活性氧生成并激活ERK1/2,但这些作用均可被mKATP阻滞剂5-羟基癸酸及氧自由基清除剂MGP所阻断.二氮嗪的细胞保护作用还可被ERK1/2阻滞剂PD98059所抵消. 结论 mKATP可通过诱导活性氧的生成而促进预处理期ERK1/2的激活,从而启动缺氧预处理延迟保护.     

丝裂素活化蛋白激酶参与心肌缺氧预处理的延迟保护作用

目的　探讨丝裂素活化蛋白激酶 (mitogen- activated protein kinase,MAPK)在心肌缺氧预处理延迟保护中的作用。方法　在培养乳鼠心肌细胞缺氧预处理的模型上 ,检测预处理后即刻、1h、6 h和 12 h的 MAPK活性变化 ,观察细胞缺氧 /复氧损伤后、延迟预处理后及蛋白激酶 C(PKC)抑制剂 chelerythrine(Ch)干预后的细胞存活率、L DH的释放、MDA含量和 SOD活性。结果　 MAPK活性在预处理后即刻明显增加 (P<0 .0 1) ,在 6 h后降至或接近对照水平。与未预处理组心肌细胞缺氧 /复氧损伤相比较 ,预处理后 2 4 h心肌细胞存活率增高 [(5 8.6 4± 5 .5 3) %vs (44 .2 9± 4 .2 7) % ,P<0 .0 1],L DH[(5 9.5 0± 11.0 8) U/ L vs(83.17± 13.6 9) U/ L,P<0 .0 1]和 MDA含量[(2 .33± 0 .4 9) nmol/ L vs(3.2 9± 0 .2 6 ) nmol/ L,P<0 .0 1]均降低 ,SOD活性增加 [(2 1.5 3± 3.6 3) n U/ m l vs(12 .86± 2 .6 8) n U/ ml,P<0 .0 1]。PKC抑制剂 chelerythrine可消除预处理的延迟保护作用。结论　预处理 2 4 h心肌细胞对再次缺氧 /复氧有保护作用 ,PKC、MAPK均参与心肌细胞预处理后的延迟保护作用     

缺血预适应抗心律失常机制的实验研究

研究缺血预适应 (IPC)抗心律失常的机制。采用玻璃微电极技术 ,观察 IPC对模拟缺血 复氧灌流过程中大鼠乳头肌动作电位 (AP)、有效不应期 (ERP)的影响。结果 :IPC过程中 AP进行性缩短。IPC使其后缺血 1～ 5 min时静息膜电位 (RMP)、AP幅度 (APA)、复极 90 %的 AP时程 (APD90 )、ERP、零相最大上升速度 (Vmax)迅速缩短或降低 ,IPC促进了缺血 3 0 min时上述参数的明显改善 ;IPC限制了复氧灌流时 APD90 的过度延长 ,并使 RMP超极化。ATP敏感性钾通道的加速开放与上述改变有关。结论 :IPC加速 ATP敏感性钾通道的开放 ,迅速缩短 APD和 ERP,降低膜电位 ,导致心肌细胞兴奋性降低及丧失 ,此可能是其抗缺血早期心律失常的机制之一。通过改善缺血晚期不应期离散度及缓慢传导的程度 ,减少 相折返 ,则是其抗缺血晚期心律失常的可能机制。IPC限制了复氧灌流时 APD90 的过度延长 ,从而减少内向离子流及触发活性 ,此可能与其抗再灌流心律失常有关。     

银杏叶提取物对模拟缺血条件下兔心室肌细胞动作电位及ATP敏感性钾通道的影响

研究银杏叶提取物 (EGb)对模拟缺血条件下兔心室肌细胞三磷酸腺苷敏感性钾通道电流 (IKATP)及跨膜动作电位时程 (APD)的影响 ,以探讨其抗缺血性心律失常作用的电生理机制。采用酶解法分离获取兔心室肌细胞 ,将其分为正常对照、持续缺血、缺血预处理以及含EGb液 (15 ,30 ,6 0 ,12 0 μg/L)灌流 4组。用全细胞膜片钳技术 ,记录不同条件下的IKATP和跨膜动作电位。结果 :①与持续缺血组比较 ,缺血预处理以及EGb(12 0 μg/L)可使单个心室肌细胞APD50 、APD90 明显缩短 (n =5 ,P <0 .0 5 )。EGb处理组与缺血预处理组相比较 ,对APD的影响无显著差异 ;②与持续缺血组比较 ,缺血预处理和EGb(12 0 μg/L)均可以使IKATP由 112 4± 15 3pA增至 344 0± 2 0 5和 2 95 9± 12 9pA(n =5 ,P <0 .0 5 ) ,使得IKATPI V曲线抬高 ;③增大的电流均可被Glibenclimide阻断。结论 :EGb可开放细胞膜ATP敏感性钾通道、缩短APD ,产生类似心肌缺血预适应的病理生理过程。     

Effect of high concentration of glucose on dopamine release from pheochromocytoma-12 cells

To investigate the mechanism of the action of high concentration of glucose on transmitter release from neuronal cells, we examined the effect of high concentration of glucose on dopamine release from pheochromocytoma-12 (PC12) cells. When the cells were incubated with 9.0 or 13.5 mg/mL glucose (2- or 3-fold of the optimum glucose concentration for PC12 cells), dopamine release was increased in a dose-related manner. Glucose-induced increase in dopamine release was blunted by nicardipine, a Ca2+ channel blocker. Following addition of 13.5 mg/mL glucose, intracellular Ca2+ concentration was increased, which was eliminated by nicardipine. Administration of 9.0 or 13.5 mg/mL glucose induced membrane depolarization in a dose-related manner. Glucose-induced dopamine release was inhibited by pinacidil or diazoxide, adenosine triphosphate (ATP)-sensitive K+ channel (KATP channel) openers. These results suggest that a high concentration of glucose induced ATP production, which blocked the KATP channel to induce membrane depolarization, and increased intracellular Ca2+ concentration and dopamine release. When the cells were cultured with 9.0 or 13.5 mg/mL glucose for 7 days, high potassium chloride (KCl)-induced dopamine release and 45Ca2+ uptake were increased. These results suggest that long-term incubation with a high concentration of glucose increased the capacity of Ca2+ uptake to enhance depolarization-induced dopamine release from PC12 cells. These data taken together suggest that a high concentration of glucose induced activation of the Ca2+ channel to stimulate dopamine release from PC12 cells.     

The Electrogenic Sodium/Potassium Pump and Passive Sodium Influx of Isolated Guinea Pig Ventricular Myocytes

Electrogenic Sodium/Potassium Pump in Ventricular Myocytes. Introduction: The membrane current generated by the sodium/potassium pump was measured in ventricular myocytes isolated from guinea pig hearts. Methods and Results: Cells were impaled with microelectrodes to minimize dialysis of the intracellular milieu and thus maintain normal physiological conditions. To selectively record pump current, it was necessary to suppress potassium currents with external barium and to buffer intracellular calcium with a calcium chelator. Exposure to potassium-free solution was used to inhibit the sodium/potassium (Na/K) pump and load the cell with intracellular sodium (Na _i). When the pump was subsequently reactivated by replacing extracellular potassium, there was a sharp increase of a transient outward membrane current, which declined back to control with a time constant of 50.7 ±12.9 seconds (mean ± SD, n = 11). The increase of outward membrane current became larger after longer periods of potassium removal and was abolished by the pump inhibitor strophanthidin. These features show that the transient outward current was generated by the Na/K pump. The pump current of quiescent myocytes was assessed by two different methods. First, it was evaluated directly by measuring the loss of outward pump current upon exposure to a maximal blocking dose of strophanthidin. The loss of outward current was 20.3 ± 5.5 pA (n = 11), which is equivalent to a pump current density of 0.164 /μA/cm². The second method was to estimate passive Na influx into quiescent myocytes, by measuring the charge extruded by the pump after a period of potassium removal. The charge extruded during recovery reflects the magnitude of Na _i accumulation during potassium removal. Passive Na influx was found to be 4.58 ± 2.4 pmol/cm² per second. If passive Na influx is normally balanced fully by Na extrusion on the Na/K pump, this will result in an average pump current of 18.0 pA (equivalent to a pump current density of 0.147 μA/cm²). Thus, although the two methods for estimating the pump current of quiescent myocytes were different, their results agreed well. A pump current of 20 pA will contribute only about 0.4 mV to the resting potential of ventricular myocytes. This conclusion was confirmed experimentally by showing that pump inhibition depolarized the resting potential of myocytes by only 0.45 ± 0.05mV(n = 10). Conclusion: An important finding of this study is that the electrogenic Na/K pump makes a much lower contribution to the resting potential of ventricular muscle than was previously supposed. (J Cardiovasc Electrophysiol, Vol. 3, pp. 225–238, June 1992)     

Association of diabetic neuropathy with Na/K ATPase gene polymorphism

Summary Diabetes mellitus induces a decrease in Na/K ATpase activity in man and animals, and this decrease plays a role in the development of diabetic neuropathy. Na/K ATPase is encoded by various genes, of which the ATP1 A1 gene is expressed predominantly in peripheral nerves and in erythrocytes. To investigate whether a polymorphism in the Na/K ATPase genes could explain the predisposition of some patients with insulin-dependent diabetes mellitus (IDDM) to develop polyneuropathy, a restriction fragment length polymorphism (RFLP) of the ATP1 A1 gene was studied together with erythrocyte Na/K ATPase activity in 81 Caucasian patients with more than 10 years' duration of IDDM. Associations with diabetic neuropathy, retinopathy and nephropathy were sought. Digestion of the first intron of the ATP1 A1 gene by the Bgl II restriction enzyme revealed a dimorphic allelism. Frequency of the restricted allele was 0.18 in this selected series (however, it was 0.10 in representative samples of IDDM patients and of normal subjects in our area). Mean erythrocyte Na/K ATPase activity was lower in diabetic patients than in 42 control subjects (292 ± 10, vs 402 ± 13 nmol Pi · mg protein^{− 1}· h^{− 1}, p < 0.0001) and was not related to HbA_1c value or to diabetes duration. It was lower in the group of the 28 patients bearing the restricted allele (241 ± 10 vs 319 ± 11 nmol Pi · mg protein^{− 1}· h^{− 1}, p < 0.0001). Neuropathy was absent in 50 patients, mild in 15 and severe in 16. When classified accordingly the three groups of patients did not differ with respect to sex, age and duration of diabetes. The respective frequency of the restricted allele among the groups was 10, 73 and 81 %, (p < 0.0001) and mean erythrocyte Na/K ATPase activity was respectively: 322 ± 10.7 nmol Pi · mg protein^{− 1}· h^{− 1}, 268 ± 15 and 229 ± 17, (p < 0.001). A borderline association between renal status or retinal status and repartition of polymorphism and a borderline correlation between renal status and Na/K ATPase activity were found, but significance disappeared after checking for the presence or absence of neuropathy. IDDM patients bearing the ATP1 A1 variant detected by Bgl II RFLP are much more frequently affected by neuropathy (relative risk 6.5, with 95 % CI 3.3–13). Identification of this risk factor may help to prevent this complication. It is suggested that the restricted allele is in linkage disequilibrium with a genomic mutation allowing diabetes to induce a greater impairment of Na/K ATPase activity which could in turn favour the development of neuropathy. [Diabetologia (1997) 40: 506–511] Received: 26 August 1996 and in revised form: 28 January 1997     

硝苯地平阻断豚鼠心室肌细胞L型钙流特性的研究

选择性L型钙通道阻断剂硝苯地平 (Nif)为常用的工具药 ,因此必须了解它对L型钙流 (ICa(L) )浓度、状态依赖性阻断 ,使用和非使用依赖性阻断等特性。以豚鼠分离的单个心室肌细胞为对象 ,采用膜片钳全细胞记录技术 ,给予 35℃的各种含药物细胞外液快速灌流 ,记录ICa(L) 。结果 :①保持电位 - 80mV ,使用含铯离子的细胞内、外液 ,在 +10mV的钳制电压 ,Nif抑制ICa(L) 的IC50 为 0 .3μmol·L-1;当保持电位为 - 40mV时 ,IC50 为 0 .0 5 μmol·L-1,显示Nif优先选择与失活态钙通道结合。②使用富含钾离子的细胞内、外液 ,对ICa(L) 的非使用依赖性阻断 ,随Nif使用浓度 (30～ 10 0 μmol·L-1)的增加和药物作用时间的延长而加强 ,同时对ICa(L) 的使用依赖性阻断则减小。③在 10s的静息间隔药物作用时间后的第一个实验刺激 ,Nif 3μmol·L-1或 30 μmol·L-1加速ICa(L) 的失活 ,提示Nif对ICa(L) 可能存在激活态阻断。结论 :在生理条件下 ,Nif对ICa(L) 的阻断呈浓度、状态依赖性 ,对ICa(L) 的非使用依赖性阻断随使用浓度的增加和作用时间的延长而加强 ,对ICa(L) 的使用依赖性阻断则随之减弱。     

门冬氨酸钾对肝细胞内钾离子浓度及细胞膜Na+·K+-ATP酶活性的影响

目的 了解门冬氨酸钾对肝细胞内钾离子浓度及细胞膜Na+·K+-ATP酶活性的影响.方法 人和大鼠肝细胞株培养传代,通过CCK-8测细胞活力确定门冬氨酸钾和氯化钾分别作用于两株肝细胞的合适浓度,利用该浓度处理细胞,培养0、24和48 h后破碎细胞取上清液测定两株肝细胞内钾离子浓度,提取细胞膜定磷法测定细胞膜Na+· K+-ATP酶活性.统计学处理采用t检验,方差分析及LSD法.结果 与空白组和氯化钾组相比,门冬氨酸钾组K+进入细胞内的量明显增多(P＜0.05或P＜0.01),在24 h、48 h两个时间点,L02细胞内K+浓度比KCl组分别升高了31％和38％,比空白组分别升高了62％和73％;BRL细胞内K+浓度比KCI组分别升高了21％和40％,空白组分别升高了52％和68％.且其细胞膜Na+· K+ -ATP酶活性升高(P＜0.01),但两株细胞间均无明显差异.结论 门冬氨酸钾能促进K+进入细胞内,并提高了肝细胞膜Na+· K+-ATP酶的活性.     

Functional Communication Between Cardiac ATPSensitive K+ Channel and Na/K ATPase

K_ATP Channel and Na/K ATPase. Introduction: Functional interaction between K_ATP channel and Na/K ATPase was studied in single guinea pig ventricular myocytes because both membrane molecules are known to he involved in ischemic episodes. Methods and Results: K_ATP channel currents were recorded at 36°C by using whole cell, cell attached, inside-out, and open cell-attached modes of patch clamp techniques on enzymatically isolated ventricular myocytes. In the whole cell mode, ouabain (1 μM) reversibly inhibited the K_ATP currents induced by metabolic stress (ATP-free pipette solution and 1 mM NaCN), but not those activated by cromakalim (100 μM), a K_ATP channel opener. In the cell-attached mode, ouabain concentration dependently inhibited K_ATP, channel opening induced by metabolic suppression (5.5 μM 2-deoxyglucose and 1 mM CN). Half-inhibition concentration for ouabain was 21.0 ± 5.5 nM and the Hill coefficient was 0.8 ± 0.1 (n = 26). However, ouabain did not have an effect on the channel activity induced by cromakalim (100 μM). In the inside-out mode, ouabain applied to the internal side of membrane did not affect the channel. In the open cell-attached mode made by preincubation with streptolysin-0 (0.08 U/mL), the K_ATP channels were not activated by the metabolic inhibitors but were by reducing extracellular ATP concentrations, because subsarcolenimal ATP concentration could he controlled through tiny membrane holes. The channels thus activated were not suppressed by ouabain. Conclusion: The inhibition of Na/K ATPase by ouahain appeared to block the K_ATP channels by accumulating subsarcolemmal ATP caused by a decrease of the transition from ATP to ADP. In the presence of ischemic episodes, the administration of digitalis compounds may affect the opening of K_ATP channels, which is primarily protective against the development of irreversible myocardial damage.     

Role of ONO-3144, a new cardioplegic agent, in the reoxygenation injury in the anoxic myocardium

We have investigated the effect of ONO-3144 (2-aminomethyl-4-tert-butyl-6-propionylphenol), which facilitates the conversion of prostaglandin G2 to H2 and acts as a scavenger of free radicals, on the reoxygenation injury in the anoxic heart. Rat hearts were perfused retrogradely with Krebs-Henseleit (KH) medium for 30 min (n = 8) in Group I. In Group II, the hearts which were perfused with anoxic KH medium for 40 min were reoxygenated for 30 min (n = 8). Group III hearts were similar to those in Group II except that 4 mg ONO-3144/liter was added to both anoxic and reoxygenation media (n = 8). Coronary effluent was collected for creatine kinase (CK) loss. Four rats hearts in each group were fixed for electron microscopic study and the remaining hearts were frozen in liquid nitrogen for measurement of adenosine triphosphate (ATP). A six-fold increase in CK leakage, observed after reoxygenation of anoxic heart, was prevented by ONO-3144. Tissue ATP was reduced from 22.2 +/- 0.9 mumol/g dry weight (Group I) to 5.5 +/- 1.1 mumol/g dry weight (Group II). A significant amount of ATP (9.05 +/- 1.22 mumol/g dry weight) was preserved in the treated Group III. The number of normal cells obtained by morphometrical analysis increased significantly from 56.7 +/- 7.8% (Group II) to 86.2 +/- 1.0% (Group III) and moderately injured cells were reduced to 3% in Group III as compared to 16% in the untreated Group I. Injury to the severely injured cells was not prevented by the drug treatment. At electron microscopic level, the cellular membranes, mitochondria and glycogen deposits were well preserved in Group III. Thus, ONO-3144 treatment provides a protection against reoxygenation injury in the anoxic myocardium by scavenging. .OH or other closely related species of free radicals. Therefore, free radicals generated through the conversion of prostaglandin G2 to H2 might play an important role in the reoxygenation injury of the anoxic myocardium.     

Low calcium reperfusion of ischemic myocardium

The mechanisms underlying protection of ischemic myocardium by reperfusion with solutions containing reduced concentrations of calcium (Ca) were studied in isolated vascularly perfused rabbit interventricular septa at 37°C. After 45 min of total ischemia adenosine triphosphate (ATP), and phosphocreatine (PC) contents fell to 6.8 ± 0.82, and 12.4 ± 2.0 μm/g dry tissue (dt) (±s.e.m.). After 5 min of reperfusion with 0.75 mm Ca these values had recovered significantly to 10.3 ± 1.0, and 33.3 ± 3.6, (μm/g dt), while reperfusion with 2.5 mm Ca produced 5.9 ± 1.0 and 22.0 ± 2.6 (μm/g dt). The significantly greater recovery of ATP and PC after 0.75 mm Ca reperfusion persisted after an additional 25 min of reperfusion with 2.5 mm Ca when compared to septa reperfused for the entire 30 min with 2.5 mm Ca. When mechanical work was reduced by cessation of stimulation during the first 5 min of reperfusion, ATP, PC and total creatine (TC) recovery were significantly improved in muscles exposed to 2.5 mm Ca but showed no additional improvement in muscles reperfused with 0.75 mm Ca. A further reduction to 100 μm Ca reperfusion in quiescent muscles did significantly improve ATP, PC and TC recovery. Quiescence achieved with 16.0 mm K impaired ATP, PC and TC recovery. This effect was reversed by 100 μm Ca reperfusion. Measurements of mitochondrial oxygen consumption, respiratory control and $\text{ADP}\text{O}$ showed results parallel to the ATP, PC and TC determinations. Both mechanical work and Ca itself influence mitochondrial respiration, ATP, PC and TC content after ischemia. As previously demonstrated for mechanical function, metabolic recovery can be improved by interventions limited to the first 5 min of reperfusion.