钙离子载体ionomycin引起内耳毛细胞胞内游离钙变化的研究

为研究听毛细胞的钙信号转导 ,观察了钙离子载体 ionomycin引起毛细胞胞内游离钙变化的过程。分离的豚鼠耳蜗外毛细胞经钙敏荧光染料 fluo-3和 fura-red染色后 ,用激光扫描共聚焦显微镜进行检测 ,以 fluo-3 /fura-red荧光比值指示胞内游离钙浓度高低。 5～ 10μmol/L ionomycin作用后 ,外毛细胞胞内游离钙由静态比值 1.69± 0 .5 9增至峰值时的 2 .78± 1.43 (x± s,n=10 ,P<0 .0 5 )。外毛细胞胞内游离钙增高的时程有两种模式 ,一种为胞内游离钙在药物作用后 10～ 3 5 s即有明显增加 (n=6) ,部分呈双相升高 ;另一种在药物作用后经过约 2 0 0～ 3 0 0 s的潜伏期 ,胞内游离钙方出现明显增加 (n=4)。一个外毛细胞在ionomycin作用后有钙波形成。ionomycin作用后外毛细胞胞内游离钙动态变化的多种模式显示了其作用机制和内耳毛细胞钙调控过程的复杂性 ,保证了钙在复杂的听觉外周过程中起重要的调节作用     

急性低氧大鼠肺动脉平滑肌内质网钙信号的变化及意义

目的：观察急性低氧大鼠肺动脉平滑肌内质网钙信号的变化及意义。方法:细胞水平：钙荧光探针(Fura-2/AM)负载大鼠肺动脉平滑肌细胞（PASMCs），荧光分光光度法，细胞外液无Ca2+及含Ca2+，在常氧（37 ℃、5% CO2、21 %O2、74 %N2 ）和急性低氧(37 ℃、5% CO2、2% O2、93 %N2)时，检测理阿诺碱（RD）和环匹阿尼酸（CPA）等对细胞浆内游离钙离子浓度（［Ca2+］i）的影响；离体血管环水平：相同条件，离体血管灌流方法检测肺动脉环张力变化。结果:(1)急性低氧时［Ca2+］i升高：常氧组［Ca2+］i为（96.99±7.16） nmol/L，低氧组为(257.06±32.48) nmol/L (P<0.01)。(2)与低氧组比较，预先用RD或普鲁卡因(procain)抑制内质网理阿诺碱受体敏感钙库，随后再给予低氧刺激时［Ca2+］i不升高，为（100.91±11.21） nmol/L (P<0.01)；而用CPA或thapsigargin（TG）抑制内质网摄取Ca2+，再给低氧刺激时［Ca2+］i呈升高状态(P>0.05)，而在细胞外液含钙及低氧下CPA及TG引起［Ca2+］i进一步升高(P<0.05)。(3)低氧引起肺动脉环收缩：常氧对基础张力无影响，低氧引起肺动脉环收缩，最大收缩张力达（49.28±8.64） g/g,P<0.01。(4)与低氧组比较，预先用RD或procain抑制内质网理阿诺碱受体敏感钙库，再给予低氧刺激，肺动脉环不收缩，最大收缩张力（3.75±1.14） g/g, P<0.01；而用CPA或TG后，再给予低氧刺激，肺动脉环呈收缩状态(P>0.05)，而在细胞外液含钙及低氧下CPA及TG引起肺动脉环进一步收缩(P<0.05)。结论:急性低氧可以引起内质网释放Ca2+，至少来自理阿诺碱受体敏感钙库的Ca2+释放参与了低氧肺血管收缩的发病机制；这可能是PASMCs自身具有的，既不依赖细胞外Ca2+内流，也不依赖血管内皮。     

地塞米松诱导小鼠腹腔巨噬细胞凋亡过程中[Ca^2＋]i的变化

目的 研究地塞米松诱导调亡小鼠巨噬细胞[Ca^2 ]i的变化及其对调亡的影响以及信使分子对凋亡和[Ca^2 ]i变化的影响。方法 激光扫描共聚焦显微术、流式细胞术和荧光标记术。结果 1、凋亡巨噬细胞内fluo-3荧光强度逐渐增强,胞内钙库受体抑制剂,尤其是Ca^2 内流阴断剂抑制钠fluo-3荧光强度变化,同时使细胞凋亡率降低;2、staurosporine 和DFcAMP显著降低巨噬细胞凋亡率并明显抑制fluo-3荧光强度改变。genistein和亚甲蓝落降低巨噬细胞凋亡率,并降低fluo-3荧光强度升高幅度。结论 1、胞外Ca^2 内流和内源性Ca^2 释放,主要是胞外Ca^2 内流使[Ca^2 ]i逐渐升高并促进巨噬细胞凋亡;2、PKC促进[Ca^2 ]i升高和巨噬细胞凋亡。cAMP抑制[Ca^2 ]i升高和巨噬细胞凋亡。cGMP、TPK降低[Ca^2 ]i升高幅度并稍抑制巨噬细胞凋亡。结果提示,[Ca^2 ]i是信使分子调控巨噬细胞凋亡的主要靶点。     

血管疾病治疗新靶标：STIM1和Orai1

钙离子(Ca2+)是常见的第二信使,不同于其它第二信使,其主要位于细胞外或存储于内质网(endoplasmic reticulum,ER)等细胞器内.静息状态下细胞内游离Ca2+浓度(intracellular Ca2+ concentration,[Ca2+]i)约为细胞外的1/20000,受体激活或生物信号刺激可通过改变[Ca2+]i进一步发挥生物放大效应.[Ca2+]i的升高主要通过胞内Ca2+释放和胞外Ca2+内流两大途径.随着胞内钙库的排空,位于质膜上的Ca2+内流通道被激活,使Ca2+由胞外进入胞质内,这个过程称为钙库操纵的钙内流(store-operated calcium entry,SOCE),其通道称为钙库操纵的钙通道(store-operated calcium channel,SOCC).近来研究证实组成SOCC的主要蛋白是:Ca2+感受蛋白基质相互作用分子1(stromal interaction molecule 1,STIM1)[1-2]和Ca2+通道蛋白Orai1[3-4].     

血管平滑肌细胞钙库Ca~(2+)动员的变化在休克血管低反应性形成中的作用

目的:探讨大鼠腹主动脉血管平滑肌细胞(VSMCs)在缺氧培养时胞内钙库钙释放的变化情况,并探讨胞内钙库钙释放在休克血管对去甲肾上腺素(NE)低反应性中的可能作用,为进一步阐明休克血管低反应性的发生机制提供依据。方法:建立大鼠失血性休克(40mmHg,2h)的在体模型及大鼠VSMCs缺氧细胞模型;采用Fura-3/AM钙离子成像方法测定VSMCs胞浆钙离子浓度([Ca2+])的变化情况及其与IP3受体(IP3R)及雷诺定受体(RyR)介导的钙释放通道的关系;采用离体器官张力测定技术,检测不同内钙释放依赖信号通路在休克血管低反应性形成中的可能作用。结果:在细胞外液无Ca2+的前提下,NE可通过动员内钙释放引起VSMCs胞浆[Ca2+]的明显升高;缺氧培养后VSMCs胞浆[Ca2+]较正常对照组有所升高,由NE诱导的VSMCs胞浆[Ca2+]较对照组有所降低,但均无显著差别;但在缺氧培养的VSMCs,细胞内钙库钙释放功能明显改变,表现为与正常对照组比较,缺氧VSMCs由IP3R敏感钙释放通道开放剂adenophostinA(10-5mol/L)及ATP-Na2(10-4mol/L)诱导的VSMCs胞浆[Ca2+]升高不显著,但RyR敏感钙释放通道开放剂caffeine可诱导缺氧VSMCs胞浆[Ca2+]的明显升高;失血性休克(40mmHg,2h)可引起大鼠腹主动脉血管对由NE诱导的收缩反应性明显降低,IP3R激动剂ATP-Na2(10-4mol/L)并不明显提高休克血管对NE的收缩反应性,但IP3R阻断剂heparin(104U/L)可明显抑制休克血管对NE的收缩反应性;此外,在无钙及含钙的K-H液中,RyR阻断剂ryanodine(10-5mol/L)可部分恢复休克血管对NE的收缩反应性,而RyR激动剂caffeine(10-3mol/L)可进一步降低休克血管对NE诱导的收缩反应性。结论:失血性休克后由RyR介导的内钙释放被激活部分参与了休克血管低反应性的形成。     

地塞米松诱导小鼠腹腔巨噬细胞凋亡过程中[Ca~(2 )]i的变化

目的　研究地塞米松诱导凋亡小鼠腹腔巨噬细胞 [Ca2 ]i的变化及其对凋亡的影响以及信使分子对凋亡和 [Ca2 ]i变化的影响。方法　激光扫描共聚焦显微术、流式细胞术和荧光标记术。结果　 1 凋亡巨噬细胞内fluo 3荧光强度逐渐增强。胞内钙库受体抑制剂 ,尤其是Ca2 内流阻断剂抑制细胞内fluo 3荧光强度变化 ,同时使细胞凋亡率降低 ;2 .staurosporine和DcAMP显著降低巨噬细胞凋亡率并明显抑制fluo 3荧光强度改变。genistein和亚甲蓝稍降低巨噬细胞凋亡率 ,并降低fluo 3荧光强度升高幅度。结论　 1 胞外Ca2 内流和内源性Ca2 释放 ,主要是胞外Ca2 内流使[Ca2 ]i逐渐升高并促进巨噬细胞凋亡 ;2 .PKC促进 [Ca2 ]i升高和巨噬细胞凋亡。cAMP抑制[Ca2 ]i升高和巨噬细胞凋亡。cGMP、TPK降低 [Ca2 ]i升高幅度并稍抑制巨噬细胞凋亡。结果提示 ,[Ca2 ]i是信使分子调控巨噬细胞凋亡的主要靶点。     

甘氨酸对内毒素刺激大鼠腹腔巨噬细胞释放TNF-α和升高[Ca~(2 )]_i的抑制作用特征

利用TNF-α敏感的细胞系L929,用MTT法检测细胞释放TNF-α的量和用单细胞钙荧光成像系统检测[Ca2 ]i变化动力学,研究甘氨酸对内毒素(LPS)刺激的大鼠腹腔巨噬细胞(PMψ)释放肿瘤坏死因子α(TNF-α)和升高[Ca2 ]i的影响。结果显示,LPS剂量依赖性的刺激PMψ[Ca2 ]i呈单峰状升高。甘氨酸对正常PMψ基本无激活作用而能显著抑制LPS刺激PMψ过度释放TNF-α和升[Ca2 ]i,两种效应都呈现相似的剂量依赖性,其对[Ca2 ]i升高的抑制作用主要表现为抑制胞外钙内流。结果表明,甘氨酸通过抑制LPS刺激的PMψ胞外钙内流抑制[Ca2 ]i升高,是其抑制LPS刺激PMψ过度释放TNF-α的信号传导的关键环节。     

次声对视网膜微血管内皮细胞胞浆[Ca2+]i水平的影响

目的通过检测次声暴露前后视网膜微血管内皮细胞(BREC)胞浆[Ca2 ]i水平的改变,对次声致大鼠血-视网膜屏障通透性改变的细胞内信号转导途径进行初步探讨.方法采用荧光探针Flu-3研究次声及BKca开放剂NS-1619干预下BREC胞浆[Ca2 ]i水平的改变.结果次声暴露导致细胞内钙库释放Ca2 ,同时观察到NS-1619外钙依赖性地增加[Ca2 ]i.结论次声引起[Ca2 ]i升高,通过细胞收缩成份引起内皮细胞主动收缩,从而达到对血-视网膜屏障的调节效应.     

磷脂酶C-三磷酸肌醇途径参与β-淀粉样肽(25-35)诱导的海马神经元[Ca~(2+)]i的增加

目的探讨内质网(ER)钙库在β-淀粉样肽(25-35)(Aβ25-35)诱导的细胞内钙超载中的作用。方法用钙高度特异性荧光探针Flou-3/AM负载海马神经元,进行荧光染色,利用激光扫描共焦显微镜观察在不同药物干预条件下海马神经元内游离钙离子荧光强度的变化。结果 2、10、20μmol/L各浓度组凝聚态Aβ25-35均增加了海马神经元胞内[Ca2+]i(n=5,P0.05);三磷酸肌醇受体(IP3R)的特异性抑制剂2-APB明显地抑制了胞内[Ca2+]i的增加,蓝尼碱受体(RyR)的特异性抑制剂硝苯呋海因(dantrolene)却无法抑制。Aβ25-35作用1h后内质网钙容量即有明显下降,作用24h后降低更为明显。在无细胞外钙情况下,磷脂酶C(PLC)的抑制剂U73122部分抑制了Aβ25-35诱导的胞内[Ca2+]i的增加。结论凝聚态Aβ25-35可通过IP3途径产生,IP3作用于IP3R而引起内质网钙的释放,磷脂酶C的活化可能参与了上述过程。     

氯通道ClC-3对脑血管平滑肌细胞Ca2+池操纵性Ca2+内流的影响

目的 :研究ClC - 3Cl-通道在培养的牛脑血管平滑肌细胞 (CSMC)Ca2 + 池操纵性Ca2 + 内流中的作用。方法 :采用培养的CSMC ,用硫代磷酸修饰的ClC - 3反义寡核苷酸与脂质体一起转染入平滑肌细胞 4 8h ,以正义和随机序列寡核苷酸作为对照 ,在生物荧光双波长影像分析系统用Fura- 2 /Am荧光探针测定单个细胞内游离Ca2 + 浓度。结果 :(1)采用Ca2 + ImageSystem测定脑血管平滑肌细胞静息 [Ca2 + ]i ,以 340 / 380nm波长的比值表示为 0 6 84± 0 0 0 2 ,内皮素 - 1(ET - 1) 10 -7mol·L-1刺激细胞引起 [Ca2 + ]i呈双相升高反应 ,先是快…     

Caffeine affects Ca uptake and Ca release from intracellular stores: fura-2 measurements in isolated snail neurones

Using the fluorescent probe fura-2, the average cytoplasmic concentration of free Ca2+ [( Ca]i) was measured in isolated voltage-clamped neurons of the snail Helix pomatia. In normal Ringer solution [Ca]i transients elicited by membrane depolarizations lasting 30-100 s have a voltage dependence similar to that of the calcium current. In the presence of caffeine [Ca]i transients did not depend on the testing voltage, indicating Ca release from intracellular stores. In both cases [Ca]i decayed after the transient increase. The rate of [Ca]i decline was monoexponential and independent of the membrane potential. In caffeine-containing solution the decline was 3 times faster. Steady membrane depolarization in the presence of caffeine induced periodic changes in [Ca]i. A simple model to describe these oscillations on the basis of Ca release from and Ca uptake into intracellular stores predicted that the oscillations could be initiated and modulated by Ca influx into the cytoplasm, which is in line with experimental data.     

Caffeine stimulates the reverse mode of Na+/Ca2+ exchanger in ferret ventricular muscle

This study investigated the effect of caffeine on the sarcolemmal mechanisms involved in intracellular calcium control. Ferret cardiac preparations were treated with ryanodine and thapsigargin in order to eliminate the sarcoplasmic reticulum (SR) function. This treatment abolished caffeine contracture irreversibly in normal solution. The perfusion with K‐free medium that blocked the Na⁺–K⁺ pump resulted in a recovery of slow relaxing caffeine contractures similar to Na‐free contractures. The amplitude of caffeine contractures was dependent on the bathing [caffeine]o and [Ca²⁺]_o. Divalent cations Ni²⁺ and Cd²⁺, which have an inhibitory effect on the Na⁺/Ca²⁺ exchanger, produced dose‐dependent inhibition of caffeine responses with apparent Ki of 780 ± 19 and 132 ± 5 μM , respectively. Caffeine also caused dose‐dependent inhibition of Na‐free contractures (Ki=4.62 ± 1.5 mM ), and the reduction or removal of [Na⁺]_o exerted an inhibitory effect on caffeine contractures (Ki=73.5 ± 17.12 mM ). These experiments indicate that the increase in resting tension following exposure to caffeine was mediated by Na⁺/Ca²⁺ exchanger, which represents an additional element of complexity in caffeine action on cardiac muscle.     

Calcium and voltage-dependent alterations of cell volume in neuroblastoma×glioma hybrid NG108-15 cells

Intracellular calcium ([Ca2+](i)), cell volume, membrane potential and currents were measured in neuroblastomaxglioma hybrid cells to gain insight into how [Ca2+](i) controls cell volume. [Ca2+](i) was increased by fluid shear stress, mechanical stimulation of the cells, the Ca2+ ionophore A23187, caffeine and thapsigargin. The increase in [Ca2+](i) induced by mechanical stimulation was decreased by about 50% by caffeine and abolished after incubation of the cells in a Ca2+-free solution. Mechanical stimulation by stirring the cell suspension induced cell shrinkage that was abolished by caffeine, but induced cell swelling in Ca2+-free solution. In the presence of caffeine, A23187 induced cell shrinkage whereas thapsigargin induced cell swelling. Both cell volume changes were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid. The cells were hyperpolarized by fluid shear stress and A23187 and depolarized by caffeine, thapsigargin and intracellular EGTA. Under all these conditions, the membrane input resistance was decreased. Voltage-clamp experiments suggested that, in addition to an increased anionic current, fluid shear stress and A23187 increased a K+ current, whereas caffeine and intracellular Ca2+ chelation increased a non-selective cation current and thapsigargin increased both a K+ and a non-selective cation current. Taken together, these results suggest that, if cell volume is closely dependent on [Ca2+](i) and the activity of Cl- channels, its relative value is dependent on the ionic selectivity of co-activated channels and the membrane potential.     

Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms.

The roles of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Na(+)-Ca2+ exchange in Ca2+ removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration ([Ca2+]i) were measured in indo-1-loaded cells. Na(+)-Ca2+ exchange was inhibited by replacement of external Na+ by Li+. To avoid net changes in cell or SR Ca2+ load during a twitch in 0 Na+ solution, intracellular Na+ (Na+i) was depleted using a long pre-perfusion with 0 Na+, 0 Ca2+ solution. SR Ca2+ accumulation was inhibited by caffeine or thapsigargin (TG). Relaxation of steady-state twitches was 2-fold faster in rat than in rabbit (before and after Na+i depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half-time for relaxation of caffeine contractures 15- and 5-fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. Inhibition of the Na(+)-Ca2+ exchange during a twitch increased the [Ca2+]i transient amplitude (delta[Ca2+]i) by 50% and the time constant of [Ca2+]i decline (tau) by 45% in rabbit myocytes. A smaller increase in tau (20%) and no change in delta[Ca2+]i were observed in rat cells in 0 Na+ solution. [Ca2+]i transients remained more rapid in rat cells. Inhibition of the SR Ca(2+)-ATPase during a twitch enhanced delta[Ca2+]i by 25% in both species. The increase in tau after TG exposure was greater in rat (9-fold) than in rabbit myocytes (2-fold), which caused [Ca2+]i decline to be 70% slower in rat compared with rabbit cells. The time course of [Ca2+]i decline during twitch in TG-treated cells was similar to that during caffeine application in control cells. Combined inhibition of these Ca2+ transport systems markedly slowed the time course of [Ca2+]i decline, so that tau was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca2+ transport mechanisms (mitochondrial Ca2+ uptake and sarcolemmal Ca(2+)-ATPase) is similar in these species. We conclude that during the decline of the [Ca2+]i transient, the Na(+)-Ca2+ exchange is about 2- to 3-fold faster in rabbit than in rat, whereas the SR Ca(2+)-ATPase is 2- to 3-fold faster in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones

Experiments were done using guinea-pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium-induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca²⁺]_i) with the Ca²⁺-sensitive dye fluo-3, we demonstrated that 10 m m caffeine activated ryanodine receptors and caused a rise in [Ca²⁺]_i in both Ca²⁺-containing and Ca²⁺-deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μ m thapsigargin or caffeine and 20 μ m ryanodine blocked subsequent caffeine-induced elevations of [Ca²⁺]_i. Treatment with thapsigargin, ryanodine or 200 μ m Cd²⁺ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μ m tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca²⁺ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea-pig sympathetic stellate neurones.     

Caffeine stimulates the reverse mode of Na/Ca2+ exchanger in ferret ventricular muscle.

This study investigated the effect of caffeine on the sarcolemmal mechanisms involved in intracellular calcium control. Ferret cardiac preparations were treated with ryanodine and thapsigargin in order to eliminate the sarcoplasmic reticulum (SR) function. This treatment abolished caffeine contracture irreversibly in normal solution. The perfusion with K-free medium that blocked the Na+--K+ pump resulted in a recovery of slow relaxing caffeine contractures similar to Na-free contractures. The amplitude of caffeine contractures was dependent on the bathing [caffeine]o and [Ca2+]o. Divalent cations Ni2+ and Cd2+, which have an inhibitory effect on the Na+/Ca2+ exchanger, produced dose-dependent inhibition of caffeine responses with apparent Ki of 780 +/- 19 and 132 +/- 5 microM, respectively. Caffeine also caused dose-dependent inhibition of Na-free contractures (Ki=4.62 +/- 1.5 mM), and the reduction or removal of [Na+]o exerted an inhibitory effect on caffeine contractures (Ki=73.5 +/- 17.12 mM). These experiments indicate that the increase in resting tension following exposure to caffeine was mediated by Na+/Ca2+ exchanger, which represents an additional element of complexity in caffeine action on cardiac muscle.     

Intracellular calcium store depletion and acrosome reaction in human spermatozoa: role of calcium and plasma membrane potential

We evaluated the presence and role of internal calcium stores in human uncapacitated spermatozoa by determining the effects of two inhibitors of Ca2+ ATPase of the sarco-endoplasmic reticulum (SERCA-ATPase), thapsigargin and cyclopiazonic acid (CPA) on intracellular calcium concentrations, [Ca2+](i), plasma membrane potential and acrosome reaction. Using a fluorescent conjugate of thapsigargin, we localized internal Ca2+ stores on the acrosome, post-acrosomal region and sperm midpiece. SERCA-ATPase inhibitors induced a rise in [Ca2+](i) both in Ca2+ and Ca2+-free media but under these latter conditions it was reduced with a progressive decline to baseline values; the re-addition of Ca2+-stimulated a rise in [Ca2+](i). This demonstrated that internal Ca2+ store depletion can evoke the opening of Ca2+-channels on sperm plasma membrane, thus showing the existence of "capacitative" Ca2+ entry into these specialized cells. The addition of thapsigargin to human spematozoa induced a dose-dependent increase in acrosome reaction percentages, but only when Ca2+ was present in the external medium. Plasma membrane potential monitoring showed that these inhibitors induced a depolarization dependent on Ca2+ influx from external medium and that this was preceded by a transient hyperpolarization caused by activation of Ca2+-dependent K+ channels. When K+-dependent plasma membrane hyperpolarization was inhibited, the thapsigargin- and CPA-stimulated rise in [Ca2+](i) plasma membrane depolarization and acrosome reaction were abolished. In conclusion, the present study demonstrates that human spermatozoa possess internal Ca2+ stores and that the capacitative Ca2+ entry pathway present in these cells regulates important biological processes that are fundamental for the acrosome reaction.     

卡托普利对缺氧/复氧乳鼠心室肌细胞游离钙的影响及其离子通道机制

目的：观察卡托普利晚期预处理对缺氧/复氧乳鼠心室肌细胞游离钙的影响及其离子通道机制。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、缺氧预适应组和卡托普利组。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(［Ca²⁺］i)；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）缺氧/复氧时，［Ca²⁺］i和Na⁺/Ca²⁺交换电流高于正常对照组（P<0.01），L-型钙电流（I_Ca-L）峰值下降，I-V曲线上移，半数失活电压(V_0.5)减小，ICa-L失活曲线左移。（2）晚期预处理和卡托普利使缺氧/复氧时［Ca²⁺］i低于缺氧/复氧组（P<0.01）；I_Ca-L增加，I-V曲线下移，V_0.5增大及稳态失活曲线右移；Na⁺/Ca²⁺ 交换电流减少；但［Ca²⁺］i和Na⁺/Ca²⁺交换电流高于对照组（P<0.05）。（3）卡托普利组与缺氧预适应组比较上述指标均无显著差异 。结论：心肌细胞缺氧/复氧，通过Na⁺/Ca²⁺交换电流的异常增加可引起［Ca²⁺］i的异常升高及其钙超载；卡托普利通过轻度增加Na⁺/Ca²⁺交换电流及其［Ca²⁺］i而触发晚期预处理，抑制后续缺氧/复氧引起的Na⁺/Ca²⁺交换电流及其［Ca²⁺］i的异常增加。