缝隙连接在大鼠缺血预适应心肌保护中的作用

目的探讨心肌缝隙连接在缺血预适应中的调控机制以及在预适应信号传导通路中的地位和作用。方法Sprague—Dawley大鼠进行30min的冠状动脉缺血和4h再灌注,根据是否使用缺血预适应、二氮嗪、18β-甘草次酸和5-羟基奎酸分为7组,即对照组（单纯缺血再灌注）、缺血预适应组、缺血预适应和5-羟基奎酸合用组、二氮嗪组、二氮嗪和5-羟基奎酸合用组、甘草次酸组、甘草次酸和5-羟基奎酸合用组。测量各组的血流动力学指标、心肌梗死面积以及缝隙连接蛋白43的磷酸化和内化。结果与对照组相比,缺血预适应、二氮嗪、18β-甘草次酸能降低心肌梗死面积（P〈0．001）,5-羟基奎酸可以阻断缺血预适应和二氮嗪的心肌保护作用（P〉0．05）,但是对缝隙连接失耦联剂甘草次酸没有影响（P〈0．001）。缺血预适应和线粒体三磷酸腺苷敏感性钾通道开放剂二氮嗪能促进缝隙连接蛋白43磷酸化,抑制去磷酸化和内化（P〈0．001,P〈0．001）,线粒体三磷酸腺苷敏感性钾通道阻断剂5-羟基奎酸能阻断该作用（P〉0．05,P〉0．0051）。结论缺血预适应通过开放线粒体三磷酸腺苷敏感性钾通道来调节缝隙连接蛋白43的磷酸化和内化,进而调控缝隙连接的化学转运,减少心肌梗死面积。     

心肌缝隙连接在缺血预适应中的作用

目的探讨心肌缝隙连接（GJ）在缺血预适应（IPC）心肌保护中的地位和作用。方法将72只大鼠分为缺血再灌注组（对照组）、IPC组、IPC＋5-羟基奎酸（5-HD）组、二氮嗪（Dia）组、Dia＋5-HD组、甘草次酸（GA）组、GA＋5-HD组、GA＋IPC组,测量各组的血流动力学指标、心肌梗死面积和心律失常发生情况。结果与对照组相比,IPC组、Dia组、GA组能降低心肌梗死面积,减少心律失常,5-HD能阻断IPC和Dia的心肌保护作用,对GA无影响。IPC加用GA不能抑制IPC的保护作用。结论在IPC信号传导通路中,GJ位于线粒体ATP敏感性钾通道的下游,可能是IPC的终末效应器,但在IPC触发阶段不起作用。     

Prostacyclin protects ischemic reperfused myocardium in the dog by inhibition of neutrophil activation

Prostacyclin (PGI2) and the stable PGI2 analogue SC39902 (6,9 alpha-epoxy,5S-fluoro-11 alpha, 15S-dehydroxyprosta-6,13E-dien-1-oic acid, sodium salt) were studied in anesthetized open-chest dogs subjected to 90 minutes of left circumflex coronary artery (LCCA) occlusion and 6 hours of reperfusion. PGI2 (50 ng/kg/min, infused into the left atrium) reduced infarct mass by 59% compared to control, but SC39902 (1.5 micrograms/kg/min) failed to produce a significant reduction in infarct size. Both PGI2 and SC39902 reduced mean arterial blood pressure, heart rate, and rate-pressure product to the same extent. Regional myocardial blood flow measured with radiolabelled tracer microspheres did not demonstrate an increase in regional blood flow to the ischemic myocardium during the 90 minutes of LCCA occlusion in the PGI2 and control treatment groups. Canine neutrophils were isolated from whole blood and activated with opsonized zymosan. PGI2 produced a concentration-dependent inhibition of neutrophil activation as measured by superoxide production in vitro, whereas SC39902 failed to effectively inhibit neutrophil activation. Neutrophil migration into inflammatory skin lesions was effectively attenuated when dogs were pretreated with PGI2 (50 ng/kg/min, intravenously). Therefore, it is suggested that the cytoprotective effect of PGI2 during myocardial ischemia and reperfusion is related to an inhibition of neutrophil migration and the production of cytotoxic activated oxygen species.     

缝隙连接对心肌肥厚兔室性心律失常的影响

目的研究兔慢性压力超负荷模型中缝隙连接(G J)对室性心律失常的影响。方法30只兔随机分为假手术组(Sham组)、心肌肥厚组(LVH组)和抗心律失常肽组(AAP10组)。LVH组和AAP10组通过缩窄腹主动脉制备兔左室压力超负荷心肌肥厚模型,Sham组仅游离腹主动脉未进行缩窄。动物饲养3个月后制备兔左室楔形心肌块的灌注模型,Sham组和LVH组灌流台氏液,AAP10组灌流含AAP10的台氏液,记录不同起搏周长下容积心电图、跨室壁离散度(TDR)及刺激反应间期(SR I),并观察早期后除极(EAD)及室性心律失常的发生率。结果在不同频率起搏下,LVH组SR I和TDR与Sham组比较均明显增加(P<0.05)。而AAP10组的SR I和TDR与LVH组比较明显减小(P<0.05)。Sham组无1例诱发EAD和室性心律失常;在5 000 m s起搏时LVH组和AAP10组EAD的发生率分别为10/10、3/10,室性心律失常发生率分别为4/10,1/10,两组比较差异有显著性(P<0.05)。结论G J激动剂AAP10减轻了心肌肥厚时SR I的延长和TDR增加,相应的减少了EAD和室性心律失常的发生率。     

Altering ventricular activation remodels gap junction distribution in canine heart

INTRODUCTION: Prolonged arrhythmic or paced ventricular activation causes persistent changes in myocardial conduction and repolarization that may result from altered electrotonic current flow, for which gap junctional coupling is the principal determinant. Remodeling of gap junctions and their constituent connexins modifies conduction and has been causally implicated in reentrant arrhythmogenesis. We hypothesized conversely that altering the pattern of ventricular activation causes gap junctional remodeling. METHODS AND RESULTS: Seven dogs were paced from the left ventricular (LV) epicardium (VVO, approximately 120 beats/min) for 21 days before excision of transmural LV samples that were divided into endomyocardial, mid-myocardial, and epimyocardial layers. Another five paced dogs had recording electrodes attached to multiple LV sites. All 12 dogs developed characteristic pacing-induced persistent T wave changes of cardiac memory. After 21 days of pacing, the ventricularly paced QRS duration prolonged by a mean of 4 msec over baseline (P < 0.05), a change that was associated with significant slowing of intraventricular conduction to local sites. These changes in QRS duration and repolarization were associated with a reduction in epimyocardial connexin43 expression on quantitative Western blotting of LV myocardium from close to, but not distant from, the pacing site (61.7+/-18.4 vs 100.9+/-34.0; P < 0.02) and a marked disruption in immunolabeled connexin43 distribution in epimyocardium only. CONCLUSION: Spatially distinct transmural and regional gap junctional remodeling is a consequence of abnormal ventricular activation and is associated with consistent changes in activation that may alter patterns of repolarization and facilitate reentrant arrhythmogenesis.     

依普利酮对大鼠肥厚心肌缝隙连接蛋白重构及心电图指标变化的作用

目的观察白发性高血压大鼠(SHR)左心室心肌肥厚时心肌缝隙连接蛋白43(Cx43)和心电学指标的变化及依普利酮的作用。方法将24只SHR随机分为依普利酮治疗组(sHR-epl组,12只)、末治疗组(SHR-c组,12只),12只Wistar-Kyoto(WKY)大鼠作为正常血压对照组(正常组)。饲养10周(26周龄)后处死,用放射免疫法测定血浆和心脏组织醛固酮(ALD)浓度,体表心电图记录Q-Tc问期,免疫组化法观察大鼠心室肌Cx43分布特征。结果未治疗组血浆和心脏组织ALD高于对照组(均为P0.05),未治疗组Cx43表达[(0.163±0.053)DM]较对照组[(0.307±0.095)DM]明显减少(均为P0.01),且分布紊乱,同时伴Q-Yc间期延长;治疗组Cx43表达[(0.288±0.089)DM]较末治疗组明显增多(P0.01),且分布规则,同时伴Q-Tc问期缩短。结论 SHR肥厚心肌存在Cx43重构现象,ALD升高可能导致肥厚心肌Cx43重构及心室肌除复极延长,依普利酮能有效改善Cx43重构及心室肌除复极时间。     

Modulation of conduction slowing in ischemic rabbit myocardium by calcium-channel activation and blockade

Ventricular action potentials and longitudinal conduction times over short distances of epicardium were recorded in isolated rabbit hearts. Global ischemia produced a progressive decrease of resting membrane potential, depression of action potentials, and conduction slowing to approximately 50% of control values over 8 to 10 min. Verapamil (2 X 10(-6) M) markedly attenuated ischemia-induced conduction slowing in association with less depression of maximum upstroke velocity (Vmax) and slightly less change in resting membrane potential. In contrast, Bay K 8644 (10(-7) M), a calcium-channel agonist, exacerbated ischemia-induced conduction slowing and depression of Vmax but did not significantly affect resting membrane potential. Regression analysis of Vmax vs resting membrane potential and the square of conduction velocity vs Vmax demonstrated that verapamil and Bay K 8644 shifted these relationships in opposite directions. The results indicate that the calcium-channel activation state can modulate slowing of conduction during early ischemia. This is most likely due to alterations in calcium influx before and/or during ischemia. There appear to be three possible components to this effect: (1) a small alteration in the magnitude of ischemia-induced depolarization, (2) alterations of membrane responsiveness at depolarized values of resting membrane potential, and (3) alterations in "nonactive" components of conduction during ischemia, such as changes in excitability or cell-to-cell coupling.     

膜离子通道及缝隙连接在心律失常中的作用

心肌细胞膜离子通道是其电活动的基础，各种原因所致的Na^＋、K^＋、Ca^2＋通道异常改变可产生多种心律失常。近年研究发现，心肌细胞间缝隙连接功能障碍同样是引起心律失常的一个重要因素。对缝隙连接进行恰当的药物干预，也能取得明显的抗心律失常作用。     

Canine neutrophil activation by cardiac lymph obtained during reperfusion of ischemic myocardium

Cardiac lymph from a canine model of myocardial ischemia and reperfusion was examined for evidence of chemotactic activity. Lymph was continuously collected from awake animals before and during a 60-minute coronary artery occlusion and up to 6 hours after the initiation of reperfusion. It was assessed for the ability to activate the following proinflammatory functions in neutrophils isolated from the blood of healthy dogs: 1) morphological changes characteristic of chemotactic stimulation, which were assessed by phase contrast microscopy, 2) orientation of canine neutrophils in a gradient of cardiac lymph, which was assessed in Zigmond chambers, 3) the binding of monoclonal antibodies reactive with CD11b and CD18 adherence glycoproteins, which was assessed by flow cytometry, and 4) adherence of canine neutrophils to monolayers of canine jugular vein endothelium, which was assessed in vitro by a visual assay. Lymph samples collected after 1 hour of reperfusion in animals demonstrating ECG evidence of ischemia and histological evidence of infarction exhibited significant stimulatory activity for each of the functions tested. Shape change-inducing activity was evaluated at more frequent intervals than other functions and was found to peak at 1 hour after initiation of reperfusion and to disappear by 6 hours. In addition, the CD11b/CD18 levels on neutrophils isolated from cardiac lymph collected during reperfusion were significantly greater than neutrophils obtained before or during occlusion. Animals that failed to exhibit evidence of infarction also failed to exhibit increased stimulatory activity in lymph collected during reperfusion, and surface levels of CD11b/CD18 on neutrophils collected from reperfusion lymph were not elevated. This study provides direct evidence supporting the hypothesis that chemotactic activity is generated in ischemic and reperfused myocardium.     

Single-channel events and gating behavior of the cardiac gap junction channel.

The activity of gap junction channels between pairs of neonatal rat heart cells in culture was studied under control conditions and during uncoupling procedures by using dual whole-cell voltage clamp techniques. Under control conditions gap junctional conductance ranged from 0.05 to 35 nS. In cell pairs exhibiting low gap junctional conductance (less than 500 pS), single-channel events with a unitary conductance of 53 +/- 2 pS (5 experiments; 186 events) were apparent. Event duration and open-time probability were estimated to be 0.95 sec and 0.17, respectively. When the junctional conductance in well-coupled cell pairs (with initial junctional conductance, greater than 5 nS) was reduced by cytoplasmic acidification or application of heptanol, single-channel events could be visualized. Compared to low-conductance controls, unitary channel conductance was unaltered (for acidification the conductance was 58 +/- 3 pS in 11 experiments with 253 events; for heptanol the conductance was 61 +/- 1 pS in 2 experiments with 171 events), while the probability of channels being open was decreased. The constancy of unitary channel conductance under control conditions and during uncoupling procedures suggests that opening and closing of the gap junction channel are all-or-none processes during which no stable subconductance states are formed.     

缺血预适应减少心肌缺血损伤机制的研究

目的　通过对心肌缺血预适应的动物模型观察 ,探讨细胞凋亡在其中的作用 ,以及p5 3,bcl 2 ,Bax基因对其发生进行的调控。方法　采用TUNEL标记技术研究心肌缺血预适应心肌细胞中细胞凋亡现象 ,并采用免疫组化染色技术及原位分子杂交技术研究p5 3,bcl 2及Bax基因的蛋白及mRNA的表达。结果　缺血预适应组 (P)及非缺血预适应组 (NP)非缺血区均未见凋亡细胞 ,但在P组缺血区可见散在的凋亡细胞 ,而在NP组缺血区则多见。P组p5 3蛋白表达显著低于NP组 ,bcl 2蛋白表达在P组显著高于NP组 ,Bax蛋白表达在P组显著低于NP组 ,并且bcl 2 /Bax的比值P组与NP组相比显著升高。P组p5 3基因mRNA表达显著低于NP组 ,bcl 2基因mRNA表达在P组显著高于NP组。结论　心肌缺血预适应对心肌的保护可通过抑制细胞凋亡来实现 ,并且通过bcl 2表达增加 ,p5 3、Bax表达减少对其进行调控。     

乌拉地尔干预急性心肌缺血的细胞机制

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Mechanism and selectivity of the effects of halothane on gap junction channel function

Volatile anesthetics alter tissue excitability by decreasing the extent of gap junction-mediated cell-cell coupling and by altering the activity of the channels that underlie the action potential. In the present study, we demonstrate, using dual whole-cell voltage-clamp techniques, that coexpression of connexin (Cx) 40 and Cx43 rendered cells more sensitive to uncoupling by halothane than cells that express only Cx40 or only Cx43. The halothane-induced reduction in junctional conductance was caused by decreased channel mean open time and increased channel mean closed time. The magnitude of the effect of halothane on channel open time was least for Cx40-like channels and greatest for heteromeric channels. Thus, the data indicate that halothane gates gap junction channels to the closed state in a dose-dependent and connexin-specific manner. One consequence of the selectivity of halothane is that the profile of single-channel events observed in the presence of halothane may not be quantitatively representative of the population of channels contributing to macroscopic conductance in cells that express more than one connexin. In addition, in tissues that express multiple connexins, such as heart and blood vessels, the capacity of the gap junctions to transmit electrical and chemical signals in the presence of halothane could vary according to the pattern of connexin expression.     

Carvedilol inhibits the mitochondrial permeability transition by an antioxidant mechanism

It was previously shown that carvedilol, a β-adrenergic receptor antagonist with antioxidant properties, was able to inhibit the mitochondrial permeability transition (MPT). In the present work, the hypothesis was that the negative impact of carvedilol on the MPT was specifically the result of its antioxidant effect. For the current investigation, we used three different MPT inducers. MPT-associated events were tested to study the protective effect of both carvedilol and cyclosporin-A, the known MPT inhibitor. Carvedilol inhibited mitochondrial swelling with calcium plus phosphate and with calcium plus t-butylhydroperoxide, but not with calcium plus carboxyatractyloside. Carvedilol inhibited the oxidation of thiol groups with calcium plus phosphate (p<0.01) and with calcium plus t-butylhydroperoxide (p<0.05), but not with calcium plus carboxyatractyloside—in opposition to the full protection afforded by cyclosporin-A when using calcium and carboxyatractyloside. Our results showed that carvedilol was effective only when the MPT was triggered by a primary oxidative process. This finding implies that the antioxidant properties of carvedilol are crucial for the observed effects and reinforces the advantageous use of carvedilol in cardiac pathologies associated with enhanced cellular oxidative stress.     

卡维地洛对大鼠心肌间隙连接通讯的影响及作用机制研究

目的研究卡维地洛对大鼠心肌缺血再灌注损伤、心肌间隙连接通讯（GJIC）及连接蛋白43（CX43）的影响,验证卡维地洛可通过改变CX43磷酸化状态抑制GJIC起到防止心肌再灌注损伤的假设.方法将大鼠随机分为假手术组、缺血再灌注组和卡维地洛组.结扎左冠状动脉前降支致缺血30 min后复灌4 h,建立心肌缺血再灌注损伤模型,于再灌注4 h末测定心肌酶及心肌梗死范围变化;制作Langendorf心脏灌流模型,随机分为假手术组、缺血再灌注组、卡维地洛组和庚醇组,于整体缺血30 min末期用改良的划痕标记染料示踪技术测定GJIC;用Western blot技术检测缺血30 min CX43磷酸化状态的改变.结果与假手术组相比,缺血再灌注组心肌酶及心肌梗死范围明显增加,GJIC无明显变化,非磷酸化CX43升高.与缺血再灌注组比较,卡维地洛组心肌酶及心肌梗死范围显著减少,同间隙连接抑制剂庚醇作用相似,GJIC也明显受到抑制,伴非磷酸化CX43水平明显上升.结论卡维地洛具有使CX43去磷酸化进而抑制GJIC防止心肌再灌注损伤的作用.     

Lindane, a gap junction blocker, suppresses FSH and transforming growth factor beta1-induced connexin43 gap junction formation and steroidogenesis in rat granulosa cells

The present study was designed to explore the role of gap junctions in follicle-stimulating hormone (FSH) and transforming growth factor beta1 (TGF beta1)-stimulated steroidogenesis in ovarian granulosa cells of gonadotropin-primed immature rats. There were three specific aims. First, we investigated the effect of FSH and TGF beta1 as well as lindane (a general gap junction blocker) on the level of connexin43 (Cx43), the major gap junction constituent in granulosa cells, and on gap junction function. The second aim was to determine the effect of lindane on FSH and TGF beta1-stimulated progesterone production and the levels of two critical players, cytochrome P450 side-chain cleavage (P450scc) enzyme and steroidogenic acute regulatory (StAR) protein. The third aim was to further investigate the specific involvement of Cx43 gap junctions in FSH and TGF beta1-stimulated steroidogenesis using a Cx43 mimetic peptide blocker. Immunoblotting analysis showed that FSH plus TGF beta1 dramatically increased the levels of phosphorylated Cx43 without significantly influencing the level of nonphosphorylated Cx43, and this stimulatory effect was completely suppressed by lindane. Also, immunofluorescence analysis showed that Cx43 immuno-reactivity increased in the FSH plus TGF beta1-treated group and predominantly appeared in a punctate pattern at cell-cell contact sites, and lindane reduced such cell periphery immunostaining. Furthermore, TGF beta1 enhanced the FSH-induced gap junction intercellular communication and lindane completely suppressed this effect. In addition, lindane suppressed the FSH and TGF beta1-stimulated increases in progesterone production and the levels of P450scc enzyme and StAR protein. This study demonstrates a clear temporal association between the Cx43 protein level/gap junction communication and progesterone production in rat ovarian granulosa cells in response to FSH and TGF beta1 as well as lindane. Furthermore, a specific Cx43 gap junction blocker suppressed FSH plus TGF beta1-stimulated progesterone production. In conclusion, this study suggests that Cx43 gap junctions may play a critical role in FSH plus TGF beta1-stimulated progesterone production in rat ovarian granulosa cells.     

Stretch-activated ion channel blocker gadolinium attenuates ischemic ST-segment elevation in canine myocardium.

Regional transmural ischemia causes both ST-segment elevation and systolic elongation (bulging) of the myocardium. Mechanical stretch might alter the transmembrane potential via stretch-activated ion channels (SAC); however, the role of SAC on ischemic ST-segment elevation has not yet fully studied. The present study investigated the role of SAC in the genesis of ischemic ST-segment elevation in the in-vivo canine heart. In 6 anesthetized dogs, an extracorporeal conduit connected to the left anterior descending coronary artery was occluded for 5 min before and after the intracoronary infusion of gadolinium (Gd, 500 micromol/min). To eliminate the effect of ischemic preconditioning, the bypass was occluded for 5 min before the experiment. Percent systolic shortening (%SS) and percent systolic elongation (%bulging) were measured using a pair of ultrasonic dimension crystals. A unipolar epicardial ECG was monitored at the center of the ischemic area for the measurement of the ST-segment level. At the end of coronary occlusion, there was no difference in the reduction of %SS or the increase of %bulging between before and after infusion of Gd. ST-segment elevation, however, was significantly attenuated by the infusion of Gd. These data demonstrated that the activation of SAC is one cause of ischemic ST-segment elevation.