Isoflurane Postconditioning Prevents Opening of the Mitochondrial Permeability Transition Pore through Inhibition of Glycogen Synthase Kinase 3β

Background: Postischemic administration of volatile anesthetics activates reperfusion injury salvage kinases and decreases myocardial damage. However, the mechanisms underlying anesthetic postconditioning are unclear.

Methods: Isolated perfused rat hearts were exposed to 40 min of ischemia followed by 1 h of reperfusion. Anesthetic postconditioning was induced by 15 min of 2.1 vol% isoflurane (1.5 minimum alveolar concentration) administered at the onset of reperfusion. In some experiments, atractyloside (10 [mu]m), a mitochondrial permeability transition pore (mPTP) opener, and LY294002 (15 [mu]m), a phosphatidylinositol 3-kinase inhibitor, were coadministered with isoflurane. Western blot analysis was used to determine phosphorylation of protein kinase B/Akt and its downstream target glycogen synthase kinase 3[beta] after 15 min of reperfusion. Myocardial tissue content of nicotinamide adenine dinucleotide served as a marker for mPTP opening. Accumulation of MitoTracker Red 580 (Molecular Probes, Invitrogen, Basel, Switzerland) was used to visualize mitochondrial function.

Results: Anesthetic postconditioning significantly improved functional recovery and decreased infarct size (36 +/- 1% in unprotected hearts vs. 3 +/- 2% in anesthetic postconditioning; P < 0.05). Isoflurane-mediated protection was abolished by atractyloside and LY294002. LY294002 inhibited isoflurane-induced phosphorylation of protein kinase B/Akt and glycogen synthase kinase 3[beta] and opened mPTP as determined by nicotinamide adenine dinucleotide measurements. Atractyloside, a direct opener of the mPTP, did not inhibit phosphorylation of protein kinase B/Akt and glycogen synthase kinase 3[beta] by isoflurane but reversed isoflurane-mediated cytoprotection. Microscopy showed accumulation of the mitochondrial tracker in isoflurane-protected functional mitochondria but no staining in mitochondria of unprotected hearts.     

Postconditioning Prevents Reperfusion Injury by Activating [delta]-Opioid Receptors

Background: While postconditioning has been proposed to protect the heart by targeting the mitochondrial permeability transition pore (mPTP), the detailed mechanism underlying this action is unknown. The authors hypothesized that postconditioning stimulates opioid receptors, which in turn protect the heart from reperfusion injury by targeting the mPTP.

Methods: Rat hearts (both in vivo and in vitro) were subjected to 30 min of ischemia and 2 h of reperfusion. Postconditioning was elicited by six cycles of 10-s reperfusion and 10-s ischemia. To measure nitric oxide concentration, cardiomyocytes loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein were imaged using confocal microscopy. Mitochondrial membrane potential was determined by loading cardiomyocytes with tetramethylrhodamine ethyl ester.

Results: In open chest rats, postconditioning reduced infarct size, an effect that was reversed by both naloxone and naltrindole. The antiinfarct effect of postconditioning was also blocked by the mPTP opener atractyloside. In isolated hearts, postconditioning reduced infarct size. Morphine mimicked postconditioning to reduce infarct size, which was abolished by both naltrindole and atractyloside. N-nitro-l-arginine methyl ester and guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one blocked the action of morphine. Further experiments showed that morphine produces nitric oxide in cardiomyocytes by activating [delta]-opioid receptors. Moreover, morphine could prevent hydrogen peroxide-induced collapse of mitochondrial membrane potential in cardiomyocytes, which was reversed by naltrindole, N-nitro-l-arginine methyl ester, and the protein kinase G inhibitor KT5823.     

Inhibition of mitochondrial permeability transition enhances isoflurane-induced cardioprotection during early reperfusion: the role of mitochondrial KATP channels

Inhibition of the mitochondrial permeability transition pore (mPTP) mediates the protective effects of brief, repetitive ischemic episodes during early reperfusion after prolonged coronary artery occlusion. Brief exposure to isoflurane immediately before and during early reperfusion also produces cardioprotection, but whether mPTP is involved in this beneficial effect is unknown. We tested the hypothesis that mPTP mediates isoflurane-induced postconditioning and also examined the role of mitochondrial KATP (mKATP) channels in this process. Rabbits (n = 102) subjected to a 30-min coronary occlusion followed by 3 h reperfusion received 0.9% saline (control), isoflurane (0.5 or 1.0 MAC) administered for 3 min before and 2 min after reperfusion, or the mPTP inhibitor cyclosporin A (CsA, 5 or 10 mg/kg) in the presence or absence of the mPTP opener atractyloside (5 mg/kg) or the selective mK(ATP) channel antagonist 5-hydroxydecanoate (5-HD; 10 mg/kg). Other rabbits received 0.5 MAC isoflurane plus 5 mg/kg CsA in the presence and absence of atractyloside or 5-HD. Isoflurane (1.0 but not 0.5 MAC) and CsA (10 but not 5 mg/kg) reduced (P < 0.05) infarct size (21% +/- 4%, 44% +/- 6%, 24% +/- 3%, and 43% +/- 6%, respectively, mean +/- sd of left ventricular area at risk; triphenyltetrazolium staining) as compared with control (42% +/- 7%). Isoflurane (0.5 MAC) plus CsA (5 mg/kg) was also protective (27% +/- 4%). Neither atractyloside nor 5-HD alone affected infarct size, but these drugs abolished protection by 1.0 MAC isoflurane, 10 mg/kg CsA, and 0.5 MAC isoflurane plus 5 mg/kg CsA. The results indicate that mPTP inhibition enhances, whereas opening abolishes, isoflurane-induced postconditioning. This isoflurane-induced inhibition of mitochondrial permeability transition is dependent on activation of mitochondrial KATP channels in vivo.     

Inhibition of glycogen synthase kinase enhances isoflurane-induced protection against myocardial infarction during early reperfusion in vivo

Inhibition of glycogen synthase kinase (GSK)-beta protects against ischemia-reperfusion injury. Brief exposure to isoflurane before and during early reperfusion after coronary artery occlusion also protects against infarction. Whether GSK-beta mediates this action is unknown. We tested the hypothesis that GSK inhibition enhances isoflurane-induced postconditioning. Rabbits (n = 88; 6 to 7 per group) subjected to a 30-min coronary occlusion followed by 3 h reperfusion received saline, isoflurane (0.5 or 1.0 minimum alveolar concentration [MAC]) administered for 3 min before and 2 min after reperfusion, the selective GSK inhibitor SB216763 (SB21; 0.2 or 0.6 mg/kg), or 0.5 MAC isoflurane plus 0.2 mg/kg SB21. Other groups of rabbits pretreated with phosphatidylinositol-3 kinase (PI3K) inhibitor wortmannin (0.6 mg/kg), 70-kDa ribosomal protein s6 kinase (p70s6K) inhibitor rapamycin (0.25 mg/kg), or mitochondrial permeability transition pore (mPTP) opener atractyloside (5 mg/kg) received 0.6 mg/kg SB21 or 0.5 MAC isoflurane plus 0.2 mg/kg SB21. Additional groups received the mPTP inhibitor, cyclosporin A (5 mg/kg), plus 0.2 mg/kg SB21 with or without atractyloside pretreatment. Isoflurane (1.0 but not 0.5 MAC) and SB21 (0.6 but not 0.2 mg/kg) reduced (P < 0.05) infarct size (21% +/- 5%, 44% +/- 7%, 23% +/- 4%, and 46% +/- 2%, respectively, of left ventricular area at risk, mean+/- sd; triphenyltetrazolium staining) as compared with control (42% +/- 6%). Isoflurane (0.5 MAC) plus 0.2 mg/kg SB21 and cyclosporin A plus 0.2 mg/kg SB21 produced similar degrees of protection (24% +/- 4% and 27% +/- 6%, respectively). Atractyloside but not wortmannin or rapamycin abolished protection produced by 0.6 mg/kg SB21 and 0.5 MAC isoflurane plus 0.2 mg/kg SB21. Thus, GSK inhibition enhances isoflurane-induced protection against infarction during early reperfusion via a mPTP-dependent mechanism.     

ROS和PI3K-Akt在缺血后处理或控制性低压灌注减轻大鼠脊髓缺血再灌注损伤中的作用

目的 探讨活性氧自由基(ROS)和磷酸酰肌醇3激酶-蛋白激酶B(PI3K-Akt)在缺血后处理或控制性低压灌注减轻大鼠脊髓缺血再灌注损伤中的作用.方法 雄性SD大鼠126只,体重300～350 g,随机分为7组(n=18),缺血再灌注组(I/R组)阻断胸主动脉同时维持MAP40 mm Hg持续9 min进行脊髓缺血,胸主动脉开放后使MAP升至100 mm Hg进行脊髓再灌注;缺血后处理组(IP组)开放主动脉后,进行再灌注30 s缺血30 s,重复3次,同时维持MAP 100 mm Hg;控制性低压灌注组(LR)开放主动脉后维持MAP 40 mm Hg持续5 min后升高至100 mm Hg;缺血后处理+PI3K抑制剂LY-294002组(IP+L组)和控制性低压灌注+LY-294002组(LR+L组)分别于实施缺血后处理和控制性低压后立即动脉注射LY-294002 25 mg/kg;缺血后处理+氧自由基清除剂N-乙酰半胱氨酸组(IP+N组)和控制性低压灌注+N-乙酰半胱氨组(LR+N组)分别于实施缺血后处理和控制性低血压后立即动脉注射N-乙酰半胱氨酸100 mg/kg.于再灌注2 h时,各组处死12只大鼠,取腰段脊髓组织,测定胞浆Akt磷酸化水平和线粒体通透性转换孔(mPTP)开放程度.分别于再灌注4、12、24、48 h进行神经行为学评分,然后处死大鼠,取腰段脊髓组织,分别进行脊髓前角正常神经元和凋亡神经元的计数.结果 与I/R组比较,IP组和LR组Akt磷酸化水平升高,mPTP开放程度和神经元凋亡计数降低,神经行为学评分和正常神经元计数升高(P＜0.01);IP组与LR组各指标比较差异无统计学意义(P＞0.05).LY294002和N-乙酰半胱氨酸均可逆转缺血后处理和控制性低压灌注对脊髓的保护作用,引起mPTP开放程度升高(P＜0.01).结论 ROS激活PI3K-Akt进而降低线粒体通透性是缺血后处理或控制性低压灌注减轻大鼠脊髓缺血再灌注损伤的机制.     

右美托咪啶后处理对大鼠离体心脏缺血再灌注时线粒体损伤的影响

目的 探讨右美托咪啶后处理对大鼠离体心脏缺血再灌注时线粒体损伤的影响.方法 健康雌性Wistar大鼠,体重220～250 g,成功制备Langendorff离体灌注模型的40个心脏随机分为5组(n=8):缺血再灌注组(A组)、右美托咪啶10 nmol/L组(B组)、右美托咪啶100 nmol/L组(C组)、线粒体通透性转换孔开放剂苍术苷组(D组)及右美托咪啶联合苍术苷组(E组).离体心脏经K-H液平衡灌注20 min后,采用全心停灌40 min再灌注60 min的方法制备离体心脏缺血再灌注模型.于再灌注即刻B组、C组、D组和E组分别灌注含10 nmol/L右美托咪啶、100 nmol/L右美托咪啶、20μmol/L苍术苷、100 nmol/L右美托咪啶和20 μmol/L苍术苷的K-H液10 min.再灌注结束即刻取心尖组织,分离线粒体,测定SOD、Na+ -K+ -ATP酶、Ca2+-ATP酶活性和MDA和Ca2+含量.结果 与A组比较,B组和C组线粒体SOD、Na+ -K+ -ATP酶和Ca2+ -ATP酶活性升高,MDA和Ca2+含量降低(P＜0.05),D组和E组上述指标比较差异无统计学意义(P＞0.05);与C组比较,D组和E组线粒体SOD、Na+-K+-ATP酶和Ca2+ -ATP酶活性降低,MDA和Ca2+含量升高(P＜0.05),B组上述指标比较差异无统计学意义(P＞0.05).结论 右美托咪啶后处理可减轻大鼠离体心脏缺血再灌注时的线粒体损伤,其机制可能与抑制线粒体通透性转换孔开放有关.     

PI3K、ERK1/2、mito-K_(ATP)通道及mPTP在七氟醚后处理减轻大鼠离体心脏缺血再灌注损伤中的作用

目的 评价磷脂酰肌醇-3-激酶(PI3K)及细胞外信号调节激酶1/2(ERK1/2)、线粒体ATP敏感性钾(mito-K_(ATP))通道及线粒体膜通透性转换孔(mPTP)在七氟醚后处理减轻大鼠离体心脏缺血再灌注损伤中的作用.方法 雄性清洁级SD大鼠,周龄7～10周,体重250～300 g,采用Langendorff法建立大鼠离体心脏灌注模型,取模型制备成功的心脏180个,随机分为12组(n=15),对照组(C组):持续灌注90 min;缺血再灌注组(IR组):停止灌注K-H液30 min,再灌注60 min;IR+LY组、IR+PD组、IR+ATR组、IR+5-HD组和IR+DMSO组:于再灌注即刻分别灌注PI3K特异性抑制剂LY294002(LV)15μmol/L、ERK1/2特异性抑制剂PD98059(PD)20 μmol/L、mPTP开放剂苍术甙(ATR)20 μmol/L、mito-K_(ATP)通道抑制剂5-羟癸酸(5-HD)100 μmol/L和溶剂二甲基亚砜(DMSO)0.02%15 min;七氟醚后处理组(S组):于再灌注即刻灌注经3%七氟醚饱和的K-H液15 min,随后更换正常K-H液再灌注45 min;S+LY组、S+PD组、S+ATR组和S+5-HD组:于再灌注即刻灌注经七氟醚饱和的K-H液同时分别灌注LY 15 μmol/L、PD 20 μmol/L、ATR 20 μmol/L、5-HD 100 μmol/L 15 min,随后更换为正常K-H液再灌注45 min.于平衡灌注20 min、停灌前即刻、再灌注15、30和60 min(T_(0～4))时测定冠状动脉流量(CF),记录心功能指标;C组、IR组和s组分别于T_0和T_4时收集冠状动脉流出液测定乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)活性和肌钙蛋白I(cTnI)浓度;于T_4时取左心室,测定心肌梗死面积,C组、IR组、IR+ATR组、IR+5.HD组、IR+DMSO组、S组、S+ATR组和S+5-HD组检测细胞凋亡情况,计算凋亡指数(AI),C组、IR组、IR+LY组、IR+PD组、IR+DMSO组、S组、S+LY组和S+PD组测定心肌烟酰胺腺嘌呤二核苷酸(NAD~+)含量.结果 与C组比较,其余各组再灌注时心功能降低,CF降低,心肌梗死面积增大,IR组、IR+ATR组、IR+5-HD组、IR+DMSO组、S组、S+ATR组和S+5-HD组AI升高,IR组、IR+LY组、IR+PD组、IR+DMSO组、S组、S+LY组和S+PD组NAD~+含量降低,IR组和S组LDH、CK-MB活性和cTnI浓度升高(P<0.05);与IR组比较,S组再灌注时心功能提高,CF升高,心肌梗死面积减小,AI降低,NAD~+含量升高,LDH、CK-MB活性和cTnI浓度降低(P<0.05),其余组上述指标差异无统计学意义(P>0.05).结论 七氟醚后处理可能通过激活PI3K及ERK1/2、促进mito-K_(ATP)通道开放、抑制mPTP开放,从而减轻大鼠离体心脏缺血再灌注损伤.     

The influence of B-cell lymphoma 2 protein, an antiapoptotic regulator of mitochondrial permeability transition, on isoflurane-induced and ischemic postconditioning in rabbits

Brief exposure to isoflurane or repetitive, transient ischemia during early reperfusion after prolonged coronary artery occlusion protects against myocardial infarction by inhibiting the mitochondrial permeability transition pore (mPTP). Inhibition of mPTP during delayed ischemic preconditioning occurred concomitant with enhanced expression of the antiapoptotic protein B cell lymphoma-2 (Bcl-2). We tested the hypothesis that Bcl-2 mediates myocardial protection by isoflurane or brief ischemic episodes during reperfusion in rabbits (n = 91) subjected to a 30-min left anterior descending coronary artery occlusion followed by 3 h reperfusion. Rabbits received 0.9% saline, isoflurane (0.5 or 1.0 minimum alveolar concentration, MAC) administered for 3 min before and 2 min after reperfusion, 3 cycles of postconditioning ischemia (10 or 20 s each) during early reperfusion, 0.5 MAC isoflurane plus 3 cycles of postconditioning ischemia (10 s), or the direct mPTP inhibitor cyclosporin A (CsA, 10 mg/kg) in the presence or absence of the selective Bcl-2 inhibitor HA14-1 (2 mg/kg, i.p.). Isoflurane (1.0, but not 0.5, MAC) and postconditioning ischemia (20 s but not 10 s) significantly (P < 0.05) reduced infarct size (mean +/- sd, 21% +/- 4%, 43% +/- 7%, 19% +/- 7%, and 39% +/- 11%, respectively, of left ventricular area at risk) as compared with control (44% +/- 4%). Isoflurane (0.5 MAC) plus 10 s postconditioning ischemia and CsA alone also exerted protection. HA14-1 alone did not affect infarct size nor block protection produced by CsA but abolished reductions in infarct size caused by 1.0 MAC isoflurane, 20 s postconditioning ischemia, and 0.5 MAC isoflurane plus 10 s postconditioning ischemia. The results suggest that Bcl-2 mediates isoflurane-induced and ischemic postconditioning by indirectly modulating mPTP activity in vivo.     

Leucine‐rich glioma inactivated 3 promotes HaCaT keratinocyte migration

Our finding that human skin expresses leucine‐rich glioma inactivated 3 (LGI3) raises the question of the function of this cytokine in keratinocytes. We have shown that LGI3 stimulates human HaCaT keratinocyte migration without affecting viability or proliferation. Western blot analysis showed that LGI3 induced focal adhesion kinase activation, Akt phosphorylation, and glycogen synthase kinase 3β (GSK3β) phosphorylation in these cells. Using the scratch wound assay and a modified Boyden chamber, we found that LY294002, a selective phosphatidylinositol 3‐kinase inhibitor, and LiCl, a selective GSK3β inhibitor, abolished LGI3‐induced cell migration. We tested β‐catenin levels after LGI3 treatment because the Akt‐GSK3β pathway regulates β‐catenin accumulation, and β‐catenin promotes cell migration. LGI3 treatment increased β‐catenin protein and nuclear localization, whereas LY294002 prevented LGI3‐induced focal adhesion kinase and Akt activation as well as β‐catenin accumulation. Overall, these data suggest that LGI3 stimulates HaCaT cell migration following β‐catenin accumulation through the Akt pathway.     

The influence of Phosphatidylinositol 3-kinase/Akt Pathway on the Ischemic Injury during Rat Liver Graft Preservation

We aimed to investigate the role of phosphatidylinositol 3 (PI3)-kinase/Akt pathway on ischemic injury. Rat liver grafts were preserved in UW solution with different treatments and were compared by 1-week survival rates and morphological changes with those of the control group. PI3-kinase/Akt was significantly activated at the sites of Thr 308 and Ser 473 in the preserved grafts. Downstream target proteins, glycogen synthase kinase-3beta (GSK-3beta) and caspase-9, were inactivated. However, survival signal transduction from Akt to Bad was blocked by calcium release after activation of PI3-kinase/Akt. Significant activation of caspase-12, -3 and -7 contributed to cell apoptosis and severe ischemic injury was shown after 7 h of preservation by UW solution with insulin. Downregulation of phospho-Akt at Thr 308 and Ser 473 was due to partial inhibition of PI3-kinase/Akt pathway by LY294002. Activation of GSK-3beta and inactivation of caspase-12 and Bad could be found in the LY294002 groups in which the liver grafts showed less ischemic injury. Higher 1-week survival rates in the heparin, LY294002, and glucagon groups confirmed the dysregulation of the pathway. In conclusion, PI3-kinase/Akt pathway was dysregulated and contributed to ischemic injury during preservation. Heparin and LY294002 could improve graft viability by maintaining calcium homeostasis during preservation.     

麻醉药物后处理心肌的胞内信号转导研究进展

背景 麻醉药物后处理是最近提出的一种心肌保护的新策略,具有很好的临床应用前景.目的 麻醉药物后处理对心肌再灌注损伤的保护是多因素参与的复杂过程,对麻醉药物后处理中心肌胞内信号转导的作用机制及研究进展作一综述.内容 麻醉药物后处理除了通过减少活性氧类物质的产生、抑制线粒体内钙超载、减轻内皮功能失调等被动作用外,还可主动激...     

Cardioprotection induced by olprinone,a phosphodiesterase III inhibitor,involves phosphatidylinositol-3-OH kinase-Akt and a mitochondrial permeability transition pore during early reperfusion

Purpose Ischemic preconditioning is mediated by the activation of phosphatidylinositol-3-OH kinase-Akt (PI3K-Akt) and by the inhibition of the opening of a mitochondrial permeability transition pore (mPTP) during early reperfusion. Preischemic administration of the phosphodiesterase type III inhibitor olprinone protects the myocardium against infarction, but its mechanism has not been fully clarified. We hypothesized that this olprinone-induced cardioprotective effect was mediated by the activation of PI3K-Akt and by the inhibition of mPTP during early reperfusion. Methods Pentobarbital-anesthetized rats (n = 42) subjected to 30-min coronary occlusion followed by 2-h reperfusion, received olprinone (20 μg·kg⁻¹) or saline (control) in the preischemic phase in the presence or absence of the PI3K-Akt inhibitor wortmannin (0.6 mg·kg⁻¹) or the mPTP opener atractyloside (5 mg·kg⁻¹) before 5 min of reperfusion. The myocardial infarct size was expressed as a percentage of the area at risk. All values were expressed as means ± SD. Statistical comparisons within groups were made using repeated-measures analysis of variance (ANOVA), followed by a paired t-test, and comparisons among groups were analyzed using a two-way ANOVA, followed by the Tukey-Kramer test. Results Mean arterial pressure and heart rate showed no significant differences within or among groups. The preischemic administration of olprinone significantly reduced the infarct size (12 ± 4%) as compared with that in the control group (43 ± 4%). Wortmannin or atractyloside abolished the protective effect of olprinone (42 ± 11% or 41 ± 10%). Conclusion The olprinone-induced cardioprotective effect could be exerted via the activation of PI3K-Akt and the inhibition of mPTP during early reperfusion.     

PI3K/Akt 信号通路在舒芬太尼后处理减轻在体大鼠心肌缺血-再灌注损伤中的作用

目的探讨PI3K/Akt信号通路在舒芬太尼后处理减轻在体大鼠心肌缺血-再灌注损伤时细胞凋亡中的作用。方法取90只健康成年雄性SD大鼠,采用随机数字表法分为五组:假手术组(Sham组),只穿线不结扎;缺血-再灌注组(IR组),结扎左冠状动脉前降支造成心肌缺血30min,再灌注120 min;舒芬太尼后处理组(Sufen组),在灌注即刻,给予舒芬太尼1.0μg/kg输注3min,再灌注处理同IR组;舒芬太尼后处理~+LY294002(PI3K抑制剂)组(SL组),再灌注前10min给予LY294002 0.3mg/kg,并行舒芬太尼后处理;LY294002组(IL组),再灌注前10 min给予LY294002 0.3mg/kg,再灌注120min。在缺血前即刻(T_0)、缺血30min(T_1)、再灌注60min(T_2)和再灌注120min(T_3)时记录HR和MAP;再灌注末,测定心肌梗死面积(IS/AAR);再灌注15 min时,采用Western blot法测定心肌组织总的Akt和磷酸化的Akt蛋白含量;在再灌注末,用RT_-PCR检测Bax和Bcl-2mRNA的表达。结果五组大鼠组间组内HR差异无统计学意义;T_2、T_3时IR组、SL组和IL组MAP明显低于Sham组(P0.05);Sufen组IS/AAR明显低于IR、SL和IL组(P0.05);五组心肌总的Akt蛋白含量表达差异无统计学意义;与Sufen组比较,sham、IR、SL和IL组磷酸化的Akt表达明显下调(P0.05),IR组、SL和IL组Bax mRNA的表达明显升高,Bcl-2mRAN的表达明显降低(P0.05)。结论舒芬太尼后处理可减轻心肌缺血-再灌注损伤,可能与激活PI3K/Akt信号通路,降低Bax和增加Bcl-2蛋白表达而达到抑制心肌细胞的凋亡有关。     

Isoflurane Protects against Myocardial Infarction during Early Reperfusion by Activation of Phosphatidylinositol-3-Kinase Signal Transduction: Evidence for Anesthetic-induced Postconditioning in Rabbits

Background: Brief episodes of ischemia during early reperfusion after coronary occlusion reduce the extent of myocardial infarction. Phosphatidylinositol-3-kinase (PI3K) signaling has been implicated in this "postconditioning" phenomenon. The authors tested the hypothesis that isoflurane produces cardioprotection during early reperfusion after myocardial ischemia by a PI3K-dependent mechanism.

Methods: Pentobarbital-anesthetized rabbits (n = 80) subjected to a 30-min coronary occlusion followed by 3 h reperfusion were assigned to receive saline (control), three cycles of postconditioning ischemia (10 or 20 s each), isoflurane (0.5 or 1.0 minimum alveolar concentration), or the PI3K inhibitor wortmannin (0.6 mg/kg, intravenously) or its vehicle dimethyl sulfoxide. Additional groups of rabbits were exposed to combined postconditioning ischemia (10 s) and 0.5 minimum alveolar concentration isoflurane in the presence and absence of wortmannin. Phosphorylation of Akt, a downstream target of PI3K, was assessed by Western blotting.

Results: Postconditioning ischemia for 20 s, but not 10 s, reduced infarct size (P < 0.05) (triphenyltetrazolium staining; 20 +/- 3% and 34 +/- 3% of the left ventricular area at risk, respectively) as compared with control (41 +/- 2%). Exposure to 1.0, but not 0.5, minimum alveolar concentration isoflurane decreased infarct size (21 +/- 2% and 43 +/- 3%, respectively). Wortmannin abolished the protective effects of postconditioning (20 s) and 1.0 minimum alveolar concentration isoflurane. Combined postconditioning (10 s) and 0.5 minimum alveolar concentration isoflurane markedly reduced infarct size (17 +/- 5%). This action was also abolished by wortmannin (44 +/- 2%). Isoflurane (1.0 minimum alveolar concentration) increased Akt phosphorylation after ischemia (32 +/- 6%), and this action was blocked by wortmannin.     

Isoflurane protects against myocardial infarction during early reperfusion by activation of phosphatidylinositol-3-kinase signal transduction: evidence for anesthetic-induced postconditioning in rabbits

BACKGROUND: Brief episodes of ischemia during early reperfusion after coronary occlusion reduce the extent of myocardial infarction. Phosphatidylinositol-3-kinase (PI3K) signaling has been implicated in this "postconditioning" phenomenon. The authors tested the hypothesis that isoflurane produces cardioprotection during early reperfusion after myocardial ischemia by a PI3K-dependent mechanism. METHODS: Pentobarbital-anesthetized rabbits (n = 80) subjected to a 30-min coronary occlusion followed by 3 h reperfusion were assigned to receive saline (control), three cycles of postconditioning ischemia (10 or 20 s each), isoflurane (0.5 or 1.0 minimum alveolar concentration), or the PI3K inhibitor wortmannin (0.6 mg/kg, intravenously) or its vehicle dimethyl sulfoxide. Additional groups of rabbits were exposed to combined postconditioning ischemia (10 s) and 0.5 minimum alveolar concentration isoflurane in the presence and absence of wortmannin. Phosphorylation of Akt, a downstream target of PI3K, was assessed by Western blotting. RESULTS: Postconditioning ischemia for 20 s, but not 10 s, reduced infarct size (P < 0.05) (triphenyltetrazolium staining; 20 +/- 3% and 34 +/- 3% of the left ventricular area at risk, respectively) as compared with control (41 +/- 2%). Exposure to 1.0, but not 0.5, minimum alveolar concentration isoflurane decreased infarct size (21 +/- 2% and 43 +/- 3%, respectively). Wortmannin abolished the protective effects of postconditioning (20 s) and 1.0 minimum alveolar concentration isoflurane. Combined postconditioning (10 s) and 0.5 minimum alveolar concentration isoflurane markedly reduced infarct size (17 +/- 5%). This action was also abolished by wortmannin (44 +/- 2%). Isoflurane (1.0 minimum alveolar concentration) increased Akt phosphorylation after ischemia (32 +/- 6%), and this action was blocked by wortmannin. CONCLUSIONS: Isoflurane acts during early reperfusion after prolonged ischemia to salvage myocardium from infarction and reduces the threshold of ischemic postconditioning by activating PI3K.     

Isoflurane alters energy substrate metabolism to preserve mechanical function in isolated rat hearts following prolonged no-flow hypothermic storage

BACKGROUND: Isoflurane enhances mechanical function in hearts subject to normothermic global or regional ischemia. The authors examined the effectiveness of isoflurane in preserving mechanical function in hearts subjected to cardioplegic arrest and prolonged hypothermic no-flow storage. The role of isoflurane in altering myocardial glucose metabolism during storage and reperfusion during these conditions and the contribution of adenosine triphosphate-sensitive potassium (K(atp)) channel activation in mediating the functional and metabolic effects of isoflurane preconditioning was determined. METHODS: Isolated working rat hearts were subjected to cardioplegic arrest with St. Thomas' II solution, hypothermic no-flow storage for 8 h, and subsequent aerobic reperfusion. The consequences of isoflurane treatment were assessed during the following conditions: (1) isoflurane exposure before and during storage; (2) brief isoflurane exposure during early nonworking poststorage reperfusion; and (3) isoflurane preconditioning before storage. The selective mitochondrial and sarcolemmal K(atp) channel antagonists, 5-hydroxydecanoate and HMR 1098, respectively, were used to assess the role of K(atp) channel activation on glycogen consumption during storage in isoflurane-preconditioned hearts. RESULTS: Isoflurane enhanced recovery of mechanical function if present before and during storage. Isoflurane preconditioning was also protective. Isoflurane reduced glycogen consumption during storage under the aforementioned circumstances. Storage of isoflurane-preconditioned hearts in the presence of 5-hydroxydecanoate prevented the reduction in glycogen consumption during storage and abolished the beneficial effect of isoflurane preconditioning on recovery of mechanical function. CONCLUSIONS: Isoflurane provides additive protection of hearts subject to cardioplegic arrest and prolonged hypothermic no-flow storage and favorably alters energy substrate metabolism by modulating glycolysis during ischemia. The effects of isoflurane preconditioning on glycolysis during hypothermic no-flow storage appears to be associated with activation of mitochondrial K(atp) channels.     

Bile salt exposure causes phosphatidyl-inositol-3-kinase-mediated proliferation in a Barrett's adenocarcinoma cell line

BACKGROUND: The mechanisms by which gastroesophageal reflux promotes malignant progression in Barrett's esophagus are poorly understood. The phosphatidylinositol-3-kinase (PI3 kinase)/Akt pathway regulates proliferation and apoptosis. We hypothesized that the PI3 kinase/Akt pathway mediates the pro-proliferative and antiapoptotic effects of bile. METHODS: The Barrett's adenocarcinoma cell line, SEG-1, was exposed to the conjugated bile salt, glycochenodeoxycholic acid (GCDA). Cell number was measured by the MTT incorporation assay and by Coulter counter. PI3 kinase/Akt activity was inferred from Western blots of phosphorylated and total Akt. Proliferation and apoptosis were determined by BrdU incorporation and cell death ELISA. RESULTS: A dose-dependent cell number increase was seen with a 20-minute exposure to GCDA. On Western blot, 200 micromol/L GCDA caused a 3-fold increase in Akt phosphorylation within 20 minutes, which was inhibited by 90% with the addition of PI3 kinase inhibitor, LY294002. LY294002 produced dose-dependent inhibition of GCDA-induced cell number increases. 200 micromol/L GCDA decreased apoptosis by 25%. Addition of LY294002 did not completely inhibit the antiapoptotic effect of bile. CONCLUSIONS: Bile salts activate the PI3 kinase/Akt signaling pathway and stimulate cell growth in SEG-1. The majority of this PI3 kinase-mediated effect is secondary to increases in proliferation rather than to decreases in apoptosis.     

Isoflurane Alters Energy Substrate Metabolism to Preserve Mechanical Function in Isolated Rat Hearts following Prolonged No-Flow Hypothermic Storage

Background: Isoflurane enhances mechanical function in hearts subject to normothermic global or regional ischemia. The authors examined the effectiveness of isoflurane in preserving mechanical function in hearts subjected to cardioplegic arrest and prolonged hypothermic no-flow storage. The role of isoflurane in altering myocardial glucose metabolism during storage and reperfusion during these conditions and the contribution of adenosine triphosphate-sensitive potassium (KATP) channel activation in mediating the functional and metabolic effects of isoflurane preconditioning was determined.

Methods: Isolated working rat hearts were subjected to cardioplegic arrest with St. Thomas' II solution, hypothermic no-flow storage for 8 h, and subsequent aerobic reperfusion. The consequences of isoflurane treatment were assessed during the following conditions: (1) isoflurane exposure before and during storage; (2) brief isoflurane exposure during early nonworking poststorage reperfusion; and (3) isoflurane preconditioning before storage. The selective mitochondrial and sarcolemmal KATP channel antagonists, 5-hydroxydecanoate and HMR 1098, respectively, were used to assess the role of KATP channel activation on glycogen consumption during storage in isoflurane-preconditioned hearts.

Results: Isoflurane enhanced recovery of mechanical function if present before and during storage. Isoflurane preconditioning was also protective. Isoflurane reduced glycogen consumption during storage under the aforementioned circumstances. Storage of isoflurane-preconditioned hearts in the presence of 5-hydroxydecanoate prevented the reduction in glycogen consumption during storage and abolished the beneficial effect of isoflurane preconditioning on recovery of mechanical function.     

Preconditioning with sevoflurane reduces changes in nicotinamide adenine dinucleotide during ischemia-reperfusion in isolated hearts: reversal by 5-hydroxydecanoic acid

BACKGROUND: Ischemia causes an imbalance in mitochondrial metabolism and accumulation of nicotinamide adenine dinucleotide (NADH). We showed that anesthetic preconditioning (APC), like ischemic preconditioning, improved mitochondrial NADH energy balance during ischemia and improved function and reduced infarct size on reperfusion. Opening adenosine triphosphate-sensitive potassium (K(atp)) channels may be involved in triggering APC. The authors tested if effects of APC on NADH concentrations before, during, and after ischemia are reversible by 5-hydroxydecanoate (5-HD), a putative mitochondrial K channel blocker. METHODS: Nicotinamide adenine dinucleotide fluorescence was measured in 60 guinea pig Langendorff-prepared hearts assigned into five groups: (1) no treatment before ischemia; (2) APC by exposure to 1.3 mm sevoflurane for 15 min; (3) 200 microm 5-HD from 5 min before to 15 min after sevoflurane exposure; (4) 35 min 5-HD alone; and (5) no treatment and no ischemia. Sevoflurane was washed out for 30 min, and 5-HD for 15 min, before 30-min ischemia and 120-min reperfusion. RESULTS: Nicotinamide adenine dinucleotide was reversibly increased during sevoflurane exposure before ischemia, and the increase and rate of decline in NADH during ischemia were reduced after APC. 5-HD abolished these changes in NADH. On reperfusion, function was improved and infarct size reduced after APC compared with other groups. CONCLUSION: Anesthetic preconditioning was evidenced by improved mitochondrial bioenergetics as assessed from NADH concentrations during ischemia and by attenuated reperfusion injury. Reversal of APC by bracketing sevoflurane exposure with 5-HD suggests that APC is triggered by mitochondrial K channel opening or, alternatively, by attenuated mitochondrial respiration without direct involvement of mitochondrial K channel opening.