Intramitochondrial signaling: interactions among mitoKATP, PKCepsilon, ROS, and MPT

Activation of protein kinase Cepsilon (PKCepsilon), opening of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)), and increased mitochondrial reactive oxygen species (ROS) are key events in the signaling that underlies cardioprotection. We showed previously that mitoK(ATP) is opened by activation of a mitochondrial PKCepsilon, designated PKCepsilon1, that is closely associated with mitoK(ATP). mitoK(ATP) opening then causes an increase in ROS production by complex I of the respiratory chain. This ROS activates a second pool of PKCepsilon, designated PKCepsilon2, which inhibits the mitochondrial permeability transition (MPT). In the present study, we measured mitoK(ATP)-dependent changes in mitochondrial matrix volume to further investigate the relationships among PKCepsilon, mitoK(ATP), ROS, and MPT. We present evidence that 1) mitoK(ATP) can be opened by H(2)O(2) and nitric oxide (NO) and that these effects are mediated by PKCepsilon1 and not by direct actions on mitoK(ATP), 2) superoxide has no effect on mitoK(ATP) opening, 3) exogenous H(2)O(2) or NO also inhibits MPT opening, and both compounds do so independently of mitoK(ATP) activity via activation of PKCepsilon2, 4) mitoK(ATP) opening induced by PKG, phorbol ester, or diazoxide is not mediated by ROS, and 5) mitoK(ATP)-generated ROS activates PKCepsilon1 and induces phosphorylation-dependent mitoK(ATP) opening in vitro and in vivo. Thus mitoK(ATP)-dependent mitoK(ATP) opening constitutes a positive feedback loop capable of maintaining the channel open after the stimulus is no longer present. This feedback pathway may be responsible for the lasting protective effect of preconditioning, colloquially known as the memory effect.     

一氧化氮和线粒体ATP敏感性钾通道介导血管紧张素转换酶抑制剂加强阈下预处理的作用

实验采用离体大鼠心脏Langendorff灌流模型,观察含巯基（卡托普利）和不含巯基（培哚普利拉）的两种血管紧张素转换酶抑制剂（angiotensin-converting enzyme inhibitors,ACEI）对抗心肌缺血的作用,并探讨一氧化氮（nitric oxide,NO）和线粒体ATP敏感性钾通道（mimchondrial ATP-sensitive potassium channel,mitoKATP channel）是否参与ACEI的心肌保护作用。结果表明：（1）给予大鼠心脏2min全心停灌和10min复灌作为闽下缺血预处理（subthreshold preconditioning,sPC）、卡托普利或培哚普利拉单独使用,均不能改善长时间缺血复灌（缺血30min＋复灌120min）引起的心肌损伤。（2）当两种ACEI分别和sPC联合使用时,与sPC组相比,缺血心脏在长时间缺血后的复灌期问左室舒张末压（left ventricular end-diastolic pressure,LVEDP）明显降低,左宦发展压（left ventricular developed pressure,LVDP）和冠脉流量明显增高,乳酸脱氢酶（lactate dehydrogenase,LDH）的释放量和心肌梗死面积明显低于sPC组。（3）利用NOS抑制剂L-NAME和mitoKATP通道的抑制剂5-HD灌流10min后,可明显抑制卡托普利／培哚普利拉和sPC联合使用引起的LVEDP降低,并使LVDP和冠脉流量降低,LDH的释放量和心肌梗死面积明显增高（P〈0．05）。（4）sPC、卡托普利或培哚普利拉单独使用,心脏NO的产生增加。ACEI和sPC联合使用,与三者单独使用相比NO的浓度亦明显增高（P〈0．05）。结果提示：含与不含巯基的ACEI与闽下缺血预处理联合使用均可使大鼠心脏功能明显改善,其心肌保护作用的机制可能通过NO途径,并和mitoKATP通道的激活有关。     

mitoKATP通道参与心肌缺血预处理保护作用的机制

目的：探讨血管紧张素转换酶抑制剂（ACEI）和阈下缺血预处理联合预处理诱导的心肌保护作用中mi-toKatp通道激动后的作用机制：方法：采用离体大鼠心脏Langendorff灌流模型,观察心脏电脱耦联发生时间、细胞膜Na^＋／K^＋-ATPase和Ca^2＋/Mg^2＋-ATPase活性的改变：结果：单独使用卡托普利、或给予大鼠心脏2min缺血／10min复灌作为阈下缺血预处理,均不能改善长时间缺血／复灌引起的心脏收缩功能下降？而卡托普利和阂下缺血预处理联合使用可增高心脏收缩功能。mitoKatp通道特异性阻断剂5-HD可取消这一联合预处理的作用一联合预处理可引起缺血后电脱耦联发生时间延长,缺血心肌细胞膜Na^＋／K^＋-ATPase和Ca^2＋/Mg^2＋-ATPase活性增高;5-HD可取消此作用结论：mitoKatp通道参与了联合预处理延迟缺血引起的细胞间脱耦联和促进细胞膜离子通道稳定性维持的作用。     

Isoflurane activates rat mitochondrial ATP-sensitive K+ channels reconstituted in lipid bilayers

Activation of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels is critical in myocardial protection induced by preconditioning with volatile anesthetics or brief periods of ischemia. In this study, we characterized rat mitoK(ATP) channels reconstituted in lipid bilayers and examined their direct regulation by isoflurane. Mitochondria and the inner membrane fraction were isolated from rat ventricles and fused into lipid bilayers. On the basis of their inhibition by 5-hydroxydecanoate (5-HD)/ATP or activation by diazoxide, mitoK(ATP) channels of several conductance states were observed in symmetrical (150 mM) potassium glutamate (26, 47, 66, 83, and 105 pS). Isoflurane (0.8 mM) increased the cumulative open probability from 0.09 +/- 0.02 at baseline to 0.50 +/- 0.09 (P < 0.05, n = 5), which was inhibited by 5-HD. Isoflurane caused a dose-dependent rightward shift in ATP inhibition of mitoK(ATP) channels, which increased the IC(50) for ATP from 335 +/- 4 to 940 +/- 34 microM at 0.8 mM (P < 0.05, n = 5 approximately 8). We conclude that direct activation of the mitoK(ATP) channel by isoflurane is likely to contribute to volatile anesthetic-induced myocardial preconditioning.     

Mitochondrial ATP-sensitive K+ channels are redox-sensitive pathways that control reactive oxygen species production

Pharmacological mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) opening protects against ischemic damage and mimics ischemic preconditioning. However, physiological and pathological signaling events that open this channel are still not fully understood. We found that catalase, which removes H(2)O(2), is capable of reversing the beneficial effects of ischemic preconditioning but not of mitoK(ATP) agonist diazoxide. On the other hand, 2-mercaptopropionylglycine prevented cardioprotection in both cases, suggesting that this compound may present effects other than scavenging of reactive oxygen species. Indeed, 2-mercaptopropionylglycine and a second thiol-reducing agent, dithiothreitol, impair diazoxide-mediated activation of mitoK(ATP) in isolated heart mitochondria. This demonstrates that mitoK(ATP) activity is regulated by thiol redox status. Furthermore, stimulating the generation of endogenous mitochondrial reactive oxygen species or treating samples with H(2)O(2) strongly enhances mitoK(ATP) activity, in a manner probably dependent on redox sensors located in the channel's sulfonylurea receptor. We also demonstrate that mitoK(ATP) channel activity effectively prevents mitochondrial reactive oxygen release. Collectively, our results suggest that mitoK(ATP) acts as a reactive oxygen sensor that decreases mitochondrial free radical generation in response to enhanced local levels of oxidants. As a result, these channels regulate mitochondrial redox state under physiological conditions and prevent oxidative stress under pathological conditions such as ischemia/reperfusion.     

Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain.

Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and K(ATP) channel openers is known to involve the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). Brain is also protected by ischemic preconditioning and K(ATP) channel openers, and it has been suggested that mitoK(ATP) may also play a key role in brain protection. However, it is not known whether mitoK(ATP) exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoK(ATP). We report partial purification and reconstitution of a new mitoK(ATP) from rat brain mitochondria. We measured K(+) flux in proteoliposomes and found that brain mitoK(ATP) is regulated by the same ligands as those that regulate mitoK(ATP) from heart and liver. We also examined the effects of opening and closing mitoK(ATP) on brain mitochondrial respiration, and we estimated the amount of mitoK(ATP) by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoK(ATP) from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoK(ATP) per milligram of mitochondrial protein than liver or heart.     

Chronic preconditioning: a novel approach for cardiac protection

Ischemic preconditioning is the most powerful protective mechanism known against lethal ischemia. Unfortunately, the protection lasts for only a few hours. Here we tested the hypothesis that the heart can be kept in a preconditioned state for constant protection against ischemia. In this study we chose BMS-191095 (BMS), a highly selective opener of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels. BMS (1 mg/kg ip) was administered to rats every 24 h until 96 h. In other groups, BMS plus wortmannin (WTN, 15 microg/kg ip), an inhibitor of the phosphatidylinositol 3-kinase (PI3-K), or BMS plus 5-hydroxydecanoic acid (5-HD, 5 mg/kg ip), an inhibitor of mitoK(ATP), or BMS plus N(omega)-nitro-L-arginine methyl ester (L-NAME) (30 microg/kg ip), an inhibitor of nitric oxide (NO) synthase, were administered to rats. Rats were then subjected to 30-min left anterior descending coronary artery occlusion and 120-min reperfusion. Cardiac function, infarct size, pathological changes, and apoptosis were assessed at the end of treatments. Saline-treated hearts displayed marked contractile dysfunction and underwent pathological changes. BMS-treated rats showed significant improvement in cardiac function, and infarct size was significantly reduced in BMS-treated hearts. However, protection by BMS was abolished by 5-HD, WTN, or L-NAME. These data demonstrate that hearts can be chronically preconditioned and retain their ability to remain resistant against lethal ischemia and that this protection is mediated by activation of mitoK(ATP) via NO and PI3-K/Akt signaling pathways.     

ATP-sensitive potassium channel: a novel target for protection against UV-induced human skin cell damage

Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP-sensitive potassium (K(ATP)) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV-induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV-induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of K(ATP) channels are functionally expressed in HaCaT cells and both non-selective K(ATP) channel opener pinacidil and mitoK(ATP) (mitochondrial K(ATP)) channel opener diazoxide attenuated UV-induced keratinocytes cell death. The protective effects were abolished by both non-selective K(ATP) channel blocker glibenclamide and selective mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). Also, activation of K(ATP) channel with pinacidil or diazoxide resulted in suppressive effects on UV-induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV-induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5-HD. Taken together, we contend that mitoK(ATP) is likely to contribute the protection against UV-induced keratinocytes cell damage. Our findings suggest that K(ATP) openers such as pinacidil and diazoxide may be utilized to prevent from UV-induced skin aging.     

Mitochondrial ATP dependent potassium channels mediate non-ischemic preconditioning by tachycardia in dogs

Brief episodes of tachycardia without myocardial ischemia prior to a coronary occlusion decrease myocardial infarct size in dogs. This non-ischemic preconditioning is mediated by adenosine. Because ischemic preconditioning is mediated through ATP dependent potassium channels, particularly the mitochondrial ones, we studied whether non-ischemic preconditioning is also mediated through these channels. In anesthetized dogs heart rate was kept constant at 120 cycles/min and aortic pressure changes were damped. Myocardial infarction was induced by occlusion of the anterior descending coronary artery for 60 min and reperfusion for 270 min. In a control group the infarct size (necrotic volume/risk region volume × 100) was 15.8 ± 1.5%. Preconditioning with five periods of tachycardia, 5 min in duration each at 213 cycles/min with intervening periods of 5 min of basal heart rate at 120 cycles/min, reduced the infarct size by 45.6% (p < 0.05) with respect to the control group. This effect was completely reverted by the blockade of ATP dependent potassium channels with glibenclamide or 5 hydroxydecanoate (a specific blocker of mitochondrial ATP dependent potassium channels) prior to preconditioning. These effects were not due to differences in collateral flow, risk region size or hemodynamic variables between the groups. These results show that mitochondrial ATP dependent potassium channels mediate non-ischemic preconditioning by tachycardia in dogs.     

Desferoxamine and ethyl-3,4-dihydroxybenzoate protect myocardium by activating NOS and generating mitochondrial ROS

Protection from a prolyl hydroxylase domain-containing enzyme (PHD) inhibitor, desferoxamine (DFO), was recently reported to be dependent on production of reactive oxygen species (ROS). Ischemic preconditioning triggers the protected state by stimulating nitric oxide (NO) production to open mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels, generating ROS required for protection. We tested whether DFO and a second PHD inhibitor, ethyl-3,4-dihydroxybenzoate (EDHB), might have similar mechanisms. EDHB and DFO increased ROS generation by 50-75% (P < 0.001) in isolated rabbit cardiomyocytes. This increase after EDHB exposure was blocked by N(omega)-nitro-L-arginine methyl ester (L-NAME), an NO synthase (NOS) inhibitor; ODQ, a guanylyl cyclase antagonist; and Rp-8-bromoguanosine-3',5'-cyclic monophosphorothioate Rp isomer, a PKG blocker, thus implicating the NO pathway in EDHB's signaling. Glibenclamide, a nonselective K(ATP) channel blocker, or 5-hydroxydecanoate, a selective mitoK(ATP) channel antagonist, also prevented EDHB's ROS production, as did blockade of mitochondrial electron transport with myxothiazol. NOS is activated by Akt. However, neither wortmannin, an inhibitor of phosphatidylinositol-3-kinase, nor Akt inhibitor blocked EDHB-induced ROS generation, indicating that EDHB initiates signaling downstream of Akt. DFO also increased ROS production, and this effect was blocked by ODQ, 5-hydroxydecanoate, and N-(2-mercaptopropionyl)glycine, an ROS scavenger. DFO increased cardiomyocyte production of nitrite, a metabolite of NO, and this effect was blocked by an inhibitor of NOS. DFO also spared ischemic myocardium in intact hearts. This infarct-sparing effect was blocked by ODQ, L-NAME, and N-(2-mercaptopropionyl)glycine. Hence, DFO and EDHB stimulate NO-dependent activation of PKG to open mitoK(ATP) channels and produce ROS, which act as second messengers to trigger entrance into the preconditioned state.     

Ouabain protects rat hearts against ischemia-reperfusion injury via pathway involving src kinase, mitoKATP, and ROS

We showed recently that mitochondrial ATP-dependent K(+) channel (mitoK(ATP)) opening is required for the inotropic response to ouabain. Because mitoK(ATP) opening is also required for most forms of cardioprotection, we investigated whether exposure to ouabain was cardioprotective. We also began to map the signaling pathways linking ouabain binding to Na(+)-K(+)-ATPase with the opening of mitoK(ATP). In Langendorff-perfused rat hearts, 10-80 microM ouabain given before the onset of ischemia resulted in cardioprotection against ischemia-reperfusion injury, as documented by an improved recovery of contractile function and a reduction of infarct size. In skinned cardiac fibers, a ouabain-induced protection of mitochondrial outer membrane integrity, adenine nucleotide compartmentation, and energy transfer efficiency was evidenced by a decreased release of cytochrome c and preserved half-saturation constant of respiration for ADP and adenine nucleotide translocase-mitochondrial creatine kinase coupling, respectively. Ouabain-induced positive inotropy was dose dependent over the range studied, whereas ouabain-induced cardioprotection was maximal at the lowest dose tested. Compared with bradykinin (BK)-induced preconditioning, ouabain was equally efficient. However, the two ligands clearly diverge in the intracellular steps leading to mitoK(ATP) opening from their respective receptors. Thus BK-induced cardioprotection was blocked by inhibitors of cGMP-dependent protein kinase (PKG) or guanylyl cyclase (GC), whereas ouabain-induced protection was not blocked by either agent. Interestingly, however, ouabain-induced inotropy appears to require PKG and GC. Thus 5-hydroxydecanoate (a selective mitoK(ATP) inhibitor), N-(2-mercaptopropionyl)glycine (MPG; a reactive oxygen species scavenger), ODQ (a GC inhibitor), PP2 (a src kinase inhibitor), and KT-5823 (a PKG inhibitor) abolished preconditioning by BK and blocked the inotropic response to ouabain. However, only PP2, 5-HD, and MPG blocked ouabain-induced cardioprotection.     

Role of the mitochondrial ATP-sensitive K+ channels in cardioprotection

The mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel was discovered more than a decade ago. Since then, several pharmacological studies have identified agents that target this channel some of which selectively target mitoK(ATP). These and other studies have also suggested that mitoK(ATP) plays a key role in the process of ischemic preconditioning (IPC) and prevention of apoptosis. The mechanism by which mitoK(ATP) exerts its protective effects is unclear, however, changes in mitochondrial Ca(2+) uptake and levels of reactive oxygen species, and mitochondrial matrix swelling are believed to be involved. Despite major advances, several important issues regarding mitoK(ATP) remain unanswered. These questions include, but are not limited to: the molecular structure of mitoK(ATP), the downstream and upstream mechanisms that leads to IPC and cell death, and the pharmacological profile of the channel. This review attempts to provide an up-to-date overview of the role of mitoK(ATP) in cardioprotection.     

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane

The ATP-sensitive K(+) (K(ATP)) channels in both sarcolemmal (sarcK(ATP)) and mitochondrial inner membrane (mitoK(ATP)) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcK(ATP) contains Kir6.2 and sulfonylurea receptor (SUR)2A, the molecular identity of mitoK(ATP) remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing K(ATP) into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing K(ATP) channels were localized in mitochondria and this mitochondrial localization was significantly increased by PKC activation with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy transfer microscopy further revealed that a significant number of Kir6.2-containing K(ATP) channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by Western blotting showing that the Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment and this increase was inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional K(ATP) channels in mitochondria was significantly increased by PMA, as shown by K(ATP)-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing K(ATP) channels was blocked by a selective PKCepsilon inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the K(ATP) channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and that the Kir6.2 content in mitochondria is increased by activation of PKCepsilon. PKC isoform-regulated mitochondrial import of K(ATP) channels may have significant implication in cardioprotection of IPC.     

二氮嗪在长时程心脏低温保存中的作用

延长心脏的体外有效保存时间对临床心脏移植具有重要意义。本文旨在研究线粒体ATP敏感性钾通道开放剂二氮嗪(diazoxide,DE)在离体大鼠心脏长时程低温保存中的作用。SD大鼠随机分成5组,包括对照组(单纯Celsior保存液),DE组(Celsior液中含15、30或45μmol／L的DE)和DE 5-HD组[Celsior液中含30μmol／L的DE和100μmol／L的5-羟基葵酸盐(5-hydroxydecanoate,5-HD)]。利用Langendorff离体鼠心灌注法,观察心脏在4℃条件下保存10h后,复灌期血流动力学恢复、冠脉流出液中心肌酶漏出量及心肌水含量变化,并做心肌超微结构检查。结果显示：与对照组比较,DE处理后,复灌期的左心室舒张末期压力明显降低,心率、左心室发展压、左心室压力变化率、冠脉流出量等的恢复率在多个复灌时间点上优于对照组,且能显著减少复灌过程中心肌酶(乳酸脱氢酶、磷酸肌酸激酶及谷草转氨酶)的漏出量,降低心肌水含量;其中30和45μmol／LDE组的保护作用优于15μmol／LDE组;电镜结果显示DE对长时程低温保存心脏的超微结构有较好的保护作用。DE的上述作用可被线粒体ATP敏感性钾通道的特异性阻断剂5-HD所取消。以上结果提示：DE可通过激活线粒体ATP敏感性钾通道显著改善离体大鼠心脏长时程低温保存效果。     

Regulation of the mitochondrial ATP-sensitive potassium channel in rat uterus cells by ROS

In previous study we demonstrated the presence of ATP-sensitive potassium current in the inner mitochondrial membrane, which was sensitive to diazoxide and glybenclamide, in mitochondria isolated from the rat uterus. This current was supposed to be operated by mitochondrial ATP-sensitive potassium channel (mitoK(ATP)). Regulation of the mitoK(ATP) in uterus cells is not studied well enough yet. It is well known that the reactive oxygen species (ROS) can play a dual role. They can damage cells in high concentrations, but they can also act as messengers in cellular signaling, mediating survival of cells under stress conditions. ROS are known to activate mitoK(ATP) during the oxidative stress in the brain and heart, conferring the protection of cells. The present study examined whether ROS mediate the mitoK(ATP) activation in myometrium cells. Oxidative stress was induced by rotenone. ROS generation was measured by 2',7'-dichlorofluorescin diacetate. The massive induction of ROS production was demonstrated in the presence of rotenone. Hyperpolarization of the mitochondrial membrane was also detected with the use of the potential-sensitive dye DiOC6 (3,3'-dihexyloxacarbocyanine iodide). Diazoxide, a selective activator of mitoK(ATP), depolarized mitochondrial membrane either under oxidative stress or under normal conditions, while mitoK(ATP) blocker glybenclamide effectively restored mitochondrial potential in rat myocytes. Estimated value for diazoxide to mitoK(ATP) under normoxia was four times higher than under oxidative stress conditions: 5.01 +/- 1.47-10(-6) M and 1.24 +/- 0.21 x 10(-6) M respectively. The ROS scavenger N-acetylcysteine (NAC) successfully eliminates depolarization of mitochondrial membrane by diazoxide under oxidative stress. These results suggest that elimination of ROS by NAC prevents the activation of mitoK(ATP) under oxidative stress. Taking into account the higher affinity of diazoxide to mitoK(ATP) under stress conditions than under normoxia, we conclude that the oxidative stress conditions are more favourable than normoxia for the activation of mitoK(ATP). Thus we hypothesize that the ROS regulate the activity of the mitoK(ATP) in myocytes.     

Cardiac protection by mitoKATP channels is dependent on Akt translocation from cytosol to mitochondria during late preconditioning

This investigation elucidates the Akt/mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel signaling pathway in late pharmacological preconditioning, using the mitoK(ATP) channel openers BMS-191095 (BMS) and diazoxide (DE). BMS (1 mg/kg ip) and DE (7 mg/kg ip) alone or BMS plus wortmannin (WTN, 15 microg/kg ip), an inhibitor of phosphatidylinositol 3-kinase, and BMS plus 5-hydroxydecanoic acid (5-HD, 5 mg/kg ip), an inhibitor of mitoK(ATP) channels, were administered to male mice. Twenty-four hours later, hearts were isolated and subjected to 40 min of ischemia and 120 min of reperfusion via Langendorff's apparatus. Both BMS and DE reduced left ventricular end-diastolic pressure and increased left ventricular developed pressure as well as reduced LDH release. Coadministration of BMS and WTN abolished the beneficial effects of BMS on cardiac function. Moreover, BMS and DE accelerated Akt phosphorylation in cardiac tissue as determined by Western blot analysis and also significantly reduced apoptosis compared with ischemic control. WTN significantly suppressed BMS-induced Akt phosphorylation, whereas 5-HD had no effect on Akt phosphorylation in cytosol, and the effect of BMS on apoptosis was abolished. It is concluded that the cardioprotective effect by mitoK(ATP) channels is attributed to the translocation of phosphorylated Akt from cytosol to mitochondria.     

二氮嗪对长时程低温保存大鼠心脏Fas/FasL蛋白表达的影响

本文旨在研究线粒体ATP敏感性钾(mitochondrial ATP-sensitive potassium channel,mitoKATP)通道开放剂二氮嗪(diazoxide,DE)对离体长时程低温保存的大鼠心脏促凋亡蛋白Fas和FasL表达的影响.利用Langendorff离体大鼠心脏灌注法,观察心脏在4 oC含或不含(对照组)DE的Celsior保存液保存8 h后,复灌期心脏作功量(rate-pressure product,RPP)变化情况,采用原位末端标记(TdT-mediated dUTP nick end labeling,TUNEL)染色法检测心肌细胞凋亡和免疫组织化学方法检测Fas和FasL蛋白表达情况.结果显示,在Celsior保存液中加入DE(30 pmol/L),复灌期RPP的恢复率在多个复灌时间点上优于对照组;同时可降低长时程低温保存心脏心肌细胞凋亡指数,减少Fas和FasL蛋白的表达.DE的上述作用可被mitoKAxr通道特异性阻断剂5.羟基葵酸盐(5-hydroxydecanoate,5-HD)所取消.以上结果提示,DE可能通过激活mitoKATP通道来减少Fas和FasL蛋白表达,从而减轻大鼠心肌缺血/再灌注损伤后的心肌细胞凋亡.     

Exogenous NO triggers preconditioning via a cGMP- and mitoKATP-dependent mechanism

Exogenous nitric oxide (NO) triggers a preconditioning-like effect in heart via a pathway that is dependent on reactive oxygen species. This study examined the signaling pathway by which the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 2 microM) triggers its anti-infarct effect. Isolated rabbit hearts experienced 30 min of regional ischemia and 120 min of subsequent reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining. Infarct size was reduced from 30.5 +/- 3.0% of the risk zone in control hearts to 10.2 +/- 2.0% in SNAP-treated hearts. Bracketing the SNAP infusion with either the guanylyl cyclase blocker 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (2 microM) or the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel blocker 5-hydroxydecanoate (200 microM) completely blocked the infarct-sparing effect of SNAP (34.3 +/- 3.8 and 32.2 +/- 1.6% infarction, respectively). Pretreatment of hearts with 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (10 microM), which is a cell-permeable cGMP analog that activates protein kinase G, mimicked the preconditioning effect of SNAP by reducing infarct size to 7.5 +/- 1.1% of the risk zone. This salutary effect was abolished by either the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) or 5-hydroxydecanoate (100 microM; 28.9 +/- 2.7 and 33.6 +/- 5.0% infarction of the risk zone, respectively). To confirm these functional data and the effect of SNAP on the guanylyl cyclase-protein kinase G signaling pathway, cGMP levels were measured. SNAP increased the level from 0.18 +/- 0.04 to 0.61 +/- 0.14 pmol/mg of protein (P < 0.05). These data suggest that exogenous NO triggers the preconditioning effect by initiating a cascade of events including stimulation of guanylyl cyclase to make cGMP, activation of protein kinase G, opening of mitoK(ATP) channels, and, finally, production of reactive oxygen species.     

ROS and NO trigger early preconditioning: relationship to mitochondrial KATP channel

Reactive oxygen species (ROS) and nitric oxide (NO) are implicated in induction of ischemic preconditioning. However, the relationship between these oxidant signals and opening of the mitochondrial ATP-dependent potassium (K(ATP)) channel during early preconditioning is not fully understood. We observed preconditioning protection by hypoxia, exogenous H(2)O(2), or PKC activator PMA in cardiomyocytes subjected to 1-h ischemia and 3-h reperfusion. Protection was abolished by K(ATP) channel blocker 5-hydroxydecanoate (5-HD) in each case, indicating that these triggers must act upstream from the K(ATP) channel. Inhibitors of NO synthase abolished protection in preconditioned cells, suggesting that NO is also required for protection. DAF-2 fluorescence (NO sensitive) increased during hypoxic triggering. This was amplified by pinacidil and inhibited by 5-HD, indicating that NO is generated subsequent to K(ATP) channel activation. Exogenous NO during the triggering phase conferred protection blocked by 5-HD. Exogenous NO also restored protection abolished by 5-HD or N(omega)-nitro-l-arginine methyl ester in preconditioned cells. Antioxidants given during pinacidil or NO triggering abolished protection, confirming that ROS are generated by K(ATP) channel activation. Coadministration of H(2)O(2) and NO restored PMA-induced protection in 5-HD-treated cells, indicating that ROS and NO are required downstream from the K(ATP) channel. We conclude that ROS can trigger preconditioning by causing activation of the K(ATP) channel, which then induces generation of ROS and NO that are both required for preconditioning protection.