Redox regulation of the mitochondrial KATP channel in cardioprotection

The mitochondrial ATP-sensitive potassium channel (mK_ATP) is important in the protective mechanism of ischemic preconditioning (IPC). The channel is reportedly sensitive to reactive oxygen and nitrogen species, and the aim of this study was to compare such species in parallel, to build a more comprehensive picture of mK_ATP regulation. mK_ATP activity was measured by both osmotic swelling and Tl⁺ flux assays, in isolated rat heart mitochondria. An isolated adult rat cardiomyocyte model of ischemia-reperfusion (IR) injury was also used to determine the role of mK_ATP in cardioprotection by nitroxyl. Key findings were as follows: (i) mK_ATP was activated by O₂⁻ and H₂O₂ but not other peroxides. (ii) mK_ATP was inhibited by NADPH. (iii) mK_ATP was activated by S-nitrosothiols, nitroxyl, and nitrolinoleate. The latter two species also inhibited mitochondrial complex II. (iv) Nitroxyl protected cardiomyocytes against IR injury in an mK_ATP-dependent manner. Overall, these results suggest that the mK_ATP channel is activated by specific reactive oxygen and nitrogen species, and inhibited by NADPH. The redox modulation of mK_ATP may be an underlying mechanism for its regulation in the context of IPC. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

The endogenous mitochondrial complex II inhibitor malonate regulates mitochondrial ATP-sensitive potassium channels: implications for ischemic preconditioning

Ischemic preconditioning (IPC) affords cardioprotection against ischemia-reperfusion (IR) injury, and while the molecular mechanisms of IPC are debated, the mitochondrial ATP-sensitive K(+) channel (mK(ATP)) has emerged as a candidate effector for several IPC signaling pathways. The molecular identity of this channel is unknown, but significant pharmacologic overlap exists between mK(ATP) and mitochondrial respiratory complex II (succinate dehydrogenase). In this investigation, we utilized isolated cardiac mitochondria, Langendorff perfused hearts, and a variety of biochemical methods, to make the following observations: (i) The competitive complex II inhibitor malonate is formed in mitochondria under conditions resembling IPC. (ii) IPC leads to a reversible inhibition of complex II that has likely been missed in previous investigations due to the use of saturating concentrations of succinate. (iii) Malonate opens mK(ATP) channels even when mitochondria are respiring on complex I-linked substrates, suggesting an effect of this inhibitor on the mK(ATP) channel independent of complex II inhibition. Together, these observations suggest that complex II inhibition by endogenously formed malonate may represent an important activation pathway for mK(ATP) channels during IPC.     

Ischemic preconditioning prevents endothelial dysfunction, P-selectin expression, and neutrophil adhesion by preventing endothelin and O2- generation in the post-ischemic guinea-pig heart.

Evidence indicates that ischemia/reperfusion (IR) results in endothelial dysfunction and neutrophil adhesion in the post-ischemic myocardium and that ischemic preconditioning (IPC), superoxide dismutase (SOD), and anti-endothelin-1 (ET-1) interventions prevent these effects. We tested the hypothesis that ET-1-induced superoxide (O(2)(-)) generation mediates endothelial injury and neutrophil accumulation in the IR heart, that IPC protects the endothelium and prevents the adhesion by attenuating post-ischemic ET-1, and thus O(2)(-), generation, and that the mitochondrial ATP-dependent potassium channel (mK(ATP)) triggers the IPC-induced protection. Langendorff-perfused guinea-pig hearts were subjected either to 30 min ischemia/35 min reperfusion (IR) or were preconditioned prior to IR with three cycles of either 5 min ischemia/5 min reperfusion or 5 min infusion/5 min wash-out of mK(ATP) opener diazoxide (0.5 microM). Neutrophils were infused to the hearts at 15-25 min of the reperfusion. Coronary flow responses to acetylcholine (ACh) and nitroprusside (SNP) served as measures of endothelium-dependent and -independent vascular function, respectively. Myocardial outflow of ET-1 and O(2)(-), P-selectin expression, neutrophil adhesion and functional recoveries were followed during reperfusion. IR augmented ET-1 and O(2)(-) outflow, P-selectin expression, and neutrophil adhesion, and impaired ACh response. These effects were attenuated or prevented by IPC and diazoxide, and 5-hydroxydecanoate (a selective mK(ATP) blocker) abolished the effects of IPC and diazoxide. SOD (150 U/ml) and tezosentan (5 nM, a mixed ET-1-receptor antagonist) mimicked the effects of IPC, although they had no effect on the ET-1 generation. The preventive effect of IPC, SOD and tezosentan on P-selectin expression preceded their effect on neutrophil adhesion. These data suggest that in guinea-pig heart: (i) ET-1-induced O(2)(-) generation mediates the post-ischemic endothelial dysfunction, P-selectin expression and neutrophil adhesion; (ii) IPC and diazoxide afford protection by attenuating the ET-1, and thus O(2)(-) generation; (iii) the mK(ATP) opening triggers the IPC protection; (iv) endothelial injury promotes post-ischemic neutrophil adhesion, but not vice versa.     

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.     

Potassium channel-oxidative phosphorylation relationship in durum wheat mitochondria from control and hyperosmotic-stressed seedlings

Durum wheat mitochondria (DWM) possess an ATP-inhibited K(+) channel, the plant mitoK(ATP) (PmitoK(ATP) ), which is activated under environmental stress to control mitochondrial ROS production. To do this, PmitoK(ATP) collapses membrane potential (ΔΨ), thus suggesting mitochondrial uncoupling. We tested this point by studying oxidative phosphorylation (OXPHOS) in DWM purified from control seedlings and from seedlings subjected both to severe mannitol and NaCl stress. In severely-stressed DWM, the ATP synthesis via OXPHOS, continuously monitored by a spectrophotometric assay, was about 90% inhibited when the PmitoK(ATP) was activated by KCl. Contrarily, in control DWM, although PmitoK(ATP) collapsed ΔΨ, ATP synthesis, as well as coupling [respiratory control (RC) ratio and ratio between phosphorylated ADP and reduced oxygen (ADP/O)] checked by oxygen uptake experiments, were unaffected. We suggest that PmitoK(ATP) may play an important defensive role at the onset of the environmental/oxidative stress by preserving energy in a crucial moment for cell and mitochondrial bioenergetics. Consistently, under moderate mannitol stress, miming an early stress condition, the channel may efficiently control reactive oxygen species (ROS) generation (about 35-fold from fully open to closed state) without impairing ATP synthesis. Anyway, if the stress significantly proceeds, the PmitoK(ATP) becomes fully activated by decrease of ATP concentration (25-40%) and increase of activators [free fatty acids (FFAs) and superoxide anion], thus impairing ATP synthesis.     

Myocardial preconditioning against ischemia-reperfusion injury is abolished in Zucker obese rats with insulin resistance

Insulin resistance (IR) precedes the onset of Type 2 diabetes, but its impact on preconditioning against myocardial ischemia-reperfusion injury is unexplored. We examined the effects of diazoxide and ischemic preconditioning (IPC; 5-min ischemia and 5-min reperfusion) on ischemia (30 min)-reperfusion (240 min) injury in young IR Zucker obese (ZO) and lean (ZL) rats. ZO hearts developed larger infarcts than ZL hearts (infarct size: 57.3 +/- 3% in ZO vs. 39.2 +/- 3.2% in ZL; P < 0.05) and also failed to respond to cardioprotection by IPC or diazoxide (47.2 +/- 4.3% and 52.5 +/- 5.8%, respectively; P = not significant). In contrast, IPC and diazoxide treatment reduced the infarct size in ZL hearts (12.7 +/- 2% and 16.3 +/- 6.7%, respectively; P < 0.05). The mitochondrial ATP-activated potassium channel (K(ATP)) antagonist 5-hydroxydecanoic acid inhibited IPC and diazoxide-induced preconditioning in ZL hearts, whereas it had no effect on ZO hearts. Diazoxide elicited reduced depolarization of isolated mitochondria from ZO hearts compared with ZL (73 +/- 9% in ZL vs. 39 +/- 9% in ZO; P < 0.05). Diazoxide also failed to enhance superoxide generation in isolated mitochondria from ZO compared with ZL hearts. Electron micrographs of ZO hearts revealed a decreased number of mitochondria accompanied by swelling, disorganized cristae, and vacuolation. Immunoblots of mitochondrial protein showed a modest increase in manganese superoxide dismutase in ZO hearts. Thus obesity accompanied by IR is associated with the inability to precondition against ischemic cardiac injury, which is mediated by enhanced mitochondrial oxidative stress and impaired activation of mitochondrial K(ATP).     

The mitochondrial complex II and ATP-sensitive potassium channel interaction: quantitation of the channel in heart mitochondria

The mitochondrial ATP-sensitive potassium channel (mK(ATP)) is important in cardioprotection, although the channel remains molecularly undefined. Several studies have demonstrated that mitochondrial complex II inhibitors activate the mK(ATP), suggesting a potential role for complex II in channel composition or regulation. However, these inhibitors activate mK(ATP) at concentrations which do not affect bulk complex II activity. Using the potent complex II inhibitor Atpenin A5, this relationship was investigated using tight-binding inhibitor theory, to demonstrate that only 0.4 % of total complex II molecules are necessary to activate the mK(ATP). These results estimate the mK(ATP) content at 15 channels per mitochondrion.     

Reactive oxygen species (ROS) mediate the effects of leucine on translation regulation and type I collagen production in hepatic stellate cells

The amino acid leucine causes an increase of collagen alpha1(I) synthesis in hepatic stellate cells through the activation of translational regulatory mechanisms and PI3K/Akt/mTOR and ERK signaling pathways. The aim of the present study was to evaluate the role played by reactive oxygen species on these effects. Intracellular reactive oxygen species levels were increased in hepatic stellate cells incubated with leucine 5 mM at early time points, and this effect was abolished by pretreatment with the antioxidant glutathione. Preincubation with glutathione also prevented 4E-BP1, eIF4E and Mnk-1 phosphorylation induced by leucine, as well as enhancement of procollagen alpha1(I) protein levels. Inhibitors for MEK-1 (PD98059), PI3K (wortmannin) or mTOR (rapamycin) did not affect leucine-induced reactive oxygen species production. However, preincubation with glutathione prevented ERK, Akt and mTOR phosphorylation caused by treatment with leucine. The mitochondrial electron chain inhibitor rotenone and the NADPH oxidase inhibitor apocynin prevented reactive oxygen species production caused by leucine. Leucine also induced an increased phosphorylation of IR/IGF-R that was abolished by pretreatment with either rotenone or apocynin. Therefore, leucine exerts on hepatic stellate cells a prooxidant action through NADPH oxidase and mitochondrial Reactive oxygen species production and these effects mediate the activation of IR/IGF-IR and signaling pathways, finally leading to changes in translational regulation of collagen synthesis.     

Sarcolemmal KATP channel triggers delayed ischemic preconditioning in rats

Previous work from our laboratory has shown that the sarcolemmal K(ATP) channel (sK(ATP)) is required as a trigger for delayed cardioprotection upon exogenous opioid administration. We also established that the mitochondrial K(ATP) (mK(ATP)) channel is not required for triggering delayed delta-opioid-induced infarct size reduction. Because mechanistic differences have been found among delta-opioids and that due to ischemic preconditioning (IPC), we determined whether the triggering mechanism of delayed IPC-induced infarct size reduction involves either the sK(ATP) or mK(ATP). Male Sprague-Dawley rats received either sham surgery or IPC (3- to 5-min cycles of ischemia and reperfusion) 24 h before being subjected to 30 min of ischemia and 2 h of reperfusion. Infarct size was determined and expressed as a percentage of the area at risk, with significance compared with sham reported at P 相似文献   

Mitochondrial Src tyrosine kinase plays a role in the cardioprotective effect of ischemic preconditioning by modulating complex I activity and mitochondrial ROS generation

Abstract

While ischemic preconditioning (IPC) and other cardioprotective interventions have been proposed to protect the heart from ischemia/reperfusion (I/R) injury by inhibiting mitochondrial complex I activity upon reperfusion, the exact mechanism underlying the modulation of complex I activity remains elusive. This study was aimed to test the hypothesis that IPC modulates complex I activity at reperfusion by activating mitochondrial Src tyrosine kinase, and induces cardioprotection against I/R injury. Isolated rat hearts were preconditioned by three cycles of 5-min ischemia and 5-min reperfusion prior to 30-min index ischemia followed by 2 h of reperfusion. Mitochondrial Src phosphorylation (Tyr⁴¹⁶) was dramatically decreased during I/R, implying inactivation of Src tyrosine kinase by I/R. IPC increased mitochondrial Src phosphorylation upon reperfusion and this was inhibited by the selective Src tyrosine kinase inhibitor PP2. IPC's anti-infarct effect was inhibited by the selective Src tyrosine kinase inhibitor PP2. Complex I activity was significantly increased upon reperfusion, an effect that was prevented by IPC in a Src tyrosine kinase-dependent manner. In support, Src and phospho-Src were found in complex I. Furthermore, IPC prevented hypoxia/reoxygenation-induced mitochondrial reactive oxygen species (ROS) generation and cellular injury in rat cardiomyocytes, which was revoked by PP2. Finally, IPC reduced LDH release induced by both hypoxia/reoxygenation and simulated ischemia/reperfusion, an effect that was reversed by PP2 and Src siRNA. These data suggest that mitochondrial Src tyrosine kinase accounts for the inhibitory action of IPC on complex I and mitochondrial ROS generation, and thereby plays a role in the cardioprotective effect of IPC.     

Mitochondrial ATP-sensitive K+ channel opening decreases reactive oxygen species generation

Mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) opening was shown previously to slightly increase respiration and decrease the membrane potential by stimulating K(+) cycling across the inner membrane. Here we show that mitoK(ATP) opening reduces reactive oxygen species generation in heart, liver and brain mitochondria. Decreased H(2)O(2) release is observed when mitoK(ATP) is active both with respiration stimulated by oxidative phosphorylation and when ATP synthesis is inhibited. In addition, decreased H(2)O(2) release is observed when mitochondrial Delta pH is enhanced, an effect expected to occur when mitoK(ATP) is open. We conclude that mitoK(ATP) is an effective pathway to trigger mild uncoupling, preventing reactive oxygen species release.     

Regulation of ionizing radiation-induced apoptosis by a manganese porphyrin complex

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Therefore, compounds that scavenge reactive oxygen species may confer regulatory effects on apoptosis. Superoxide dismutase (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. We investigated the effects of the manganese (III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP), a cell-permeable SOD mimetic, on ionizing radiation-induced apoptosis. Upon exposure to 2 Gy of gamma-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 5 microM MnTMPyP for 2 h with regard to apoptotic parameters, cellular redox status, mitochondria function, and oxidative damage to cells. MnTMPyP effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular reactive oxygen species were higher and the [NADPH]/[NADP(+)+NADPH] ratio was lower in control cells compared to MnTMPyP-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of reactive oxygen species, and the reduction of ATP production were significantly higher in control cells compared to MnTMPyP-treated cells. MnTMPyP pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that MnTMPyP may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of reactive oxygen species.     

K+-independent actions of diazoxide question the role of inner membrane KATP channels in mitochondrial cytoprotective signaling

Activation by diazoxide and inhibition by 5-hydroxydecanoate are the hallmarks of mitochondrial ATP-sensitive K+ (K(ATP)) channels. Opening of these channels is thought to trigger cytoprotection (preconditioning) through the generation of reactive oxygen species. However, we found that diazoxide-induced oxidation of the widely used reactive oxygen species indicator 2',7'-dichlorodihydrofluorescein in isolated liver and heart mitochondria was observed in the absence of ATP or K+ and therefore independent of K(ATP) channels. The response was blocked by stigmatellin, implying a role for the cytochrome bc1 complex (complex III). Diazoxide, though, did not increase hydrogen peroxide (H2O2) production (quantitatively measured with Amplex Red) in intact mitochondria, submitochondrial particles, or purified cytochrome bc1 complex. We confirmed that diazoxide inhibited succinate oxidation, but it also weakly stimulated state 4 respiration even in K+-free buffer, excluding a role for K(ATP) channels. Furthermore, we have shown previously that 5-hydroxydecanoate is partially metabolized, and we hypothesized that fatty acid metabolism may explain the ability of this putative mitochondrial K(ATP) channel blocker to inhibit diazoxide-induced flavoprotein fluorescence, commonly used as an assay of K(ATP) channel activity. Indeed, consistent with our hypothesis, we found that decanoate inhibited diazoxide-induced flavoprotein oxidation. Taken together, our data question the "mitochondrial K(ATP) channel" hypothesis of preconditioning. Diazoxide did not evoke superoxide (which dismutates to H2O2) from the respiratory chain by a direct mechanism, and the stimulatory effects of this compound on mitochondrial respiration and 2',7'-dichlorodihydrofluorescein oxidation were not due to the opening of K(ATP) channels.     

A manganese porphyrin complex is a novel radiation protector

Exposure of cells to ionizing radiation leads to formation of reactive oxygen species, which are associated with radiation-induced cytotoxicity. Therefore, compounds that scavenge reactive oxygen species may confer radioprotective effects. Superoxide dismutase (SOD) mimetics have been shown to be protective against cell injury caused by reactive oxygen species. The objective of this study was to investigate the effects of manganese(III) tetrakis(N-methyl-2-pyridyl)porphyrin (MnTMPyP), a cell-permeable SOD mimetic, on radiation-dependent toxicity. We investigated the protective role of MnTMPyP against ionizing radiation in U937 cells and mice. On exposure to ionizing radiation, there was a distinct difference between control cells and cells pretreated with MnTMPyP with respect to viability, cellular redox status, and oxidative damage to cells. Lipid peroxidation, oxidative DNA damage, and protein oxidation were significantly lower in the cells treated with MnTMPyP when the cells were exposed to ionizing radiation. The [GSSG]/[GSH + GSSG] ratio and the generation of intracellular reactive oxygen species were higher and the [NADPH]/[NADP+ + NADPH] ratio was lower in control cells compared with MnTMPyP-treated cells. Ionizing radiation-induced mitochondrial damage, as reflected by the altered mitochondrial permeability transition, increase in accumulation of reactive oxygen species, reduction of ATP production, and morphological change, was significantly higher in control cells than in MnTMPyP-treated cells. MnTMPyP administration for 14 days at a daily dosage of 5 mg/kg provided substantial protection against killing and oxidative damage in mice exposed to whole-body irradiation. These data indicate that MnTMPyP may have great application potential as a new class of in vivo, non-sulfur-containing radiation protectors.     

Tumour necrosis factor‐α promotes liver ischaemia‐reperfusion injury through the PGC‐1α/Mfn2 pathway

Tumour necrosis factor (TNF)‐α has been considered to induce ischaemia‐reperfusion injury (IRI) of liver which is characterized by energy dysmetabolism. Peroxisome proliferator–activated receptor‐γ co‐activator (PGC)‐1α and mitofusion2 (Mfn2) are reported to be involved in the regulation of mitochondrial function. However, whether PGC‐1α and Mfn2 form a pathway that mediates liver IRI, and if so, what the underlying involvement is in that pathway remain unclear. In this study, L02 cells administered recombinant human TNF‐α had increased TNF‐α levels and resulted in down‐regulation of PGC‐1α and Mfn2 in a rat liver IRI model. This was associated with hepatic mitochondrial swelling, decreased adenosine triphosphate (ATP) production, and increased levels of reactive oxygen species (ROS) and alanine aminotransferase (ALT) activity as well as cell apoptosis. Inhibition of TNF‐α by neutralizing antibody reversed PGC‐1α and Mfn2 expression, and decreased hepatic injury and cell apoptosis both in cell culture and in animals. Treatment by rosiglitazone sustained PGC‐1α and Mfn2 expression both in IR livers, and L02 cells treated with TNF‐α as indicated by increased hepatic mitochondrial integrity and ATP production, reduced ROS and ALT activity as well as decreased cell apoptosis. Overexpression of Mfn2 by lentiviral‐Mfn2 transfection decreased hepatic injury in IR livers and L02 cells treated with TNF‐α. However, there was no up‐regulation of PGC‐1α. These findings suggest that PGC‐1α and Mfn2 constitute a regulatory pathway, and play a critical role in TNF‐α‐induced hepatic IRI. Inhibition of the TNF‐α or PGC‐1α/Mfn2 pathways may represent novel therapeutic interventions for hepatic IRI.     

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.     

Constitutive NADPH oxidase and increased mitochondrial respiratory chain activity regulate chemokine gene expression

Alveolar macrophages, which generate high levels of reactive oxygen species, especially O(2)(*-), are involved in the recruitment of neutrophils to sites of inflammation and injury in the lung, and the generation of chemotactic proteins triggers this cellular recruitment. In this study, we asked whether O(2)(*-) generation in alveolar macrophages had a role in the expression of chemokines. Specifically, we hypothesized that O(2)(*-) generation is necessary for chemokine expression in alveolar macrophages after TNF-alpha stimulation. We found that alveolar macrophages have high constitutive NADPH oxidase activity that was not increased by TNF-alpha, but TNF-alpha increased the activity of the mitochondrial respiratory chain. In addition, the mitochondrial respiratory chain increased O(2)(*-) generation if the NADPH oxidase was inhibited. O(2)(*-) generation was necessary for macrophage inflammatory protein-2 (MIP-2) gene expression, because inhibition of NADPH oxidase or the mitochondrial respiratory chain or overexpression of Cu,Zn-superoxide dismutase significantly inhibited expression of MIP-2. TNF-alpha activated the ERK MAP kinase, and ERK activity was essential for chemokine gene expression. In addition, overexpression of the MEK1-->ERK pathway significantly increased IL-8 expression, and a small interfering RNA to the NADPH oxidase inhibited ERK- and TNF-alpha-induced chemokine expression. Collectively, these results suggest that in alveolar macrophages, O(2)(*-) generation mediates chemokine expression after TNF-alpha stimulation in an ERK-dependent manner.     

Different mechanisms of mitochondrial proton leak in ischaemia/reperfusion injury and preconditioning: implications for pathology and cardioprotection

The mechanisms of mitochondrial proton (H+) leak under various pathophysiological conditions are poorly understood. In the present study it was hypothesized that different mechanisms underlie H+ leak in cardiac IR (ischaemia/reperfusion) injury and IPC (ischaemic preconditioning). Potential H(+) leak mechanisms examined were UCPs (uncoupling proteins), allosteric activation of the ANT (adenine nucleotide translocase) by AMP, or the PT (permeability transition) pore. Mitochondria isolated from perfused rat hearts that were subjected to IPC exhibited a greater H+ leak than did controls (202+/-27%, P<0.005), and this increased leakage was completely abolished by the UCP inhibitor, GDP, or the ANT inhibitor, CAT (carboxyattractyloside). Mitochondria from hearts subjected to IR injury exhibited a much greater amount of H+ leak than did controls (411+/-28%, P<0.001). The increased leakage after IR was weakly inhibited by GDP, but was inhibited, >50%, by carboxyattractyloside. In addition, it was inhibited by cardioprotective treatment strategies including pre-IR perfusion with the PT pore inhibitors cyclosporin A or sanglifehrin A, the adenylate kinase inhibitor, AP5A (diadenosine pentaphosphate), or IPC. Together these data suggest that the small increase in H+ leak in IPC is mediated by UCPs, while the large increase in H+ leak in IR is mediated by the ANT. Furthermore, under all conditions studied, in situ myocardial O2 efficiency was correlated with isolated mitochondrial H+ leak (r2=0.71). In conclusion, these data suggest that the modulation of H+ leak may have important implications for the outcome of IR injury.