Mitochondrial Uncoupling Proteins: Subtle Regulators of Cellular Redox Signaling.

SIGNIFICANCE: Mitochondria are the energetic, metabolic, redox, and information signaling centers of the cell. Substrate pressure, mitochondrial network dynamics, and cristae morphology state are integrated by the protonmotive force Δp or its potential component, ΔΨ, which are attenuated by proton backflux into the matrix, termed uncoupling. The mitochondrial uncoupling proteins (UCP1-5) play an eminent role in the regulation of each of the mentioned aspects, being involved in numerous physiological events including redox signaling. Recent Advances: UCP2 structure, including purine nucleotide and fatty acid (FA) binding sites, strongly support the FA cycling mechanism: UCP2 expels FA anions, whereas uncoupling is achieved by the membrane backflux of protonated FA. Nascent FAs, cleaved by phospholipases, are preferential. The resulting Δp dissipation decreases superoxide formation dependent on Δp. UCP-mediated antioxidant protection and its impairment are expected to play a major role in cell physiology and pathology. Moreover, UCP2-mediated aspartate, oxaloacetate, and malate antiport with phosphate is expected to alter metabolism of cancer cells. CRITICAL ISSUES: A wide range of UCP antioxidant effects and participations in redox signaling have been reported; however, mechanisms of UCP activation are still debated. Switching off/on the UCP2 protonophoretic function might serve as redox signaling either by employing/releasing the extra capacity of cell antioxidant systems or by directly increasing/decreasing mitochondrial superoxide sources. Rapid UCP2 degradation, FA levels, elevation of purine nucleotides, decreased Mg2+, or increased pyruvate accumulation may initiate UCP-mediated redox signaling. FUTURE DIRECTIONS: Issues such as UCP2 participation in glucose sensing, neuronal (synaptic) function, and immune cell activation should be elucidated. Antioxid. Redox Signal. 29, 667-714.

Equipping Physiologists with an Informatics Tool Chest: Toward an Integerated Mitochondrial Phenome.

Understanding the complex involvement of mitochondrial biology in disease development often requires the acquisition, analysis, and integration of large-scale molecular and phenotypic data. An increasing number of bioinformatics tools are currently employed to aid in mitochondrial investigations, most notably in predicting or corroborating the spatial and temporal dynamics of mitochondrial molecules, in retrieving structural data of mitochondrial components, and in aggregating as well as transforming mitochondrial centric biomedical knowledge. With the increasing prevalence of complex Big Data from omics experiments and clinical cohorts, informatics tools have become indispensable in our quest to understand mitochondrial physiology and pathology. Here we present an overview of the various informatics resources that are helping researchers explore this vital organelle and gain insights into its form, function, and dynamics.

Mitochondrial reactive oxygen species: which ROS signals cardioprotection?

Mitochondria are the major effectors of cardioprotection by procedures that open the mitochondrial ATP-sensitive potassium channel (mitoKATP), including ischemic and pharmacological preconditioning. MitoKATP opening leads to increased reactive oxygen species (ROS), which then activate a mitoKATP-associated PKCε, which phosphorylates mitoKATP and leaves it in a persistent open state (Costa AD, Garlid KD. Am J Physiol Heart Circ Physiol 295, H874-H882, 2008). The ROS responsible for this effect is not known. The present study focuses on superoxide (O2(·-)), hydrogen peroxide (H2O2), and hydroxyl radical (HO(·)), each of which has been proposed as the signaling ROS. Feedback activation of mitoKATP provides an ideal setting for studying endogenous ROS signaling. Respiring rat heart mitochondria were preincubated with ATP and diazoxide, together with an agent being tested for interference with this process, either by scavenging ROS or by blocking ROS transformations. The mitochondria were then assayed to determine whether or not the persistent phosphorylated open state was achieved. Dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, Trolox, and bromoenol lactone each interfered with formation of the ROS-dependent open state. Catalase did not interfere with this step. We also found that DMF blocked cardioprotection by both ischemic preconditioning and diazoxide. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO(·) excludes H2O2 as the endogenous signaling ROS. Taken together, the results support the conclusion that the ROS message is carried by a downstream product of HO(·) and that it is probably a product of phospholipid oxidation.

Mitochondrial ROMK channel is a molecular component of mitoK(ATP).

RATIONALE: Activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) has been implicated in the mechanism of cardiac ischemic preconditioning, yet its molecular composition is unknown. OBJECTIVE: To use an unbiased proteomic analysis of the mitochondrial inner membrane to identify the mitochondrial K(+) channel underlying mitoK(ATP). METHODS AND RESULTS: Mass spectrometric analysis was used to identify KCNJ1(ROMK) in purified bovine heart mitochondrial inner membrane and ROMK mRNA was confirmed to be present in neonatal rat ventricular myocytes and adult hearts. ROMK2, a short form of the channel, is shown to contain an N-terminal mitochondrial targeting signal, and a full-length epitope-tagged ROMK2 colocalizes with mitochondrial ATP synthase ß. The high-affinity ROMK toxin, tertiapin Q, inhibits mitoK(ATP) activity in isolated mitochondria and in digitonin-permeabilized cells. Moreover, short hairpin RNA-mediated knockdown of ROMK inhibits the ATP-sensitive, diazoxide-activated component of mitochondrial thallium uptake. Finally, the heart-derived cell line, H9C2, is protected from cell death stimuli by stable ROMK2 overexpression, whereas knockdown of the native ROMK exacerbates cell death. CONCLUSIONS: The findings support ROMK as the pore-forming subunit of the cytoprotective mitoK(ATP) channel.

The mitochondrial K(ATP) channel--fact or fiction?

The mitochondrial ATP-dependent K(+) channel (mitoK(ATP)) is widely considered by many to play a central role in cardioprotection by ischemic and pharmacological preconditioning and by ischemic postconditioning. Nevertheless, several laboratories have questioned the existence of mitoK(ATP). This article summarizes the evidence for and against and addresses two key questions: How strong is the evidence for the presence of a K(ATP) channel in mitochondria? Are the pharmacological agents used to modulate mitoK(ATP) activity sufficiently specific to allow the role of these channels in cardioprotection to be established?

Channel character of uncoupling protein-mediated transport.

Mitochondrial uncoupling proteins (UCPs) are pure anion uniporters, which mediate fatty acid (FA) uniport leading to FA cycling. Protonated FAs then flip-flop back across the lipid bilayer. An existence of pure proton channel in UCPs is excluded by the equivalent flux-voltage dependencies for uniport of FAs and halide anions, which are best described by the Eyring barrier variant with a single energy well in the middle of two peaks. Experiments with FAs unable to flip and alkylsulfonates also support this view. Phylogenetically, UCPs took advantage of the common FA-uncoupling function of SLC25 family carriers and dropped their solute transport function.

Preconditioning by subinotropic doses of ouabain in the Langendorff perfused rabbit heart.

Short exposure to low concentrations of digitalis drugs like ouabain protects the rat heart against ischemia/reperfusion injury through the activation of the Na/K-adenosine triphosphatase (ATPase)/Src receptor complex and subsequent stimulation of key intracellular cardioprotective signals. Rat Na/K-ATPase, however, is relatively insensitive to digitalis, and it is not known if similar results could be obtained in species with higher sensitivity. Thus, to determine whether ouabain pretreatment protects against ischemic injury and activates the Na/K-ATPase signaling cascade in a species with cardiac glycoside sensitivity comparable to humans, the present study was conducted in the rabbit model. In Langendorff perfused rabbit hearts, 20-minute exposure to 500-nM ouabain resulted in positive inotropy as evidenced by a significant increase in +dP/dt, and this increase was accompanied by the activation of several well-characterized downstream mediators of the cardiac Na/K-ATPase receptor pathway, including Src, Akt, ERK1/2, and protein kinase Cepsilon. A short (4 minutes) administration of a subinotropic dose of ouabain (100 nM) followed by an 8-minute washout before 30 minutes of global ischemia and 120 minutes of reperfusion resulted in protection against cell death, as evidenced by a significant decrease in infarct size. These data indicate that ouabain administration activates the Na/K-ATPase signaling cascade and protects against ischemic injury in a species with high cardiac Na/K-ATPase sensitivity.

MitoKATP activity in healthy and ischemic hearts.

In addition to their role in energy transduction, mitochondria play important non-canonical roles in cell pathophysiology, several of which utilize the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). In the normal heart, mitoK(ATP) regulates energy transfer through its regulation of intermembrane space volume and is accordingly essential for the inotropic response during periods of high workload. In the ischemic heart, mitoK(ATP) is the point of convergence of protective signaling pathways and mediates inhibition of the mitochondrial permeability transition, and thus necrosis. In this review, we outline the experimental evidence that support these roles for mitoK(ATP) in health and disease, as well as our hypothesis for the mechanism by which complex cardioprotective signals that originate at plasma membrane receptors traverse the cytosol to reach mitochondria and activate mitoK(ATP).

Cardioprotective signaling to mitochondria.

Mitochondria are central players in the pathophysiology of ischemia-reperfusion. Activation of plasma membrane G-coupled receptors or the Na,K-ATPase triggers cytosolic signaling pathways that result in cardioprotection. Our working hypothesis is that the occupied receptors migrate to caveolae, where signaling enzymes are scaffolded into signalosomes that bud off the plasma membrane and migrate to mitochondria. The signalosome-mitochondria interaction then initiates intramitochondrial signaling by opening the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). MitoK(ATP) opening causes an increase in ROS production, which activates mitochondrial protein kinase C epsilon (PKCvarepsilon), which inhibits the mitochondrial permeability transition (MPT), thus decreasing cell death. We review the experimental findings that bear on these hypotheses and other modes of protection involving mitochondria.

Conditioning the heart induces formation of signalosomes that interact with mitochondria to open mitoKATP channels.

Perfusion of the heart with bradykinin triggers cellular signaling events that ultimately cause opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels, increased H2O2 production, inhibition of the mitochondrial permeability transition (MPT), and cardioprotection. We hypothesized that the interaction of bradykinin with its receptor induces the assembly of a caveolar signaling platform (signalosome) that contains the enzymes of the signaling pathway and that migrates to mitochondria to induce mitoKATP channel opening. We developed a novel method for isolating and purifying signalosomes from Langendorff-perfused rat hearts treated with bradykinin. Fractions containing the signalosomes were found to open mitoKATP channels in mitochondria isolated from untreated hearts via the activation of mitochondrial PKC-epsilon. mitoKATP channel opening required signalosome-dependent phosphorylation of an outer membrane protein. Immunodetection analysis revealed the presence of the bradykinin B2 receptor only in the fraction isolated from bradykinin-treated hearts. Immunodetection and immunogold labeling of caveolin-3, as well as sensitivity to cholesterol depletion and resistance to Triton X-100, attested to the caveolar nature of the signalosomes. Ischemic preconditioning, ischemic postconditioning, and perfusion with ouabain also led to active signalosome fractions that opened mitoKATP channels in mitochondria from untreated hearts. These results provide initial support for a novel mechanism for signal transmission from a plasma membrane receptor to mitoKATP channels.

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.

Differential increase of mitochondrial matrix volume by sevoflurane in isolated cardiac mitochondria.

BACKGROUND: Mitochondrial (m) adenosine triphosphate sensitive potassium (K(ATP)) channel opening has been reported to trigger and/or mediate cardioprotection by volatile anesthetics. However, the effects of volatile anesthetics on mitochondrial function are not well understood. Prevention of mitochondrial matrix volume (MMV) contraction during ischemia may contribute to cardioprotection against ischemia/reperfusion injury. We investigated whether sevoflurane increases MMV and if this increase is mediated by mK(ATP) channel opening. METHODS: Mitochondria from fresh guinea pig hearts were isolated and diluted in buffer that included oligomycin and ATP to inhibit ATP synthesis. Changes in MMV by diazoxide, a known mK(ATP) channel opener, and by different sevoflurane concentrations, were measured by light absorption at 520 nm in the absence or presence of the mK(ATP) channel blocker, 5-hydroxydecanoate. RESULTS: Compared with control, 30-300 microM sevoflurane (approximately 0.2-2.1 vol %) increased MMV by 30%-55%, which was similar to the effect of diazoxide. These increases were blocked by 5-hydroxydecanoate. Higher sevoflurane concentration (1000 microM; 7.1 vol %), however, had no effect on MMV. CONCLUSIONS: In clinically relevant concentrations, sevoflurane increases MMV via mK(ATP) channel opening. Preservation of mitochondrial integrity may contribute to the cardioprotective effects of sevoflurane against ischemia/reperfusion injury. Impaired mitochondrial function at supraclinical anesthetic concentrations may explain the observed biphasic response. These findings add to our understanding of the intracellular mechanisms of volatile anesthetics as cardioprotective drugs.

cGMP signalling in pre- and post-conditioning: the role of mitochondria.

Much of cell death from ischaemia/reperfusion in heart and other tissues is generally thought to arise from mitochondrial permeability transition (MPT) in the first minutes of reperfusion. In ischaemic pre-conditioning, agonist binding to G(i) protein-coupled receptors prior to ischaemia triggers a signalling cascade that protects the heart from MPT. We believe that the cytosolic component of this trigger pathway terminates in activation of guanylyl cyclase resulting in increased production of cGMP and subsequent activation of protein kinase G (PKG). PKG phosphorylates a protein on the mitochondrial outer membrane (MOM), which then causes the mitochondrial K(ATP) channel (mitoK(ATP)) on the mitochondrial inner membrane to open, leading to increased production of reactive oxygen species (ROS) by the mitochondria. This implies that the protective signal is somehow transmitted from the MOM to its inner membrane. This is accomplished by a series of intermembrane signalling steps that includes protein kinase C (PKCepsilon) activation. The resulting ROS then activate a second PKC pool which, through another signal transduction pathway termed the mediator pathway, causes inhibition of MPT and reduction in cell death.

Interactions of K+ATP channel blockers with Na+/K+-ATPase.

Two K(+) (ATP) channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na(+)/K(+)-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na(+)/K(+)-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na(+)/K(+)-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K(+) (ATP) channels. 5-HD did not affect the ouabain sensitivity of Na(+)/K(+)-ATPase. Glyburide had both activating and inhibitory effects on Na(+)/K(+)-ATPase at concentrations used to block plasma membrane K(+) (ATP) channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na(+)/K(+)-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K(+) (ATP) channels, when the relation of this channel to Na(+)/K(+)-ATPase is being studied.

Sarcoplasmic ATP-sensitive potassium channel blocker HMR1098 protects the ischemic heart: implication of calcium, complex I, reactive oxygen species and mitochondrial ATP-sensitive potassium channel.

The aim of this study was to investigate the effects of HMR1098, a selective blocker of sarcolemmal ATP-sensitive potassium channel (sarcK(ATP)), in Langendorff-perfused rat hearts submitted to ischemia and reperfusion. The recovery of heart hemodynamic and mitochondrial function, studied on skinned fibers, was analyzed after 30-min global ischemia followed by 20-min reperfusion. Infarct size was quantified on a regional ischemia model after 2-h reperfusion. We report that the perfusion of 10 microM HMR1098 before ischemia, delays the onset of ischemic contracture, improves recovery of cardiac function upon reperfusion, preserves the mitochondrial architecture, and finally decreases infarct size. This HMR1098-induced cardioprotection is prevented by 1 mM 2-mercaptopropionylglycine, an antioxidant, and by 100 nM nifedipine, an L-type calcium channel blocker. Concomitantly, it is shown that HMR1098 perfusion induces (i) a transient and specific inhibition of the respiratory chain complex I and, (ii) an increase in the averaged intracellular calcium concentration probed by the in situ measurement of indo-1 fluorescence. Finally, all the beneficial effects of HMR1098 were strongly inhibited by 5-hydroxydecanoate and abolished by glibenclamide, two mitoK(ATP) blockers. This study demonstrates that the HMR1098-induced cardioprotection occurs indirectly through extracellular calcium influx, respiratory chain complex inhibition, reactive oxygen species production and mitoK(ATP) opening. Taken together, these data suggest that a functional interaction between sarcK(ATP) and mitoK(ATP) exists in isolated rat heart ischemia model, which is mediated by extracellular calcium influx.

Ouabain triggers preconditioning through activation of the Na+,K+-ATPase signaling cascade in rat hearts.

OBJECTIVE: Because ouabain activates several pathways that are critical to cardioprotective mechanisms such as ischemic preconditioning, we tested if this digitalis compound could protect the heart against ischemia-reperfusion injury through activation of the Na+,K+-ATPase/c-Src receptor complex. METHODS AND RESULTS: In Langendorff-perfused rat hearts, a short (4 min) administration of ouabain 10 muM followed by an 8-minute washout before 30 min of global ischemia and reperfusion improved cardiac function, decreased lactate dehydrogenase release and reduced infarct size by 40%. Western blot analysis revealed that ouabain activated the cardioprotective phospholipase Cgamma1/protein kinase Cepsilon (PLC-gamma1/PKCepsilon) pathway. Pre-treatment of the hearts with the Src kinase family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) blocked not only ouabain-induced activation of PLC-gamma1/PKCepsilon pathway, but also cardiac protection. This protection was also blocked by a PKCepsilon translocation inhibitor peptide (PKCepsilon TIP). CONCLUSION: Short exposure to a low concentration of ouabain protects the heart against ischemia/reperfusion injury. This effect of ouabain on the heart is most likely due to the activation of the Na+,K+-ATPase/c-Src receptor complex and subsequent stimulation of key mediators of preconditioning, namely PLC-gamma1 and PKCepsilon.

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.

Mitochondrial PKC epsilon and mitochondrial ATP-sensitive K+ channel copurify and coreconstitute to form a functioning signaling module in proteoliposomes.

Mitochondria are key mediators of the cardioprotective signal and the mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) plays a crucial role in originating and transmitting that signal. Recently, protein kinase C epsilon (PKC epsilon) has been identified as a component of the mitoK(ATP) signaling cascade. We hypothesized that PKC epsilon and mitoK(ATP) interact directly to form functional signaling modules in the inner mitochondria membrane. To examine this possibility, we studied K+ flux in liposomes containing partially purified mitoK(ATP). The reconstituted proteins were obtained after detergent extraction of isolated mitochondria, 200-fold purification by ion exchange chromatography, and reconstitution into lipid vesicles. Immunoblot analysis revealed the presence of PKC epsilon in the reconstitutively active fraction. Addition of the PKC activators 12-phorbol 13-myristate acetate, hydrogen peroxide, and the specific PKC epsilon peptide agonist, psi epsilonRACK, each activated mitoK(ATP)-dependent K+ flux in the reconstituted system. This effect of PKC epsilon was prevented by chelerythrine, by the specific PKC epsilon peptide antagonist, epsilonV(1-2), and by the specific mitoK(ATP) inhibitor 5-hydroxydecanoate. In addition, the activating effect of PKC agonists was reversed by exogenous protein phosphatase 2A. These results demonstrate persistent, functional association of mitochondrial PKC epsilon and mitoK(ATP).

Opening mitoKATP increases superoxide generation from complex I of the electron transport chain.

Opening the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)) increases levels of reactive oxygen species (ROS) in cardiomyocytes. This increase in ROS is necessary for cardioprotection against ischemia-reperfusion injury; however, the mechanism of mitoK(ATP)-dependent stimulation of ROS production is unknown. We examined ROS production in suspensions of isolated rat heart and liver mitochondria, using fluorescent probes that are sensitive to hydrogen peroxide. When mitochondria were treated with the K(ATP) channel openers diazoxide or cromakalim, their ROS production increased by 40-50%, and this effect was blocked by 5-hydroxydecanoate. ROS production exhibited a biphasic dependence on valinomycin concentration, with peak production occurring at valinomycin concentrations that catalyze about the same K(+) influx as K(ATP) channel openers. ROS production decreased with higher concentrations of valinomycin and with all concentrations of a classical protonophoretic uncoupler. Our studies show that the increase in ROS is due specifically to K(+) influx into the matrix and is mediated by the attendant matrix alkalinization. Myxothiazol stimulated mitoK(ATP)-dependent ROS production, whereas rotenone had no effect. This indicates that the superoxide originates in complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain.

The mechanism by which the mitochondrial ATP-sensitive K+ channel opening and H2O2 inhibit the mitochondrial permeability transition.

Myocardial infarction is a manifestation of necrotic cell death as a result of opening of the mitochondrial permeability transition (MPT). Receptor-mediated cardioprotection is triggered by an intracellular signaling pathway that includes phosphatidylinositol 3-kinase, endothelial nitric-oxide synthase, guanylyl cyclase, protein kinase G (PKG), and the mitochondrial K(ATP) channel (mitoK(ATP)). In this study, we explored the pathway that links mitoK(ATP) with the MPT. We confirmed previous findings that diazoxide and activators of PKG or protein kinase C (PKC) inhibited MPT opening. We extended these results and showed that other K(+) channel openers as well as the K(+) ionophore valinomycin also inhibited MPT opening and that this inhibition required reactive oxygen species. By using isoform-specific peptides, we found that the effects of K(ATP) channel openers, PKG, or valinomycin were mediated by a PKCepsilon. Activation of PKCepsilon by phorbol 12-myristate 13-acetate or H(2)O(2) resulted in mitoK(ATP)-independent inhibition of MPT opening, whereas activation of PKCepsilon by PKG or the specific PKCepsilon agonist psiepsilon receptor for activated C kinase caused mitoK(ATP)-dependent inhibition of MPT opening. Exogenous H(2)O(2) inhibited MPT, because of its activation of PKCepsilon, with an IC(50) of 0.4 (+/-0.1) microm. On the basis of these results, we propose that two different PKCepsilon pools regulate this signaling pathway, one in association with mitoK(ATP) and the other in association with MPT.