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 PKC⑀. Activation of PKC⑀ by phorbol 12-myristate 13-acetate or H 2 O 2 resulted in mitoK ATPindependent inhibition of MPT opening, whereas activation of PKC⑀ by PKG or the specific PKC⑀ agonist ⑀ 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 PKC⑀, with an IC 50 of 0.4 (؎0.1) M. On the basis of these results, we propose that two different PKC⑀ pools regulate this signaling pathway, one in association with mitoK ATP and the other in association with MPT.A major component of ischemia-reperfusion injury is necrotic cell death, manifested at the organ level as infarction. Necrotic cell death is widely thought to be the consequence of opening the mitochondrial permeability transition (MPT), 2 as proposed originally by Crompton et al.(1) and recently by Di Lisa et al. (2). Strong protection against ischemia-reperfusion injury is afforded by a brief preliminary ischemic period, a phenomenon known as ischemic preconditioning, which was also shown to reduce MPT opening (3, 4). However, it is unclear how MPT opening is prevented when cardioprotection is triggered by ischemic preconditioning or pharmacological mitoK ATP openers such as diazoxide. In this regard, Korge et al. (5) have made important progress. They simulated ischemia in isolated mitochondria with anoxia and then exposed them to elevated Ca 2ϩ and phosphate (P i ). These conditions caused MPT opening and cytochrome c loss, which were blocked by diazoxide or phorbol 12-myristate 13-acetate (PMA). Both effects were negated by 5-hydroxydecanoate (5-HD), indicating that the protective effect of PKC in blocking MPT pore opening operates through mitoK ATP opening. Kim et al. (6) showed that MPT was inhibited by cyclic GMP in the presence of a cytosolic extract, implicating a guanylyl cyclase-dependent signaling pathway. Baines et al. (7) showed that mice with cardiac-specific expression of recombinant PKC⑀ showed inhibition of MPT opening in heart mitochondria and identified a multiprotein complex in mitochondria containing PKC⑀, adenine nucleotide translocator, the vol...

Abstract-Mitochondria are key mediators of the cardioprotective signal and the mitochondrial ATP-sensitive K ϩ channel (mitoK ATP ) plays a crucial role in originating and transmitting that signal. Recently, protein kinase C ⑀ (PKC) has been identified as a component of the mitoK ATP signaling cascade. We hypothesized that PKC and mitoK ATP interact directly to form functional signaling modules in the inner mitochondria membrane. To examine this possibility, we studied K ϩ 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 in the reconstitutively active fraction. Addition of the PKC activators 12-phorbol 13-myristate acetate, hydrogen peroxide, and the specific PKC peptide agonist, RACK, each activated mitoK ATP -dependent K ϩ flux in the reconstituted system. This effect of PKC was prevented by chelerythrine, by the specific PKC peptide antagonist, V 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 and mitoK ATP . (Circ Res. 2006;99:878-883.)Key Words: ATP-sensitive K ϩ channel Ⅲ protein kinase C⑀ Ⅲ protein phosphatase Ⅲ reactive oxygen species Ⅲ reconstitution Ⅲ cardioprotection Ⅲ mitochondria I schemic preconditioning is a powerful method for reducing ischemia/reperfusion injury in the heart, and it is widely considered that opening of the mitochondrial ATP-sensitive K ϩ channel (mitoK ATP ) plays a crucial role in this process. Cardioprotection by ischemic preconditioning can be mimicked by administering G i -coupled surface receptor agonists, such as bradykinin, and there has been progress in understanding the mechanisms by which receptor agonists open mitoK ATP . Recent findings by Oldenburg et al 1 led those authors to conclude that protein kinase G (PKG) is the terminal cytosolic kinase in this pathway, with the next step being opening of mitoK ATP . We then showed that mitoK ATP opening could be induced by adding exogenous PKGϩcGMP to isolated heart mitochondria. 2 We also showed that an endogenous, mitochondrial protein kinase C ⑀ (PKC) is an obligatory intermediate between PKG and mitoK ATP in this process. 2 These findings raise the question of PKC localization in mitochondria. It seemed unlikely that PKC would be required to probe the entire inner membrane surface area to find its substrate, given that mitoK ATP is a very low abundance protein. Accordingly, we hypothesized that these 2 proteins are joined in a complex.To examine this possibility, we studied K ϩ flux in proteoliposomes containing partially purified mitoK ATP . We found that the reconstitutively active fraction contains PKC, as revealed by immunoblot. We observed that addition of the PKC activator...

Coronatine is a phytotoxin produced by some plant-pathogenic bacteria. It has been shown that coronatine mimics the action of methyl jasmonate (MeJA) in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack. In Arabidopsis thaliana, MeJA is essential for pollen grain development. The coi1 (for coronatine-insensitive) mutant of Arabidopsis, which is insensitive to coronatine and MeJA, produces sterile male flowers and shows an altered response to wounding. When the differential display technique was used, a message that was rapidly induced by coronatine in wild-type plants but not in coi1 was identified and the corresponding cDNA was cloned. The coronatine-induced gene ATHCOR1 (for A. thaliana coronatineinduced) is expressed in seedlings, mature leaves, flowers, and siliques but was not detected in roots. The expression of this gene was dramatically reduced in coi1 plants, indicating that COI1 affects its expression. ATHCOR1 was rapidly induced by MeJA and wounding in wild-type plants. The sequence of ATHCOR1 shows no strong homology to known proteins. However, the predicted polypeptide contains a conserved amino acid sequence present in several bacterial, animal, and plant hydrolases and includes a potential ATP/GTP-binding-site motif (P-loop).

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.

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