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...

1 Previous studies have shown that¯upirtine, a centrally acting, non-opioid analgesic agent, also exhibits neuroprotective activity in focal cerebral ischaemia in mice and reduces apoptosis induced by NMDA, gp 120 of HIV, prior protein fragment or lead acetate as well as necrosis induced by glutamate or NMDA in cell culture. To study the potential mechanism of the neuroprotective action of¯upirtine, we investigated whether¯upirtine is able to modulate potassium or NMDA-induced currents in rat cultured hippocampal neurones by use of the whole-cell con®guration of the patch-clamp technique. 2 We demonstrated that 1 mM¯upirtine activated an inwardly rectifying potassium current (K ir ) in hippocampal neurones (DI=739+18 pA at 7130 mV; n=10). This e ect was dose-dependent (EC 50 =0.6 mM). The reversal potential for K ir was in agreement with the potassium equilibrium potential predicted from the Nernst equation showing that K ir was predominantly carried by K + . Furthermore, the induced current was blocked completely by Ba 2+ (1 mM), an e ect typical for K ir . 3 The activation of K ir by¯upirtine was largely prevented by pretreatment of the cells with pertussis toxin (PTX) indicating the involvement of a PTX-sensitive G-protein in the transduction mechanism (DI=73+6 pA at 7130 mV; n=8). Inclusion of cyclic AMP in the intracellular solution completely abolished the activation of K ir (n=7). 4 The selective a 2 -adrenoceptor antagonist SKF-86466 (10 mM), the selective 5-HT 1A antagonist NAN 190 as well as the selective GABA B antagonist 2-hydroxysaclofen (10 mM) failed to block the¯upirtine e ect on the inward recti®er. 5 Flupirtine (1 mM) could not change the current induced by 50 mM NMDA. 6 These results show that in cultured hippocampal neurones¯upirtine activates an inwardly rectifying potassium current and that a PTX-sensitive G-protein is involved in the transduction mechanism.

Mitochondrial Ca2+ controls numerous cell functions, such as energy metabolism, reactive oxygen species generation, spatiotemporal dynamics of Ca2+ signaling, cell growth and death in various cell types including neurons. Mitochondrial Ca2+ accumulation is mainly mediated by the mitochondrial Ca2+ uniporter (MCU), but recent reports also indicate that mitochondrial Ca2+-influx mechanisms are regulated not only by MCU, but also by multiple channels/transporters. We previously reported that ryanodine receptor (RyR), which is a one of the main Ca2+-release channels at endoplasmic/sarcoplasmic reticulum (SR/ER) in excitable cells, is expressed at the mitochondrial inner membrane (IMM) and serves as a part of the Ca2+ uptake mechanism in cardiomyocytes. Although RyR is also expressed in neuronal cells and works as a Ca2+-release channel at ER, it has not been well investigated whether neuronal mitochondria possess RyR and, if so, whether this mitochondrial RyR has physiological functions in neuronal cells. Here we show that neuronal mitochondria express RyR at IMM and accumulate Ca2+ through this channel in response to cytosolic Ca2+ elevation, which is similar to what we observed in another excitable cell-type, cardiomyocytes. In addition, the RyR blockers dantrolene or ryanodine significantly inhibits mitochondrial Ca2+ uptake in permeabilized striatal neurons. Taken together, we identify RyR as an additional mitochondrial Ca2+ uptake mechanism in response to the elevation of [Ca2+]c in neurons, suggesting that this channel may play a critical role in mitochondrial Ca2+-mediated functions such as energy metabolism.