Effects of ATP-sensitive potassium channel activators diazoxide and BMS-191095 on membrane potential and reactive oxygen species production in isolated piglet mitochondria.

Mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel openers protect the piglet brain against ischemic stress. Effects of mitoK(ATP) channel agonists on isolated mitochondria, however, have not been directly examined. We investigated the effects of K(ATP) channel openers and blockers on membrane potential and on the production of reactive oxygen species (ROS) in isolated piglet mitochondria. Diazoxide and BMS-191095, putative selective openers of mitoK(ATP), decreased the mitochondrial membrane potential (delta psi(m)). On a molar basis, diazoxide was less effective than BMS-191095. In contrast, diazoxide but not BMS-191095 increased ROS production by mitochondria. Since diazoxide also inhibits succinate dehydrogenase (SDH), we examined the effects of 3-nitropropionic acid (3-NPA), an inhibitor of SDH. 3-NPA failed to change the delta psi(m) but increased ROS production. Inhibitors of K(ATP) channels did not affect resting delta psi(m) or ROS production, but glibenclamide and 5-hydroxydecanoate (5-HD) blocked effects of diazoxide and BMS-191095 on delta psi(m) and diazoxide effects on ROS production. We conclude that BMS-191095 has selective effects on mitoK(ATP) channels while diazoxide also increases ROS production probably via inhibition of SDH.

Diazoxide preconditioning protects against neuronal cell death by attenuation of oxidative stress upon glutamate stimulation.

We examined the effects of diazoxide, the putative mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channel opener, against glutamate excitotoxicity in primary cultures of rat cortical neurons. Cells were treated with diazoxide for 24 hr and then exposed to 200 microM glutamate. Cell viability was measured 24 hr after glutamate exposure. We found that treatment 24 hr before glutamate exposure with 250 and 500 microM diazoxide but not with another mitoK(ATP) channel opener, nicorandil, increased neuronal viability from 54 +/- 2% to 84 +/- 2% and 92 +/- 3%, respectively (n = 25-40). These effects were not inhibited by the putative mitoK(ATP) channel blocker 5-hydroxydecanoic acid. Diazoxide application increased production of reactive oxygen species (ROS) and coapplication of M40401, a superoxide dismutase mimetic, prevented delayed preconditioning. The 24 hr preconditioned neurons showed significantly reduced ROS production upon glutamate stimulation compared to that in untreated cells. These results suggest that diazoxide induces delayed preconditioning in cultured cortical neurons via increased ROS production and attenuation of oxidative stress upon glutamate stimulation.

The mitochondrial K(ATP) channel opener BMS-191095 induces neuronal preconditioning.

BMS-191095, reportedly a selective mitoK(ATP) channel opener which is free from the known side effects of the prototype mitoK(ATP) channel opener diazoxide, induced acute and delayed preconditioning against glutamate excitotoxicity and delayed preconditioning against oxygen-glucose deprivation in primary cultures of rat cortical neurons. BMS-191095 dose dependently depolarized the mitochondria, increased the phosphorylation of PKC isoforms, but had no detectable effects on the activation of MAP kinases and did not influence the expressions of HSP70 and Mn-SOD. In BMS-191095-preconditioned neurons the glutamate-induced free-radical production was abolished. Our data give the first evidence that selective opening of mitoK(ATP) channels with BMS-191095 leads to remarkable neuroprotection via mechanisms that involve mitochondrial depolarization, PKC activation and attenuated free radical production during neuronal stress.

Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons.

We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygen-glucose deprivation (OGD). Diazoxide pre-treatment induced delayed pre-conditioning and almost entirely attenuated the OGD-induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate abolished the protective effect of diazoxide while the non-selective KATP channel blocker glibenclamide did not. The non-selective KATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese-superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxide-pre-conditioned neurons after OGD. In pre-conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3-nitropropionic acid also induced pre-conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre-conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase-inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP-sensitive potassium channels.