Keita Mayanagi

The Mitochondrial KATP Channel Opener BMS-191095 Reduces Neuronal Damage after Transient Focal Cerebral Ischemia in Rats

Activation of mitochondrial ATP-sensitive potassium (mitoKATP) channels protects the brain against ischemic or chemical challenge. Unfortunately, the prototype mitoKATP channel opener, diazoxide, has mitoKATP channel-independent actions. We examined the effects of BMS-191095, a novel selective mitoKATP channel opener, on transient ischemia induced by middle cerebral artery occlusion (MCAO) in rats. Male Wister rats were subjected to 90 mins of MCAO. BMS-191095 (25 μg; estimated brain concentration of 40 μmol/L) or vehicle was infused intraventricularly before the onset of ischemia. In addition, the effects of BMS-191095 on plasma and mitochondrial membrane potentials and reactive oxygen species (ROS) production in cultured neurons were examined. Finally, we determined the effects of BMS-191095 on cerebral blood flow (CBF) and potassium currents in cerebrovascular myocytes. Treatment with BMS-191095 24 h before the onset of ischemia reduced total infarct volume by 32% and cortical infarct volume by 38%. However, BMS-191095 administered 30 or 60 mins before MCAO had no effect. The protective effects of BMS-191095 were prevented by co-treatment with 5-hydroxydecanoate (5-HD), a mitoKATP channel antagonist. In cultured neurons, BMS-191095 (40μmol/L) depolarized the mitochondria without affecting ROS levels, and this effect was inhibited by 5-HD. BMS-191095, similar to the vehicle, caused an unexplained but modest reduction in the CBF. Importantly, BMS-191095 did not affect either the potassium currents in cerebrovascular myocytes or the plasma membrane potential of neurons. Thus, BMS-191095 afforded protection against cerebral ischemia by delayed preconditioning via selective opening of mitoKATP channels and without ROS generation.

Acute treatment with rosuvastatin protects insulin resistant (C57BL/6J ob/ob) mice against transient cerebral ischemia

The purpose of this study was to investigate the short-term effects of rosuvastatin (RSV), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on transient, focal cerebral ischemia in C57BL/6J ob/ob mice with insulin resistance (IR). Male ob/ob, lean, or wild-type (WT) mice were treated with RSV (10 mg/kg per day, i.p.) or vehicle for 3 days. Ischemia was induced by 60 mins of middle cerebral artery occlusion (MCAO) and cortical blood flow (CBF) was monitored by laser-Doppler flowmetry. Infarct volumes were measured 24 h after reperfusion. IR mice exhibited a higher infarct volume compared with Lean or WT mice, and RSV reduced infarct volume only in obese mice (40% ± 3% versus 32% ± 3%, P < 0.05). Blood cholesterol and insulin levels were elevated in ob/ob mice but were unaffected by RSV. The CBF reductions during MCAO were similar in all groups and were not affected by RSV. Although RSV did not increase cortical endothelial NO synthase (eNOS) levels in the ob/ob mice, it attenuated the increased cortical expression of intracellular adhesion molecule-1 (ICAM-1) after MCAO from ob/ob mice. Thus, RSV protects against stroke in IR mice by a mechanism independent of effects on the lipid profile, CBF, or eNOS but dependent on suppression of post-MCAO ICAM-1 expression.

Transient glucose and amino acid deprivation induces delayed preconditioning in cultured rat cortical neurons

Several studies have demonstrated that glucose deprivation, combined either with anoxia or with the inhibition of oxidative phosphorylation, leads to the development of ischemic tolerance in neurons. The aim of our experiments was to investigate whether similar effects could be achieved by transient energy deprivation without either anoxia or the inhibition of the electron transfer chain. Preconditioning was carried out by incubating primary rat cortical neuronal cultures for 3, 6 or 9u2003h in a glucose‐ and amino acid‐free balanced salt solution supplemented with B27 in normoxic conditions. After 24u2003h, neuronal cultures were exposed to oxygen–glucose deprivation, glutamate or hydrogen peroxide. Cell viability was measured 24u2003h after the lethal insults. Potential mechanisms that can influence free radical production were also examined. Energy deprivation protected neuronal cells against lethal stimuli (e.g. cell survival after oxygen–glucose deprivation was 33.1u2003±u20030.52% in the untreated group and 80.1u2003±u20031.27% in the 9‐h energy deprivation group), reduced mitochondrial membrane potential, decreased free radical formation, attenuated the intracellular free calcium surge upon glutamate receptor stimulation, and resulted in an elevated level of GSH. Our findings show that transient energy deprivation induces delayed preconditioning and prevents oxidative injuries and neuronal cell death.

Systemic administration of diazoxide induces delayed preconditioning against transient focal cerebral ischemia in rats

Diazoxide is the prototypical opener of mitochondrial ATP-sensitive potassium channels (mitoK(ATP)) and protects neurons in vivo and in vitro against chemical and anoxic stresses. While we have previously shown that diazoxide administration induces acute preconditioning against transient cerebral ischemia in rats, the potential for delayed preconditioning of diazoxide has not been examined. The purpose of this study was to determine whether diazoxide promotes delayed preconditioning following 90 min of middle cerebral artery occlusion (MCAO) in male Wistar rats. Diazoxide (10 mg/kg) or vehicle was injected intraperitoneally 24 h before MCAO. Infarct volumes were measured 72 h after reperfusion. In animals anesthetized with halothane, treatment with diazoxide exhibited a 35% reduction (48.3+/-3.0% to 31.3+/-4.8%) and 18% reduction (35.1+/-2.2% to 28.9+/-2.1%) in cortical and subcortical infarct volumes, respectively. Administration of the mitoK(ATP) blocker 5-hydroxydecanoate attenuated this beneficial effect. In contrast, diazoxide did not induce delayed preconditioning in isoflurane-anesthetized rats. These findings support the concept that diazoxide produces delayed preconditioning via mitoK(ATP) activation but that physiological status can affect induction of preconditioning.

Neuronal preconditioning with the antianginal drug, bepridil

It has recently been shown that the antianginal drug bepridil (BEP) activates mitochondrial ATP‐sensitive potassium (mitoKATP) channels and thus confers cardioprotection. Our aim was to investigate whether BEP could induce preconditioning in cultured rat cortical neurons. Although BEP depolarized isolated and in situ mitochondria and increased reactive oxygen species generation, no acute protection was observed. However, a 3‐day BEP‐treatment elicited dose‐dependent delayed neuroprotection against 180u2003min of oxygen–glucose deprivation (cell viability: untreated, 52.5u2003± 0.85%; BEP 1u2003μmol/L, 59.6u2003±u20031.53%*; BEP 2.5u2003μmol/L, 71.9u2003±u20031.23%*; BEP 5u2003μmol/L, 95.3u2003±u20030.89%*; meanu2003±u2003SEM; *pu2003<u20030.05 vs. untreated) and 60u2003min of glutamate excitotoxicity (200u2003μmol/L; cell viability: untreated, 54.1u2003±u20030.69%; BEP 1u2003μmol/L, 61.2u2003±u20031.19%*; BEP 2.5u2003μmol/L, 78.1u2003±u20031.67%*; BEP 5u2003μmol/L, 91.2u2003±u20031.20%*; meanu2003±u2003SEM; *pu2003<u20030.05 vs. untreated), and inhibited the reactive oxygen species surge upon glutamate exposure. The protection was antagonized with co‐application of the superoxide dismutase mimetic M40401, but not with reduced glutathione, catalase, or with the mitoKATP blocker 5‐hydroxydecanoate. Furthermore, BEP treatment resulted in increased levels of phosphorylated protein kinase C, manganese‐dependent superoxide dismutase, glutathione peroxidase, and Bcl‐2. Our results indicate that BEP induces delayed neuronal preconditioning which is dependent on superoxide generation but perhaps not on direct mitoKATP activation.