mitoKATP Channel Research Articles

Chronic preconditioning: a novel approach for cardiac protection

Ischemic preconditioning is the most powerful protective mechanism known against lethal ischemia. Unfortunately, the protection lasts for only a few hours. Here we tested the hypothesis that the heart can be kept in a preconditioned state for constant protection against ischemia. In this study we chose BMS-191095 (BMS), a highly selective opener of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels. BMS (1 mg/kg ip) was administered to rats every 24 h until 96 h. In other groups, BMS plus wortmannin (WTN, 15 microg/kg ip), an inhibitor of the phosphatidylinositol 3-kinase (PI3-K), or BMS plus 5-hydroxydecanoic acid (5-HD, 5 mg/kg ip), an inhibitor of mitoK(ATP), or BMS plus N(omega)-nitro-L-arginine methyl ester (L-NAME) (30 microg/kg ip), an inhibitor of nitric oxide (NO) synthase, were administered to rats. Rats were then subjected to 30-min left anterior descending coronary artery occlusion and 120-min reperfusion. Cardiac function, infarct size, pathological changes, and apoptosis were assessed at the end of treatments. Saline-treated hearts displayed marked contractile dysfunction and underwent pathological changes. BMS-treated rats showed significant improvement in cardiac function, and infarct size was significantly reduced in BMS-treated hearts. However, protection by BMS was abolished by 5-HD, WTN, or L-NAME. These data demonstrate that hearts can be chronically preconditioned and retain their ability to remain resistant against lethal ischemia and that this protection is mediated by activation of mitoK(ATP) via NO and PI3-K/Akt signaling pathways.

Cardiac ATP-Sensitive Potassium Channels: A Potential Target for an Anti-Ischaemic Pharmacological Strategy

Brief periods of ischaemia induce in the myocardium an increased resistance to the injury due to a subsequent, more prolonged ischaemic episode. This phenomenon, known as ischaemic pre-conditioning (IPC), articulated in two distinct phases (an early and a delayed one), is ensured by different biological mechanisms. Although an exhaustive comprehension of these mechanisms has not yet been reached, it is widely accepted that among the various signals involved as triggers and/or end-effectors, an important role is undoubtedly played by the activation of cardiac ATP-sensitive potassium channels (K(ATP)). In the myocardial cells, K(ATP) channels have been identified both in the sarcolemmal membrane (sarc-K(ATP)) and in the mitochondrial inner membrane (mito-K(ATP)). Although many experimental findings suggest that a role of sarc-K(ATP) channel activation in IPC cannot be excluded, in the last few years, many authors have indicated that this phenomenon could be attributed to the exclusive (or at least prevalent) activation of the mito-K(ATP) channels. Conversely, drugs modulating the K(ATP) channels (as activators or blockers), on one hand, have been employed as useful experimental tools for basic studies on IPC. On the other hand, K(ATP)-openers have been viewed as promising possible therapeutic agents for limiting the myocardial injury due to ischaemic episodes. In particular, those molecules exhibiting a good degree of selectivity towards the mito-K(ATP) channels have been indicated as potential anti-ischaemic cardio-protective pharmacological tools, devoid of other biological effects (such as negative inotropic activity, hypotension or hyperglycaemia) linked to the activation of cardiac and non-cardiac sarcK(ATP) channels. In this paper, we wish to report the experimental evidence supporting the role of sarc- and mito-K(ATP) channels in IPC, the relative signalling pathways potentially involved in the mechanisms of cardio-protection and, finally, an overview of the most important molecules acting as activators or blockers of K(ATP) channels, with their selectivity profiles.

Testosterone confers cardioprotective effect on rat heart via mitochondrial ATP‐senstitive potassium channel

In this study, we examined whether testosterone might improve myocardial tolerance to ischemia via activating mitochondrial KATP channels (mitoKATP) and depressing mitochondrial permeability transition pore (mitoPTP). Sprague-Dawley rats were approximately eight weeks old at the time of surgery. For the gonadectomized group, the vas deferens were ligated bilaterally and the testes removed. The replacement of testosterone propionate (200 g/mg/d) began 1 week after gonadectomy. Eight weeks later, in isolated hearts subjected to regional ischemia, testosterone treatment improved the recovery of rate-pressure product and coronary flow during reperfusion, and reduced the infarct size and release of lactate dehydrogenase. Administration of 20 μmol/L atractyloside, a mitoPTP opener, for the last 5 min of ischemia and first 15 min of reperfusion, or pretreatment with 100 μmol/L 5-HD, an inhibitor of the mitoKATP channel, for 10 min before ischemia, attenuated the beneficial effect by testosterone. In cultured cardiomyocytes pretreated with 10 μmol/L testosterone for 20 min, a significant inhibition of H2O2-induced cell death was observed, while 5- HD attenuated the inhibition. These findings indicate that testosterone protects the myocardium against ischemia and reperfusion injury by inhibiting mitochondrial permeability transition pore opening as well as activating mitoKATP channels.

Mitochondrial contribution to the molecular mechanism of heart acclimatization to chronic hypoxia: role of nitric oxide

A remarkable number of adaptive responses; including changes in the cardiovascular, respiratory and hematologic systems; takes place during acclimatization to natural or simulated high altitude. This adaptation to chronic hypoxia confers the heart an improved tolerance to all major deleterious consequences of acute O2 deprivation, not only reducing infarct size but also alleviating post-ischemic contractile dysfunction and ventricular arrhythmias. There is growing evidence about the involvement of mitochondria and NO in the establishment of cardioprotection. This review focuses on evidence about the putative role of different effectors of heart acclimatization to chronic hypoxia. Along with classical parameters, we consider NO, specially that generated by mtNOS, mitochondrial respiratory chain, mitoK(ATP) channels, reactive oxygen species and control of gene expression by HIF-1.

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 confers cardioprotection. Our aim was to investigate whether BEP could induce preconditioning in cultured rat cortical neurons. Although BEP depolarized mitochondria and increased free radical generation, no acute protection was observed. However, a 3-day BEP treatment elicited dose-dependent delayed neuroprotection against 180 min of oxygen-glucose deprivation (cell viability: untreated, 52.5±0.85%; BEP 1 μM, 59.6±1.53%∗; BEP 2.5 μM, 71.9±1.23%∗; BEP 5 μM, 95.3±0.89%∗; mean ± SEM; ∗p<0.001 vs. untreated) and 60 min of glutamate excitotoxicity (200 μM; cell viability: untreated, 54.1±0.69%; BEP 1 μM, 61.2±1.19%∗; BEP 2.5 μM, 78.1±1.67%∗; BEP 5 μM, 91.2±1.20%∗; mean ± SEM; ∗p<0.001 vs. untreated). The protection was antagonized with the superoxide dismutase mimetic M40401, but not with glutathione, catalase, or the mitoKATP blocker 5-hydroxydecanoate. BEP treatment resulted in increased levels and activity of protein kinase C, manganese-dependent superoxide dismutase, catalase, 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. Supported by NIH grants (HL-50587, HL-65380, and HL-77731) for D. W. Busija.

Oxytocin preconditions rabbit heart against infarction via activation of mitochondrial ATP-sensitive K+ channels

Oxytocin (OXY) is a nonapeptide hormone and classically related to reproductive functions. Recently, increasing evidence supports the role of OXY as a regulator of cardiovascular function. However, the cardiac effects of OXY have not been much studied. OXY receptors have been found in the heart and activate protein kinase C (PKC). PKC has been shown to modulate ATP-sensitive K+ channels in cardiac mitochondria (mitoKATP channels), a key element of cardioprotection. Therefore, we assessed the hypothesis that OXY confers cardioprotection by opening of mitoKATP channels.

Signaling Mechanisms in Ischemic Preconditioning

See related article, pages 878–883 The cardiac “warm up” phenomenon, described more than 50 years ago in patients with coronary artery disease, refers to improvement in cardiac symptoms and physical performance following exposure to short periods of ischemia.1 Several mechanisms, such as adaptive reduction in oxygen consumption by the ischemic myocardial region, improved oxygen supply via collateral recruitment or dilation of the stenotic vessel, and activation of an intrinsic phenomenon called ischemic preconditioning (IPC) have been proposed to account for this phenomenon. IPC refers to a process in which brief periods of ischemia improves the ability of the heart to tolerate subsequent prolonged ischemic periods.3 It was first identified in the heart in 1986 by Murry et al,2 and has since been demonstrated in various experimental and animal models.3 Several triggers have been proposed for IPC, including adenosine, bradykinin, protaglandins, opiod receptors, nitric oxide, and Ca2+.4 These triggers lead to the activation of several intracellular pathways that ultimately protect myocardial cells against injury. Although the details of these pathways have not been totally characterized, mitochondria have been shown to be key mediators of IPC. Specifically, the opening of a mitochondrial channel, called the mitochondrial ATP-sensitive potassium channel or mitoKATP is believed to be critical for the induction of IPC; drugs that activate this channel protect against ischemia and inhibitors of mitoKATP reverse these protective effects.5 The signaling pathways that lead to the activation of mitoKATP are still under investigation.5 In a recent article, Oldenburg et al demonstrated that in isolated rabbit adult cardiomyocytes, bradykinin increased the levels of reactive oxygen species (ROS) and this effect was reversed by inhibitors of both mitoK …

Iptakalim protects PC12 cell against H 2O 2-induced oxidative injury via opening mitochondrial ATP-sensitive potassium channel

The final common pathway in the demise of dopaminergic neurons in Parkinson’s disease may involve oxidative stress and excitotoxicity. In this study, we examined the neuroprotective effects of a novel ATP-sensitive potassium channel (K ATP) opener, iptakalim (IPT), against H 2O 2-induced cytotoxicity in rat dopaminergic PC12 cells. Pretreatment with IPT could attenuate increased extracellular glutamate levels and inhibit calcium influxing induced by H 2O 2. Moreover, IPT regulated the expressions of bcl-2 and bax which were responsible for inhibiting apoptosis in PC12 cells. These protective effects of IPT were abolished by selective mitoK ATP channel blocker 5-hydroxydecanoate. Therefore, IPT can protect PC12 cells against H 2O 2-induced oxidative injury via activating mitoK ATP channel.

Mechanism of reactive oxygen species generation after opening of mitochondrial KATP channels

opening of the ATP-sensitive K+ (KATP) channel mediates the cardioprotective effect induced by pathophysiological stressors such as ischemic preconditioning (IPC) ([15][1], [29][2]), heat shock ([19][3]), and pharmacological agents, including adenosine ([5][4]), ACh ([26][5]), opioids ([12][6]),

Diazoxide-induced cardioprotection via ΔΨm loss depending on timing of application

Although the role of mitochondrial ATP-sensitive potassium (mitoK ATP) channels in cardioprotection is widely accepted, it remains unclear when their opening is critical for protection. We tested the hypothesis that the mitoK ATP channel acts as a trigger or mediator of protection against apoptosis through loss of mitochondrial inner membrane potential (ΔΨm). Exposure of neonatal rat cardiomyocytes to H 2O 2 (0.5 mmol/L) resulted in apoptosis associated with severe ΔΨm loss. Pretreatment with diazoxide (20 to 100 μmol/L) prevented H 2O 2-induced apoptosis and ΔΨm loss at 2 but not 18 h after exposure, while the latter was prevented by cotreatment with diazoxide. Lack of protection by pretreatment with diazoxide was observed in cardiomyocytes cultured in a medium containing H 2O 2 for 2 h and then not containing for 16 h. The slopes of the regression lines of the relationship between the proportion of apoptotic cells and ΔΨm loss ( y = − 0.89 vs. − 0.42) and the proportion of cells with high side scatter signal differed between cardiomyocytes exposed H 2O 2 for 2 and 18 h. Diazoxide per se caused a transient ΔΨm loss (within 30 min) with a recovery followed by persistent ΔΨm loss (after 6 h). Inhibition of the former by 5-hydroxydecanoate (5-HD, 0.5 mmol/L) abolished protection of pretreatment with diazoxide (trigger phase), while that of the latter prevented the protection of cotreatment with diazoxide (mediator phase). Our results suggest that mitoK ATP channels act as a trigger and mediator of cardioprotection through a transient or persistent ΔΨm loss depending on phenotypic consequence in response to oxidants.

A29. Nicorandil opens mitochondrial KATP channels both directly and through a NO-PKG-dependent pathway

Nicorandil, a hybrid nitrate and potassium channel opener, protects ischemic myocardium by opening mitochondrial ATP-sensitive potassium (mitoKATP) channels. We previously reported that nitric oxide (NO) also opened KATP channels in rabbit hearts by a protein kinase G (PKG) mechanism. This study examined whether the NO-donor property of nicorandil also contributes to opening mitoKATP channels through PKG. MitoKATP channel opening was monitored in adult rabbit cardiomyocytes by measuring reactive oxygen species (ROS) production with reduced MitoTracker Red. Nicorandil increased ROS production in a dose-dependent manner. The selective mitoKATP channel inhibitor 5-hydroxydecanoate (200 μM) completely blocked ROS production by nicorandil at all doses. The PKG inhibitor 8-bromoguanosine-3′, 5′-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-cGMPs, 50 μM) shifted the dose-ROS production curve rightward with an increase of the EC50 from 2.4 × 10−5 M to 6.9 × 10−5 M. Rp-8-Br-cGMPs did not affect the increase in ROS production by the selective mitoKATP channel opener diazoxide, whereas it completely blocked increased ROS production from the NO donor S-nitroso-N-acetylpenicillamine (1 μM). These results indicate that nicorandil does open mitoKATP channels indirectly via a NO-PKG signaling pathway in addition to its direct effect on the channels.

Cardiac protection by mitoKATP channels is dependent on Akt translocation from cytosol to mitochondria during late preconditioning

This investigation elucidates the Akt/mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel signaling pathway in late pharmacological preconditioning, using the mitoK(ATP) channel openers BMS-191095 (BMS) and diazoxide (DE). BMS (1 mg/kg ip) and DE (7 mg/kg ip) alone or BMS plus wortmannin (WTN, 15 microg/kg ip), an inhibitor of phosphatidylinositol 3-kinase, and BMS plus 5-hydroxydecanoic acid (5-HD, 5 mg/kg ip), an inhibitor of mitoK(ATP) channels, were administered to male mice. Twenty-four hours later, hearts were isolated and subjected to 40 min of ischemia and 120 min of reperfusion via Langendorff's apparatus. Both BMS and DE reduced left ventricular end-diastolic pressure and increased left ventricular developed pressure as well as reduced LDH release. Coadministration of BMS and WTN abolished the beneficial effects of BMS on cardiac function. Moreover, BMS and DE accelerated Akt phosphorylation in cardiac tissue as determined by Western blot analysis and also significantly reduced apoptosis compared with ischemic control. WTN significantly suppressed BMS-induced Akt phosphorylation, whereas 5-HD had no effect on Akt phosphorylation in cytosol, and the effect of BMS on apoptosis was abolished. It is concluded that the cardioprotective effect by mitoK(ATP) channels is attributed to the translocation of phosphorylated Akt from cytosol to mitochondria.

A41. Differential Age and Gender Response to Ischemia: Efficacy of cardioprotection and the opening of mitoKATP channels

A41. Differential Age and Gender Response to Ischemia: Efficacy of cardioprotection and the opening of mitoKATP channels

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

Activation of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels protects the brain against ischemic or chemical challenge. Unfortunately, the prototype mitoK(ATP) channel opener, diazoxide, has mitoK(ATP) channel-independent actions. We examined the effects of BMS-191095, a novel selective mitoK(ATP) 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 microg; estimated brain concentration of 40 micromol/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 mitoK(ATP) channel antagonist. In cultured neurons, BMS-191095 (40 micromol/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 mitoK(ATP) channels and without ROS generation.

Mitochondrial ATP-sensitive potassium channels and mitochondrial protein kinase C: sometimes it's good to have a close neighbor

the fine study by Jiang et al. ([8][1]) in this issue of the American Journal of Physiology Heart and Circulatory Physiology is an important milestone in the ongoing studies on the role of the mitochondrial ATP-sensitive K+ (mitoKATP) channels in myocardial ischemia. It is not particularly

The role of ATP sensitive K + channels and of nitric oxide synthase on myocardial ischemia/reperfusion-induced apoptosis

During ischemia, ATP-sensitive K+ channels (KATP channels) open, and this triggers necrotic processes and apoptosis. In this study, we investigated whether selective sarcoplasmic and mitochondrial KATP channel blockers affected myocardial apoptosis and nitric oxide synthase (NOS) activity in a rat model of myocardial ischemia/reperfusion in vitro. Isolated rat hearts were subjected to 30 min of coronary artery occlusion followed by 30 min of reperfusion. A selective sarcKATP channel blocker, HMR1098 and a selective mitoKATP channel blocker, 5-hydroxydecanoate, were added to the perfusion fluid 10 min before occlusion. Myocardial apoptosis was detected immunohistochemically using the TUNEL method. Myocardial inducible NOS (iNOS) and endothelial NOS (eNOS) were determined immunohistochemically. In control hearts, apoptosis induction was associated with a greater immunoreactivity of iNOS than eNOS. Treatment with HMR1098, at a concentration of 3 micromol/l, significantly reduced the TUNEL-positive cardiomyocytes and this was associated with decreased iNOS and increased eNOS immunoreactivity. When this drug was administered at a higher concentration, at 30 micromol/l, a more marked reduction in apoptosis was observed but, in contrast to the effects observed at the lower drug concentration, eNOS immunoreactivity was almost completely abolished while iNOS was strong. Moreover, ischemia-induced cardiac dysfunction (e.g. contractile force and recovery of coronary flow) was increased by the higher concentration of HMR 1098. In hearts treated with 5-hydroxydecanoate, myocyte apoptosis was slightly reduced, and this was associated with an almost equal increase in both iNOS and eNOS immunoreactivity. These findings suggest that iNOS appears to be more important than eNOS in the reduction of apoptosis. However, the further inhibition of apoptosis by the higher concentration of HMR 1098 was associated with poorer cardiac function.

MitoKATP channel activation suppresses gap junction permeability in the ischemic myocardium by an ERK-dependent mechanism

Ischemic preconditioning accelerates suppression of gap junction (GJ) permeability during myocardial ischemia, and GJ blockers reduce infarct size. We hypothesized that the mitochondrial ATP-sensitive K+ (mitoKATP) channel is one of the mechanisms regulating GJ permeability through the mitogen-activated protein kinase ERK, leading to cardioprotection. In isolated rabbit hearts, tissues were sampled before and after infusion of diazoxide, a selective mitoKATP channel opener, and their intercalated disc-rich fractions were obtained for immunoblotting of mitogen-activated protein kinases. GJ permeability in the myocardium was assessed by using Lucifer yellow as a tracer of GJ communication. Infarction was induced by 30-min global ischemia/2 h reperfusion, and infarct size was expressed as a percent of area-at-risk (%IS/AR). Diazoxide (100 microM) induced phosphorylation of ERK1/2 and 279Ser/282Ser of connexin-43, a GJ subunit protein, and phospho-ERK1/2 was co-immunoprecipitated with connexin-43 in the diazoxide-treated myocardium. This ERK1/2 phosphorylation by diazoxide was inhibited by N-2-mercaptopropionyl-glycine, a free radical scavenger. Diazoxide at 10 and 100 microM reduced intercellular transport of Lucifer yellow during ischemia by 44% and 69%, respectively, and this effect of diazoxide on GJ communication was abolished by PD98059, an ERK inhibitor. Pretreatment with 10 microM and 100 microM diazoxide reduced %IS/AR from 57.1+/-3.7% to 21.5+/-10.5% and 5.0+/-1.3%, respectively. PD98059 abolished cardioprotection by 10 microM diazoxide but not that by 100 microM diazoxide. Opening of the mitoKATP channel activates ERK1/2 via free radicals and induces ERK-mediated suppression of GJ permeability. This suppression of GJ permeability may partly contribute to cardioprotection afforded by mitoKATP channel activation.

Activation of PKC‐ε occurs upstream of mitoK ATP channel activation in the mechanism of diazoxide‐induced cardioprotection

In this study, we examined which isoforms of protein kinase C (PKC) family are modulated by diazoxide (DZX) and evaluated their roles in hypoxia-induced apoptotic cell death mediated via mitochondrial death pathway. We further investigated the interrelationship between PKC isoforms and the mitochondrial ATP-sensitive potassium (mitoKATP) channel in the mechanism of cardioprotection by DZX in rat heart-derived H9c2 cell lines and neonatal rat cardiomyocytes. Treatment with 100 μM DZX induced isoform-specific translocation of PKC-ε from the cytosolic to the mitochondrial fraction in both H9c2 and neonatal cardiomyocytes. A specific blockade of PKC-ε translocation by εV1-2 peptide abrogated the anti-apoptotic effect of DZX, suggesting an essential role of PKC-ε in cardioprotection. DZX-induced flavoprotein oxidation was completely inhibited by 10 μM εV1-2, and DZX-induced translocation of PKC-ε remained unaltered after treatment with 100 μM 5-hydroxydecanoate (5-HD). A direct PKC activation by phorbol ester mimicked flavoprotein-oxidizing effect of DZX, and this effect was completely blocked by εV1-2 or 5-HD. During hypoxia, the treatment with 100 μM DZX prevented the increase in mitochondrial Ca2+, mitochondrial depolarization and cytochrome c release induced by hypoxia, and all these effects of DZX were almost completely blocked by εV1-2 as well as 5-HD. In summary, these results suggest that DZX induces translocation of PKC-ε as an upstream signaling molecule for the mitoKATP channel, leading to cardioprotection through inhibition of mitochondrial death pathway.

The mitochondrial K ATP channel opener BMS‐191095 reduces neuronal damage after transient focal cerebral ischemia in rats

In our recent study, BMS-191095, a newly developed selective mitoKATP channel opener induced preconditioning in primary cultures of rat cortical neurons (B. Kis et al. NeuroReport 15:345–349, 2004). However whether BMS-191095 protects the brain against ischemic stress has not been studied. We investigated the effect of BMS-191095 against 90 min transient middle cerebral artery occlusion (MCAO) in male Wistar rats. BMS-191095 was infused into the left lateral ventricle 30 min (0.4 mM, n=11, or 4 mM, n=6), 60 min (4 mM, n=7) and 24 hr (4 mM, n=12, in 15 μL dimethyl sulfoxide) before MCAO. Infarct volumes were measured 72 hr after reperfusion. Treatment with BMS-191095 24 hr before MCAO but not 30–60 min exhibited a 29% reduction (206.2 ± 16.5 vs 149.2 ± 18.2 mm3) and a 34% reduction (144.2 ± 15.4 vs 95.2 ± 14.4 mm3) in infarct volumes for total brain and cortex, respectively. The protective effect of BMS-191095 was completely prevented by the pretreatment with 5-hydroxydecanoate (5-HD), a selective blocker of mitoKATP channels (40mg/kg IP; n=8). Intraventricular administration of BMS-191095 did not changed local cerebral blood flow measured with laser-Doppler flowmetry. BMS-191095 depolarized the mitochondrial membrane potential without increasing the ROS production in cultured rat cortical neurons, which was dose dependently inhibited by 5-HD. These results suggest that opening of mitoKATP channels exhibits delayed preconditioning in the rat brain and this effect is dissociated from ROS production. Supported by NIH grants (HL-30260, HL-46558, HL-50587, DK-62372) for D. W. Busija.

Delayed preconditioning with BMS‐191095 increases the expression of catalase in cultured rat cortical neurons

Selective opening of the mitochondrial adenosine triphosphate sensitive potassium (mitoKATP) channels with BMS-191095 is an effective way to induce tolerance against ischemia and other lethal stimuli. Previous works from our laboratory demonstrated that glutamate-induced free radical production was completely abolished in BMS-191095 preconditioned neurons, suggesting that improved free radical scavenging capacity plays an important role in the neuroprotective effect of BMS-191095. The objective of our present in vitro study was to identify potential effectors of BMS-191095 induced neuroprotection. Cortical neurons of 18-day old Spraque-Dawley rat fetuses were cultured in B27 supplemented Neurobasal medium. After 14 days in vitro the neuronal cultures were treated with BMS-191095 (50 μM) once a day for 1, 2, or 3 days, then the cells were harvested, and protein was isolated. The expression of catalase, manganese dependent superoxide dismutase (Mn-SOD), Bcl-2, and heat shock protein 70 (HsP-70) was analyzed using Western blotting. Treatment with BMS-191095 resulted in a dose-dependent elevation of catalase expression, whereas the protein level of Mn-SOD, Bcl-2, and HsP-70 did not change. Our results indicate that catalase may play a role in the improved free radical scavenging capacity of neurons after treatment with the mitoKATP opener BMS-191095. Supported by NIH grants (HL-50587, HL-65380, HL-77731) for D. W. Busija.