Mitochondrial ATP-Sensitive Potassium Channel: A Novel Site for Neuroprotection

Abstract

purpose. It has been shown that bradykinin (BK) protects retinal neurons against glutamate excitotoxicity, but it was not clear how BK inhibits glutamate excitotoxicity. The purpose of this study was to investigate the effect of opening the mitochondrial adenosine triphosphate (ATP)–sensitive potassium (Mit K (ATP)) channel on glutamate excitotoxicity and the protective effect of BK using cultured retinal neurons.

methods. Primary cultures were obtained from the retina of fetal rats (gestation days 17–19). Glutamate neurotoxicity was assessed by 10-minute exposure to 1 mM glutamate followed by 1-hour incubation in glutamate-free medium, using the trypan blue exclusion method. BK, diazoxide (the opener of the Mit K (ATP) channel), 5HD, and glibenclamide (blockers of the Mit K (ATP) channel) were applied simultaneously with glutamate. Mitochondrial membrane potential was measured as the ratio of 590:527 nm fluorescence of JC-1.

results. Cell viability was markedly reduced by 10-minute exposure to 1 mM glutamate followed by 1-hour incubation in glutamate-free medium, and glutamate induced mitochondrial depolarization of retinal neurons. BK and diazoxide protected retinal neurons against glutamate excitotoxicity and inhibited glutamate-induced mitochondrial depolarization. These actions of BK and diazoxide were inhibited by the coapplication of 5HD and glibenclamide. Furthermore, diazoxide inhibited the sodium nitroprusside (SNP, NO donor) toxicity, but did not inhibit the 3-morpholinosydnonimine (SIN-1, NO, and superoxide donor) toxicity.

conclusions. These results suggest that BK and diazoxide protect retinal neurons against glutamate excitotoxicity by opening the Mit K (ATP) channel. It is suggested that opening of the Mit K (ATP) channel inhibited glutamate-induced generation of superoxide.

Glutamate is the primary excitatory neurotransmitter in the retina. However, excessive overactivation of glutamate receptors leads to excitotoxic neuronal cell death. Previous in vivo pathophysiological studies have shown that the selective vulnerability of inner retinal layers to ischemic injury is due to the massive release of glutamate and subsequent activation of glutamate receptors. ¹ Among all, the N-methyl d-aspartate (NMDA) subtype glutamate receptor has been demonstrated to play a central role in the glutamate neurotoxicity that is responsible for ischemic injury in the retina. ² Compelling evidence from in vitro studies indicates that excessive intracellular Ca²⁺ influx, mediated predominantly by NMDA receptors, triggers glutamate-induced neuronal death. ³ For example, according to the temporal profile of Ca²⁺ images obtained from rat retinal cultures, the intracellular Ca²⁺ level was increased immediately after a brief exposure to glutamate. ⁴ Retinal neurons survive when challenged by NMDA in the absence of extracellular Ca^{2+ 5 6} or in the presence of MK-801, ² a selective NMDA receptor blocker, or either lomerizine or flunarizine, Ca²⁺ channel blockers. ⁵ Furthermore, we have demonstrated in cultured retinal neurons that Ca²⁺ influx resulting from stimulation of NMDA receptor activates nitric oxide synthase (NOS) and that an excess amount of nitric oxide (NO) produced by NOS mediates glutamate neurotoxicity. ⁷ NO alone has no toxic action on retinal neurons but peroxynitrite formed by the interaction of NO and superoxide (O₂ ^•−) mediates excitotoxicity. NO is freely permeable but O₂ ^•− may pass through membranes only through anion channels. ⁸ Thus, the site of O₂ ^•− formation secondary to NMDA receptor stimulation determines which neurons die ⁹ in our retinal culture. 

Bradykinin (BK) is a 9-amino-acid peptide with a wide range of biological actions, of which are mediated through at least two subtypes of receptors, B1 and B2. We found that BK-B2 receptors were abundantly distributed in rat retinal neurons and that BK acting at the BK-B2 receptor protects retinal neurons against glutamate neurotoxicity. ¹⁰ BK-induced protection against glutamate neurotoxicity is considered to occur downstream to NO generation and upstream to O₂ ^•− formation. Although BK is widely used to increase intracellular Ca²⁺ concentration, ¹¹ BK protects retinal neurons against glutamate and even inhibits Ca²⁺ ionophore-induced cell death. Recently, it has become apparent that a large increase in intracellular Ca²⁺ concentration after Ca²⁺ influx is not in itself the primary determinant of subsequent cell death ^{12 13 14} and that mitochondrial Ca²⁺ uptake is necessary to trigger glutamate excitotoxicity. ^{15 16 17 18} Mitochondrial Ca²⁺ accumulation after activation of NMDA receptor causes an immediate mitochondrial membrane depolarization in cultured brain neurons ^{19 20 21 22} and further results in production of reactive oxygen species Ca²⁺ dependently in various cultured neurons. ^{21 23 24 25} Activation of BK-B2 receptors is thus suggested to inhibit the formation of O₂ ^•− in mitochondria after glutamate exposure. 

The mitochondrial ATP-sensitive potassium (Mit K (ATP)) channel is a potassium channel in the inner mitochondrial membrane that is inactivated by ATP. Cardioprotective action of BK is now considered to be mediated by opening the Mit K (ATP) channel. Mit K (ATP) channel has been extensively studied in the heart over the past 5 years. Recently, Bajgar et al. ²⁶ have identified Mit K (ATP) channel in rat brain cortical neurons and found that brain mitochondria contained seven times more Mit K (ATP) channel per milligram of mitochondrial protein than the liver or heart. Despite the abundance, little is known about its function in the neurons of the brain or those of the retina. In the current study, the protective action of BK against glutamate neurotoxicity was mediated through Mit K (ATP) channel. We showed that opening the Mit K (ATP) channel protected retinal neurons against glutamate neurotoxicity by inhibiting formation of O₂ ^•− in the mitochondria, by using diazoxide, a specific Mit K (ATP) channel opener and its blockers, 5-hydroxydecanoate (5HD) and glibenclamide. These results suggest Mit K (ATP) channel is the novel site for neuroprotection in the retinal glutamate neurotoxicity. 

Materials and Methods

All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Cell Culture

Primary cultures were obtained from fetal Wistar rat retinas (17 to 19 days’ gestation). The procedures have been described previously. ^{2 4 5 6 7 10 27} In brief, retinal tissues were mechanically dissociated, and single cell suspensions were plated on plastic coverslips (1.0 × 10⁶ cells/mL). Ten coverslips were placed in a 60-mm dish. Approximately 15 to 20 dishes were obtained and used for a single experiment. Retinal cultures were incubated with Eagle’s minimum essential medium (EMEM; Nissui, Tokyo, Japan) containing 2 mM glutamine, 11 mM glucose (total), 24 mM sodium bicarbonate, and 10 mM HEPES with 10% heat-inactivated fetal calf serum added during the first week and then supplemented with 10% horse serum for the remaining 10 to 11 days. Ten micromolar cytosine arabinoside (Ara-C) was added to the culture on the sixth day to eliminate proliferating cells. Only those cultures maintained for 9 to 10 days in vitro were used. Only isolated cells were used; clusters of cells were excluded from the results. A previous immunocytochemical study demonstrated that these isolated cells are mainly amacrine cells. ²⁷  

Drug Application

In our previous study using cultured rat retinal neurons, we demonstrated that cell viability was markedly reduced by exposure to glutamate (1 mM) for 10 minutes followed by postincubation in glutamate-free medium for more than 1 hour, ^{2 4 5 6 7 10 27} and that there was no significant difference between the reduction in cell viability for 1-hour incubation and 24-hour incubation. ²⁷ Therefore, in this study, cultures were exposed to drugs as follows. Glutamate neurotoxicity was assessed by 10-minute exposure to 1 mM glutamate followed by 1-hour incubation in glutamate-free medium. Sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1) were tested using a method similar to that used to test glutamate. According to our previous study on the dose–response relationship in the neurotoxic effects of NO-generating agents, the concentrations at 500 μM consistently reduced cell viability to 30% to 40%. ⁷ Therefore, we used this concentration to examine NO-induced neurotoxicity. Effects of BK, diazoxide, 5HD, and glibenclamide were assessed by simultaneous application of these drugs with glutamate, SNP, and SIN-1. To investigate the effects of simultaneous drug application, we added drugs to the incubation medium during glutamate exposure and removed them from culture medium during the postincubation period. 

Measurement of Neurotoxicity

The neurotoxic effects of glutamate and the protective effects of drugs on retinal cultures were quantitatively assessed by the trypan blue exclusion, method as described previously. ^{2 4 5 6 7 10 27} At each session of the experiment, we randomly picked five coverslips from different dishes that constituted the number of samples (n = 5) for measurement of neurotoxicity. All experiments were performed in EMEM at 37°C. After completion of drug treatment, cell cultures were stained with 1.5% trypan blue solution at room temperature for 10 minutes and fixed with isotonic formalin (pH 7.0, 2–4°C). The fixed cultures were rinsed with physiological saline and examined under Hoffman modulation microscopy at ×400. More than 200 cells on each of five coverslips were randomly counted to determine the viability of cell culture. The cells were counted by a masked observer. Viability of culture was calculated as the percentage of the ratio of the number of unstained cells (viable cells) to the total number of cells counted (viable cells plus nonviable cells). In each experiment, five coverslips were used to obtain mean ratios ± SEM of cell viability. The significance of data was determined by the Dunnett two-tailed test. 

Measurement of Mitochondrial Membrane Potential

Mitochondrial membrane potential was assessed with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1), as described previously. ¹⁹ Dye loading was achieved by incubation in EMEM containing 1 μg/mL JC-1 and 0.1% dimethyl sulfoxide (DMSO) at 37°C for 20 minutes. After they were loaded, coverslips were mounted on the 1 mL chamber on the microscope stage. Fields were illuminated with the 490-nm line of a xenon laser, and emission at 527 and 590 nm was monitored by microscope (Diaphot 300; Nikon, Tokyo, Japan), with an image intensifier (IIC-200; Princeton Scientific Instruments, Monmouth Junction, NJ), filters (530DF30 and OG590; Omega Optical, Brattleboro, VT), and a charge-coupled device (CCD) camera system (PentaMAX System; Princeton Scientific Instruments). The obtained signal was analyzed with image-analysis software (MetaMorph Imaging System; Universal Imaging Corp., West Chester, PA). The ratio was calculated by dividing the signal at 590 nm by the signal 527 nm after background subtraction. Figure 4a is a digitally synthesized image of the signal at 590 nm and the signal 527 nm. The coverslips were not continuously perfused. Ten microliters (1% volume of the chamber) of glutamate and other drugs were added to the chamber. Then the fluorescence of JC-1 was measured. 

Results

Effect of 5HD on the Protective Effect of Bradykinin

Cell viability was markedly reduced by a 10-minute exposure to 1 mM glutamate followed by a 1-hour incubation in glutamate34-free medium. Coapplication of MK-801, a selective blocker of the NMDA receptor, with glutamate markedly restored cell viability, indicating that the toxic action of glutamate is primarily mediated through NMDA receptors in our cultured retinal neurons (Fig. 1) . Simultaneous application of BK (10 μM) with glutamate induced the recovery of cell viability. To investigate the role of Mit K (ATP) channel in BK-induced protection against glutamate neurotoxicity, 5HD, a specific inhibitor of Mit K (ATP) channel, was applied simultaneously with glutamate and BK. Figure 2 demonstrates the effect of 5HD on the protective effect of BK against glutamate-induced neurotoxicity. Simultaneous application of 5HD with glutamate and BK reduced cell viability. 5HD alone did not affect cell viability, and 5HD had no effect on glutamate neurotoxicity (data not shown). Another Mit K (ATP) channel blocker, glibenclamide, also blocked the protective effect of bradykinin (Fig. 3) . These results suggest that the protective effect of BK against glutamate neurotoxicity is mediated by Mit K (ATP) channel. We therefore examined the membrane potential of mitochondria, because it is the central parameter that controls mitochondrial respiration, ATP synthesis, and Ca²⁺ accumulation. ^{17 18 19 20 21 22 28 29}  

Effect of Bradykinin and 5HD on Glutamate-Induced Mitochondrial Depolarization

Figure 4 demonstrates the fluorescence of neurons loaded with JC-1. JC-1 monomers accumulate selectively in mitochondria and subsequently aggregate as a function of the membrane potential. ³⁰ When mitochondrial membrane is hyperpolarized, JC-1 forms J-aggregates in mitochondria which emit 590-nm fluorescence (red-orange fluorescence) under the illumination of 490-nm light. However, when mitochondrial membrane potential is depolarized, JC-1 emits 527-nm fluorescence (green fluorescence). 

First, we used FCCP, a proton ionophore of the mitochondrial inner membrane, that is well known to depolarize mitochondrial membrane potential. Five minutes after application of FCCP, fluorescence of JC-1 shifted from red-orange to greenish yellow, indicating that the mitochondrial membrane in the retinal neurons was depolarized by FCCP. Next, we investigated the effect of glutamate on the mitochondrial membrane potential. By the application of glutamate, the 590:527-nm ratio of fluorescence decreased (Fig. 4b) . When BK was applied simultaneously with glutamate, the shift in fluorescence of JC-1 was markedly suppressed. We then investigated the effect of 5HD on glutamate-induced mitochondrial depolarization. When 5HD was applied simultaneously with glutamate and BK, the fluorescence of JC-1 shifted from red-orange to greenish yellow. However, 5HD alone did not affect JC-1 fluorescence. These results suggest that 5HD inhibits the effect of BK on mitochondrial membrane potential, and that BK suppresses glutamate-induced mitochondrial membrane depolarization through Mit K (ATP). 

Protective Action of Diazoxide on Glutamate Neurotoxicity

These results suggest that BK protects neurons by opening the Mit K (ATP) channel. We therefore examined the effects of opening the Mit K (ATP) channel on glutamate neurotoxicity, by using diazoxide, which opens the Mit K (ATP) channel. Figure 5a demonstrates the dose–response effect of diazoxide on glutamate-induced neurotoxicity, and a typical example is demonstrated in Figure 1e . Cell viability was markedly reduced by a 10-minute exposure to 1 mM glutamate followed by a 1-hour incubation in glutamate-free medium. Simultaneous application of diazoxide at concentrations of 1 to 100 μM with glutamate demonstrated a dose-dependent recovery of viability. 

Figure 5b shows the effect of 5HD on diazoxide-induced action against glutamate neurotoxicity. Thereafter, 5HD and diazoxide were added to the incubation medium during glutamate exposure and removed from culture medium followed by 1-hour incubation. 5HD (10 μM) reduced cell viability when applied simultaneously with glutamate and BK. Another Mit K (ATP) channel blocker, glibenclamide, also reduced cell viability when applied simultaneously with glutamate and diazoxide (Fig. 3) . These results strongly suggest that opening the Mit K (ATP) channel protects neurons against glutamate neurotoxicity. 

Effect of Diazoxide on Neurotoxicity Induced by SNP and SIN1

Previously, we reported that BK inhibited neurotoxicity induced by ionomycin (a calcium ionophore), and SNP (an NO-generating agent), but it did not affect neurotoxicity induced by SIN-1 (an NO- and O₂ ^•−-generating agent). ¹⁰ BK-induced protection against glutamate neurotoxicity is thus suggested to take place downstream of NO generation and upstream of O₂ ^•− generation. We therefore examined the effects of diazoxide on SNP- and SIN-1–induced neurotoxicity to elucidate the site of action in the cascade of glutamate neurotoxicity. Figure 6 demonstrates the effect of diazoxide on SNP- and SIN-1–induced neurotoxicity. Cell viability was markedly reduced by 10-minute exposure to SNP (500 μM) or SIN-1 (10 μM) followed by a 1-hour incubation in SNP-free or SIN-1–free medium. Simultaneous application of diazoxide at concentrations of 0.1 to 10 μM with SNP demonstrated dose-dependent recovery of viability (Fig. 6a) . By contrast, diazoxide did not induce recovery of cell viability when applied with SIN-1 (Fig. 6b) . These results suggest that diazoxide exerts its protective action against glutamate neurotoxicity by inhibiting the generation of O₂ ^•−. 

Effect of Diazoxide on Glutamate-Induced Mitochondrial Depolarization

Finally, we examined the effect of diazoxide on glutamate induced mitochondrial depolarization. Figure 7 demonstrates the 590:527-nm ratio of JC1 fluorescence. By simultaneous application of diazoxide with glutamate, the decrease of the 590:527-nm ratio was completely suppressed. By applying 5HD simultaneously with glutamate and diazoxide this action of diazoxide was inhibited. These results suggest that the opening of the Mit K (ATP) channel inhibits glutamate-induced mitochondrial depolarization. 

Discussion

This is the first report to show that opening the Mit K (ATP) channel protects retinal neurons against glutamate toxicity. BK protected neurons from glutamate toxicity by opening the Mit K (ATP) channel, and that diazoxide, which opens the Mit K (ATP) channel, protected neurons from glutamate toxicity. 

Previously, we found that BK protects retinal neurons against glutamate neurotoxicity through BK-B2 receptors by inhibiting generation of O₂ ^•−. ¹⁰ Although BK-B2 receptors are abundantly distributed in retinal neurons throughout the rat retina, their functional role in the retina is not yet known. The protective action of BK against glutamate neurotoxicity was antagonized by simultaneous application of 5HD, a selective Mit K (ATP) channel blocker and glibenclamide, which is also known as a blocker of the Mit K (ATP) channel, with BK. Furthermore, BK inhibited glutamate-induced mitochondrial membrane depolarization, and this inhibitory action was also blocked by coapplication of 5HD. These results suggest that the protective action of BK is mediated by the opening of the Mit K (ATP) channel. 

Diazoxide was used as an opener of cell membrane K (ATP) channel before Garlid et al. ^{31 32} first showed that low concentrations (1–10 μM) of diazoxide are specific to opening of the Mit K (ATP) channel, by using a reconstituted mitochondrial vesicle, and that a higher concentration (>10 μM) of diazoxide is not specific to Mit K (ATP) channel but activates cell membrane K (ATP). ³¹ Higher concentrations of diazoxide (16–164 μM ³³ and 150 μM ³⁴ ) are also known to inhibit succinate dehydrogenase, one of the subunits composing complex II. As for 5HD, McCullough et al. ³⁵ and Garlid et al. ³² showed that 5HD blocks the Mit K (ATP) channel without any effect on cell membrane K (ATP) channel. Since they reported the specific action of 5HD on mitochondria, it has been widely used as a specific blocker of the Mit K (ATP) channel at doses of 10 to 300 μM. Recently, Hanley et al. ³⁶ showed that 5HD could be a substrate of acyl-coenzyme A (CoA) synthase and converted to 5HD-CoA, using a high concentration of 5HD (1.4 mM) in a cell-free system. They also showed that a relatively high dose of diazoxide (10–100 μM) inhibits succinate oxidation, by using submitochondrial particles of myocytes. They suggest that the cardioprotective effect of diazoxide is mediated by inhibition of complex II, and that 5HD-CoA antagonizes this effect by supplying electrons to ubiquinone. By contrast, in our cultured retinal neurons, a much lower concentration (1 μM) of diazoxide was sufficient to inhibit glutamate neurotoxicity. Furthermore, in our present study, not only 5HD but also glibenclamide suppressed the protective effect of diazoxide. Glibenclamide is a less specific but more potent Mit K (ATP) channel blocker than diazoxide. ³² Because glibenclamide is not a fatty acid, it cannot affect mitochondrial electron transport by acting as a substrate of acyl-CoA synthase as can 5HD. Therefore, we suggest that the protective effect of diazoxide was mediated through Mit K (ATP) channel activation in our retinal culture. 

Exposure of the neurons to glutamate has been demonstrated to result in mitochondrial depolarization. ^{20 22 28} The glutamate-induced Ca²⁺-dependent decrease in mitochondrial membrane potential (ΔΨ_m) is a well characterized property of neuronal mitochondria. ^{37 38} White and Reynolds ¹⁹ have demonstrated in rat embryonic cultured forebrain neurons that NMDA receptor-dependent Ca²⁺ influx is required for mitochondrial depolarization. The present study demonstrated that simultaneous application of diazoxide with glutamate completely inhibited glutamate-induced mitochondrial membrane depolarization. The inhibitory action of diazoxide on mitochondrial depolarization induced by glutamate was antagonized by addition of 5HD, thus suggesting that opening of the Mit K (ATP) channel is responsible for the observed maintenance of polarized states of mitochondrial membrane potentials during glutamate challenge. Because acute depolarization after glutamate addition reflects accumulation by the mitochondria of Ca²⁺ entering through the NMDA receptor, ²⁰ it is thus suggested that opening the Mit K (ATP) channel inhibited Ca²⁺ influx into the mitochondrial inner membrane, which would have caused acute depolarization of ΔΨ_m after addition of glutamate. 

The present cytological study suggests that opening the Mit K (ATP) channel appeared to protect cultured retinal neurons from glutamate-induced cell death by inhibiting production of ROS. Glutamate receptor agonists were shown to elicit the generation of ROS. ⁸ Inhibition of ROS production has been demonstrated to attenuate glutamate receptor-mediated neurotoxicity. ^{23 39} In our cultured retinal neurons, inhibition of O₂ ^•− formation by superoxide dismutase, a radical scavenger, markedly reduced NMDA-induced neuronal death. ⁷ The main source of NMDA-induced production of O₂ ^•− has been indicated as present in mitochondria. ^{8 23 25 40} Several recent studies on cultured central nervous system neurons have demonstrated that Ca²⁺-induced mitochondrial dysfunction is responsible for increased generation of ROS, which mediates glutamate-induced neuronal death. ^{41 42} According to a recent study on the effects of ΔΨ_m on ROS production by isolated rat brain cortical mitochondria, ²⁹ the optimal conditions for ROS generation require either a hyperpolarized ΔΨ_m or a substantial level of complex I inhibition. In cultured rat hippocampal neurons, concurrent increases in O₂ ^•− production and depolarization of ΔΨ_m have been noted immediately after brief exposure of cultured neurons to NMDA at excitotoxic concentrations. ²¹ Taking together the evidence of the inhibitory action of mitochondrial electron transport inhibitors on glutamate-induced ROS production, ²³ Votyakova and Reynolds suggest that Ca²⁺-mediated inhibition of complex I or complex III is responsible for the generation of ROS consequent to activation of the NMDA receptor. ²⁹ Therefore, the present study suggests that opening the Mit K (ATP) channel inhibited the initial Ca⁺ entry into mitochondria after NMDA receptor activation, which would cause ROS generation as well as depolarization of 1 ΔΨ_m. This is consistent with recent reports in the heart, which suggest that opening the Mit K (ATP) channel inhibits mitochondrial Ca²⁺ uptake, resulting in cardioprotection. ^{43 44}  

Glutamate-induced ROS production and mitochondrial membrane depolarization, both require Ca²⁺ entry into the mitochondria. Because diazoxide and BK showed inhibitory effects on both processes and their actions were blocked by 5HD and glibenclamide, opening of the Mit K (ATP) channel is thus suggested to inhibit O₂ ^•− production by inhibiting mitochondrial Ca²⁺ uptake, leading to neuroprotection of retinal neurons against glutamate toxicity. 

 Supported in part by a Grant-in-Aid (13470366 and 14370780) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

 Submitted for publication August 12, 2002; revised January 14, 2003; accepted February 14, 2003.

 Disclosure: T. Yamauchi, None; S. Kashii, None; H. Yasuyoshi, None; S. Zang, None; Y. Honda, None; A. Akaike, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Trypan blue staining. Photomicrographs showing the effect of MK801, bradykinin (BK), and diazoxide on glutamate (Glt.)-induced neuronal death. Cultures were photographed using Hoffman modulation microscopy after trypan blue staining followed by formalin fixation. (a) Nontreated cells (control). (b) Cells were treated with glutamate for 10 minutes and further incubated with glutamate-free medium for 1 hour. Marked cell death was observed. (c–e) Cells were incubated with glutamate and MK801 10 μM (c), BK 10 μM (d), or diazoxide 10 μM (e) for 10 minutes and further incubated without glutamate and drugs.

Figure 2.

 

Effect of 5HD on protective effect of BK. Simultaneous application of BK (10 μM) along with glutamate reduced glutamate-induced cell death, whereas simultaneous application of 5HD inhibited this protective effect of BK. However, 5HD alone did not demonstrate neurotoxicity.

Figure 3.

 

Effect of glibenclamide on the protective effect of BK and diazoxide. Simultaneous application of glibenclamide (5 μM) inhibited the protective effect of BK and diazoxide against glutamate neurotoxicity.

Figure 4.

 

Effect of BK and 5HD on glutamate-induced mitochondrial depolarization. (a) JC-1 fluorescence (overlay image of 590- and 527-nm fluorescence). (aA) FCCP (750 nM), (aB) glutamate (1 mM), (aC) glutamate+BK (10 μM), and (aD) glutamate+BK (10 μM)+5HD (10 μM). (aA) By the application of FCCP, which is well known to depolarize mitochondrial membrane potential, JC-1 fluorescence shifted from 590 (orange) to 527 nm (green). (aB) By the application of glutamate (1 mM), mitochondrial membrane was depolarized. (aC) Simultaneous application of BK (10 μM) with glutamate suppressed glutamate-induced mitochondrial depolarization. (aD) 5HD (10 μM), applied simultaneously with glutamate and BK, inhibited the suppressive effect of BK to glutamate-induced mitochondrial depolarization. Bar, 25 μm. (b) 590:527-nm ratio of JC-1 fluorescence. By simultaneous application of BK with glutamate, glutamate-induced mitochondrial depolarization was suppressed. Simultaneous application of 5HD with BK and glutamate inhibited the suppressive effect of BK on glutamate-induced mitochondrial depolarization. Numbers are used to denote significant differences. (○) 1: glutamate (n = 7); (▪) 2: glutamate+diazoxide (n = 7); (▴) 3: glutamate+diazoxide+5HD (n = 7). 2 vs. 1 and 3 vs. 2: P < 0.01, at every time point, by the Dunnett test.

Figure 5.

 

Protective effect of diazoxide against glutamate neurotoxicity. (a) Simultaneous application of diazoxide suppressed glutamate-induced neuronal death in a dose-dependent manner. (b) Simultaneous application of 5HD suppressed the effect of diazoxide.

Figure 6.

 

Effect of diazoxide on NO-induced neurotoxicity. (a) Simultaneous application of diazoxide suppressed SNP (NO donor)-induced neuronal death. (b) Diazoxide did not suppress SIN-1 (NO and O₂ ^•− donor)-induced neuronal death.

Figure 7.

 

Effect of diazoxide and 5HD on glutamate-induced mitochondrial depolarization. Simultaneous application of diazoxide (10 μM) with glutamate (1 mM), completely suppressed glutamate-induced mitochondrial depolarization. Simultaneous application of 5HD (10 μM) with diazoxide and glutamate, inhibited the suppressive effect of diazoxide on glutamate-induced mitochondrial depolarization. Numbers are used to denote significant differences. (○) 1: glutamate (n = 7); (▪) 2: glutamate+diazoxide (n = 7); (▴) 3: glutamate+diazoxide+5HD (n = 7). 2 vs. 1 and 3 vs. 2: P < 0.01 at every time point by the Dunnett test.