The effects of ATP-sensitive mitochondrial K + channel(mitoK ATP) on mitochondrial membrane potential(Δψm),cell proliferation and protein kinase C alpha(PKCα) expression in airway smooth muscle cells(ASMCs) were in...The effects of ATP-sensitive mitochondrial K + channel(mitoK ATP) on mitochondrial membrane potential(Δψm),cell proliferation and protein kinase C alpha(PKCα) expression in airway smooth muscle cells(ASMCs) were investigated.Thirty-six Sprague-Dawley(SD) rats were immunized with saline(controls) or ovalbumin(OVA) with alum(asthma models).ASMCs were cultured from the lung of control and asthma rats.ASMCs were treated with diazoxide(the potent activator of mitoK ATP) or 5-hydroxydencanote(5-HD,the inhibitor of mitoK ATP).Rhodamine-123(R-123) was used to detect Δψm.The expression of PKCα protein was examined by using Western blotting,while PKCα mRNA expression was detected by using real-time PCR.The proliferation of ASMCs was measured by MTT assay and cell cycle analysis.In diazoxide-treated normal ASMCs,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls.The ratio of G 0 /G 1 cells was decreased(P<0.05) in diazoxide-treated ASMCs from normal rats.However,there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group.In untreated and diazoxide-treated ASMCs of asthmatic rats,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group.Furthermore,in comparison to ASMCs from asthmatic rats,these values were considerably increased in asthmatic group treated with diazoxide(P<0.05).After exposure to 5-HD for 24 h,these values were decreased as compared with asthma control group(P<0.05).In ASMCs of asthma,the signal transduction pathway of PKCα may be involved in cell proliferation,which is induced by the opening of mitoK ATP and the depolarization of Δψm.展开更多
Mitochondrial K＋-ATP （mito-KATP） channels play an important role in cellular function and survival following ischemic stress. The present results revealed that intervention with diazoxide, a mito-KATP channel opene...Mitochondrial K＋-ATP （mito-KATP） channels play an important role in cellular function and survival following ischemic stress. The present results revealed that intervention with diazoxide, a mito-KATP channel opener, led to an increase in Bcl-2 expression in the cerebral cortex of rats subjected to cerebral ischemia reperfusion injury. In addition, the intervention also led to clear improvements in neuronal mitochondrial morphology and consciousness post-injury. Glibenclamide a mito-KATP channel blocker, exhibited the converse effects. Both diazoxide and glibenclamide exerted dose-dependent effects （in particular, at 18 mg/kg diazoxide and 25 mg/kg glibenclamide）. These findings suggest that diazoxide exerts a neuroprotective effect on cerebral ischemia reperfusion injury by opening mito-KATP channels and upregulating Bcl-2 expression.展开更多
Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels(K-ATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus K-ATP channel opene...Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels(K-ATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus K-ATP channel openers(KCOs) have been proposed as potential neuroprotectants. Our previous studies demonstrated that K-ATP channels could regulate glutamate uptake activity in PC12 cells as well as in synaptosomes of rats. Since glutamate transporters(GluTs) of astrocytes play crucial roles in glutamate uptake and KATP channels are also expressed in astrocytes, the present study showed whether and how KATP channels regulated the function of GluTs in primary cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood-brain barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium(MPP+). These potentiated effects were completely abolished by mitochondrial K-ATP blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could inhibit MPP+-induced elevation of reactive oxygen species (ROS) and phosphorylation of protein kinases C(PKC). These findings are the first to demonstrate that activation of K-ATP channel, especially mitochondrial K-ATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phosphorylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also provides a new strategy for neuroprotection.展开更多
BACKGROUND ATP-sensitive K^+(KATP)channels were originally found in cardiac myocytes by Noma in 1983.KATP channels were formed by potassium ion-passing poreforming subunits(Kir6.1,Kir6.2)and regulatory subunits SUR1,S...BACKGROUND ATP-sensitive K^+(KATP)channels were originally found in cardiac myocytes by Noma in 1983.KATP channels were formed by potassium ion-passing poreforming subunits(Kir6.1,Kir6.2)and regulatory subunits SUR1,SU2A and SUR2B.A number of cells and tissues have been revealed to contain these channels including hepatocytes,but detailed localization of these subunits in different types of liver cells was still uncertain.AIM To investigate the expression of KATP channel subunits in rat liver and their localization in different cells of the liver.METHODS Rabbit anti-rat SUR1 peptide antibody was raised and purified by antigen immunoaffinity column chromatography.Four of Sprague-Dawley rats were used for liver protein extraction for immunoblot analysis,seven of them were used for immunohistochemistry both for the ABC method and immunofluorescence staining.Four of Wistar rats were used for the isolation of hepatic stellate cells(HSCs)and Kupffer cells for both primary culture and immunocytochemistry.RESULTS Immunoblot analysis showed that the five kinds of KATP channel subunits,i.e.Kir6.1,Kir6.2,SUR1,SUR2A,and SUR2B,were detected in liver.Immunohistochemical staining showed that Kir6.1 and Kir6.2 were weakly to moderately expressed in parenchymal cells and sinusoidal lining cells,while SUR1,SUR2A,and SUR2B were mainly localized to sinusoidal lining cells,such as HSCs,Kupffer cells,and sinusoidal endothelial cells.Immunoreactivity for SUR2A and SUR2B was expressed in the hepatocyte membrane.Double immunofluorescence staining further showed that the pore-forming subunits Kir6.1 and/or Kir6.2 colocalized with GFAP in rat liver sections and primary cultured HSCs.These KATP channel subunits also colocalized with CD68 in liver sections and primary cultured Kupffer cells.The SUR subunits colocalized with GFAP in liver sections and colocalized with CD68 both in liver sections and primary cultured Kupffer cells.In addition,five KATP channel subunits colocalized with SE-1 in sinusoidal endothelial cells.CONCLUSION Observations from the present study indicated that KATP channel subunits expressed in rat liver and the diversity of KATP channel subunit composition might form different types of KATP channels.This is applicable to hepatocytes,HSCs,various types of Kupffer cells and sinusoidal endothelial cells.展开更多
BACKGROUND： Recent studies have suggested that mitochondrial ATP-sensitive K＋ channel openers could reduce myocardium infarct size, and protect the function of the mitochondria. OBJECTIVE： To investigate the change...BACKGROUND： Recent studies have suggested that mitochondrial ATP-sensitive K＋ channel openers could reduce myocardium infarct size, and protect the function of the mitochondria. OBJECTIVE： To investigate the changes of cerebral infarction volume and the activity of marker enzymes in brain mitochondria of rats given the ATP-sensitive K＋ channel opener, nicorandil, before focal cerebral ischemia/reperfusion （I/R）. DESIGN, TIME AND SETTING： Randomized, controlled animal experiment, completed at the Brain Scientific Research Center of the Affiliated Hospital of Qingdao University from July to November 2007. MATERIALS： Sixty healthy male Wistar rats weighing 280-300 g. Nicorandil, 5-hydroxydecanoate （5-HD） and cytochrome C were purchased from Sigma in the USA. Standard malondialdehyde （MDA） and protein were purchased from Nanjing Jiancheng Biotechnology Institute. METHODS： Sixty rats were randomly divided into a sham operation group, a middle cerebral artery occlusion （MCAO） group, a nicorandil group and a nicorandil＋5-HD group. MCAO for 2 hours was performed in the MCAO group, nicorandil group and nicorandil＋5-HD group. A total of 5 mL saline were given to the MCAO group before MCAO. The nicorandil group was injected with the ATP-sensitive K＋ channel opener nicorandil 10 mg/kg intraperitoneally 30 minutes before MCAO. The nicorandil＋5-HD group was injected with 5-HD 10 mg/kg intravenously 15 minutes before the same treatment as the nicorandil group. MAIN OUTCOME MEASURES： Infarct volume by total brain slice calculation, activities of succinate dehydrogenase （SDH） and cytochrome oxidase （CO）, and content of MDA were observed at 22 hours of reperfusion after 2 hours MCAO. RESULTS： Sixty rats were included in the final analysis, without any loss. （1） Infarct volume： compared with the MCAO group and nicorandil＋5-HD group, the percentage of infarct volume was significantly decreased in the nicorandil group （P 〈 0.01）. （2） The content of MDA, expression of SDH and CO in brain： the expressions of SDH and CO in the sham operation group were significantly lower than those in the MCAO, nicorandil and nicorandil＋5-HD groups （P 〈 0.01）. The expressions of SDH and CO in the nicorandil group were significantly higher than those in the MCAO and nicorandil＋5-HD groups （P 〈 0.05）. The content of MDA in the brain of the nicorandil group was significantly lower than those in the MCAO and nicorandil＋5-HD groups （P 〈 0.01）. CONCLUSION： Nicorandil can significantly reduce the infarct volume in a rat MCAO model, increase the activity of the mitochondria and protect against cerebral I/R injury.展开更多
基金supported by grants from Natural Science Foundation of Hubei Province,China (No. 2010CDB096)the National Key Technology R&D Program of the 12th National Five-year Development Plan of China (No. 2012BAI05B01)
文摘The effects of ATP-sensitive mitochondrial K + channel(mitoK ATP) on mitochondrial membrane potential(Δψm),cell proliferation and protein kinase C alpha(PKCα) expression in airway smooth muscle cells(ASMCs) were investigated.Thirty-six Sprague-Dawley(SD) rats were immunized with saline(controls) or ovalbumin(OVA) with alum(asthma models).ASMCs were cultured from the lung of control and asthma rats.ASMCs were treated with diazoxide(the potent activator of mitoK ATP) or 5-hydroxydencanote(5-HD,the inhibitor of mitoK ATP).Rhodamine-123(R-123) was used to detect Δψm.The expression of PKCα protein was examined by using Western blotting,while PKCα mRNA expression was detected by using real-time PCR.The proliferation of ASMCs was measured by MTT assay and cell cycle analysis.In diazoxide-treated normal ASMCs,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and percentage of cells in S phase were markedly increased as compared with untreated controls.The ratio of G 0 /G 1 cells was decreased(P<0.05) in diazoxide-treated ASMCs from normal rats.However,there were no significant differences between the ASMCs from healthy rats treated with 5-HD and the normal control group.In untreated and diazoxide-treated ASMCs of asthmatic rats,the R-123 fluorescence intensity,protein and mRNA levels of PKCα,MTT A values and the percentage of cells in S phase were increased in comparison to the normal control group.Furthermore,in comparison to ASMCs from asthmatic rats,these values were considerably increased in asthmatic group treated with diazoxide(P<0.05).After exposure to 5-HD for 24 h,these values were decreased as compared with asthma control group(P<0.05).In ASMCs of asthma,the signal transduction pathway of PKCα may be involved in cell proliferation,which is induced by the opening of mitoK ATP and the depolarization of Δψm.
文摘Mitochondrial K＋-ATP （mito-KATP） channels play an important role in cellular function and survival following ischemic stress. The present results revealed that intervention with diazoxide, a mito-KATP channel opener, led to an increase in Bcl-2 expression in the cerebral cortex of rats subjected to cerebral ischemia reperfusion injury. In addition, the intervention also led to clear improvements in neuronal mitochondrial morphology and consciousness post-injury. Glibenclamide a mito-KATP channel blocker, exhibited the converse effects. Both diazoxide and glibenclamide exerted dose-dependent effects （in particular, at 18 mg/kg diazoxide and 25 mg/kg glibenclamide）. These findings suggest that diazoxide exerts a neuroprotective effect on cerebral ischemia reperfusion injury by opening mito-KATP channels and upregulating Bcl-2 expression.
文摘Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels(K-ATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus K-ATP channel openers(KCOs) have been proposed as potential neuroprotectants. Our previous studies demonstrated that K-ATP channels could regulate glutamate uptake activity in PC12 cells as well as in synaptosomes of rats. Since glutamate transporters(GluTs) of astrocytes play crucial roles in glutamate uptake and KATP channels are also expressed in astrocytes, the present study showed whether and how KATP channels regulated the function of GluTs in primary cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood-brain barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium(MPP+). These potentiated effects were completely abolished by mitochondrial K-ATP blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could inhibit MPP+-induced elevation of reactive oxygen species (ROS) and phosphorylation of protein kinases C(PKC). These findings are the first to demonstrate that activation of K-ATP channel, especially mitochondrial K-ATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phosphorylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also provides a new strategy for neuroprotection.
基金Supported by the Program of the network-type joint Usage/Research Center for Radiation Disaster Medical Science of Hiroshima University,Nagasaki University,and Fukushima Medical University
文摘BACKGROUND ATP-sensitive K^+(KATP)channels were originally found in cardiac myocytes by Noma in 1983.KATP channels were formed by potassium ion-passing poreforming subunits(Kir6.1,Kir6.2)and regulatory subunits SUR1,SU2A and SUR2B.A number of cells and tissues have been revealed to contain these channels including hepatocytes,but detailed localization of these subunits in different types of liver cells was still uncertain.AIM To investigate the expression of KATP channel subunits in rat liver and their localization in different cells of the liver.METHODS Rabbit anti-rat SUR1 peptide antibody was raised and purified by antigen immunoaffinity column chromatography.Four of Sprague-Dawley rats were used for liver protein extraction for immunoblot analysis,seven of them were used for immunohistochemistry both for the ABC method and immunofluorescence staining.Four of Wistar rats were used for the isolation of hepatic stellate cells(HSCs)and Kupffer cells for both primary culture and immunocytochemistry.RESULTS Immunoblot analysis showed that the five kinds of KATP channel subunits,i.e.Kir6.1,Kir6.2,SUR1,SUR2A,and SUR2B,were detected in liver.Immunohistochemical staining showed that Kir6.1 and Kir6.2 were weakly to moderately expressed in parenchymal cells and sinusoidal lining cells,while SUR1,SUR2A,and SUR2B were mainly localized to sinusoidal lining cells,such as HSCs,Kupffer cells,and sinusoidal endothelial cells.Immunoreactivity for SUR2A and SUR2B was expressed in the hepatocyte membrane.Double immunofluorescence staining further showed that the pore-forming subunits Kir6.1 and/or Kir6.2 colocalized with GFAP in rat liver sections and primary cultured HSCs.These KATP channel subunits also colocalized with CD68 in liver sections and primary cultured Kupffer cells.The SUR subunits colocalized with GFAP in liver sections and colocalized with CD68 both in liver sections and primary cultured Kupffer cells.In addition,five KATP channel subunits colocalized with SE-1 in sinusoidal endothelial cells.CONCLUSION Observations from the present study indicated that KATP channel subunits expressed in rat liver and the diversity of KATP channel subunit composition might form different types of KATP channels.This is applicable to hepatocytes,HSCs,various types of Kupffer cells and sinusoidal endothelial cells.
基金This work was supported by the Natural Science Foundation of Jiangsu Province(No.BK2002138).
文摘利用离体海马脑片缺氧无糖(oxygen-glucose deprivation,OGD)损伤模型,探讨七氟醚预处理对神经细胞的保护作用及陔作用与线粒体内膜ATP敏感钾通道(mitochondrial ATP-sensitive potassium channels,mitoK_(ATP)channels)的关系,随机将脑片用2%、4%、6%七氟醚,以及6%七氟醚复合mitoK_(ATP)通道阻滞剂5-羟基奎酸盐(5-hydroxydecanoic acid,5-HD)预处理30min,观察OGD损伤14min复氧1h期间顺向群峰电位(orthodromic population spike,OPS)的变化,并应用透射电镜观察细胞超微结构的改变。结果表明,与单纯OGD组相比,七氟醚预处理可使海马脑片OPS消失时间明显延长(P＜0．01),使OPS明显恢复,其中4%、6%七氟醚组的恢复率均为71．4%(P＜0．05 vs OGD),相应恢复程度为(61．0±42．3)%和(78．7±21．1)%(P＜0．01),而且6%七氟醚的保护作用可被5-HD取消。OGD组的海马CA1区锥体细胞明显水肿,核膜皱缩、破裂,染色质聚集,线粒体肿胀畸形,嵴断裂或消失,而4%和6%七氟醚组仅见海马CA1区锥体细胞轻度水肿,核膜皱缩不明显,染色质均匀,线粒体轻度肿胀。结果提示,七氟醚预处理对大鼠海马脑片OGD损伤有一定的保护作用,且七氟醚对神经细胞的保护作用与激活mitoK_(ATP)通道有关。
文摘BACKGROUND： Recent studies have suggested that mitochondrial ATP-sensitive K＋ channel openers could reduce myocardium infarct size, and protect the function of the mitochondria. OBJECTIVE： To investigate the changes of cerebral infarction volume and the activity of marker enzymes in brain mitochondria of rats given the ATP-sensitive K＋ channel opener, nicorandil, before focal cerebral ischemia/reperfusion （I/R）. DESIGN, TIME AND SETTING： Randomized, controlled animal experiment, completed at the Brain Scientific Research Center of the Affiliated Hospital of Qingdao University from July to November 2007. MATERIALS： Sixty healthy male Wistar rats weighing 280-300 g. Nicorandil, 5-hydroxydecanoate （5-HD） and cytochrome C were purchased from Sigma in the USA. Standard malondialdehyde （MDA） and protein were purchased from Nanjing Jiancheng Biotechnology Institute. METHODS： Sixty rats were randomly divided into a sham operation group, a middle cerebral artery occlusion （MCAO） group, a nicorandil group and a nicorandil＋5-HD group. MCAO for 2 hours was performed in the MCAO group, nicorandil group and nicorandil＋5-HD group. A total of 5 mL saline were given to the MCAO group before MCAO. The nicorandil group was injected with the ATP-sensitive K＋ channel opener nicorandil 10 mg/kg intraperitoneally 30 minutes before MCAO. The nicorandil＋5-HD group was injected with 5-HD 10 mg/kg intravenously 15 minutes before the same treatment as the nicorandil group. MAIN OUTCOME MEASURES： Infarct volume by total brain slice calculation, activities of succinate dehydrogenase （SDH） and cytochrome oxidase （CO）, and content of MDA were observed at 22 hours of reperfusion after 2 hours MCAO. RESULTS： Sixty rats were included in the final analysis, without any loss. （1） Infarct volume： compared with the MCAO group and nicorandil＋5-HD group, the percentage of infarct volume was significantly decreased in the nicorandil group （P 〈 0.01）. （2） The content of MDA, expression of SDH and CO in brain： the expressions of SDH and CO in the sham operation group were significantly lower than those in the MCAO, nicorandil and nicorandil＋5-HD groups （P 〈 0.01）. The expressions of SDH and CO in the nicorandil group were significantly higher than those in the MCAO and nicorandil＋5-HD groups （P 〈 0.05）. The content of MDA in the brain of the nicorandil group was significantly lower than those in the MCAO and nicorandil＋5-HD groups （P 〈 0.01）. CONCLUSION： Nicorandil can significantly reduce the infarct volume in a rat MCAO model, increase the activity of the mitochondria and protect against cerebral I/R injury.
