Effects of Mitochondrial ATP-sensitive K^+ Channel on Protein Kinase C Pathway and Airway Smooth Muscle Cell Proliferation in Asthma

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.

ATP-sensitive potassium channels:novel potential roles in Parkinson's disease

The ATP-sensitive potassium（KATP）channels which extensively distribute in diverse tissues（e.g.vascular smooth muscle,cardiac cells,and pancreas）are well-established for characteristics like vasodilatation,myocardial protection against ischemia,and insulin secretion.The aim of this review is to get insight into the novel roles of KATPchannels in Parkinson＇s disease（PD）,with consideration of the specificities KATPchannels in the central nervous system（CNS）, such as the control of neuronal excitability,action potential,mitochondrial function and neurotransmitter release.

Localization of ATP-sensitive K^+ channel subunits in rat liver

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.

Expression levels of K_(ATP)channel subunits and morphological changes in the mouse liver after exposure to radiation

BACKGROUND ATP sensitive K+(K_(ATP))channels are ubiquitously distributed in various of cells and tissues,including the liver.They play a role in the pathogenesis of myocardial and liver ischemia.AIM To evaluate the radiation-induced changes in the expression of K_(ATP)channel subunits in the mouse liver to understand the potential role of K_(ATP)channels in radiation injury.METHODS Adult C57BL/6 mice were randomly exposed toγ-rays at 0 Gy(control,n=2),0.2 Gy(n=6),1 Gy(n=6),or 5 Gy(n=6).The livers were removed 3 and 24 h after radiation exposure.Hematoxylin and eosin staining was used for morphological observation;immunohistochemical staining was applied to determine the expression of K_(ATP)channel subunits in the liver tissue.RESULTS Compared with the control group,the livers exposed to 0.2 Gyγ-ray showed an initial increase in the expression of Kir6.1 at 3 h,followed by recovery at 24 h after exposure.Exposure to a high dose of 5.0 Gy resulted in decreased expression of Kir6.1 and increased expression of SUR2B at 24 h.However,the expression of Kir6.2,SUR1,or SUR2A had no remarkable changes at 3 and 24 h after exposure to any of these doses.CONCLUSION The expression levels of Kir6.1 and SUR2B in mouse liver changed differently in response to different radiation doses,suggesting a potential role for them in radiation-induced liver injury.

Effect of G_(αq/11) Protein and ATP-sensitive Potassium Channels on Ischemic Preconditioning in Rat Hearts

Objectives To investigate the effect of Gαq/11 signaling pathway and ATP-sensitive potassium channel ( KATP channel ) on ischemic preconditioning (IPC) protection in rat hearts. Methods Two series of experiments were performed in Wistar rat hearts. In the first series of experiment, ischemic preconditioning was induced by left anterior descending occlusion (three, 5 min episodes separated by 5 min of reperfusion), ischemia-reperfusion injury was induced by 30 min coronary artery occlusion followed by 90 min reperfusion. Hemodynamics, infarct size and scores of ventricular arrhythmias were measured. The expression of Gαq/11 protein in the heart was measured by Western blot analysis in the second series. Results Ischemic preconditioning rats showed decreased infarct size and scores of ventricular arrhythmia vs non-IP control rats. The effect of IPC was significantly attenuated by glibenclamide (1 mg/kg, ip), a nonselective KATP channel inhibitor. IPC caused a significant increase in the expression of Gαq/11 protein. Conclusions Activations of Gαq/11 signal pathway and KATP channel played significant roles in the classical cardioprotection of ischemic precon-ditioning rat heart and might be an important mechanism of signal transduction pathway during the ischemic preconditioning.

MODELING AND SIMULATION OF E1784K MUTATION AND SODIUM IONIC CHANNEL DISEASES

In the clinical reports, the E1784K mutation in SCN5A is recognized as a phenotypic overlap between the long QT syndrome （LQT3） and the Brugada syndrome （BrS） in the characteristics of electrocardiograms （ECGs） since the mutation can influence sodium channel functions. However it is still unclear if the E1784K mutation-induced sodium ionic channel alterations account for the overlap at tissue level. Thsu, a detailed computational model is developed to underpin the functional impacts of the E1784K mutation on the action potential （AP）, the effective refractory period （ERP） and the abnormal ECG. Simulation results stlggest＇that the E1784K mutation-induced sodium channel alterations are insufficient to produce the phenotypic overlap between LQT3 and BrS, and the overlap may arise from the complicated effects of the E1784K mutation-induced changes in sodium channel currents with an increase of the transient outward current ITo or a decrease of the L-type calcium current ICaL .

TRPC1与BK-α的表达对大鼠糖尿病肾病的影响

目的 探究瞬时受体电位C1(transient receptor potential channel 1,TRPC1)蛋白和大电导钙离子激活钾通道α亚单位(large conductance Ca^(2+)-activated K^(+)channel α subunit,BK-α)蛋白对大鼠糖尿病肾病(diabetic kidney disease,DKD)的影响。方法 将SD大鼠随机分为对照组(n=15)和模型组(n=15)。利用高脂饲料和链脲佐菌素(streptozocin,STZ)构建DKD模型。采用血糖分析仪检测大鼠血糖变化;采用全自动生化分析仪检测大鼠肾功能水平;HE染色检测肾组织的病理变化以确定造模成功。实时荧光定量PCR(RT-qPCR)和蛋白免疫印迹分别检测肾组织TRPC1和BK-α的mRNA和蛋白表达水平;免疫组化检测TRPC1和BK-α的分布和表达情况。结果 模型组大鼠空腹血糖(fasting plasma glucose,FPG)、尿白蛋白排泄率(urinary albumin excretion rates,UAER)、血尿素氮(blood urea nitrogen,BUN)和肌酐(creatinine,Cr)均显著高于对照组(P <0.01);模型组大鼠肾小管内壁细胞出现膨胀现象,部分细胞脱离;可见肾小管发生病变或死亡;此外,在许多肾小管及肾间质区域发现有中性白细胞及其残骸;以上HE染色结果提示,DKD模型复制成功。TRPC1和BK-α在肾小球部位最为丰富,且模型组大鼠肾组织中TRPC1和BK-α的mRNA和蛋白水平都显著高于对照组(P <0.05)。结论 大鼠糖尿病肾病影响TRPC1和BK-α在肾组织中的分布和表达。

Beneficial effects of adenosine triphosphate-sensitive K^+ channel opener on liver ischemia/reperfusion injury

AIM: To investigate the effect of diazoxide administration on liver ischemia/reperfusion injury.

K^+channels inhibited by hydrogen peroxide mediate abscisic acid signaling in Vicia guard cells

A number of studies show that environmental stress conditions increase abscisic acid (ABA) and hydrogen peroxide (H2O2) levels in plant cells. Despite this central role of ABA in altering stomatal aperture by regulating guard cell ion transport, little is known concerning the relationship between ABA and H2O2 in signal transduction leading to stomatal movement. Epidermal strip bioassay illustrated that ABA- inhibited stomatal opening and ABA-induced stomatal closure were abolished partly by externally added catalase (CAT) or diphenylene iodonium (DPl), which are a H2O2 scavenger and a NADPH oxidase inhibitor respectively. In contrast, internally added CAT or DPI nearly completely or partly reversed ABA-induced closure in half-stoma. Consistent with these results, whole-cell patch-clamp analysis showed that intracellular application of CAT or DPI partly abolished ABA-inhibited inward K+ current across the plasma membrane of guard cells. H2O2 mimicked ABA to inhibit inward K+ current, an effect which was reversed by the addition of ascorbic acid (Vc) in patch clamping micropipettes. These results suggested that H2O2 mediated ABA-induced stomatal movement by targeting inward K+ channels at plasma membrane.

Inhibitory Effects of Blockage of Intermediate Conductance Ca^(2+) -Activated K^+ Channels on Proliferation of Hepatocellular Carcinoma Cells

The roles of intermediate conductance Ca2＋-activated K＋ channel （IKCal） in the pathogene- sis of hepatocellular carcinoma （HCC） were investigated. Immunohistochemistry and Western blotting were used to detect the expression of IKCal protein in 50 HCC and 20 para-carcinoma tissue samples. Real-time PCR was used to detect the transcription level of IKCal mRNA in 13 HCC and 11 para-carcinoma tissue samples. The MTT assay was used to measure the function of IKCal in human HCC cell line HepG2 in vitro. TRAM-34, a specific blocker of IKCal, was used to intervene with the function of IKCal. As compared with para-carcinoma tissue, an over-expression of IKCal protein was detected in HCC tissue samples （P〈0.05）. The mRNA expression level of IKCal in HCC tissues was 2.17 times higher than that in para-carcinoma tissues. The proliferation of HepG2 cells was suppressed by TRAM-34 （0.5, 1.0, 2.0 and 4.0 pxnol/L） in vitro （P〈0.05）. Our results suggested that IKCal may play a role in the proliferation of human HCC, and IKCal blockers may represent a potential therapeutic strategy for HCC.

Nitric oxide activation of a potassium channel (BK_(Ca)) in feline lower esophageal sphincter

AIM:To assess the effect of nitric oxide (NO) on the large conductance potassium channel (BKCa) in isolated circular (CM) and sling (SM) muscle cells and muscle strips from the cat lower esophageal sphincter (LES) to determine its regulation of resting tone and relaxation.METHODS:Freshly enzymatically-digested and isolated circular smooth muscle cells were prepared from each LES region.To study outward K + currents,the perforated patch clamp technique was employed.To assess LES resting tone and relaxation,muscle strips were mounted in perfused organ baths.RESULTS:(1) Electrophysiological recordings from isolated cells:(a) CM was more depolarized than SM (-39.7 ± 0.8mV vs-48.1 ± 1.6 mV,P < 0.001),and maximal outward current was similar (27.1 ± 1.5 pA/pF vs 25.7 ± 2.0 pA/pF,P > 0.05);(b) The NO donor sodium nitroprusside (SNP) increased outward currents only in CM (25.9 ± 1.9 to 46.7 ± 4.2 pA/pF,P < 0.001) but not SM (23.2 ± 3.1 to 27.0 ± 3.4 pA/pF,P > 0.05);(c) SNP added in the presence of the BK Ca antagonist iberiotoxin (IbTX) produced no increase in the outward current in CM (17.0 ± 2.8 vs 13.7 ± 2.2,P > 0.05);and (d) L-NNA caused a small insignificant inhibition of outward K + currents in both muscles;and (2) Muscle strip studies:(a) Blockade of the nerves with tetrodotoxin (TTX),or BK Ca with IbTX had no significant effect on resting tone of either muscle;and (b) SNP reduced tone in both muscles,and was unaffected by the presence of TTX or IbTX.CONCLUSION:Exogenous NO activates BK Ca only in CM of the cat.However,as opposed to other species,exogenous NO-induced relaxation is predominantly by a non-BK Ca mechanism,and endogenous NO has minimal effect on resting tone.

Ion channels in neurodevelopment:lessons from the Integrin-KCNB1 channel complex

Ion channels modulate cellular excitability by regulating ionic fluxes across biological membranes.Pathogenic mutations in ion channel genes give rise to epileptic disorders that are among the most frequent neurological diseases affecting millions of individuals worldwide.Epilepsies are trigge red by an imbalance between excitatory and inhibitory conductances.However,pathogenic mutations in the same allele can give rise to loss-of-function and/or gain-of-function va riants,all able to trigger epilepsy.Furthermore,certain alleles are associated with brain malformations even in the absence of a clear electrical phenotype.This body of evidence argues that the underlying epileptogenic mechanisms of ion channels are more diverse than originally thought.Studies focusing on ion channels in prenatal cortical development have shed light on this apparent paradox.The picture that emerges is that ion channels play crucial roles in landmark neurodevelopmental processes,including neuronal migration,neurite outgrowth,and synapse formation.Thus,pathogenic channel mutants can not only cause epileptic disorders by alte ring excitability,but further,by inducing morphological and synaptic abnormalities that are initiated during neocortex formation and may persist into the adult brain.

HERG K+ channels expression in gastric cancers and analysis of its regulation in tumor cell proliferation and apoptosis

Objective： To investigate the expression of hergl gene in tumor tissues from gastric carcinomas and gastric carcinoma cell lines, and study the relationship between HERG K＋ channel expressions and tumor cell proliferation and apoptosis. Methods： RT-PCR and PCR assays were used to detect the expression of hergl gene in 64 gastric carcinomas and the gastric cancer cell line SGC-7901. Blocking the HERG K＋ channels was used to evaluate their effects on tumor cell proliferation and apoptosis. Results：The statistically significant expression of hergl gene was detected in all the gastric cancers and SGC-7901 cells, but not in normal tissues. The HERG K＋ channel blocker, E-4031, increased the cell population in G0/G1（P 〈 0.05） and the number of apoptotic tumor cells（P 〈 0.05）. Conclusion： HERG K＋ channels were expressed in all gastric carcinomas tested and these channels appear to modulate tumor cell proliferation and apoptosis.

Adenosine triphosphate-sensitive potassium channel opener protects PC12 cells against hypoxia-induced apoptosis through PI3K/Akt and Bcl-2 signaling pathways

Although previous studies have shown the neuroprotective effects of the adenosine triphosphate （ATP）-sensitive potassium （KATP） channel opener against ischemic neuronal damage, little is known about the mechanisms involved. Phosphatidylinositol-3 kinase （PI3K）/v-akt murine thy-moma viral oncogene homolog （Akt） and Bcl-2 are thought to be important factors that mediate neuroprotection. The present study investigated the effects of KATP openers on hypoxia-induced PC12 cell apoptosis, as well as mRNA and protein expression of Akt and Bcl-2. Results demon-strated that pretreatment of PC12 cells with pinacidil, a KATP opener, resulted in decreased PC12 cell apoptosis following hypoxia, as detected by Annexin-V fluorescein isothiocyanate/ propidium iodide double staining flow cytometry. In addition, mRNA and protein expression of phosphorylated Akt （p-Akt） and Bcl-2 increased, as detected by immunofluorescence, Western blot analysis, and reverse-transcription polymerase chain reaction. The protective effect of this preconditioning was attenuated by glipizide, a selective KATP blocker. These results demonstrate for the first time that the protective mechanisms of KATP openers on PC12 cell apoptosis following hypoxia could result from activation of the PI3K/Akt signaling pathway, which further activates expression of the downstream Bcl-2 gene.

Involvement of leak K^+ channels in neurological disorders

TWIK-related acid-sensitive K+(TASK) channels give rise to leak K+ currents which influence the resting membrane potential and input resistance. The wide expression of TASK1 and TASK3 channels in the central nervous system suggests that these channels are critically involved in neurological disorders. It has become apparent in the past decade that TASK channels play critical roles for the development of various neurological disorders. In this review, I describe evidence for their roles in ischemia, epilepsy, learning/memory/cognition and apoptosis.

Neuroprotective effects of a mitochondrial K^+-ATP channel opener (diazoxide) are mediated by Bcl-2 expression upregulation

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.

K X-ray fluorescent source for energy-channel calibration of the spectrometer

A new K X-ray fluorescent source for calibrating the X or γ-ray multichannel analyzer spectrometer is introduced. A detailed description of the K fluorescent source device is given. The calibration method used and experimental results obtained are presented. The purity and efficiency of K fluorescence photons from this device are discussed. This new fluorescent source may be used as a substitute for radioactive nuclides for the energy-channel calibration of some MCA spectrometers.

K-ATP channel openers facilitate glutamate uptake by GluTs in rat primary cultured Astrocytes

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.

Numerical analysis of turbulent mixed convection air flow in inclined plane channel with k-εtype turbulence model

Numerical study on turbulent mixed convection in inclined plane channels,from 15° to 90° (vertical),was carried out to examine the effect of inclination on fluid flow and heat transfer distributions.The turbulent air flows upward or downward into the duct with one wall heated from bottom.Calculation results with several kinds of k-εtype turbulence models were used to compare the experimental data with those in literatures to determine suitable model.The dependents of Nusselt number on the inclination angle of both the buoyancy-aided and buoyancy-opposed flow are discussed.

Voltage-dependent K^+-channel Responses during Activation and Damage in Alveolar Macrophages Induced by Quartz Particles

The roles of voltage-dependent K^＋ channels during activation and damage in alveolar macrophages （AMs） exposed to different silica particles were examined. Rat AMs were collected by means of bronchoalveolar lavage, and were adjusted to 5× 10^5/mL. After AMs were exposed to different concentrations （0, 25, 50, 100, 200 μg/mL） of quartz particles and 100 μg/mL amorphous silica particles for 24 h, the voltage-depended K^＋ current in AMs was measured by using patch clamp technique. Meanwhile the leakage of lactate dehydrogenase （LDH） and the viability of AMs were detected respectively. Patch clamp studies demonstrated that AMs possessed outward delayed and inward rectifying K^＋ current. Exposure to quartz particles increased the outward delayed K^＋ current but it had no effect on inward rectifier K^＋ current in AMs. Neither of the two K^＋ channels in AMs was affected by amorphous silica particles. Cytotoxicity test showed that both silica particles could damage AM membrane and result in significant leakage of LDH （P〈0.05）. MTT studies, however, showed that only quartz particles reduced viability of AMs （P〈0.05）. It is concluded that quartz parti- cles can activate the outward delayed K^＋ channel in AMs, which may act as an activating signal in AMs to initiate an inflammatory response during damage and necrosis in AMs induced by exposure to quartz particle. K^＋ channels do not contribute to the membrane damage of AMs.