吡那地尔及二氮嗪对长期低氧大鼠肺动脉平滑肌ＫＡＴＰ通道蛋白表达的影响

目的:研究慢性低氧对大鼠肺动脉平滑肌ATP敏感钾通道(KATP)蛋白表达的影响及KATP开放剂吡那地尔及二氮嗪的作用.方法:SD雄性大鼠35只随机分成对照组、低氧组、吡那地尔干预组[吡那地尔2.0 mg/(kg·d),ig]、二氮嗪干预组[二氮嗪1.5 ms/(ks·d),ig]、5-羟癸酸(5-HD) 二氮嗪干预组[5-HD 3.0 ms/(kg·d),ig;二氮嗪1.5 ms/(ks·d),ig],每组7只.将低氧组和各干预组大鼠放入常压低氧舱内[O2(10.0%±0.5%)],每周6天,每天6 h 4周后测定平均肺动脉压(mPAP)并采用Western-blot技术,分析各组肺动脉主干平滑肌KATP蛋白表达.结果:①慢性低氧组大鼠的mPAP显著高于正常对照组,吡那地尔干预组肺动脉压较低氧组显著下降.二氮嗪干预组加重慢性低氧所致的肺动脉压力升高,5.HD Z.氮嗪干预组的mPAP显著低于二氮嗪干预组,P均<0.05.②低氧组调节亚基磺酰脲受体2B(SUR2B)蛋白水平显著低于正常组,吡那地尔干预组SUR2B显著高于低氧组,二氮嗪干预组SUR2B蛋白水平显著低于低氧组,5-HD 二氮嗪干预组SUR2B显著高于二氮嗪于预组,与低氧组亦有显著统计学差异,P均<0.05.各组内向整流性孔区6.1(Kir6.1)蛋白没有显著差异(P<0.05).结论:慢性低氧抑制KATP通道蛋白的表达,而吡那地尔能提高表达,对慢性低氧所致的肺动脉高压和肺血管壁重构具有较好的预防和逆转作用;二氮嗪能加重低氧性肺动脉压升高,并抑制KATP通道蛋白的表达.     

吡那地尔对人肺动脉平滑肌细胞KATP通道mRNA表达的影响

目的:研究经典KATP开放剂吡那地尔对原代培养人肺动脉平滑肌细胞 KATP 通道mRNA表达的影响,以探讨KATP通道在肺动脉高压发生发展中的分子生物学机制.方法:分离人3～4级肺小动脉,原代培养人肺动脉平滑肌细胞.分成ET-1组、ET-1 吡那地尔组及ET-1 吡那地尔 格列本脲组.以Trizol法抽提总RNA,逆转录为cDNA,采用Real-time PCR方法检测各组细胞KATP通道SUR2B和Kir亚单位mRNA表达量.结果:ET-1 组 KATP通道SUR2B mRNA表达量较对照组明显减少,为对照组的0.09±0.01倍(P<0.05,n=3).加用吡那地尔可拮抗ET-1作用,提高SUR2B mRNA表达量,为ET-1组的10.94±1.13倍,为对照组的0.97±0.03倍(P>0.05,n=3).格列本脲可拈抗吡那地尔作用,减少SUR2B mRNA表达量,为ET-1 吡那地尔组的0.10±0.01倍,为对照组的0.07±0.02倍(P<0.05,n=3).各组细胞KATP通道 Kir6.1 mRNA 表达量无统计学差异.结论:KATP 开放剂吡那地尔可增加原代培养人肺动脉平滑肌细胞KATP通道SUR2B mRNA表达量,这可能是其调节肺动脉平滑肌细胞KATP通道表达和功能的机制.     

埃他卡林对ＥＴ-１诱导的人肺动脉平滑肌细胞ＫＡＴＰ通道蛋白表达的影响

目的:研究新型ATP敏感性钾通道(KATP)开放剂埃他卡林对内皮素-1(ET-1)诱导的人肺动脉平滑肌细胞(HPASMCs)上KATP蛋白表达的影响.方法:原代培养人肺动脉平滑肌细胞,随机分成对照组,ET-1组,ET-1 埃他卡林组,ET-1 吡那地尔组,ET-1 埃他卡林 格列本脲组,ET-1 吡那地尔 格列本脲组,用Western-blot方法分析各组KATP蛋白磺酰脲受体亚单位(SUR2B)和内向整流性孔区亚单位(Kir6.1)表达变化情况.结果:与ET-1的作用相反,埃他卡林能使ET-1诱导下的SUR2B亚基表达升高,特异性KATP阻断剂格列本脲可逆转埃他卡林引起的SUR2B亚基表达升高;但各组对Kir6.1亚基表达无明显影响.结论:埃他卡林通过上调KATP的SUR2B亚基表达而发挥其在治疗低氧性肺动脉高压(HPH)中的作用,可望成为治疗低氧性肺动脉高压的新药.     

二氮嗪预处理对Aβ1-42作用神经元KATP各亚基表达的影响

目的探讨ATP敏感的钾离子(KATP)通道开放药物二氮嗪防治A1-42细胞毒性作用的分子机制。方法采用细胞原代培养的方法,培养大鼠皮层神经元并进行鉴定。将原代培养的细胞随机分为对照组、单纯Aβ1-42干预组、二氮嗪预处理1 h后Aβ1-42干预组、单纯二氮嗪预处理组和单纯Aβ42-1干预组(Aβ1-42反序列对照),各组又分为24、72 h两个亚组。采用免疫荧光双染及免疫印迹法,观察干预后不同培养时间(24、72 h)细胞KATP通道各亚基Kir6.1、Kir6.2、SUR1、SUR2蛋白表达水平的变化。结果与对照组比较,单纯A1-42处理24 h组Kir6.1、SUR2显著升高(P<0.05),二氮嗪预处理后Aβ1-42作用细胞24 h组各亚基表达均无明显变化;二氮嗪预处理后Aβ1-42作用细胞72 h组与单纯A1-42处理72 h组KATP通道各亚基表达均明显升高(P<0.05),而二氮嗪预处理后Aβ1-42作用细胞72 h组与单纯A1-42处理72 h组相比Kir6.1、Kir6.2、SUR2表达显著下调(P<0.05)。结论二氮嗪预处理可完全逆转A1-42作用神经元24 h所引起的Kir6.2及SUR1的表达上调,只能部分逆转A1-42作用神经元72 h所引起的Kir6.1、Kir6.2、SUR2的表达增加,可能会维持神经细胞正常生理功能,起到防治A1-42细胞毒性作用。     

吡那地尔对大鼠心肌缺血/再灌注时细胞凋亡及fas基因表达的影响

目的　探讨 ATP敏感性钾通道 (KATP)开放剂吡那地尔对大鼠心肌缺血 /再灌注时心肌细胞凋亡及 fas基因蛋白表达的调节作用 .方法　 4 0只大鼠随机分成 4组 :1假手术组 (仅穿线而不结扎 ,观察 6 .5 h) ;2缺血 /再灌注组(缺血 30 min,再灌注 6 h) ;3吡那地尔组 (缺血前 10 m in静推吡那地尔 0 .2 mg· kg- 1 ,然后缺血 30 min/再灌注 6 h) ;4吡那地尔 +优降糖组 (缺血前 10 min静推吡那地尔 0 .2 m g·kg- 1 +优降糖 3mg·kg- 1 ,然后缺血 30 min/再灌注 6 h) .称质量法计算心肌梗死范围 ,以缺口末端标记法 (TUNEL)检测凋亡细胞 ,以 SABC免疫组化法检测 fas蛋白的表达 .结果　缺血 /再灌注组出现显著的心肌细胞凋亡 ,伴有 fas蛋白表达指数升高 (P<0 .0 1vs假手术组 ) ;吡那地尔可抑制心肌细胞凋亡 ,并显著下调 fas蛋白的表达 (P<0 .0 5 ) ,而优降糖可取消吡那地尔的作用 .结论　细胞凋亡及 fas基因表达改变参与了心肌缺血 /再灌注损伤过程 ,KATP开放剂 (吡那地尔 )可抑制心肌细胞凋亡、下调 fas基因的蛋白表达 .     

吡那地尔对大鼠局灶性脑缺血再灌注后caspase-3及Bcl-2蛋白表达的影响

目的探讨吡那地尔对大鼠局灶性脑缺血再灌注后Caspase-3及Bcl-2蛋白表达的影响。方法40只Wistar雄性大鼠随机分为假手术组、对照组、KATP开放剂治疗组(开放剂组)及KATP开放剂 阻断剂治疗组(阻断剂组)。应用线栓法制备大鼠大脑中动脉缺血模型(MCAO),应用TTC染色检测脑梗死体积,应用TUNEL法检测神经元凋亡,应用免疫组化方法检测caspase-3及Bcl-2蛋白表达。结果开放剂组脑梗死体积显著小于对照组和阻断剂组(P<0.01),对照组与阻断剂组之间差异无显著性(P>0.05);开放剂组神经元凋亡数较对照组和阻断剂组显著减少(P<0.01)。对照组与阻断剂组之间差异无显著性(P>0.05);开放剂组caspase-3蛋白表达显著少于对照组和阻断剂组(P<0.01),对照组与阻断剂组之间差异无显著性(P>0.05);开放剂组Bcl-2蛋白表达显著多于对照组和阻断剂组(P<0.01),对照组与阻断剂组之间差异无显著性(P>0.05)。结论KATP通道开放剂能显著减轻脑缺血再灌注后脑梗死体积、减少Caspase-3蛋白表达、增加Bcl-2蛋白表达、抑制神经元凋亡,对脑缺血再灌注损伤发挥保护作用。     

冠心康对大鼠缺血心肌细胞ATP敏感钾通道亚基Kir6.1、Kir6.2、SUR2A和SUR2B表达的影响

目的：通过观察中药复方冠心康对大鼠缺血心肌细胞ATP敏感钾（ATP-sensitive potassium,K_（ATP））通道亚基的影响,探讨冠心康保护心血管、抗心肌缺血的可能作用机制。方法：48只SPF级Wistar大鼠随机分为正常组、模型组、格列本脲组、吡那地尔组、冠心康组、冠心康加格列本脲组。采用酶解法分离大鼠心室肌细胞,用无钙台式液灌流10 min、停灌30 min、再灌注45 min模拟心肌缺血再灌注损伤。将药物直接加入细胞液中（格列本脲10μmol/L、吡那地尔50μmol/L、冠心康注射液1 ml/L）充分作用后,4℃放置24 h。采用实时荧光定量多聚酶链式反应法及蛋白质印迹法检测各组心肌细胞K_（ATP）亚基Kir6.1、Kir6.2、SUR2A和SUR2B的mRNA表达及蛋白含量。结果：正常大鼠心肌细胞可见SUR2A、Kir6.1和Kir6.2蛋白及mRNA的表达,而SUR2B蛋白几乎不表达。模型组K_（ATP）各个亚基蛋白及mRNA的表达均较正常组有一定程度的增加。与模型组比较,吡那地尔可显著增加K_（ATP）各亚基mRNA及蛋白的表达,格列本脲可阻断这一作用;冠心康可明显增加K_（ATP）通道各亚基mRNA及蛋白的表达。结论：复方冠心康具有明显的促进K_（ATP）开放的作用,从而起到心血管保护作用。     

吡那地尔对体外培养大鼠大脑皮层神经细胞缺氧缺糖损伤的保护作用

目的 :探讨ATP敏感性K+ 通道 (KATP)开放剂吡那地尔 (pinacidil,Pin)对缺氧缺糖再复氧损伤大鼠大脑皮层神经细胞的保护作用。方法 :体外培养大鼠大脑皮层神经细胞 ,细胞培养至 10d ,建立神经细胞缺氧缺糖损伤模型 ,观察Pin及KATP阻断剂格列苯脲对缺氧缺糖不同时间 ,再复氧 2 4h后细胞死亡率、丙二醛 (MDA)含量、超氧化物歧化酶 (SOD)活力的影响。结果 :缺氧缺糖、再复氧后大鼠的神经细胞死亡率均显著升高、MDA生成增多、SOD的活力下降 ,Pin干预后 ,细胞死亡率下降、MDA生成减少、SOD的活力升高 ;格列苯脲能拮抗Pin这种保护作用。结论 :Pin对缺氧缺糖损伤神经细胞具有保护作用 ,并与拮抗氧自由基有关     

格列吡嗪对糖尿病大鼠心肌ATP敏感性钾通道基因表达的影响

目的:观察磺脲类药物格列吡嗪对糖尿病大鼠心肌组织中ATP敏感性钾通道(KATP)基因表达的影响.方法:50只雌性大鼠随机分为正常饮食组(n=10)、高脂饮食组(高脂饲料6周,n=10)和高脂饮食 小剂量链脲佐菌素(STZ)组(糖尿病模型组,高脂饮食6周25 mg/kg的剂量一次性腹腔注射链脲佐菌素,n=30);尾静脉采血测定各组大鼠血糖、血TG、Tch和血清胰岛素,采用稳态模型(HOMA)评价胰岛素抵抗(IR).成模大鼠随机分为糖尿病组、糖尿病胰岛素治疗组和糖尿病格列吡嗪组(n=10),另设对照组10只(正常大鼠予等量生理盐水灌胃).RT-PCR检测KATP组成亚基SUR2和Kir6.2的表达.结果:STZ注射后3 d糖尿病模型组大鼠空腹血糖均≥16.67 mmol/L,糖尿病模型建立成功.糖尿病格列吡嗪治疗组KATP SUR2和Kir6.2的表达水平与糖尿病组、糖尿病胰岛素治疗组以及对照组相比无显著差异.结论:成功建立大鼠2型糖尿病模型,STZ诱导的实验性糖尿病不影响心肌KATP SUR2和Kir6.2的表达水平;治疗剂量的格列吡嗪对心肌KATP SUR2和Kir6.2的表达无影响.     

Aβ1-42对大鼠基底前脑神经元KATP通道各亚基蛋白表达的影响

目的研究β淀粉样蛋白（Aβ_1-42）对原代培养基底前脑胆碱能神经元ATP敏感性钾通道（KATP）各亚基蛋白表达的影响,探讨阿尔茨海默病发病的细胞毒性分子机制。方法 运用细胞原代培养的方法培养大鼠基底前脑胆碱能神经元并进行鉴定, 用2μmmol/L的Aβ_1-42对原代培养细胞进行干预, 免疫荧光双染及免疫印记观察干预后不同时间(分别为0, 24, 72h)细胞KATP通道各亚基Kir6.1、Kir6.2和SUR1、SUR2蛋白表达水平的变化。结果与正常对照组比较,Aβ_1-42作用胆碱能神经元24h后, KATP通道亚基Kir6.1及SUR2蛋白表达显著增多(P<0.05),而亚基Kir6.2及SUR1蛋白表达无明显变化。但Aβ_1-42作用时间达72h后, KATP通道各个亚基蛋白表达均显著升高(P<0.05)。 结论 Aβ_1-422作用胆碱能神经元不同的时间段（24h和72h）,细胞KATP通道各亚基蛋白表达有不同程度的增加,且增加速度不一致。可能由此改变KATP通道的结构和功能,从而影响Aβ_1-42的神经细胞毒性作用。     

线粒体KATP在吡那地尔预处理后失血性休克大鼠组织血流灌注和线粒体功能保护效应中的作用

目的 观察吡那地尔(pinacidil)预处理对失血性休克大鼠血流灌注和线粒体功能的保护效应,及其与线粒体ATP激活钾通道[adenosine triphosphate(ATP)-activated potassium channels,KATP]的关系.方法 Wiser大鼠60只,随机数字表法分为:正常对照组、休克组...     

Effect of mitochondrial KATP channel on voltage-gated K+ channel in 24 hour-hypoxic human pulmonary artery smooth muscle cells

Background Hypoxic pulmonary hypertension (HPH) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). The mechanism of hypoxic pulmonary hypertension has not yet been fully elucidated. The mitochondrial ATP-sensitive K+ channel (MitoKATP) is extremely sensitive to hypoxia, and is a decisive factor in the control of mitochondrial membrane potential (ΔΨm). This study investigated the changes of cell membrane potential and Kv channel in cultured human pulmonary artery smooth muscle cell (hPASMC) exposed to 24 hour-hypoxia, and explored the role of MitoKATP and ΔΨm in this condition. Methods Fresh human lung tissues were obtained from the patients undergoing a chest operation. hPASMCs were isolated, cultured, and divided into 6 groups: ① control group, cultured under normoxia; ② diazoxide group, cultured in normoxia with diazoxide, an opener of MitoKATP; ③ 5-HD group, cultured in normoxia with sodium 5-hydroxydecanoate (5-HD), an antagonist of MitoKATP; ④ 24 hour-hypoxia group; ⑤ 24 hour-hypoxia + diazoxide group; and ⑥ 24 hour-hypoxia + 5HD group. Whole-cell patch-clamp technique was used to trace the cell membrane K+ currents. The expressions of cell membrane Kv1.5 mRNA and protein were determined by RT-PCR and Western blot technique, respectively. The relative changes in mitochondrial potential were tested with rhodamine fluorescence (R-123) technique. Results After exposure to diazoxide for 24 hours, the intensity of R-123 fluorescence in normoxic hPASMCs was significantly increased compared with control group (P&lt;0.05), but there were no significant changes in these tests after the hPASMCs had been exposed to 5-HD for 24 hours. Twenty-four hour-hypoxia or 24 hour-hypoxia + diazoxide could markedly increase the intensity of R-123 fluorescence in hPASMC and the changes were more significant in 24 hour-hypoxia +diazoxide group than in 24 hour-hypoxia group (P&lt;0.05) although 5-HD could partly weaken the effect of 24 hour-hypoxia on the intensity of R-123 fluorescence. After exposure to diazoxide for 24 hours, the cell membrane K+ currents and the expression of cell membrane Kv1.5 mRNA and protein in normoxic hPASMCs were significantly decreased compared with control group (P&lt;0.05), but there were no significant changes in these tests after the hPASMCs had been exposed to 5-HD for 24 hours. Also, 24 hour-hypoxia or 24 hour-hypoxia + diazoxide decreased the cell membrane K+ currents and the expression of Kv1.5 mRNA and protein (P&lt;0.05) but the changes were more significant in 24 hour-hypoxia + diazoxide group than in 24 hour-hypoxia group (P&lt;0.05). Again, 5-HD could partly weaken the inhibitory effect of 24 hour-hypoxia on the cell membrane K+ currents and the expression of Kv1.5 mRNA or protein (P&lt;0.05). Conclusions The opening of MitoKATP followed by a depolarization of ΔΨm in hypoxia might contribute to the alterations in the expression of cell membrane Kv1.5 mRNA and protein leading to change in the cell membrane potential of hypoxic hPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension.     

Greater hypertrophy in right than left ventricles is associated with pulmonary vasculopathy in sinoaortic-denervated Wistar-Kyoto rats

1. Biventricular hypertrophy has been described in a high blood pressure variability (BPV) model of sinoaortic-denervated (SAD) rats without systemic hypertension. To explore the possible involvement of the lung in SAD-induced right ventricular hypertrophy (RVH), we examined lung morphology, in addition to systemic haemodynamics and ventricle morphology, in Wistar-Kyoto rats 32 weeks after SAD. 2. In Wistar-Kyoto rats 32 weeks after SAD, there existed a substantial elevation in BPV, with no change in the average level of arterial pressure. Biventricular hypertrophy following SAD was characterized by a greater hypertrophy in right than left ventricles; both absolute and normalized right ventricular weights were significantly increased by 22 and 27％, respectively, and only normalized left ventricular weight was significantly increased by 12％. No infarcts were found in any ventricles examined. 3. In the lung, the most prominent change following SAD was pulmonary vasculopathy, including wall thickening, perivascular fibrosis and cell infiltration. In pulmonary arteries with an internal diameter of 70-130 microm, the external diameter, wall thickness and wall thickness to internal diameter ratio were increased in SAD compared with control rats. 4. There was no correlation between right and left ventricular weights. In contrast with BPV-correlated left ventricular weight, right ventricular weight was correlated with the wall thickness of the pulmonary artery, but not with BPV. 5. These findings suggest that greater RVH following SAD is associated with pulmonary vasculopathy, but is not secondary to the left ventricular problems or high BPV.     

Cardioprotective effects of mitochondrial KATP channels activated at different time

Backgroud Recent studies in adult hearts have indicated that KATP channels in the inner mitochondrial membrance are responsible for the protection. And we investigated whether opening of mitochondrial KATP channels (mKATP) could provide myocardial protection for immature rabbits and determined its role in cardioprotection.Methods Thirty-four 3-4-week-old rabbits, weighing 300-350 g, were divided randomly into five groups: Group Ⅰ (control group, n=8); Group Ⅱ ［diazoxide preconditioning group; n=8; the hearts were pretreated with 100 μmol/L diazoxide for 5 minutes followed by 10-minute wash out with Krebs-Henseleit buffer (KHB)］; Group Ⅲ ［diazoxide+5-hydroxydeconate (5-HD) preconditioning group; n=5; the hearts were pretreated with 100 μmol/L diazoxide and 100 μmol/L 5-HD); Group Ⅳ (diazoxide+cardioplegia group; n=8; cardioplegia containing 100 μmol/L diazoxide perfused the hearts for 5 minutes before ischemia); Group Ⅴ (diazoxide+5-HD+cardioplegia group; n=5; the cardioplegia contained 100 μmol/L diazoxide and 100 μmol/L 5-HD). All hearts were excised and connected to langendrff perfusion system and passively perfused with KHB at 38℃ under a pressure of 70 cmH2O. After reperfusion, the recovery rate of left ventricular diastolic pressure (LVDP), ±dp/dtmax, coronary flow (CF), the creatinine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) in coronary sinus venous effluent and the tissue ATP were measured. Mitochondria were evaluated semiquantitatively by morphology.Results After ischemia and reperfusion (I/R), the two groups that were treated by diazoxide only (Groups Ⅱ and Ⅳ) had a significant improvement in LVDP, ±dp/dtmax, and CF recovery. AST, LDH, and CK were decreased, and the levels of tissue ATP in the two groups were higher. Mitochondria was protected better in Group Ⅳ than in other groups. Conclusions Activating mKATP channels before and during ischemia can similarly protect immature rabbit hearts, and the mechanism is related to the direct protective effect on mitochondria. Opening of mKATP channel during ischemia provides a better protection for mitochondria than it does before ischemia.     

大鼠消化道平滑肌细胞K_(ATP)通道的牵张敏感性

目的探讨大鼠结肠平滑肌细胞上是否存在ATP敏感K+通道(ATP-sensitive potassium channel,KATP)及其牵张敏感性。方法用RT-PCR方法检测SD大鼠结肠平滑肌组织KATPmRNA的表达;联合应用单通道膜片钳技术和压力钳技术,采用inside-out模式记录大鼠结肠平滑肌细胞上KATP通道的电活动。结果在大鼠结肠平滑肌组织中可检测出较高水平的KATPmRNA的表达;用inside-out模式在大鼠结肠平滑肌上可记录到KATP通道的电活动,该通道在膜电位钳制电压为+60 mV时电导值为(44.5±1.5)pS。KATP通道特异性阻断剂——glibenclamide能抑制该通道的活动。且该KATP通道在负压刺激下开放概率增加。结论大鼠结肠平滑肌细胞上分布有KATP通道,该KATP通道对牵张刺激敏感。     

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.     

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 pro-posed 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 up-take 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 phosphoryla-tion of protein kinases C(PKC). These findings are the first to demonstrate that activation of K-ATP channel, especially mitochondri-al K-ATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phospho-rylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also pro-vides a new strategy for neuroprotection.     

银杏叶提取物延迟预处理对大鼠心肌缺血再灌注时细胞色素c氧化酶表达的影响

目的:探讨银杏叶提取物(EGb761)延迟预处理对大鼠心肌缺血再灌注时细胞色素c氧化酶(cytochrome c oxidase,CcO)表达的影响。方法:健康成年Sprague-Dawley雄性大鼠40只,随机分成4组:S组(假手术组),仅开胸并分离冠状动脉左前降支,但不阻断血流150 min;IR组(缺血再灌注组),行冠状动脉左前降支阻断30 min,再灌注120 min; M组(银杏叶提取物延迟预处理组),予以静脉注射银杏叶提取物EGb761 100 mg/kg,给药后24 h同IR组处理;D组[银杏叶提取物预处理+5-羟葵酸(5-HD)组],缺血前15 min静脉注射5-HD 5 mg/kg,余同M组处理。再灌注结束后测心肌CcO的表达和心肌梗死面积,观察心肌细胞超微结构。结果:与IR组[(37.87±5.92)%]比较,M组[(23.78±4.82)%]心肌梗死面积减小(P<0.05),D组[(39.62±5.18)%]差异无统计学意义(P>0.05)。与S组比,IR组、M组和D组CcO均升高(P<0.05);与IR组比,M组CcO增高(P<0.05)。电镜下,M组心肌细胞损伤程度较IR组减轻,D组与IR组差异无统计学意义(P>0.05)。结论:银杏叶提取物延迟预处理对大鼠心肌的保护作用与上调心肌CcO表达有关。     

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.