西地那非抑制低氧对人肺动脉平滑肌细胞电压依赖性钾通道的下调作用

目的探讨西地那非对低氧下调人肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)电压依赖性钾通道(voltage-dependent potassium channels,Kv)的干预作用。方法应用膜片钳技术记录PASMCs K+电流;观察低氧刺激前后钾离子(K+)电流的变化以及西地那非(sildenafil)的干预作用;通过特异性Kv1.5抗体透析,分析西地那非作用于人PASMCs Kv分子靶点即通道亚单位;运用Real-time PCR及Western blotting技术检测低氧刺激前后及西地那非对人PASMCs上的Kv1.5通道基因转录水平和蛋白表达水平的影响。结果低氧明显抑制了人PASMCs细胞膜上的全细胞K+电流;而西地那非则可以对抗低氧对全细胞K+电流的抑制作用;而且西地那非对低氧下人PASMCs Kv的调控作用靶点主要是Kv1.5;同时西地那非部分恢复了低氧抑制的细胞上的Kv1.5 mRNA和蛋白表达。结论西地那非能够抑制低氧对人肺动脉平滑肌细胞电压依赖性钾通道功能与表达的下调作用。     

K~+v通道在缺氧性肺血管收缩中的作用

目的 探讨K+v通道在缺氧性肺血管收缩(HPV)中的作用.方法 Wistar大鼠40只随机分为两组:常氧对照组和低氧组,采用酶消化的方法获得单个肺动脉血管平滑肌细胞(PASMC),将分离细胞经a-actin免疫组化鉴定为PASMC.采用膜片钳技术记录PASMC静息膜电位(Em)和电压门控性钾通道电流(Ikv).细胞内灌流Kv1.2,Kv1.3,Kv1.5,Kv2.1,Kv9.3抗体混合液(1:125),探讨钾通道在HPV中的作用.结果 低氧组膜电位明显去极化,细胞内灌流Kv1.2,Kv1.3,Kv1.5,Kv2.1,Kv9.3抗体混合液可显著抑制常氧对照组PASMC的Ikv,使Em去极化,细胞内灌流Kv1.2,Kv1.3,Kv1.5,Kv2.1,Kv9.3抗体混合液对低氧组PASMC的Ikv和Em均无显著影响.结论 Kv1.2,Kv1.3,Kv1.5,Kv2.1,Kv9.3可能是氧敏感型通道,并可能介导了缺氧性肺血管收缩.     

K+v通道亚型在急性缺氧性肺血管收缩中的作用研究

目的 探讨K+v通道亚型在急性缺氧性肺血管收缩(HPV)中的作用.方法 Wistar大鼠30只随机分为两组:常氧对照组和低氧组,采用酶消化的方法 获得单个肺动脉血管平滑肌细胞(PASMC).将分离细胞经a-actin免疫组化鉴定为PASMC细胞.采用膜片钳技术记录PASMC静息膜电位(Em)和电压门控性钾通道电流(Ikv).细胞内灌流Kvl.2、Kvl.3、Kvl.5、Kv2.1抗体混合液(1:125),探讨钾通道在HPV中的作用.结果 低氧组膜电位明显去极化,细胞内灌流Kvl.2、Kvl.3、Kvl.5、Kv2.1抗体混合液可显著抑制常氧对照组PASMC的Ikv,使Em去极化,细胞内灌流Kvl.2、Kvl.3、Kvl.5、Kv2.1抗体混合液对低氧组PASMC的Ikv和Em均无显著影响.结论 Kvl.2、Kvl.3、Kvl.5、Kv2.1可能是氧敏感型通道,并可能介导了急性缺氧性肺血管收缩.     

K+v通道在缺氧性肺血管收缩中的作用研究

目的 探讨K+v通道在缺氧性肺血管收缩（HPV）中的作用。方法Wistar大鼠40只随机分为两组:常氧对照组和低氧组, 采用酶消化的方法获得单个肺动脉血管平滑肌细胞（PASMC）,将分离细胞经a-actin免疫组化鉴定为PASMC。采用膜片钳技术记录PASMC静息膜电位（Em）和电压门控性钾通道电流（Ikv）。细胞内灌流Kv1.2、Kv1.3、Kv1.5、Kv2.1、Kv9.3抗体混合液（1：125）,探讨钾通道在HPV中的作用。结果 低氧组膜电位明显去极化,细胞内灌流Kv1.2、Kv1.3、Kv1.5、Kv2.1、Kv9.3抗体混合液可显著抑制常氧对照组PASMC的Ikv,使Em去极化,细胞内灌流Kv1.2、Kv1.3、Kv1.5、Kv2.1、Kv9.3抗体混合液对低氧组PASMC的Ikv和Em均无显著影响。结论Kv1.2、Kv1.3、Kv1.5、Kv2.1、Kv9.3可能是氧敏感型通道,并可能介导了缺氧性肺血管收缩。     

蛋白激酶C对慢性吸烟大鼠支气管平滑肌细胞膜电压门控钾通道的作用

目的 探讨蛋白激酶C对慢性吸烟大鼠支气管平滑肌细胞（bronchial smooth muscle cells, BMSCs）膜电压门控钾通道（Kv通道）的作用，为慢性阻塞性肺疾病的防治提供理论依据。方法 建立大鼠的慢性吸烟模型。大鼠随机分为吸烟组（A组）和对照组（B组）。采用急性酶分离法获得单个大鼠的支气管平滑肌细胞。用全细胞膜片钳技术测定其膜电位和Kv通道电流。比较用蛋白激酶C激动剂12-佛波醇脂（PMA）和抑制剂GFX前后两组细胞Kv通道电流的不同变化。结果 慢性吸烟大鼠BSMC的静息膜电位和Kv通道电流明显低于对照组。PKC可抑制正常大鼠BSMC+50 mV刺激时的峰值Kv通道电流（从78.3±10.5 pA/pF 下降到50.4±8.6 pA/pF, n=10 cells, P<0.05）。该抑制作用可被GFX抵消（77.9±7.8 pA/pF vs 70.4±9.6 pA/pF, n=10 cells, P>0.05）。但是PKC对慢性吸烟大鼠的BSMC的Kv通道电流无抑制作用（58.3±7.6 pA/pF vs 56.4±6.3 pA/pF, n=10 cells, P>0.05）。结论 慢性吸烟可抑制大鼠BMSC膜电压门控钾通道（Kv通道）的电流。PKC活化后对正常大鼠的BSMC有抑制作用，但对慢性吸烟大鼠的BSMC则无抑制作用。 【关键词】吸烟 支气管 钾通道     

MitoK_(ATP)-PKC通路对人体肺动脉平滑肌细胞电压门控钾通道的影响

目的 以人体肺动脉平滑肌细胞(HPASMCs)为研究对象,研究线粒体膜上ATP敏感的钾通道(MitoKATP)对HPASMCs膜电位及电压门控钾通道(KV1.5)表达的影响,以及蛋白激酶C(PKC)在其信号转导中的可能作用.方法 从人正常肺组织分离出HPASMCs进行培养.利用激光共聚焦显微镜技术检测细胞线粒体膜电位(ΔΨm),全细胞膜片钳技术记录细胞膜电流变化,Western blot检测KV1.5蛋白和PKC-α的表达情况.结果 ①Diazoxide组肺动脉平滑肌细胞PKC-α的表达比正常对照组明显增强(P<0.05);这种作用可被5-羟基癸酸盐(5-HD)的作用所逆转;单独应用5-HD,平滑肌细胞PKC-α的表达与对照组比较差异无显著性意义.② Diazoxide作用24 h可明显抑制Kv电流以及下调Kv1.5蛋白的表达(P<0.05);佛波酯(PMA) 作用24 h也可明显抑制Kv电流以及下调Kv1.5蛋白的表达(P<0.05),而加入R0-31-8220可以逆转PMA的这一作用;Diazoxide和PMA同时应用较其中之一单独应用对人肺动脉平滑肌细胞Kv电流和Kv1.5表达有更加明显的抑制作用(均P<0.05);Diazoxide与R0-31-8220同时应用则部分逆转了Diazoxide对肺动脉平滑肌细胞Kv电流和Kv1.5表达的抑制(均P<0.05).结论 MitoKATP的开放和ΔΨm的增加可抑制Kv通道的活性和表达,可能参与并影响了肺动脉高压的发生发展, PKC在其中可能起介导作用.     

电压门控性钾通道亚型Kv2.1在低氧性肺动脉高压形成和恢复中的作用

目的 研究电压门控性钾通道亚型Kv2.1基因表达变化在大鼠低氧性肺动脉高压形成以及恢复中的作用.方法 32只雄性SD大鼠均分为正常对照组、高原慢性低压低氧组(CH组)、二氯醋酸钠(DCA)治疗组(CH+DCA组)和高原低氧返回平原恢复7 d组(CHR7组).正常对照组在常压常氧平原条件下喂养,其余各组在模拟高原5 000 m低压氧舱内喂养21 d,动物模型建成后用闭式胸腔法测平均肺动脉压(mPAP),荧光定量PCR法检测4组大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)的Kv2.1 mRNA表达;免疫组织化学方法检测大鼠PASMCs Kv2.1的表达;图像分析技术检测肺小动脉形态改变及Kv2.1表达强度变化;Western blot法检测肺动脉平滑肌细胞Kv2.1蛋白表达.结果 模拟高原5 000 m缺氧21 d后,与正常对照组相比,CH组Kv2.1 mRNA和蛋白表达明显下降,免疫组化显示PASMCs Kv2.1表达的平均光密度值(mIOD)减少,而mPAP明显增高,肺小动脉管壁增厚,管腔狭窄,肺血管重构明显.与CH组相比,DCA+CH组和CHR,组Kv2.1 mRNA和蛋白则明显恢复表达,PASMCs Kv2.1表达的mIOD增加,mPAP下降,血管重构减弱.结论 Kv2.1基因表达变化与高原低氧性肺动脉高压变化存在负相关性,表明Kv2.1在低氧性肺动脉高压的形成和恢复中可能起着一定的作用.     

哮喘大鼠肺泡巨噬细胞电压依赖性钾通道的活性研究

目的探讨哮喘模型大鼠肺泡巨噬细胞(AM)电压依赖性钾通道(Kv)活性的变化及其对巨噬细胞功能的影响。方法利用卵蛋白(OVA)致敏并激发大鼠建立哮喘模型,收集支气管肺泡灌洗液(BALF)培养AM。利用全细胞电压钳模式记录AM细胞膜上的Kv电流,观察比较哮喘模型组和对照组Kv通道活性的变化。结果病理学检测发现哮喘模型组(n=7)炎性细胞浸润明显增加,BALF细胞总数显著增高,与对照组(n=7)比较差异显著(P<0.01),但AM百分比无明显差异;应用Kv通道特异性阻断剂4-氨基吡啶(4-AP)后电流幅度明显降低,证实检测到的AM细胞膜上的电流为Kv电流;哮喘大鼠AM细胞Kv电流幅度[(243.56±133.56)pA,n=10]较对照组[(656.23±162.34)pA,n=11]明显降低(P<0.01)。结论哮喘大鼠AM细胞Kv通道活性降低,可能导致AM兴奋性增高,易被激活分泌各种炎性介质和细胞因子,从而参与哮喘炎症的形成。     

L-精氨酸调节大鼠低氧性肺血管结构重建与肺动脉平滑肌细胞凋亡

目的:探讨L-精氨酸(L-arginine,L-Arg)对低氧性肺血管结构重建大鼠肺动脉平滑肌细胞凋亡的影响及其机制.方法:将17只Wistar大鼠随机分为对照组(n=7)、低氧组(n=5)和低氧+L-Arg组(n=5).对大鼠肺血管进行显微形态学观测.通过末端转移酶介导的dUTP切口末端标记法(TUNEL)检测肺动脉平滑肌细胞凋亡,并以免疫组织化学方法研究肺动脉平滑肌细胞Fas表达.结果:低氧2周后出现大鼠肺血管结构重建.同时,肺中、小型动脉凋亡平滑肌细胞百分数均明显低于对照组(P均＜0.05),平滑肌细胞Fas表达明显降低.然而,L-Arg缓解了低氧性肺血管结构重建的形成.低氧+L-Arg组大鼠肺中、小型动脉凋亡平滑肌细胞百分数均明显高于低氧组(P均＜0.05).L-Arg使低氧大鼠肺动脉平滑肌细胞Fas表达明显增强.结论:L-Arg通过使肺动脉平滑肌细胞Fas表达上调,促进肺动脉平滑肌细胞的凋亡,从而对低氧性肺血管结构重建有重要的调节作用.     

尼氟灭酸对低氧性肺动脉高压大鼠肺动脉平滑肌细胞mCLCA mRNA及MAPK表达的影响Symbol

目的 研究氯通道阻滞剂尼氟灭酸(niflumic acid,NFA)对低氧性肺动脉高压大鼠肺动脉平滑肌细胞钙激活氯离子通道蛋白 (chloride channel calcium activated protein,mouse;mCLCA) mRNA及丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK )表达的影响.方法 健康雄性SD大鼠24只,随机分为对照组、低氧组、低氧+用药组,每组8只.建立常压低氧肺动脉高压大鼠模型,低氧组及低氧+用药组均在低氧箱中缺氧8 h/d,共21 d.于缺氧前3 d开始,低氧+用药组每天腹腔内注射NFA 3 mg/kg体重,低氧组注射同等剂量对照剂.缺氧21 d后测量右心室肥厚指数(RVHI).分离并培养原代肺动脉平滑肌细胞;采用RT-PCR方法检测肺动脉平滑肌细胞mCLCA mRNA表达的变化;免疫细胞化学方法检测肺动脉平滑肌细胞中MAPK表达的变化.结果 低氧+用药组RVHI、mCLCA mRNA表达、MAPK表达均较低氧组明显降低(均P<0.05).结论氯通道阻滞剂NFA对低氧性肺动脉高压大鼠肺动脉平滑肌细胞mCLCA mRNA及MAPK的表达有抑制作用,从而推测NFA对低氧性肺动脉高压有一定治疗作用.     

The effect of protein kinase C on voltage-gated potassium channel in pulmonary artery smooth muscle cells from rats exposed to chronic hypoxia

Background Chronic hypoxia can cause pulmonary hypertension and pulmonary heart disease with high mortality.The signal transduction pathway of protein kinase C (PKC) plays an important role in chronic pulmonary hypertension. So it is necessary to investigate the effect of PKC on voltage-gated potassium (K+) channels in pulmonary artery smooth muscle cells of rats exposed to chronic hypoxia.Methods Male Wistar rats were randomly divided into a control group (group A) and a chronic hypoxia group (group B). Group B received hypoxia ［oxygen concentration （10±1）%］ eight hours per day for four consecutive weeks. Single pulmonary artery smooth muscle cells were obtained using an acute enzyme separation method. Conventional whole cell patch clamp technique was used to record resting membrane potential, membrane capacitance and voltage-gated K+ currents. The changes in voltage-gated K+ currents before and after applying paramethoxyamphetamine (PMA) (500 nmol/L), an agonist of PKC, and PMA plus carbohydrate mixture of glucose, fructose and xylitol (GFX) (30 nmol/L), an inhibitor of PKC, were compared between the two groups.Results The resting membrane potential in group B was significantly lower than that of group A: －(29.0±4.8) mV (n=18) vs －(42.5±4.6) mV (n=35) (P&lt;0.01). But there was no change in membrane capacitance between the two groups: (17.9±4.6) pF (n=40) vs (19.7±5.8) pF (n=31) (P&gt;0.05). The voltage-gated K+ currents were significantly inhibited by PMA in group A, and this effect was reversed by GFX. However, the voltage-gated K+ currents in group B were not affected by PMA.Conclusions The resting membrane potential and voltage-gated K+ currents in pulmonary artery smooth muscle cells from rats exposed to chronic hypoxia decreased significantly. It seems that PKC has different effects on the voltage-gated K+ currents of pulmonary artery smooth muscle cells under different conditions.     

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.     

尾核神经元的原代培养及离子通道特性的研究

  目的 建立一种稳定的大鼠尾核神经元的分离和体外培养方法,用于中枢神经元离子通道特性的研究。方法 自新生24 h内的Wistar乳鼠大脑中分离出尾核,结合酶消化及机械吹打分离尾核神经元,进行体外原代神经元培养。采用全细胞膜片钳技术记录电压门控钠通道电流和电压门控钾通道（Kv）电流。结果 分离出的神经元形态正常,细胞膜光滑有弹性,保存了主要离子通道的活性。结论 本方法适合用于中枢神经系统神经元离子通道的研究。     

一氧化氮对急、慢性缺氧大鼠血流动力学及缺氧性肺血管收缩反应的影响

观察了吸入０．００４％的一氧化氮（ＮＯ）对急、慢性缺氧大鼠血流动力学、缺氧性肺血管收缩反应（ＨＰＶ）、血气及高铁血红蛋白（ＭｅｔＨｂ）的影响。结果表明：（１）常氧吸入ＮＯ时能明显降低慢性缺氧大鼠肺动脉平均压（Ｐｐａ）和肺血管阻力（ＰＶＲ），但对正常大鼠的Ｐｐａ和ＰＶＲ无明显影响；（２）慢性缺氧大鼠急性缺氧时ＨＰＶ较正常大鼠弱，吸入ＮＯ不但降低两者的急性缺氧肺动脉高压，且完全逆转两者的ＨＰＶ；（３）吸入ＮＯ对急、慢性缺氧大鼠体循环血流动力学、血气及ＭｅｔＨｂ含量无明显影响。提示吸入ＮＯ能选择性降低，急、慢性缺氧性肺动脉高血压，且逆转ＨＰＶ。     

Relationship of Intracellular Free Ca^2＋ Concentration and Calcium-activated Chloride Channels of Pulmonary Artery Smooth Muscle Cells in Rats under Hypoxic Conditions

Hypoxic pul monary hypertension ( HPH) ischaracterized by an elevation of pul monary vascularreaction and pul monary vascular reconstruction.Cytoplasmic free Ca2 concentration ([ Ca2 ]i)plays a key role in the regulation of vascular tone ,pul monary vasoconstriction and proliferation ofsmooth muscle cells[1]. The present study evalua-ted the role of [Ca2 ]iin the regulation of pul mo-nary vascular tone through regulation of calcium-activated chloride (Clca) channels in rats under a-cute h…     

Wirkung der Lipoxygenase- und Cyclooxygenase-Metaboliten auf die akute hypoxische pulmonale Vasokonstriktion beim Schweinchen

The role of lipoxygenase and cyclooxygeriase metabolites in acute hypoxic pulmonary vasoconstriction (HPV) was studied in piglets, it has been found that acute alveolar hypoxia induced remarkable pulmonary vasoconstrion, associated with an increase in cardiac output. The hypoxic pulmonary vasoconstriction response was insignificantly attenuated after infusion of DEC. indomethacin potentiated mar kedly the increase in pulmonary artery pressure and pulmonary vascular resistance and thus augmented HPV. It is inferred that hypoxic pulmonary vsaoconstriction in piglet may be mediated by other important mediators in addition to leukotrienes, but modulated by prostaglandins to prevent an excessive rise in pulmonary artery pressure.