肺动脉高压大鼠肺动脉平滑肌细胞钾通道的研究

目的：观察肺动脉高压大鼠（PHR）肺动脉平滑肌细胞膜电容(Cm)、膜电流(I)、电流密度(pA/pF)及I-V曲线，并与正常SD大鼠进行比较。观察盐酸埃他卡林对正常血压及肺动脉高压大鼠动脉平滑肌钾通道的影响。方法：SD大鼠,置于常压缺氧（10%O2）舱内，每天6 h，每周6 d，持续4周,使之平均肺动脉压升高,建立肺动脉高压大鼠模型。急性分离大鼠肺内动脉平滑肌细胞，用全细胞记录（whole cell recording）技术记录细胞钾电流、膜电容并计算电流密度。结果：PHR肺动脉平滑肌细胞Cm值、细胞膜钾电流值均显著高于正常血压SD大鼠（P<0.05）；PHR肺动脉平滑肌细胞钾电流密度值显著低于正常血压大鼠(P<0.05)。与正常SD大鼠比较，PHR钾电流I-V曲线下移。盐酸埃他卡林在10 μmol·L-1浓度下，可显著增强正常血压SD大鼠及PHR动脉平滑肌钾电流(P<0.05)。结论：PHR肺动脉平滑肌细胞的膜电容、膜钾电流比正常血压SD大鼠高；钾电流密度比正常血压SD大鼠低，I-V曲线下移。盐酸埃他卡林对正常血压大鼠及肺动脉高压大鼠动脉平滑肌钾电流都有增强作用。     

cGMP对慢性低氧大鼠肺动脉平滑肌细胞膜电压门控钾通道的作用

目的:探讨cGMP对慢性低氧大鼠肺动脉平滑肌细胞(PASMC)膜电压门控钾通道(Kv通道)的作用, 为进一步阐明慢性低氧性肺动脉高压的发病机理提供理论依据。方法: Wistar大鼠, 随机分为对照组和慢性低氧组, 低氧组大鼠每天低氧(氧浓度10%±1%)8 h, 连续4周。单个大鼠PASMC的获得采用急性酶分离法(胶原酶Ⅰ型和木瓜蛋白酶)。采用全细胞膜片钳技术测定两组PASMC的静息膜电位(Em)和电压门控钾通道的钾离子电流(I_KV), 观察并比较cGMP (1 mmol/L) 以及cGMP和蛋白激酶G(PKG)抑制剂H-8 (1 mmol/L) 应用后两组PASMC I_KV的不同变化。结果:慢性低氧大鼠PASMC的静息膜电位和电压门控钾通道电流明显低于正常对照组。cGMP可抑制正常和慢性低氧大鼠PASMC +50 mV刺激时的峰值I_KV[正常组从(118.0±5.0)pA/pF下降到(89.9±16.5) pA/pF, n=6, P<0.05;慢性低氧组则从(81.0±5.0) pA/pF 下降到(56.8±9.1) pA/pF, n=6, P<0.05], 该抑制作用可被PKG的抑制剂H-8阻断[正常组(119.2±10.3) pA/pF vs (117.8±9.1)　pA/pF, n=6, P>0.05;慢性低氧组(96.8±6.2) pA/pF vs (98.0±2.2)　pA/pF, n=6, P>0.05]。结论:慢性低氧抑制肺动脉平滑肌细胞的电压门控钾通道。cGMP可能通过磷酸化作用而抑制正常和慢性低氧肺动脉平滑肌细胞的电压门控钾通道电流。     

Differential expression of the mechanosensitive potassium channel TREK-1 in epicardial and endocardial myocytes in rat ventricle

Mechanoelectric feedback (MEF) is the process by which mechanical forces on the myocardium induce electrical responses. It is thought that MEF is important in controlling the beat to beat force of contraction in the ventricle, in response to fluctuations in load, and it may also play a role in controlling the dispersion of repolarization. The transduction mechanism for MEF is via stretch sensitive ion channels in the surface membrane of myocytes. Two types of stretch sensitive channels have been described; a non-selective cation channel, and a potassium selective channel. TREK-1 is a member of the recently cloned tandem pore potassium channels that has been shown to be mechanosensitive and to be expressed in rat heart. Here we report that the gene expression level of TREK-1, quantified using real-time RT-PCR against glyceraldehyde phosphate dehydrogenase (GAPDH) as a comparator gene, was found to be 0.34 +/- 0.14 in endocardial cells compared to 0.02 +/- 0.02 in epicardial cells (P < 0.05). To confirm that this is reflected in a different current density, whole cell TREK-1 currents, activated by chloroform, were recorded with patch clamp techniques in epicardial and endocardial cells. TREK-1 current density in epicardial and endocardial cells was 0.21 +/- 0.06 pA/pF and 0.8 +/- 0.27 pA/pF, respectively (P相似文献   

Lysophosphatidylcholine decreases single channel conductance of inward rectifier K channel in mammalian ventricular myocytes

Effect of lysophosphatidylcholine (LPC) on the inward rectifier K channel of the isolated guinea pig ventricular cell was studied using patch clamp technique. In that LPC (100 microM) decreased the magnitude of the single channel conductance from 48 +/- 5 pS (mean and S.D., n = 5) to 12 +/- 9 pS (n = 8), this event may be the prime factor related to the alleged LPC-induced depolarization of cardiac tissues.     

敲低IK1钾通道抑制ClC-3氯通道的表达和功能

 目的: 探讨ClC-3氯通道是否为IK1钾通道的调节靶点,重点研究鼻咽癌细胞IK1钾通道对ClC-3氯通道功能及蛋白表达的影响。方法: 采用siRNA转染技术抑制低分化鼻咽癌上皮细胞(CNE-2Z) IK1 基因的表达;real-time PCR技术检测ClC-3 mRNA的表达;Western blot检测ClC-3的蛋白表达;细胞免疫荧光结合激光共聚焦显微镜技术检测ClC-3和IK1蛋白在细胞内分布;全细胞膜片钳记录细胞氯电流。结果: IK1 siRNA可以成功转染CNE-2Z细胞,有效抑制鼻咽癌细胞IK1钾离子通道的表达;用IK1 siRNA抑制鼻咽癌细胞IK1钾离子通道的表达后, ClC-3的mRNA表达上调而ClC-3蛋白却表达减少:在低分化鼻咽癌上皮细胞,低渗刺激可激活氯通道,产生一个较大的氯电流,在成功转染IK1 siRNA的细胞,此氯电流明显减弱。结论: 敲低IK1钾离子通道可抑制ClC-3氯离子通道的表达和功能。     

病毒感染大鼠心室肌细胞钙通道基因表达及功能改变的研究

目的：研究大鼠心室肌细胞感染柯萨奇病毒B3（CVB3）后L型钙通道mRNA表达量及其电生理特性的变化。 方法: 用CVB3感染培养的SD大鼠心室肌细胞，采用半定量逆转录-聚合酶链式反应技术，检测病毒感染心室肌细胞L型钙通道各亚单位mRNA表达量的变化；用全细胞膜片钳技术，观察病毒感染前后心室肌细胞L型钙电流（ICa-L）的变化。结果: 病毒感染组心室肌细胞L型钙通道α1和β亚单位mRNA的表达量显著高于正常对照组（4.00±0.07 vs 2.21±0.41, P<0.01; 2.06±0.06 vs 1.22±0.30, P<0.05），而α2/δ亚单位mRNA表达量的改变不明显（4.12±0.19 vs 4.13±0.27, P>0.05）；感染组细胞ICa-L的平均电流密度明显大于正常组细胞［（-8.66±0.99) pA/pF vs (-6.97±1.75) pA/pF, P<0.01］，且前者的电流-电压曲线下移，峰电流密度增加25.74%（P<0.05）。结论: CVB3感染心室肌细胞后，使其L型钙通道α1和β亚单位mRNA的表达量增加，ICa-L增大，可能是病毒感染导致心肌出现异常电生理活动的细胞和分子机制之一。     

2型糖尿病大鼠心室肌细胞离子通道功能和表达变化

目的:探讨2型糖尿病大鼠心室肌细胞离子通道电流及其相关蛋白表达的改变。方法:采用Zucker糖尿病肥胖(Zucker diabetic fatty,ZDF)大鼠建立2型糖尿病大鼠模型,Zucker瘦型(Zucker lean,ZL)大鼠为对照组。采用急性酶解法分离大鼠单个心室肌细胞,采用全细胞膜片钳技术记录其动作电位、L型钙通道电流(I_Ca-L)及瞬时外向钾电流(I_to)的变化;提取大鼠心肌组织蛋白,采用Western blot检测心肌细胞肥大标志物β-肌球蛋白重链(β-myosin heavy chain,β-MHC)和心房钠尿肽(atrial natriuretic peptide,ANP),以及L型钙通道(Cav1.2)和钾通道(Kv4.3)的蛋白表达水平。结果:用高脂饮食诱导ZDF大鼠成功建立2型糖尿病模型;与ZL大鼠相比,ZDF大鼠心室肌细胞的动作电位时程显著延长,I_Ca-L和I_to密度显著降低[峰值分别为(?5.96±0.37)pA/pF vs(?4.92±0.30)pA/pF,(12.43±0.86)pA/pF vs(7.48±0.58)pA/pF,均P<0.05];与ZL大鼠相比,ZDF大鼠心肌组织中β-MHC和ANP的表达水平明显增加,伴有Cav1.2和Kv4.3蛋白表达下降(P<0.05)。结论:与ZL大鼠相比,2型糖尿病大鼠心肌细胞肥大,心室肌细胞动作电位时程延长,I_Ca-L和I_to密度及其相关蛋白表达水平降低,提示糖尿病大鼠心室肌细胞发生电生理重构。     

低O2高CO2对大鼠肺中小动脉可溶性鸟苷酸环化酶基因及其蛋白表达的影响

目的:探讨慢性低O₂高CO₂时大鼠肺动脉可溶性鸟苷酸环化酶(sGC)基因及其蛋白表达的变化。方法:复制大鼠低O₂高CO₂肺动脉高压模型, 免疫组织化学技术观察低O₂高CO₂肺动脉高压1、2、4周组及对照组肺中小动脉sGCα₁、β₁亚基蛋白的表达, 原位杂交技术观察肺中小动脉sGCα₁亚基mRNA的表达。结果:低O₂高CO₂1、2、4周组肺中小动脉sGCα₁、β₁亚基蛋白及α₁亚基mRNA表达弱于对照组(均P＜0.01)。结论:低O₂高CO₂抑制肺中小动脉sGCmRNA及其蛋白的表达, 参与肺动脉高压形成与发展。     

Hypoxia decreases expression of soluble guanylate cyclase in cultured rat pulmonary artery smooth muscle cells

Nitric oxide (NO) has an important role in modulating the pulmonary vascular tone. NO acts, in part, by stimulating soluble guanylate cyclase (sGC) to synthesize the intracellular second messenger cyclic GMP. In vascular smooth muscle cells, sGC is a heterodimer composed of alpha1 and beta1 subunits. The objective of this study was to test whether oxygen concentration regulates sGC expression in cultured rat pulmonary artery smooth muscle cells (rPaSMC). rPaSMC were exposed to 0, 3, and 20% oxygen for 1-48 h, and sGC subunit mRNA levels were measured. Compared with rPaSMC exposed to 20% oxygen, sGC alpha1 and beta1 subunit mRNA levels were markedly decreased in rPaSMC exposed to 0% and 3% oxygen. The decrease in sGC subunit mRNA levels in hypoxic rPaSMC was detected as early as 6 h of exposure. Compared with rPaSMC exposed to 20% oxygen, exposure of rPaSMC to 3% oxygen progressively decreased sGC subunit protein levels at 24 and 48 h. There was also a 30% and 50% decrease in sGC enzyme activity in cells exposed to hypoxia for 24 and 48 h (P < 0.05 and P < 0.001, respectively, as compared with cells maintained in normoxia). These results demonstrate that hypoxia decreases sGC expression in cultured pulmonary artery smooth muscle cells and suggest that, in hypoxic vascular smooth muscle, decreased cyclic GMP synthesis may limit the vasodilator response to NO.     

慢性缺氧对肺动脉内皮细胞Ⅲ型一氧化氮合酶mRNA表达的影响

目的和方法:采用原位杂交技术结合图象分析检测常氧(PO₂21.3kPa)及慢性缺氧(PO₂5.3±0.7kPa)培养的猪肺动脉内皮细胞Ⅲ型一氧化氮合酶mRNA的表达及其对急性缺氧刺激反应的变化。结果:常氧条件培养的肺动脉内皮细胞急性缺氧12h,NOSⅢmRNA表达明显增加(2、4、6代分别比缺氧前增加48%、125%、119%,P＜0.05);慢性缺氧条件下培养的肺动脉内皮细胞急性缺氧12h,NOSⅢmRNA表达亦明显增加(分别比急性缺氧前增加92%、245%、565%,P＜0.01)。其增加的幅度在慢性缺氧培养组均高于同代常氧组,且随着缺氧时间的延长,这种差别更显著。结论:慢性缺氧增强了肺动脉内皮细胞Ⅲ型一氧化氮合酶基因对急性缺氧的反应,这可能在慢性缺氧时肺血管对缺氧的反应性降低中,具有重要作用。     

慢性缺氧改变肺动脉内皮细胞急性缺氧时ET-1mRNA的表达

目的:检测常氧及慢性缺氧培养的猪肺动脉内皮细胞(PAEC)内皮素(ET)－1mRNA的表达及其对急性缺氧刺激的反应。方法:原位杂交技术结合图像分析。结果:常氧及慢性缺氧培养的PAEC急性缺氧后,ET－1mRNA表达均增加(P<0.05),以慢性缺氧组升高的幅度更大。结论:慢性缺氧可增强PAEC在急性缺氧时ET-1mRNA的表达。     

Regulation of gadd153 mRNA expression by hypoxia in pulmonary artery smooth muscle cells.

Hypoxia causes pulmonary hypertension and induces oxygen radicals in pulmonary artery smooth muscle cells (PASMCs). Since oxidative stress regulates gaddl53 expression, we examined gaddl53 mRNA in PASMCs cultured in a hypoxic environment. Gadd153 mRNA content was increased in PASMCs cultured for 24 hours in 1% oxygen. This increase was not abrogated by inhibition of protein synthesis. To explore the signaling pathways mediating hypoxic regulation of gaddl53 mRNA, the impact of calcium channel blockade by verapamil, G protein inhibition by pertussis toxin, and protein kinase C (PKC) down-regulation, was examined. Although none of these interventions reduced basal expression of gaddl53 mRNA in PASMCs, all of them suppressed the induction by hypoxia. In contrast, antioxidants had no effect. These observations indicate hypoxia induces gaddl53 expression in PASMCs through common signaling pathways.     

慢性低氧对大鼠肺组织电压门控钾通道表达的影响

目的:探讨慢性低氧对大鼠肺组织电压门控钾通道亚型Kv1.5、Kv2.1、Kv9.3表达的影响。方法:将12只Wistar大鼠随机分为对照组和慢性低氧组,每组6只。采用RT-PCR和Westernblot对大鼠肺组织匀浆中Kv1.5、Kv2.1、Kv9.3mRNA和Kv1.5蛋白质的表达进行观察。结果:慢性低氧组大鼠肺组织匀浆中Kv1.5、Kv2.1、Kv9.3mRNA和Kv1.5蛋白质表达明显低于对照组(P<0.01)。结论:慢性低氧可以从转录、翻译两个水平抑制大鼠肺组织中Kv1.5、Kv2.1Kv9.3的表达,Kv1.5、Kv2.1、Kv9.3表达的减少必将导致Kv数目的减少和电流的降低。Kv1.5、Kv2.1、Kv9.3可能是氧敏感钾通道亚型。     

VEGF和PCNA在慢性低氧性肺动脉及高压大鼠主动脉、肺动脉平滑肌细胞中表达

目的研究慢性低氧性肺动脉高压(PAH)发生过程中,血管内皮生长因子(VEGF)及细胞核增殖抗原(PCNA)在体循环血管、肺循环血管平滑肌细胞中的表达。方法利用低压缺氧舱建立大鼠缺氧性肺动脉高压模型。实验分为3组即正常氧组、缺氧2wk组和缺氧3wk组。用免疫组化染色和图像分析,检测主动脉、肺动脉主干及肺内小动脉平滑肌细胞中VEGF及PCNA的表达量。结果VEGF在正常氧组大鼠的主动脉、肺动脉主干及肺内小动脉平滑肌内均有表达;缺氧组大鼠肺动脉主干及肺内小动脉平滑肌细胞内VEGF的表达明显增强并随着缺氧时间的延长而增加;主动脉平滑肌内VEGF的表达量无明显变化。PCNA在正常氧组大鼠的主动脉、肺动脉主干及肺内小动脉平滑肌内均有微弱表达;但缺氧时只有肺小动脉平滑肌内其表达量增加;主动脉、肺动脉主干平滑肌内PCNA的表达量无明显差别。结论在缺氧性肺动脉高压的发生过程中,VEGF在体循环血管、肺循环血管平滑肌细胞中的表达量具有差异性,提示其可能在肺动脉高压形成过程中起重要作用。     

慢性阻塞性肺疾病合并慢性缺氧患者肺动脉平滑肌细胞电压门控钾通道亚型基因表达的变化

目的：探讨人肺动脉平滑肌细胞（PASMCs）的几种Kv通道亚型：Kv1.2、 Kv1.3、Kv1.5、Kv2.1、Kv3.1等，在COPD合并慢性缺氧时基因表达的变化。旨在探索预防人类肺源性心脏病的发生和找到新的防治方法提供试验依据。 方法： 从手术室切取人正常肺组织、单纯COPD患者和COPD合并慢性缺氧患者肺组织，将标本分为：①正常对照的PASMCs、单纯COPD和COPD合并慢性缺氧患者的PASMCs；②正常对照的PASMCs和经过慢性缺氧培养的PASMCs。利用半定量RT-PCR技术，分析Kv1.2、Kv1.3、Kv1.5、Kv2.1、Kv3.1等的基因表达。 结果： ①Kv1.2、Kv1.3、Kv1.5、Kv2.1、Kv3.1等基因在正常PASMCs和单纯COPD患者PASMCs中均有表达，而且两者无显著差异；②Kv1.2、Kv1.5、Kv2.1在患者在体慢性缺氧和离体慢性缺氧时的表达均明显降低（P<0.05）；Kv1.3在患者在体慢性缺氧时表达明显降低（P<0.05），而离体慢性缺氧时无显著变化（P>0.05）；Kv3.1在患者在体慢性缺氧和离体慢性缺氧时的表达均无显著变化（P>0.05）；③Kv1.2、Kv1.5、Kv2.1、Kv3.1等基因在单纯COPD时表达显著上调（P<0.05）。 结论： 在慢性缺氧情况下，Kv1.2、Kv1.3、Kv1.5、Kv2.1 4种亚型基因表达明显下降，提示可能在促进人肺动脉高压的形成和发展中起重要作用。而慢性缺氧对Kv3.1基因表达无显著影响，提示它们可能对缺氧不敏感，在人肺动脉高压发生中处于次要地位。至于在单纯COPD时几种亚型的表达上调，原因不清楚，需进一步研究证实。     

Hypoxia suppresses Kv 2.1 channel expression through endogenous 15-hydroxyeicosatetraenoic acid in rat pulmonary artery

We have previously reported that hypoxia activates lung 15-lipoxygenase (15-LOX), which catalyzes arachidonic acid to produce 15-HETE, leading to constriction of neonatal rabbit pulmonary arteries. Hypoxia suppresses Kv2.1 channel expression. Although the Kv channel inhibition by hypoxia is likely to be mediated through 15-HETE, direct evidence is still lacking. To explore whether 15-LOX/15-HETE pathway contributes to the hypoxia-induced down-regulation of Kv2.1 channel, we performed studies using 15-LOX blockers, semi-quantitative PCR and western blot analysis. We found that Kv2.1 channel expression at the mRNA and protein levels was greatly up-regulated in pulmonary arterial smooth muscle cells (PASMCs) and pulmonary artery (PA) after blockade of endogenous 15-HETE under hypoxic condition. 15-HETE further decreased Kv2.1 channel expression in comparison with 12-HETE and 5-HETE in cultured PASMCs and PA under normoxic conditions. These data indicate that hypoxia suppresses Kv2.1 channel expression through endogenous 15-HETE in PA.     

Characterization of the large-conductance Ca-activated K channel in myocytes of rat saphenous artery

We used the patch-clamp method to characterize the BK channel in freshly isolated myocytes from the saphenous branch of the rat femoral artery. Single-channel recordings revealed that the BK channel had a conductance of 187 pS in symmetrical 150 mM KCl, was blocked by external tetraethylammonium (TEA) with a KD(TEA) of approx. 300 microM at +40 mV, and by submicromolar charybdotoxin (CTX). The sensitivity of the BK channel to Ca was especially high (KD(ca) approx. 0.1 microM at +60 mV) compared to skeletal muscle and neuronal tissues. We also investigated the macroscopic K current, which under certain conditions is essentially sustained by BK channels. This conclusion is based on the findings that the macroscopic current activated upon depolarization follows a single exponential time course and is virtually fully blocked by 100 nM CTX and 5 mM external TEA. We made use of this occurrence to assess the voltage and Ca dependence of the macroscopic BK current. In intact myocytes, the BK channel showed a strong and voltage-dependent reduction of the outward current (62% at +40 mV), most likely due to block by intracellular Ba and polyamines. The results obtained from macroscopic and unitary current indicate that approx. 2.5% of the BK channels are active under physiological conditions, sustaining approx. 20 pA of outward current. Given the high input resistance of these cells, few BK channels are required to open in order to cause a significant membrane hyperpolarization, and thus function to limit the contraction resulting from acute increases in intravascular pressure, or in response to hypertensive pathologies.     

Effects of monocarboxylic acid-derived Cl- channel blockers on depolarization-activated potassium currents in rat ventricular myocytes

The effects of monocarboxylic acid-derived Cl(-) channel blockers on cardiac depolarization-activated K(+) currents were investigated. Membrane currents in rat ventricular myocytes were recorded using the whole-cell configuration of the patch-clamp technique. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and niflumic acid (NFA) induced an outward current at 0 mV. Both NPPB and NFA failed to induce any current when used intracellularly or after K(+) in the bath and pipette solutions was replaced by equimolar Cs(+). Voltage pulse protocols revealed that NPPB and NFA enhanced the steady-state K(+) current but inhibited the transient outward K(+) current. Genistein, a tyrosine kinase (PTK) inhibitor, inhibited NPPB- and NFA-induced outward current. Another PTK inhibitor, lavendustin A, produced a comparable effect. In contrast, the inactive analogue of genistein, daidzein, was ineffective. Orthovanadate, a tyrosine phosphatase inhibitor, markedly slowed the deactivation of the outward current induced by NPPB and NFA. The protein kinase A (PKA) inhibitor H-89 inhibited NPPB-induced outward current at 0 mV. In contrast, the protein kinase C (PKC) inhibitor H-7 was without significant effect on the action of NPPB. Pretreatment of the myocytes with genistein or H-89 prevented the enhancing effect of NPPB. Increasing intracellular Cl(-) from 22 to 132 mm slightly reduced NPPB-induced outward current at 0 mV. These results demonstrate that the monocarboxylic acid-derived Cl(-) channel blockers NPPB and NFA enhance cardiac steady-state K(+) current, and suggest that the enhancing effect of the Cl(-) channel blockers is mediated by stimulation of PKA and PTK signalling pathways.