瞬时受体电位亚族M7在小鼠心肌梗死后的表达变化及其意义

目的 观察小鼠心肌梗死后成纤维细胞(CF)上瞬时受体电位亚族M7样电流(TRPM7L)的变化及其对Ca2+信号的影响,探讨瞬时受体电位亚族(TRP)M7离子通道在CF功能中的作用.方法 制备小鼠心肌梗死模型并分离小鼠CF,培养传代小鼠CF及其基因沉默技术(SiRNA)转染,应用膜片钳技术观察小鼠CF缺血后及低pH值溶液下TRPM7L内外向电流特征,用钙荧光显像技术观察缺血心肌、酸性pH值时小鼠CF的Ca2+内流变化,测定缺血时小鼠CF细胞总胶原含量改变.结果 (1)小鼠CF上富含TRPM7表达,且TRPM7L电生理特性与TRPM7通道一致;(2)心肌缺血使小鼠CF的Ca2+内流较对照组增加[(7.4±0.7)pA/pF比(16.2±1.7)pA/pF,P<0.01],而酸中毒时增加3倍;(3)SiRNA显著减少TRPM7L的mRNA丰度和90%的电流幅值.结论 TRPM7是小鼠CF细胞中TRPM7L的分子基础;TRPM7L是CF中钙内流主要媒介;致纤维化因素(缺血、酸中毒等)通过调节TRPM7L介导的Ca2+信号机制影响小鼠CF细胞的病理生理功能,在心肌纤维化的形成中可能发挥重要作用.     

瞬时受体电位通道亚族M7样电流在小鼠心肌梗死后的变化及意义

目的 观察小鼠心肌成纤维细胞(CFs)在心肌梗死后瞬时受体电位通道亚族M7(TRPM7)样电流的变化及其对胶原生成的影响,探讨TRPM7离子通道在心脏纤维化形成中的潜在病理生理作用. 方法 (1)制备小鼠心肌梗死模型并分离CFs细胞;(2)培养传代CFs及小分子干扰RNA(SiRNA)技术感染;(3)应用膜片钳技术观察CFs缺血后TRPM7通道内外向电流特征;(4)钙荧光显像技术观察缺血心肌对CFs的钙离子内流影响;(5)测定缺血对CFs总胶原含量的影响.结果 (1)心肌缺血能使CFs的含钙离子内向电流较对照组显著增加,分别为(7.4±0.7)pA/pF和(16.25±1.7)pA/pF(P<0.05);(2)SiRNA使TRPM7样电流的mRNA水平明显下降,且电流幅值减少约90%;(3)心肌缺血组CFs的TRPM的mRNA丰度及总胶原含量较基础值增加2.3倍左右.结论 致纤维化因子心肌缺血可通过TRPM7样电流介导的钙离子信号机制调节CFs功能,在心肌组织纤维化的病理生理过程中发挥重要作用.     

氯通道参与脑血管平滑肌细胞Ca2+池操纵性Ca2+内流

研究Cl-通道在牛脑血管平滑肌细胞Ca2+池操纵性Ca2+内流中的作用.采用培养的脑血管平滑肌细胞,在生物荧光双波长影像分析系统用Fura-2/Am荧光探针测定单个细胞内游离Ca2+浓度.结果发现① Cl-通道阻断剂DIDS(0.75 μmol/L)能降低内皮素1(10-7 mol/L)刺激引起的脑血管平滑肌细胞Ca2+内流,抑制率为29.6%±3.9%,随后加入Cl-通道阻断剂NPPB(10 μmol/L)能继续降低平台相,抑制率为44.9%±8.7%;交换加药顺序,NPPB能降低内皮素1刺激引起的Ca2+内流,DIDS能进一步降低Ca2+内流.②DIDS(0.75 μmol/L)能降低三磷酸腺苷(10 μmol/L)刺激引起的脑血管平滑肌细胞Ca2+内流,抑制率为26.7%±10.5%,随后加入NPPB(10 μmol/L)能继续降低平台相,抑制率为54.3%±9.6%;交换加药顺序,NPPB能降低三磷酸腺苷刺激引起的Ca2+内流,DIDS能进一步降低Ca2+内流.③DIDS(0.75 μmol/L)能降低环匹阿尼酸(10 μmol/L)刺激引起的脑血管平滑肌细胞Ca2+内流,抑制率为26.5%±5.0%,随后加入NPPB(10 μmol/L)能继续降低平台相,抑制率为46.1%±4.2%;交换加药顺序,NPPB能降低环匹阿尼酸刺激引起的Ca2+内流,DIDS能进一步降低Ca2+内流.提示对DIDS、NPPB敏感的非同一性Cl-通道参与内皮素1、三磷酸腺苷、环匹阿尼酸触发的脑血管平滑肌细胞 Ca2+池操纵性Ca2+内流.     

肾上腺素诱导的血管平滑肌细胞C1~-电流及其与Ca2+运动的关系

为观察肾上腺素对主动脉平滑肌细胞C1-电流的影响及其与Ca2+内流的关系,采用膜片钳单离子通道(细胞贴附式)技术和Fura-2荧光法测定细胞内游离Ca2+浓度变化.结果发现,10 μmol/L肾上腺素可引起氯通道开放概率由对照组的0.061±0.0042增加到0.690±0.011;平均开放时间由1.08±0.23 ms延长到6.44±0.57 ms.此Cl-电流可被硝苯地平和EGTA抑制.肾上腺素可引起平滑肌细胞内游离Ca2+浓度由静息时77±13 nmol/L快速升高达峰值随后维持在高水平的内流平台相,达216±27 nmol/L.Cl-通道阻断剂尼氟灭酸在一定范围内呈浓度依赖性抑制肾上腺素诱发的Cl-电流及Ca2+内流,8 μmol/L 尼氟灭酸对细胞内游离Ca2+浓度的抑制率达27%±8%.结果表明,Cl-通道开放在调节平滑肌细胞Ca2+内流中起重要作用.     

肥大和衰竭心肌L型Ca2+通道研究进展

心肌细胞 L 型钙电流 （ IC a· L）触发了 Ca2 +诱导的 Ca2 +释放 （ CICR） ,调节着细胞内瞬时 Ca2 +动力学 ,从而在兴奋—收缩耦联过程中起关键性作用。ICa· L密度和通道功能的改变参与了心力衰竭的发生。作者综述了 :1L型 Ca2 +通道的结构和生理作用 ;2肥大和衰竭的心肌 ICa· L变化及其频率依赖性调节机制 ;3 Ca2 + 通道和心力衰竭的治疗关系 ;4L型 Ca2 +通道未来研究方向     

肺动脉平滑肌细胞上的钙离子通道研究进展

钙离子(Ca2+)为肺动脉平滑肌细胞(PASMC)内至关重要的第二信史,其细胞内浓度的精细变化直接受到多种Ca2+通道的调控.按照细胞内Ca2+的来源,位于细胞膜上,调控细胞外Ca2+进入细胞的通道称为钙内流通道,位于肌质网上调控内质网/肌质网内钙库的Ca2+释放的通道称为钙释放通道.根据Ca2+通道激活方式的不同,C...     

Na+/Ca2+交换体α-2(807-844)抗体的制备及其对大鼠心肌细胞Na+/Ca2+交换电流的抑制作用

目的 在制备和纯化抗心肌Na+/Ca2+交换体α-2(807- 844)肽段抗体的基础上,观察抗体对大鼠心肌细胞Na+/Ca2+交换电流及L型钙电流(ICa)、瞬时外向钾电流(Ito)和内向整流钾电流(Ik1)的影响.方法 利用人工合成的α-2(807-844)多肽免疫兔制备抗α-2(807- 844)抗体,抗血清经Protein A亲和柱纯化,利用全细胞膜片钳技术观察纯化后抗体对心肌细胞Na+/Ca2+交换电流(INa/Ca)、L型钙电流(ICa)、瞬时外向钾电流(Ito)和内向整流钾电流(Ik1)的影响.结果 经主动免疫,抗Na+/Ca2+交换体α- 2(840-877)抗血清效价达1∶243000,纯化后抗α-2(840- 877)抗体浓度为7.21 mg/mL.纯化后的抗体在SDS - PAGE时出现清晰的2条带,其分子量分别为25 kD和50 kD左右.在1 nmol/L～104 nmol/L浓度范围内,抗α-2抗体对成年大鼠心肌细胞外向和内向Na+/Ca2+变换电流均表现为剂量依赖性的抑制作用；对Ito和Ik1则均未见明显影响.此外,104 nmol/L该抗体对L型钙电流亦具有抑制作用.通过氨基酸序列比对发现,α- 2(807 - 844)肽段与L型钙通道第2结构域孔环(697～730位氨基酸)序列相似度为23.7％,可能是抗体对L型钙通道出现交叉反应的原因.结论 在1 nmol/L～103 nmol/L浓度范围内,抗心肌Na+/Ca2+变换体α-2(807- 844)抗体可特异性抑制大鼠心肌Na+/Ca2+变换活动；对L型钙通道电流、瞬时外向钾通道电流和内向整流钾通道电流均未见明显影响.     

肾上腺素诱导的血管平滑肌细胞C1~-电流及其与Ca~(2 )运动的关系

为观察肾上腺素对主动脉平滑肌细胞C1- 电流的影响及其与Ca2 内流的关系 ,采用膜片钳单离子通道(细胞贴附式 )技术和Fura 2荧光法测定细胞内游离Ca2 浓度变化。结果发现 ,10 μmol L肾上腺素可引起氯通道开放概率由对照组的 0 .0 6 1± 0 .0 0 4 2增加到 0 .6 90± 0 .0 11;平均开放时间由 1.0 8± 0 .2 3ms延长到 6 .4 4± 0 .5 7ms。此Cl- 电流可被硝苯地平和EGTA抑制。肾上腺素可引起平滑肌细胞内游离Ca2 浓度由静息时 77± 13nmol L快速升高达峰值随后维持在高水平的内流平台相 ,达 2 16± 2 7nmol L。Cl- 通道阻断剂尼氟灭酸在一定范围内呈浓度依赖性抑制肾上腺素诱发的Cl- 电流及Ca2 内流 ,8μmol L尼氟灭酸对细胞内游离Ca2 浓度的抑制率达 2 7%± 8%。结果表明 ,Cl- 通道开放在调节平滑肌细胞Ca2 内流中起重要作用。     

pH值改变对大鼠离体冠状动脉静息张力的影响及机制

目的 研究在基础张力状态下,不同pH对大鼠冠状动脉血管静息张力的影响并探讨机制.方法 采用离体血管张力记录方法,大鼠冠状动脉环的张力舒缩状态采用PowerLab和DMT系统记录.观察pH值梯度改变对大鼠冠脉血管环张力的影响.观察内外钙、Na+/Ca2+交换体抑制剂KB - R7943(1×10-6 mol/L)、氯离子通道阻断剂NPPB(3×10-5 mol/L)、钙通道阻断剂维拉帕米(1×10-5 mol/L)、Na+/H+交换体抑制剂氧氯吡咪(AM,3×10-5 mol/L)、Na+ -K+- ATP酶抑制剂哇巴因(1×10-6mol/L)、NO合酶抑制剂L - NAME(1×10 4 mol/L)对浴液pH6.4时冠脉张力的影响.结果 随胞外pH值逐渐降低,大鼠离体冠脉血管环的静息张力逐渐增强.外钙内流和内钙释放均参与pH6.4时的冠脉收缩,钙通道阻断剂Verapamil可部分阻断冠脉收缩幅度的升高.与对照组相比,KB- R7943、哇巴因对pH6.4时冠脉收缩幅度无显著影响(P＞0.05);NPPB、氨氯吡咪均可抑制pH6.4时冠脉收缩幅度的升高(P＜0.05);L - NAME可增强pH6.4时冠脉收缩幅度(P＜0.05).结论 酸中毒时,随胞外pH值降低,大鼠冠脉的静息张力升高.其作用机制可能与内外钙、氯通道、Na+/H+交换有关.     

钙离子在肿瘤坏死因子α诱导心肌肥大中的作用

目的 研究Ca2+在肿瘤坏死因子α(TNF-α)诱导心肌细胞肥大中的作用.方法 Lowry法测心肌细胞蛋白含量;计算机图像分析系统测心肌细胞体积;3H-亮氨酸掺入法测心肌细胞蛋白合成;Till阳离子测定系统观察胞内[Ca2+]i瞬变.结果 IP3R阻断剂2-APB(30 μmol/L)或RyR阻断剂ryanodine (50 μmol/L)能降低由TNF-α(100μg/L)诱导的心肌细胞蛋白合成、蛋白含量以及细胞体积增加,二者合用抑制作用更强.L型Ca2+通道阻断剂nifedipine(50 μmol)对上述反应无明显作用(P＞0.05).IP3R阻断剂2-APB(30μmol/L)或RyR阻断剂ryanodine(50 μmol/L)能降低由TNF-α(100 μg/L)诱导的心肌细胞内钙离子瞬变幅度增高,二者合用作用抑制更强.L型Ca2+通道阻断剂nifedipine(50μmol/L)对其无明显作用(P＞0.05).结论 TNF-α通过调节心肌细胞内钙离子浓度从而诱导心肌细胞肥大.而在此过程中TNF-α可能主要是通过作用IP3R和RyR促使胞内钙贮库Ca2+释放而引起胞内钙离子水平增高的,而非通过打开L型Ca2+通道.     

Evidence for functional expression of TRPM7 channels in human atrial myocytes

Transient receptor potential melastatin-7 (TRPM7) channels have been recently reported in human atrial fibroblasts and are believed to mediate fibrogenesis in human atrial fibrillation. The present study investigates whether TRPM7 channels are expressed in human atrial myocytes using whole-cell patch voltage-clamp, RT-PCR and Western blotting analysis. It was found that a gradually activated TRPM7-like current was recorded with a K(+)- and Mg(2+)-free pipette solution in human atrial myocytes. The current was enhanced by removing extracellular Ca(2+) and Mg(2+), and the current increase could be inhibited by Ni(2+) or Ba(2+). The TRPM7-like current was potentiated by acidic pH and inhibited by La(3+) and 2-aminoethoxydiphenyl borate. In addition, Ca(2+)-activated TRPM4-like current was recorded in human atrial myocytes with the addition of the Ca(2+) ionophore A23187 in bath solution. RT-PCR and Western immunoblot analysis revealed that in addition to TRPM4, TRPM7 channel current, mRNA and protein expression were evident in human atrial myocytes. Interestingly, TRPM7 channel protein, but not TRPM4 channel protein, was significantly increased in human atrial specimens from the patients with atrial fibrillation. Our results demonstrate for the first time that functional TRPM7 channels are present in human atrial myocytes, and the channel expression is upregulated in the atria with atrial fibrillation.     

TRPM7 channels in hippocampal neurons detect levels of extracellular divalent cations

Exposure to low Ca(2+) and/or Mg(2+) is tolerated by cardiac myocytes, astrocytes, and neurons, but restoration to normal divalent cation levels paradoxically causes Ca(2+) overload and cell death. This phenomenon has been called the "Ca(2+) paradox" of ischemia-reperfusion. The mechanism by which a decrease in extracellular Ca(2+) and Mg(2+) is "detected" and triggers subsequent cell death is unknown. Transient periods of brain ischemia are characterized by substantial decreases in extracellular Ca(2+) and Mg(2+) that mimic the initial condition of the Ca(2+) paradox. In CA1 hippocampal neurons, lowering extracellular divalents stimulates a nonselective cation current. We show that this current resembles TRPM7 currents in several ways. Both (i) respond to transient decreases in extracellular divalents with inward currents and cell excitation, (ii) demonstrate outward rectification that depends on the presence of extracellular divalents, (iii) are inhibited by physiological concentrations of intracellular Mg(2+), (iv) are enhanced by intracellular phosphatidylinositol 4,5-bisphosphate (PIP(2)), and (v) can be inhibited by Galphaq-linked G protein-coupled receptors linked to phospholipase C beta1-induced hydrolysis of PIP(2). Furthermore, suppression of TRPM7 expression in hippocampal neurons strongly depressed the inward currents evoked by lowering extracellular divalents. Finally, we show that activation of TRPM7 channels by lowering divalents significantly contributes to cell death. Together, the results demonstrate that TRPM7 contributes to the mechanism by which hippocampal neurons "detect" reductions in extracellular divalents and provide a means by which TRPM7 contributes to neuronal death during transient brain ischemia.     

Effects of intracellular acidosis on [Ca2+]i transients, transsarcolemmal Ca2+ fluxes, and contraction in ventricular myocytes

We examined the effects of intracellular acidosis produced by washout of NH4Cl on [Ca2+]i transients (indo-1 fluorescence), cell contraction (video motion detector), and 45Ca and 24Na fluxes in cultured chick embryo ventricular myocytes. Exposure of cells to 10 mM NH4Cl produced intracellular alkalosis (pH 7.6), and subsequent washout resulted in a transient acidosis (pH 6.5). Exposure to 10 mM NH4Cl slightly decreased [Ca2+]i transients but increased the amplitude of cell contraction. Subsequent washout of NH4Cl initially increased diastolic [Ca2+]i and decreased the peak positive and negative d[Ca2+]i/dt, while the amplitude of cell contraction was markedly decreased. Subsequently, peak systolic [Ca2+]i increased with partial recovery of contraction. A similar increase in [Ca2+]i and decrease in contraction after washout of NH4Cl was observed in single paced adult guinea pig ventricular cells. Acidosis decreased 45Ca uptake by sarcoplasmic reticulum vesicles isolated from chick embryo ventricle. However, the [Ca2+]i increase caused by intracellular acidosis was also observed in the presence of 10 mM caffeine, suggesting that altered sarcoplasmic reticulum handling of calcium is not the only mechanism involved. Intracellular acidosis only slightly increased total 24Na uptake under these conditions, an effect resulting from the combination of a stimulation of amiloride-sensitive sodium influx (Na(+)-H+ exchange) and inhibition of sodium influx via Na(+)-Ca2+ exchange, manifested by a significant decrease in 45Ca efflux. Further support for a lack of involvement of an increased [Na+]i in the observed increase in [Ca2+]i during acidosis was low-sodium, nominal 0-calcium extracellular solution, an experimental condition that minimizes the possible effects of Na(+)-H+ exchange and Na(+)-Ca2+ exchange. We conclude that the [Ca2+]i increase caused by intracellular acidosis in cultured ventricular cells is primarily due to changes in [Ca2+]i buffering and [Ca2+]i extrusion, rather than to an increase in transsarcolemmal calcium influx. Intracellular acidosis also markedly decreases the sensitivity of the contractile elements to [Ca2+]i in cultured chick embryonic and adult guinea pig ventricular myocytes.     

Transient receptor potential channels in cardiovascular function and disease

Sustained elevation in the intracellular Ca2+ concentration via Ca2+ influx, which is activated by a variety of mechanisms, plays a central regulatory role for cardiovascular functions. Recent molecular biological research has disclosed an unexpectedly diverse array of Ca(2+-entry channel molecules involved in this Ca2+ influx. These include more than ten transient receptor potential (TRP) superfamily members such as TRPC1, TRPC3-6, TRPV1, TRPV2, TRPV4, TRPM4, TRPM7, and polycystin (TRPP2). Most of them appear to be multimodally activated or modulated and show relevant features to both acute hemodynamic control and long-term remodeling of the cardiovascular system, and many of them have been found to respond not only to receptor stimulation but also to various forms of stimuli. There is good evidence to implicate TRPC1 in neointimal hyperplasia after vascular injury via store-depletion-operated Ca2+ entry. TRPC6 likely contributes to receptor-operated and mechanosensitive Ca2+ mobilizations, being involved in vasoconstrictor and myogenic responses and pulmonary arterial proliferation and its associated disease (idiopathic pulmonary arterial hypertension). Considerable evidence has also been accumulated for unique involvement of TRPV1 in blood flow/pressure regulation via sensory vasoactive neuropeptide release. New lines of evidence suggest that TRPV2 may act as a Ca2+-overloading pathway associated with dystrophic cardiomyopathy, TRPV4 as a mediator of endothelium-dependent hyperpolarization, TRPM7 as a proproliferative vascular Mg2+ entry channel, and TRPP2 as a Ca2+-entry channel requisite for vascular integrity. This review attempts to provide an overview of the current knowledge on TRP proteins and discuss their possible roles in cardiovascular functions and diseases.     

Interaction between calcium and hydrogen ions in canine coronary arteries

In vitro experiments were carried out to study the effects of "respiratory" and "metabolic" acid-base alterations on canine coronary arteries precontracted by high potassium. Alkalosis (pH 7.70 +/- 0.02) increased the coronary tone by 33 +/- 3% (P less than 0.05) and acidosis (pH 7.10 +/- 0.01) decreased it by 30 +/- 3% (P less than 0.05). The tension stabilized in approximately 15 mins and its variation was of similar extent whether changes in extracellular pH were produced by changes in PCO2 or in bicarbonate concentration. The contractile changes were not abolished neither by alpha or beta adrenergic blockade nor by endothelium denudation. The effect of pH could not be offset by altering extracellular potassium or calcium when the plateau of the relationship between contractility of the smooth muscle and concentration of these ions was reached. When tonus developed by the strips and [Ca2+]0 were plotted reciprocally, DTmax significantly increased in alkalosis and decreased in acidosis. No significant changes in Ca50 ([Ca2+]0 necessary to obtain 50% of maximal force) were detected. pH changes did not modify Ca2+ influx but a relaxation of similar extent induced by nifedipine produced a detectable decrease in Ca2+ influx. Ca2+ efflux was augmented in acidosis and depressed in alkalosis. Our data do not support the hypothesis of hydrogen ion changes inducing calcium influx alterations in a competitive fashion as the mechanism involved to contract or relax conduit coronary arteries. On the contrary, they suggest that acid-base variations alter the contractile activity of vascular smooth muscle by modulating Ca2+ efflux.     

Two-pore domain K channel, TASK-1, in pulmonary artery smooth muscle cells

Pulmonary vascular tone is strongly influenced by the resting membrane potential of smooth muscle cells, depolarization promoting Ca2+ influx, and contraction. The resting potential is determined largely by the activity of K+-selective ion channels, the molecular nature of which has been debated for some time. In this study, we provide strong evidence that the two-pore domain K+ channel, TASK-1, mediates a noninactivating, background K+ current (IKN), which sets the resting membrane potential in rabbit pulmonary artery smooth muscle cells (PASMCs). TASK-1 mRNA was found to be present in PASMCs, and the membranes of PASMCs contained TASK-1 protein. Both IKN and the resting potential were found to be exquisitely sensitive to extracellular pH, acidosis inhibiting the current and causing depolarization. Moreover, IKN and the resting potential were enhanced by halothane (1 mmol/L), inhibited by Zn2+ (100 to 200 micromol/L) and anandamide (10 micromol/L), but insensitive to cytoplasmic Ca2+. These properties are all diagnostic of TASK-1 channels and add to previously identified features of IKN that are shared with TASK-1, such as inhibition by hypoxia, low sensitivity to 4-aminopyridine and quinine and insensitivity to tetraethylammonium ions. It is therefore concluded that TASK-1 channels are major contributors to the resting potential in pulmonary artery smooth muscle. They are likely to play an important role in mediating pulmonary vascular responses to changes in extracellular pH, and they could be responsible for the modulatory effects of pH on hypoxic pulmonary vasoconstriction.