钙离子通道的分子结构及其亚单位的功能

钙离子通道是镶嵌于膜脂质中的糖蛋白或外侧糖基化的蛋白复合体，广泛存在于细胞膜表面，形成一个功能单位或小孔，对Ｃａ２＋具有高度的选择通透性，控制着离子的转运与细胞的功能。细胞内多种酶系统都对细胞内Ｃａ２＋非常敏感，Ｃａ２＋作为“第二信使”，进行着细胞调节的功能。此外，在心肌细胞的兴奋－收缩偶联中，Ｃａ２＋有着重要的作用     

通脉汤含药血清对缺氧心肌细胞游离钙及L型钙通道的作用

目的:研究通脉汤对缺氧心肌细胞钙离子超负荷的保护性作用机制。方法:采用血清药理学方法及建立心肌细胞体外缺氧模型,直接测定不同条件下心肌细胞内的游离钙离子浓度,并通过RT-PCR方法分析细胞膜上L型钙离子通道的表达。结果:在通脉汤含药血清干预下缺氧心肌细胞的游离钙离子浓度显著降低(376.89±78.78nmol/L),达到与左旋氨氯地平相当的水平,并同时降低缺氧条件下的细胞膜L型钙离子通道的表达,而左旋氨氯地平对L型钙通道的表达没有影响。结论:通脉汤可以减轻缺氧心肌细胞钙超负荷,并与钙拮抗剂起到协同作用。     

联合成像系统研究心肌细胞微区力学及细胞内钙离子变化

目的 使用联合成像系统在亚细胞水平研究心肌细胞微区力学性质及细胞内钙离子的快速变化,并探讨两者之间的关系.方法 应用原子力显微镜(AFM)和激光扫描共聚焦显微镜(LSCM)组合成的联合成像系统对急性分离的大鼠单个心室肌细胞微区力进行检测,同时测定细胞内钙离子浓度的变化,分析细胞微区力和细胞内钙离子浓度变化两者之间的相关性.结果 观察到心肌细胞内钙离子浓度的快速变化引起了钙波的传递.通过AFM微悬臂的偏转可以计算出细胞微区力,细胞微区力的大小与细胞内钙离子浓度呈正相关(r=0.701,P=0.003).结论 联合成像系统可以同步研究心肌细胞微区力学及细胞内分子快速变化,进而在亚细胞水平研究心脏疾病的发病机制.     

心肌细胞膜上的Na~+/Ca~(2+)交换机制

钙离子作为动物细胞中的信使分子,特别是作为心肌兴奋-收缩耦联的触发者,在心肌节律性活动过程中受到精细的调节。实际上,钙离子的跨膜电化学梯度在细胞所含的各种阳离子中是最高的(约+130mV);加之在静息状况下心肌细胞内钙离子浓度很低(约50—200nmol/L);这样,在一个动作电位期间,胞内钙离子浓度可以有10倍左右的变化,从而保证     

极低频磁场对胞内钙振荡影响的机理分析

基于双钙库模型,本文以离子跨过细胞膜、细胞器膜迁移的几率作为细胞对电磁场的响应因子,来调制流过膜通道的离子的速率,进而影响细胞内钙离子的浓度。数值分析表明:在极低频磁场的作用下,一定能量因子下的频率条件或是一定频率因子下的能量条件可引起钙振荡形式的变化;窗效应是频率因子和能量因子共同作用的结果。     

心肌细胞内游离钙检测方法的研究进展

人们早就了解Ca~(2 )在心肌细胞的生化代谢、收缩功能及电生理特性中的作用。近几年来,人们发现许多药物作用机理如洋地黄类、抗心绞痛和抗心律失常类药物的作用与Ca~(2 )有关;并发现细胞内游离Ca~(2 )浓度([Ca~(2 )]_i)与依赖Ca~(2 )的酶的激活、ATP的消耗及细胞膜的结构和功能完整性、心肌细胞间电耦联     

细胞外钙离子浓度与哇巴因(ouabain)的强心作用及哇巴因对钙离子通入兔心肌细胞内结构的影响

强心苷对心脏的作用研究甚多,但其对心肌收缩的增强作用与细胞内各种结构离子分布的关系尚无确切的知识。心肌兴奋与收缩偶联时必须有钙离子存在、因之学者多认为在心肌兴奋与收缩偶联时,强心苷增加钙离子的可利用性(availability),但细胞内     

小鼠胚胎干细胞分化心肌细胞钠通道、钙通道电生理特性研究

目的:研究小鼠胚胎干细胞分化心肌细胞钠通道、钙通道电生理特性。方法:通过布法罗大鼠肝细胞条件培养基和小鼠胚胎成纤维细胞饲养层培养ES-D3细胞, 经过诱导分化, 收集第12d的干细胞分化心肌细胞并和培养的胚胎期小鼠心肌细胞一起做膜片钳实验, 比较两种细胞离子通道电生理特性。结果:第12d的干细胞分化心肌细胞钠通道、钙通道电流形态和通道特性与胚胎期心肌细胞类似。结论:ES细胞分化过程中出现离子通道功能表达, 干细胞分化心肌细胞与胚胎期小鼠心肌细胞钠通道、钙通道电生理特性类似。     

钙离子运转障碍与心力衰竭

钙离子运转障碍与心力衰竭南京医学院动脉粥样硬化研究中心（南京２１００２９）朱宇，蔡海江心力衰竭是临床上较为常见的一种病理过程。由于钙离子在心肌细胞内的正常运转对于心脏的兴奋一收缩偶联起十分重要的作用，因而人们对于钙离子运转障碍在心力衰竭发病机制中的作...     

氧化苦参碱对豚鼠心室肌细胞膜钠通道的影响

目的：研究氧化苦参碱(Oxy)对豚鼠心室肌细胞膜钠通道电流的影响，探讨Oxy在离子通道水平的作用机制。 方法: 用急性酶解法分离豚鼠心室肌细胞，应用膜片钳全细胞记录技术，观察不同浓度的Oxy对钠通道的影响。 结果: 用Oxy 1、10、100、1 000 μmol/L可浓度依赖性地抑制钠电流。在100 μmol/L时，给药后电流密度抑制约40％（P<0.01），可使心肌细胞钠电流－电压曲线上移，但激活电位、峰电位及反转电位无改变；Oxy使失活曲线向负电位方向变化，并使灭活后恢复过程延迟；对激活曲线无明显影响。 结论: Oxy可通过浓度依赖性和电压依赖性地抑制心肌细胞膜钠通道电流。     

Development of excitation-contraction coupling in cardiomyocytes

The excitation-contraction (E-C) coupling system during the development of heart can be investigated because of marked progression in electrophysiology and microfluorescence studies. During the developmental period, Ca2+ influx mediating the E-C coupling is mainly through the L-type Ca2+ channels. In the fetal period, T-type Ca2+ channels and the reverse mode of the Na-Ca exchange system also contribute to Ca2+ influx. These contributions probably reduce gradually until adulthood. The contraction of fetal cardiomyocytes has been thought to depend mainly on the Ca2+ influx. However, recent studies reveal that immature sarcoplasmic reticulum (SR) already works in the early fetal period. Ca2+ spark, a local and unitary movement of Ca2+, can be observed in adult cardiomyocytes by the use of a confocal microscope. On the other hand, no Ca2+ spark is observed in fetal cardiomyocytes. The frequency of Ca(2+)-spark evocation increases during the postnatal period. Therefore a close distance between the L-type Ca2+ channel and the SR Ca(2+)-release channel is essential to the establishment of the Ca2+ spark.     

Effects of heparin on excitation-contraction coupling in skeletal muscle toad and rat.

1. Intracellularly applied heparin was found to cause a novel, use-dependent block of excitation-contraction (E-C) coupling in skinned skeletal muscle fibres of the toad. After one to four depolarizations in the presence of 100 micrograms ml-1 heparin, no further depolarization-induced responses could be elicited, even though addition of caffeine or lowering [Mg2+] could still induce massive Ca2+ release. This effect could not be reversed by extensive wash-out of the heparin (> 15 min). 2. Heparin (100 micrograms ml-1) did not abolish subsequent depolarization-induced responses if applied while the voltage sensors were in either their resting or inactivated states, that is (a) while a fibre remained fully polarized, (b) when a fibre was already chronically depolarized or (c) after a fibre had been depolarized in the presence of D600 (gallopamil) and then repolarized. 3. When a toad fibre was depolarized in heparin, with the associated Ca2+ release blocked by the presence of 10 mM intracellular Mg2+, subsequent E-C coupling was abolished. Heparin did not interrupt E-C coupling when Ca2+ release was triggered in the absence of any depolarization, by either caffeine or low [Mg2+]. Thus, the opening of the Ca2+ release channels was neither necessary nor sufficient for heparin to abolish E-C coupling. 4. Heparin had direct effects on the contractile apparatus in toad fibres, increasing the Ca2+ sensitivity and decreasing the maximum Ca(2+)-activated force. These effects could only be partly reversed by extensive wash-out of heparin. 5. At 100 micrograms ml-1, both low molecular weight heparin and pentosanpolysulphate, another highly sulphated polysaccharide, were less effective than heparin in blocking the depolarization-induced response and in changing the properties of the contractile apparatus, and these effects could be substantially reversed by wash-out. Two other polyanions, de-N-sulphated heparin (100 micrograms ml-1), which lacked N-sulphate groups, and polyglutamate (500 micrograms ml-1), had no measurable effect on either E-C coupling or the contractile apparatus. 6. In skinned fibres of the extensor digitorum longus muscle of the rat, 100 micrograms ml-1 heparin had little or no effect on E-C coupling and on the Ca2+ sensitivity of the contractile apparatus, but caused a larger reduction of the maximum Ca(2+)-activated force than in skinned fibres of the toad. 7. These results indicate that heparin blocks E-C coupling in toad muscle if, and only if, it is present when the voltage sensors are activated by depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

流体低剪切力上调内皮细胞IL-8基因表达的细胞内信号转导途径分析

目的　利用流室系统，体外研究低剪切力上调内皮细胞IL-8基因表达的胞内信号转导途径。方法　以培养的人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)为研究对象，采用定量RT-PCR检测HUVECs经低剪切力(0.42 Pa)刺激后IL-8基因的表达情况及多种阻断剂对其的干预作用。结果　(1)KT5720(蛋白激酶A抑制剂)、Neomycin（磷脂酶C抑制剂）、Calphostin C（蛋白激酶C抑制剂）、Tyrphostin-25（酪氨酸蛋白激酶抑制剂）均不同程度地抑制低剪切力对内皮细胞IL-8基因的上调；(2)Ca(上标 2+)可促进低剪切力上调内皮细胞IL-8 mRNA表达；(3)非水解的GDP类似物－GDPβS亦可抑制低剪切力诱导的内皮细胞IL-8基因的上调；(4)蛋白激酶G抑制剂－KT5823对低剪切力诱导的IL-8基因表达无明显影响。结论　低剪切力上调内皮细胞IL-8基因表达的胞内信号转导途径非常复杂，涉及到多种信号分子。     

Properties of calcium currents and contraction in cultured rat diaphragm muscle

The characterization of calcium currents and contraction simultaneously measured in cultured rat diaphragm muscle cells was carried out in the present study. Whole-cell patch-clamp experiments were designed to further elucidate the mechanism of excitation-contraction (E-C) coupling in diaphragm which, though generally considered a skeletal-type muscle, has been reported to exhibit properties indicative of a cardiac-like E-C coupling mechanism. Normalized current/voltage (I/V) curves for two concentrations of external calcium (2.5 and 5 mM) were obtained from diaphragm myoballs. Both curves showed peaks corresponding to the activation of a T-type calcium current and a dihydropyridine-sensitive L-type calcium current. The normalized curve for the voltage dependence of the activation of contraction in diaphragm myoballs followed a typical Boltzmann-type relationship to the peak of contraction. Thereafter, the curve declined in a manner that was more pronounced in diaphragm compared to that measured in additional experiments using cultured rat limb muscle myoballs. This effect could be interpreted in terms of a more pronounced participation of the L-type current in E-C coupling in cultured diaphragm muscle. An increased likelihood of cultured diaphragm muscle to undergo depletion of sarcoplasmic reticular calcium stores during repetitive stimulation, or a heightened propensity for the voltage sensor for E-C coupling in diaphragm to enter the inactive state could also explain this effect. Maximal contractile activity was only slightly affected when the L-type current was blocked by externally applied cadmium (2 mM) or cobalt (3 mM), suggesting that a pronounced calcium-current-dependent component of contraction is unlikely in cultured diaphragm muscle. These results show that T- and L-type calcium channels are expressed in cultured rat diaphragm muscle cells and that, in contrast to cardiac muscle, the entry of calcium ions via L-type voltage-dependent calcium channels is not a prerequisite for contraction. Differences in the voltage sensitivity of contraction, observed at depolarized membrane potentials in cultured rat diaphragm and limb muscle cells, suggest that the voltage sensor for E-C coupling in diaphragm might more readily enter an inactivated configuration — possibly by a mechanism which is dependent on the concentration of external calcium.     

Slowly inactivating sodium current (I(NaP)) underlies single-spike activity in rat subthalamic neurons

One-half of the subthalamic nucleus (STN) neurons switch from single-spike activity to burst-firing mode according to membrane potential. In an earlier study, the ionic mechanisms of the bursting mode were studied but the ionic currents underlying single-spike activity were not determined. The single-spike mode of activity of STN neurons recorded from acute slices in the current clamp mode is TTX-sensitive but is not abolished by antagonists of ionotropic glutamatergic and GABAergic receptors, blockers of calcium currents (2 mM cobalt or 40 microM nickel), or intracellular Ca(2+) ions chelators. Tonic activity is characterized by a pacemaker depolarization that spontaneously brings the membrane from the peak of the afterspike hyperpolarization (AHP) to firing threshold (from -57.1 +/- 0.5 mV to -42.2 +/- 0.3 mV). Voltage-clamp recordings suggest that the Ni(2+)-sensitive, T-type Ca(2+) current does not play a significant role in single-spike activity because it is totally inactivated at potentials more depolarized than -60 mV. In contrast, the TTX-sensitive, I(NaP) that activated at -54.4 +/- 0.6 mV fulfills the conditions for underlying pacemaker depolarization because it is activated below spike threshold and is not fully inactivated in the pacemaker range. In some cases, the depolarization required to reach the threshold for I(NaP) activation is mediated by hyperpolarization-activated cation current (I(h)). This was directly confirmed by the cesium-induced shift from single-spike to burst-firing mode which was observed in some STN neurons. Therefore, a fraction of I(h) which is tonically activated at rest, exerts a depolarizing influence and enables membrane potential to reach the threshold for I(NaP) activation, thus favoring the single-spike mode. The combined action of I(NaP) and I(h) is responsible for the dual mode of discharge of STN neurons.     

Evaluation of left ventricular mechanical work and energetics of normal hearts in SERCA2a transgenic rats

Cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) is responsible for most of the Ca(2+) removal during diastole and a larger Ca(2+) handling energy consumer in excitation-contraction (E-C) coupling. To understand the cardiac performance under long-term SERCA2a overexpression conditions, we established SERCA2a transgenic (TG) Wistar rats to analyze cardiac mechanical work and energetics in normal hearts during pacing at 300 beats/min. SERCA2a protein expression was increased in TGI and TGII rats (F2 and F3 of the same father and different mothers). Mean left ventricular (LV) end-systolic pressure (ESP) and systolic pressure-volume area (PVA; a total mechanical energy per beat) at midrange LV volume (mLVV) were significantly larger in TGI rats and were unchanged in TGII rats, compared to those in non-TG [wildtype (WT)] littermates. Mean myocardial oxygen consumption per minute for E-C coupling was significantly increased, and the mean slope of myocardial oxygen consumption per beat (VO(2))-PVA (systolic PVA) linear relation was smaller, but the overall O(2) cost of LV contractility for Ca(2+) is unchanged in all TG rats. Mean Ca(2+) concentration exerting maximal ESP(mLVV) in TGII rats was significantly higher than that in WT rats. The Ca(2+) overloading protocol did not elicit mitochondrial swelling in TGII rats. Tolerance to higher Ca(2+) concentrations may support the possibility for enhanced SERCA2a activity in TGII rats. In conclusion, long-term SERCA2a overexpression enhanced or maintained LV mechanics, improved contractile efficiency under higher energy expenditure for Ca(2+) handling, and improved Ca(2+) tolerance, but it did not change the overall O(2) cost of LV contractility for Ca(2+) in normal hearts of TG rats.     

Hydralazine-induced promoter demethylation enhances sarcoplasmic reticulum Ca2+ -ATPase and calcium homeostasis in cardiac myocytes

Sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) plays an essential role in Ca(2+) homeostasis and cardiac functions. The promoter region of SERCA2a has a high content of CpG islands; thus, epigenetic modification by inhibiting methylation can enhance SERCA2a expression in cardiomyocytes. Hydralazine, a drug frequently used in heart failure, is a potential DNA methylation inhibitor. We evaluated whether hydralazine can modulate Ca(2+) handling through an increase in SERCA2a expression via regulating methylation. We used indo-1 fluorescence, real-time RT-PCR, immunoblotting, and methylation-specific PCR to investigate intracellular Ca(2+), the expressions of RNA and protein, and methylation of SERCA2a in HL-1 cardiomyocytes with and without (control) the administration of hydralazine (1, 10, and 30 μM) for 72 h. Hydralazine (10 and 30 μM) increased the intracellular Ca(2+) transients and SR Ca(2+) contents. Hydralazine (10 and 30 μM) decreased methylation in the SERCA2a promoter region and increased the RNA and protein expressions of SERCA2a. Additionally, hydralazine (10 and 30 μM) decreased the expression of DNA methyltransferase 1. Moreover, treatment with hydralazine in isoproterenol-induced heart failure rats decreased the promoter methylation of SERCA2a and increased SERCA2a RNA expression. In conclusion, hydralazine-induced promoter demethylation may improve cardiac function through increasing SERCA2a and modulating calcium homeostasis in cardiomyocytes.