NCA增加心肌收缩力的作用研究

目的: 硝酰基 (HNO)作为一氧化氮 (NO) 的单电子还原产物,对活体心脏发挥正性肌力作用。我们对于这些效果是否由于乙酸1-亚硝基环已酯(NCA,HNO供体) 对肌原纤维的直接作用进行了研究。方法: 将大鼠右心室的完整的梳状肌连接在张力换能器与刺激电极之间,肌小节长度设定在2.2-2.3 μm之间,K-H液表面灌流后 (pH = 7.4,室温),Fura-2经玻璃微电极负载进行离子透入法检测[Ca²⁺]_i,同时测定心肌收缩张力的变化。稳态条件下对最大钙离子活化张力(F_max)及达到50%活性需要[Ca²⁺]_i(Ca₅₀)进行测定。Western blotting方法检测原肌球蛋白表达的变化。结果: 收缩力在NCA作用下呈剂量依赖性增加(20-100 μmol/L)。不同频率作用下(0.5-3.0 Hz,NCA 20 μmol/L,Ca²⁺ 0.5 mmol/L)收缩力的增加 (P< 0.01) 和[Ca²⁺]_i瞬变 (P>0.05) 没有受到明显的影响。舒张期作用力及[Ca²⁺]_i不受NCA的影响。与对照组相比,稳态活化过程中NCA (20 μmol/L) 能增加最大钙离子活力F_max [(124.0±15.0) mN/mm² vs (90.0± 4.2)mN/mm²,P< 0.05],降低Ca₅₀[ (0.39±0.01) μmol/L vs (0.57±0.03) μmol/L,P< 0.01],但不影响希尔系数 (3.94 ± 0.18 vs 4.92 ± 0.84, P>0.05)。同对照组相比,NCA处理去肌膜心肌收缩力明显增加 (P< 0.05)。非还原条件下Western blotting可见肌原纤维出现交联。巯基还原剂二硫苏糖醇 (DTT,5.0 mmol/L) 能够阻止并逆转NCA的活动,进一步证实氧化还原反应依赖HNO 的效应。结论: NCA提供的HNO是心脏钙离子增敏剂,心肌调节蛋白质巯基翻译后修饰可能是其靶点作用所在。     

心室肌细胞电生理-力学复合模型在心力衰竭中的仿真研究

目的:研究心力衰竭情况下快速收缩心肌和慢速收缩心肌力学特性的变化及其对心脏电生理的影响。方法:基于心衰心肌生理实验数据建立心室肌细胞电生理-力学复合模型,仿真对比正常和心衰肌细胞内钙离子浓度的变化,进而研究其对细胞收缩力学特性的影响。结果:仿真显示与正常细胞相比,心衰肌细胞电生理特性的变化导致细胞内钙离子浓度降低。进而与肌钙蛋白的钙结合亚单位(TnC)结合的钙离子比正常时大大减小,直接导致细胞收缩力减小。结论:心衰时心室肌细胞的收缩力减小,肌细胞中正常的电力学反馈作用减弱,增大了心室壁细胞动作电位的透壁梯度,从而可能诱发心律失常。仿真结果与文献报道的实验发现一致,将来在组织和器官层次上的心脏建模工作可以整合单细胞的电力学模型,有助于深入研究心力衰竭的病理机制。     

Ryanodine受体2增快胚胎发育期心肌细胞胞浆内钙瞬变

目的 :在成年心肌细胞中 ,细胞内钙瞬变是由外钙内流激发肌浆网储存钙的释放而引起的 ,位于肌浆网的钙离子释放通道ryanodine受体 2 (RyR2 )在其中起着关键的调控作用。但RyR2在发育早期心肌细胞钙瞬变形成中的作用和地位仍不清楚。本课题利用RyR2基因剔除 (RyR2 -/-)胚胎干细胞 (embryonicstemcell,ESC)体外分化的心肌细胞 ,探讨了RyR2在心肌发育过程中对钙瞬变的调控作用。方法 :在体外诱导正常R1系ESC (RyR2 + /+ )和RyR2 -/-ESC分化为心肌细胞 ,并分离出能自主收缩的心肌细胞 ,以钙离子荧光指示剂Indo - 1为探针 ,应用光谱荧…     

Changes of calcium release in myocardial sarcoplasmic reticulum during heart failure

目的:探讨慢性心力衰竭时心肌细胞肌浆网内钙离子释放的变化.方法:采用冠状动脉左前降支结扎法制备大鼠慢性心衰模型,酶解法分离成年大鼠心室肌细胞.用全细胞模式膜片钳技术和共聚焦显微镜钙成像技术同步实时记录心肌细胞的L-型钙电流以及胞质内的钙瞬变.结果:心衰组的钙诱导钙瞬变幅度小于对照组,并且心衰组的咖啡因诱导钙瞬变幅度也低于对照组.结论:心力衰竭的发生与心肌细胞内钙离子释放变化有着十分密切的关系.     

钙稳态和自噬在心肌肥厚中作用的研究进展

心肌肥厚指心脏在各种因素作用下出现的心肌质量和体积的增加。生理性心肌肥厚可以转变为病理性心肌肥厚,最终导致心力衰竭和心源性猝死。心肌肥厚病理因素较为复杂,钙稳态和自噬在心肌肥厚的发生发展中起着重要作用。钙信号主要包括钙离子/钙调蛋白/钙调素依赖蛋白激酶II/肌细胞增强因子-2(Ca²⁺/CaM/CaMKⅡ/MEF-2)和钙离子/钙调蛋白/钙调素依赖蛋白激酶II/钙调神经磷酸酶/活化T细胞核因子(Ca²⁺/CaM/CaMKⅡ/CaN/NFAT)两条通路,同时钙稳态与自噬两种途径也互相调节。本文对钙稳态和自噬在心肌肥厚中的作用作一综述。     

内皮素对哺乳动物心肌活动的影响

本文简要综述了内皮素对哺乳动物心肌的生理效应及作用机制 ,特别是着重从心肌力学、心肌收缩力、心肌动作电位及心肌超微结构等方面较为系统地阐明了适当剂量的内皮素对哺乳动物心肌活动的正性作用及较高剂量内皮素的负性影响 ,并用细胞内钙离子增加的理论对内皮素影响心肌活动的机制作了初步分析和探讨     

心力衰竭对心肌钙相关通道的影响

心力衰竭伴有心室功能的下降,神经体液因子的激活,以及致心律失常性的增加.心力衰竭的发病机制与心肌钙转运失常密切相关,而与心肌钙离子密切相关的通道——钙离子通道及相关钙调控蛋白(calcium handling proteins)随着收缩和(或)舒张功能的下降而改变,易触发心律失常.深入研究这些蛋白变化与心律失常发生的关系,将会对目前心衰心律失常防治的难题提供更好的解决方法.     

心肌细胞钙稳态失调在心力衰竭中作用的研究进展

心肌细胞的钙稳态依赖于质膜、肌/内质网(SR)、线粒体等对钙(Ca2+)转运的调节.胞质钙超载、SR钙渗漏等在内的钙稳态失调,使心肌细胞钙瞬变减低,收缩力下降是心力衰竭的主要特征之一.     

肿瘤坏死因子α通过PI3K-IP3R-Ca2+途径诱导乳鼠心肌肥大

 目的: 探讨肿瘤坏死因子α(TNF-α)是否通过PI3K-IP₃R-Ca²⁺信号途径诱导心肌肥大。方法: 以培养的乳鼠心肌细胞为模型,采用Lowry法测心肌细胞蛋白含量;[³H]-亮氨酸掺入法测定心肌细胞蛋白合成;计算机图像分析系统测心肌细胞体积;Till阳离子测定系统测定心肌细胞内Ca²⁺浓度。结果: PI3K阻断剂LY294002(50 μmol/L)明显抑制TNF-α(100 μg/L)诱导的心肌[Ca²⁺]_i增高(P<0.01),对正常心肌[Ca²⁺]_i无明显影响;其抑制程度与合用2-氨基乙基二苯硼酸盐(2-APB)组相近,但小于与兰尼碱(RYA)合用组(P<0.05)。PI3K阻断剂LY294002(50 μmol/L)明显抑制TNF-α(100 μg/L)诱导的心肌细胞蛋白含量、蛋白合成及细胞体积的增加;其抑制程度与合用2-APB组无差异,但明显大于单用2-APB组,小于合用RYA组(P<0.05)。PI3K阻断剂LY294002(50 μmol/L)对正常心肌细胞蛋白含量、蛋白合成及细胞体积无明显影响。结论: TNF-α通过PI3K-IP₃R-Ca²⁺途径诱导心肌肥大。     

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     

The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations

Astrocytes in the rat thalamus display spontaneous [Ca²⁺]_i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca²⁺]_i oscillations using slices loaded with Fluo-4 AM (5 μM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca²⁺]_i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca²⁺]_i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca²⁺]_i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca²⁺]_i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca²⁺ entry. Increasing [Ca²⁺]_o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca²⁺ ATPase by cyclopiazonic acid also induced [Ca²⁺]_i transients in astrocytes indicating a role for cytoplasmic Ca²⁺ in the induction of spontaneous oscillations. Incubation with 20 μM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases.

This study indicates that Ca²⁺ has a role in triggering Ca²⁺ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca²⁺]_i oscillations.     

Monoclonal antibody to β2 subunit of neuronal nicotinic receptor depresses the postjunctional non-contractile Ca mobilization in the mouse diaphragm muscle

The involvement of subtypes of nicotinic acetylcholine receptor (nAChR) in the postjunctional non-contractile Ca²⁺ mobilization was investigated in mouse diaphragm muscles treated with an anticholinesterase, using monoclonal antibodies (mAbs) to nAChR subunits. mAb 210 (specific for 1 subunit of muscle nAChR) depressed contractile Ca²⁺ transients without affecting non-contractile Ca²⁺ transients. mAb 270 (specific for β2 subunit of neuronal nAChR) depressed only non-contractile Ca²⁺ transients. mAb 210 did not completely block the ACh-activated channel currents in flexor digitorum brevis muscle cells. The present findings indicate that the anti-β2 mAb 270-related subtype of nAChR may postsynaptically operate the non-contractile Ca²⁺ mobilization at the neuromuscular junction, suggesting the involvement of a subtype different from the usual muscle-type nAChR.     

活性氧簇介导血管紧张素Ⅱ对延髓神经元胞内游离Ca~(2+)水平的调节作用

目的:探讨活性氧簇(reactive oxygen species,ROS)介导血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)对延髓神经元胞内游离Ca~(2+)的调节作用及其机制。方法:原代培养延髓神经元;免疫荧光双标法鉴定培养神经元的特征;给予Ang Ⅱ处理后,用二氢乙啶荧光探针法测定神经元ROS水平;同时或单独给予Ang Ⅱ和NADPH氧化酶抑制剂apocynin或自由基清除剂TEMPOL后,Fura-2/AM钙瞬变法记录神经元胞内游离Ca~(2+)的水平;CCK-8法检测神经元活性。结果:原代培养的延髓神经元多数为谷氨酸阳性的神经元;Ang Ⅱ(5μmol/L)可在10 min内显著升高神经元ROS水平(P0.01);给予Ang Ⅱ处理后延髓神经元胞内Ca~(2+)水平显著升高(P0.01);给予apocynin/TEMPOL预处理后,Ang Ⅱ引起的延髓神经元胞内Ca~(2+)的升高则被抑制(P0.05)。实验浓度的Ang Ⅱ对神经元无毒性作用。结论:ROS介导Ang Ⅱ诱导的延髓神经元胞内Ca~(2+)的升高作用,可能是Ang Ⅱ在中枢诱导氧化应激作用的潜在细胞内信号机制。     

Role of intracellular calcium in fatigue in single skeletal muscle fibers isolated from the rat

The aim of the present study was to demonstrate the role of intracellular calcium ([Ca²⁺]_i) in the performance of fatigued muscle fibers isolated from the skeletal muscle of the rat. We measured developed tension of a single myocyte during short tetanic electrical stimulation of various intensities along with [Ca²⁺]_i dynamics by fura-2. The performance of individual muscle fiber was assessed by developed tension during 100 Hz tetanic stimulation (‘100 Hz force’). We regarded the muscle fiber fatigued when, after repeated tetanic stimulations, the developed tension declined to 50% of the initial level. When fatigue was induced by maximal stimulation (100 Hz tetani), the 100 Hz force measured immediately following completion of fatigue was considerably decreased (48% of control). This change in the muscle performance was associated with significant increase in the resting [Ca²⁺]_i (280% of control) and decrease in Ca²⁺ transient (54% of control). The 50% relaxation time after cessation of tetanic stimulation (RT₅₀) was also prolonged. In contrast, when fatigue was induced by low frequency electrical stimulation (30 Hz tetani), neither the 100 Hz force, RT₅₀, nor Ca²⁺ transient in fatigue were significantly different from the controls, while the resting [Ca²⁺]_i increased only slightly. These findings suggest a tight relationship between [Ca²⁺]_i and the performance of fatigued single isolated skeletal muscles. Also, the results show that performance of the fatigued muscle fiber may in part depend on the protocol used to produce muscle fatigue.     

Effects of calcium chelators on intracellular calcium and excitotoxicity

In an attempt to probe the relationship between excitotoxicity and increases in intracellular calcium ([Ca²⁺]_i), BAPTA-AM and its analogs were applied to cultured hippocampal neurons. Chelation of [Ca²⁺]_i depressed and prolonged transient responses to glutamate and did not effect elevation of [Ca²⁺]_i by prolonged exposure. This explains the inability of the chelators to prevent glutamate-induced toxicity.     

Modulation of the dihydropyridine-insensitive Ca influx by 8-bromo-guanosine-3′:5′-monophosphate, cyclic (8-Br-cGMP) in bovine adrenal chromaffin cells

Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 μM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca²⁺]_i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Combined incubations with nifedipine 10 μM and 8-Br-cGMP reduced KCl-evoked intracellular Ca²⁺ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1μM) under conditions of low depolarization. Neomycin (0.2 mM) and θ-Aga Toxin-IVA (AgTx) (1μM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca²⁺ influx pathway in the chromaffin cell.     

Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons

Cultured cortical neurons exposed for 24 h to low concentrations of the Ca²⁺ ionophores, ionomycin (250 nM) or A-23187 (100 nM), underwent apoptosis, accompanied by early degeneration of neurites, cell body shrinkage, chromatin condensation and internucleosomal DNA fragmentation. This death could be blocked by protein synthesis inhibitors, as well as by the growth factors brain-derived neurotrophic factor or insulin-like growth factor I. If the ionomycin concentration was increased to 1–3 μM, then neurons underwent necrosis, accompanied by early cell body swelling without DNA laddering, or sensitivity to cycloheximide or growth factors. Calcium imaging with Fura-2 suggested a possible basis for the differential effects of low and high concentrations of ionomycin. At low concentrations, ionomycin induced greater increases in intracellular Ca²⁺ concentration in neurites than in neuronal cell bodies, whereas at high concentrations, ionomycin produced large increases in intracellular Ca²⁺ concentration in both neurites and cell bodies.

We hypothesize that the ability of low concentrations of Ca²⁺ ionophores to raise intracellular Ca²⁺ concentration preferentially in neurites caused early neurite degeneration, leading to loss of growth factor availability to the cell body and consequent apoptosis, whereas high concentrations of ionophores produced global cellular Ca²⁺ overload and consequent necrosis.     

Modulation of Human T-Lymphocyte Plasma Membrane Ca +2 Permeability by Imidazole Antimycotics

The role of cytochrome P-450 in the regulation of plasma membrane Ca⁺² permeability of human peripheral T-lymphocytes by intracellular Ca⁺² was examined. We assessed the effect of imidazole inhibitors of cytochrome P-450 on the intracytoplasmic free Ca⁺² ([Ca⁺²]i) response generated using the microsomal ATPase inhibitor thapsigargin (THG) to deplete the intracellular Ca⁺² stores. Econazole, miconazole and clotrimazole dramatically inhibited the THG mediated increase in [Ca⁺²]i and indud an increase in [Ca⁺²]i themselves. This inhibitory effect was previously observed in other cell systems and was attributed to inhibition of cytochrome P-450 by these agents. However, we evaluated a variety of structurally dissimilar P-450 inhibitors and found that none affected [Ca⁺²]i, indicating that the mechanism of imidazole action does not involve P-450.     

Adenosine triphosphate mobilizes cytosolic calcium and modulates ionic currents in mouse taste receptor cells

By using photometry and the patch clamp technique, we identified P_2Y-like receptors in mouse taste receptor cells (TRCs) and found them to be coupled to Ca²⁺ mobilization and ionic current modulation. Particularly, adenosine triphosphate (ATP) and the P_2Y agonist 2-methylthio-ATP increased intracellular Ca²⁺ by stimulating the phosphoinositide pathway, whereas β,γ-methylene- -ATP, a P_2X agonist, was ineffective. In a distinctive TRC subpopulation, ATP closed Ca²⁺ channels. This regulation may underlie the negative feedback tuning neurotransmitter release. By mobilizing intracellular Ca²⁺, ATP activated Ca²⁺-dependent Cl⁻ channels, the intracellular event that may universally occur upon taste stimulation triggering IP₃ formation and Ca²⁺ release in the TRC cytoplasm.