模拟微重力效应对骨细胞SOC通道功能的影响

目的探究模拟微重力效应下骨细胞钙池操纵Ca~(2+)通道(store-operated calcium channels,SOC)的活性变化以及其可能机制,阐明失重性骨丢失的发生机制。方法以小鼠骨细胞(MLO-Y4)为对象,分为回转模拟微重力效应组(simulated microgravity, SM)和正常重力组(control, CON)。分别旋转培养24、48 h后,激光共聚焦显微镜检测毒胡萝卜素引发细胞内质网钙库耗竭后胞内Ca~(2+)浓度水平,以反映SOC通道的活性;免疫荧光染色法观察膜骨架spectrin和内质网膜蛋白IP_3R的分布情况,研究SOC通道功能变化的可能机制。结果在内质网钙库释放Ca~(2+)时期,24、48 h SM组的胞内Ca~(2+)浓度水平与CON组相比均无显著差异,而在胞外Ca~(2+)经SOC通道内流时期,24 h SM组只在前4 min比CON组有显著性下降,48 h SM组在整个时期均比CON组有显著性下降。与CON组相比,SM组膜骨架spectrin向细胞边缘聚集,而ER膜蛋白IP_3R则向ER核被膜区域聚集,且48 h组更为显著。结论模拟微重力效应可抑制骨细胞SOC通道活性。骨细胞膜骨架spectrin以及内质网膜上蛋白IP_3R位置分布变化,可能影响SOC通道激活过程中蛋白间的构象耦合,进而降低骨细胞SOC通道的活性。     

阿片受体与神经细胞内Ca2+

阿片受体包括κ ,μ ,δ等多种亚型 ,属G蛋白偶联受体 ,广泛分布于神经系统。阿片受体可通过与神经细胞内第二信使Ca2 + 偶联 ,调节神经细胞的功能。κ ,δ阿片受体被激活后对神经细胞Ca2 + 通道的作用存在双向性 ,即通过多种途径或不同方式 ,抑制或激活Ca2 + 通道。δ阿片受体激活后可动员细胞内钙库 ,引起钙库释放增加 ;在多种神经细胞上 ,μ阿片受体激活后可抑制高电压依赖钙通道 ,而对低电压钙通道无明显影响。     

血管疾病治疗新靶标：STIM1和Orai1

钙离子(Ca2+)是常见的第二信使,不同于其它第二信使,其主要位于细胞外或存储于内质网(endoplasmic reticulum,ER)等细胞器内.静息状态下细胞内游离Ca2+浓度(intracellular Ca2+ concentration,[Ca2+]i)约为细胞外的1/20000,受体激活或生物信号刺激可通过改变[Ca2+]i进一步发挥生物放大效应.[Ca2+]i的升高主要通过胞内Ca2+释放和胞外Ca2+内流两大途径.随着胞内钙库的排空,位于质膜上的Ca2+内流通道被激活,使Ca2+由胞外进入胞质内,这个过程称为钙库操纵的钙内流(store-operated calcium entry,SOCE),其通道称为钙库操纵的钙通道(store-operated calcium channel,SOCC).近来研究证实组成SOCC的主要蛋白是:Ca2+感受蛋白基质相互作用分子1(stromal interaction molecule 1,STIM1)[1-2]和Ca2+通道蛋白Orai1[3-4].     

TRP离子通道

钙离子作为一种第二信使，在许多细胞功能中发挥着重要作用。短期的，如递质、腺体分泌，肌肉收缩；长期的，如细胞分化、程序死亡等。正常细胞都有一套完善的体系来维持钙的内稳定平衡。内质网／肌浆网的钙释放和来自质膜上钙通道的钙流入升高胞浆钙，而同时位于这两个部位的Ca^2 —泵也不断地将胞浆钙储存回钙库或泵出胞外。质膜上     

钙离子感受蛋白STIM1与Orai1在钙敏感受体介导钙内流及NO生成中的相互作用

目的:研究Ca~(2+)感受蛋白[基质相互作用分子1(STIM1)与钙释放激活钙通道蛋白1(Orai1)]在人脐静脉内皮细胞(HUVECs)钙敏感受体(Ca SR)介导的钙内流和一氧化氮(NO)生成中的相互作用。方法:将Ca SR激动剂精胺[钙池操纵性钙通道(SOC)和受体操纵性钙通道(ROC)均激活]单用或与ROC模拟剂12-O-十四烷酰佛波醇-13-醋酸酯(TPA)+Ca SR负性变构调节剂Calhex 231(激活ROC、阻断SOC)、蛋白激酶C(PKC)抑制剂Ro 31-8220和PKCα/β1选择性抑制剂Go 6976(激活SOC、阻断ROC)联合孵育HUVECs;利用免疫荧光技术检测HUVECs中STIM1和Orai1的蛋白表达和共定位;免疫共沉淀法检测STIM1和Orai1之间的相互作用;取2~3代HUVECs随机分为特异性的质粒转染组(sh STIM1+sh Orai1组)、空质粒组(vehicle-STIM1+vehicle-Orai1组)和未转染组(control组),将3组细胞分别加入上述4组不同药物刺激,采用荧光探针Fura-2/AM检测HUVECs中Ca~(2+)浓度的变化,NO荧光探针DAF-FM DA负载方法同步检测HUVECs中NO生成的变化。结果:STIM1和Orai1蛋白表达共定位于胞浆,与control组相比,加入Calhex 231+TPA、Ro 31-8220和Go 6976刺激后,STIM1和Orai1在胞浆中的定位均减少,且二者的相互作用均减弱;在4种不同处理因素作用下,sh STIM1+sh Orai1组细胞内Ca~(2+)浓度和NO净荧光强度均明显降低(P0.05)。结论:STIM1与Orai1以二元复合物的形式共同调节Ca SR,并通过激活SOC和ROC介导钙内流及NO生成。     

卡维地洛抑制大鼠起搏心肌细胞钙库超载诱导的钙释放*

 目的：探讨非选择性β受体阻滞剂卡维地洛对起搏心肌细胞钙库超载诱导钙释放（SOICR）的作用及其机制。方法：电刺激起搏大鼠单个心肌细胞并灌注异丙肾上腺素及咖啡因诱导钙库钙超载,从而触发肌浆网钙释放通道（兰尼碱受体2,RyR2）舒张期开放引起SOICR。应用钙离子荧光成像技术记录胞内钙浓度的实时变化。实验分为对照组、卡维地洛组、美托洛尔组、酚妥拉明组和硝苯地平组。结果：(1) 与基线刺激时比较,对照组细胞灌注异丙肾上腺素和咖啡因后,起搏细胞钙瞬变振幅显著增高（P<0.01）,SOICR的发生率显著增加（P<0.01）。(2) 在1~4 Hz起搏频率下卡维地洛组细胞SOICR发生率分别为2.00%、6.00%、10.00%和16.00%,均显著低于对照组（分别为43.59%、74.36%、87.18%和89.74%,均P<0.01）;卡维地洛对心肌细胞SOICR的抑制在不同起搏频率下无明显差异（P>0.05）。酚妥拉明、美托洛尔和硝苯地平组细胞与对照组细胞比较,SOICR发生率无差异（均P>0.05）。(3) 起搏细胞钙瞬变振幅的比较,各组间相比未见明显差异（P>0.05）;咖啡因峰值估测钙库钙总量的比较,各组间也无明显差异（P>0.05）。结论：卡维地洛可明显抑制起搏心肌细胞SOICR的发生,其作用机制可能是直接抑制RyR2的自发性开放而非源于对α1、β1受体和L型钙通道的阻滞作用。     

钙库操纵性钙通道在人循环纤维细胞中的表达及功能

 目的: 研究钙库操纵性钙通道(store-operated calcium channels,SOCC)相关功能蛋白ORAI1-3和STIM1-2在人循环纤维细胞(circulating fibrocytes)中的表达及SOCC对人循环纤维细胞分化的影响。方法: 采集健康人外周静脉血,分离出单个核细胞,体外培养分化为循环纤维细胞。采用RT-PCR和real-time PCR检测循环纤维细胞中ORAI1-3及STIM1-2的mRNA表达情况,并检测SOCC抑制剂对循环纤维细胞分化的影响。结果: Real-time PCR检测结果显示ORAI1-3和STIM1-2 mRNA在循环纤维细胞中有较高的表达水平,并且SOCC抑制剂SKF-96365对循环纤维细胞分化具有明显的抑制作用。结论: SOCC表达于循环纤维细胞中,并且影响循环纤维细胞的分化。     

幼儿教师情绪劳动策略与情绪耗竭的关系:心理资本的调节作用

目的:探索幼儿教师情绪劳动策略与情绪耗竭的关系,检验心理资本对两者关系的调节作用。方法:采用情绪劳动策略量表、职业倦怠量表中情绪耗竭分量表和本土心理资本量表对253名幼儿教师进行集体施测。结果:①情绪耗竭与自然行为、深层行为、事务型心理资本和人际型心理资本呈显著负相关,与表层行为呈显著正相关。②高人际型心理资本组中,幼儿教师的深层行为能显著预测情绪耗竭。③低人际型心理资本组中,幼儿教师的表层行为能显著预测情绪耗竭。结论:幼儿教师情绪劳动策略与情绪耗竭关系密切;人际型心理资本在深层行为与情绪耗竭、表层行为与情绪耗竭关系中起显著的调节作用。     

T细胞耗竭机制的研究进展

在慢性感染性疾病及肿瘤的进展过程中,持续性的抗原刺激会导致T细胞耗竭.耗竭T细胞的效应功能会在疾病进展过程中逐渐出现障碍直至丧失,从而影响机体对疾病的有效控制.T细胞耗竭的机制以及通过逆转耗竭T细胞来治疗慢性感染性疾病和肿瘤已成为近年的研究热点.本文将对耗竭T细胞的主要特点、耗竭现象的逆转及其与疾病之间的关系进行综述.     

慢性缺氧对大鼠肺动脉平滑肌细胞胞内钙的影响及其机制研究

目的:研究慢性缺氧对大鼠肺动脉平滑肌细胞(PASMCs)胞内钙浓度(［Ca²⁺］_i)的影响及L-型钙通道和胞内钙库的作用,为缺氧性肺动脉高压(HPH)发病机制的进一步研究提供理论依据。 方法:复制大鼠缺氧性肺动脉高压动物模型，利用Fura－2/AM钙离子成像方法测定PASMCs在不同钙离子浓度细胞外液及L-型钙通道阻滞剂nifedipine和IP3R钙通道抑制剂肝素干预前后 ［Ca²⁺］_i变化。 结果:(1)缺氧＋含钙外液组PASMCs ［Ca²⁺］_i 显著高于对照＋含钙外液组（P<0.05）。缺氧＋含钙外液组PASMCs ［Ca²⁺］_i显著高于缺氧＋无钙外液组（P<0.05）。(2)缺氧nifedipine组PASMCs［Ca²⁺］_i在加药前后无显著差异（P>0.05）。（3）缺氧未干预组与缺氧肝素组PASMCs ［Ca²⁺］_i无明显差异（P>0.05）。 结论:慢性缺氧可使PASMCs的［Ca²⁺］_i增加。慢性缺氧引起［Ca²⁺］_i增加可能与细胞外钙内流有关，L-型钙通道和IP3R钙通道在调节［Ca²⁺］_i的过程中可能不独立发挥作用。     

Lifting the fog in store-operated Ca2+ entry

Immunoreceptor signalling leads to Ca2+ release from intracellular stores, which is followed by the entry of external Ca2+ into the cell through mysterious store-operated Ca2+ (SOC) channels. Recent studies have identified stromal interaction molecules (STIMs) as having an essential role in SOC influx. Remarkably, Ca2+ store depletion induces a rapid redistribution of the endoplasmic reticulum STIM1 into puncta that accumulate near the plasma membrane. The combined data suggest that STIM1 functions as a Ca2+ sensor in the signalling pathways connecting Ca2+ store depletion to SOC entry.     

ORAI and store-operated calcium influx in human airway smooth muscle cells

The initial bronchoconstrictor response of the asthmatic airway depends on airway smooth muscle (ASM) contraction. Intracellular calcium is a key signaling molecule, mediating a number of responses, including proliferation, gene expression, and contraction of ASM. Ca(2+) influx through receptor-operated calcium (ROC) or store-operated calcium (SOC) channels is believed to mediate longer term signals. The mechanisms of SOC activation in ASM remain to be elucidated. Recent literature has identified the STIM and ORAI proteins as key signaling players in the activation of the SOC subtype; calcium release-activated channel current (I(CRAC)) in a number of inflammatory cell types. However, the role for these proteins in activation of SOC in smooth muscle is unclear. We have previously demonstrated a role for STIM1 in SOC channel activation in human ASM. The aim of this study was to investigate the expression and define the potential roles of the ORAI proteins in SOC-associated Ca(2+) influx in human ASM cells. Here we show that knockdown of ORAI1 by siRNA resulted in reduced thapsigargin- or cyclopiazonic acid (CPA)-induced Ca(2+) influx, without affecting Ca(2+) release from stores or basal levels. CPA-induced inward currents were also reduced in the ORAI1 knockdown cells. We propose that ORAI1 together with STIM1 are important contributors to SOC entry in ASM cells. These data extend the major tissue types in which these proteins appear to be major determinants of SOC influx, and suggest that modulation of these pathways may prove useful in the treatment of bronchoconstriction.     

New developments in the signaling mechanisms of the store-operated calcium entry pathway

The most ubiquitous pathway for regulated calcium (Ca²⁺) entry into the cells is the store-operated Ca²⁺ (SOC) entry pathway (also called capacitative Ca²⁺ entry) that is conserved from lower organisms such as yeast, worms, and flies to man. The SOC concept was proposed over two decades ago, and SOC channels are defined by their activation in response to depletion of the internal Ca²⁺ stores. Influx through SOC channels is necessary for the replenishment of the Ca²⁺ stores and is also involved in cell signaling to the nucleus. Despite intensive investigations, most of which are focusing on transient receptor potential (TRP) channels as molecular candidates for SOC channels, the mechanisms of activation and the identity of the key molecular players participating in this signaling pathway have long remained elusive. In the last 2–3 years, however, the improvements of RNA silencing protocols combined with high throughput platforms have yielded significant breakthroughs, with the identification of Stim1 as the Ca²⁺ store sensor and Orai1 (CRACM1) as the pore-forming subunit of the archetypical SOC channel, CRAC. This review summarizes the recent advances in the mechanisms of activation of SOC channels and their molecular composition, with emphasis on the roles of Stim, Orai, and TRP proteins.     

次声对视网膜微血管内皮细胞胞浆[Ca2+]i水平的影响

目的通过检测次声暴露前后视网膜微血管内皮细胞(BREC)胞浆[Ca2 ]i水平的改变,对次声致大鼠血-视网膜屏障通透性改变的细胞内信号转导途径进行初步探讨.方法采用荧光探针Flu-3研究次声及BKca开放剂NS-1619干预下BREC胞浆[Ca2 ]i水平的改变.结果次声暴露导致细胞内钙库释放Ca2 ,同时观察到NS-1619外钙依赖性地增加[Ca2 ]i.结论次声引起[Ca2 ]i升高,通过细胞收缩成份引起内皮细胞主动收缩,从而达到对血-视网膜屏障的调节效应.     

Dynamic regulation of sarcoplasmic reticulum Ca(2+) stores by stromal interaction molecule 1 and sarcolipin during muscle differentiation

During muscle development, the sarco/endoplasmic reticulum (SR/ER) undergoes remodeling to establish a specialized internal Ca(2+) store for muscle contraction. We hypothesized that store operated Ca(2+) entry (SOCE) is required to fill Ca(2+) stores and is, therefore, critical to creating a mature SR/ER. Stromal interaction molecule 1 (STIM1) functions as a sensor of internal Ca(2+) store content and an activator of SOCE channels. Myocytes lacking STIM1 display reduced SR Ca(2+) content and altered expression of key SR proteins. Sarcolipin (SLN), an inhibitor of the SR calcium pump, was markedly increased in the muscle of mutant STIM1 mice. SLN opposes the actions of STIM1 by limiting SOCE, reducing SR Ca(2+) content and delaying muscle differentiation. During mouse muscle development SLN is highly expressed in embryonic muscle, while the expression of STIM1 is up-regulated postnatally. These results suggest that SOCE regulates SR/ER specialization and that SLN and STIM1 act in opposing fashions to govern SOCE during myogenesis.     

Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells

Autosomal-dominant polycystic kidney disease is a multiorgan disease and its vascular manifestations are common and life-threatening. Despite this, little is known about their pathogenesis. Somatic mutations to the normal PKD allele in cystic epithelia and cyst development associated with the unstable Pkd2(WS25) allele suggest a two-hit model of cystogenesis. However, it is unclear if this model can account for the cardiovascular pathology or if haploinsufficiency alone is disease-associated. In the present study, we found a decreased polycystin-2 (PC2, protein encoded by Pkd2 gene) expression in Pkd2( +/-) vessels, roughly half the wild-type level, and an enhanced level of intracranial vascular abnormalities in Pkd2 (+/-) mice when induced to develop hypertension. Consistent with these observations, freshly dissociated Pkd2 (+/-) vascular smooth muscle cells have significantly altered intracellular Ca(2+) homeostasis. The resting [Ca(2+)](i) is 17.1% lower in Pkd2 (+/-) compared with wild-type cells (P=0.0003) and the total sarcoplasmic reticulum Ca(2+) store (emptied by caffeine plus thapsigargin) is decreased (P<0.0001). The store operated Ca(2+) (SOC) channel activity is also decreased in Pkd2 (+/-) cells (P=0.008). These results indicate that inactivation of just one Pkd2 allele is sufficient to significantly alter intracellular Ca(2+) homeostasis, and that PC2 is necessary to maintain normal SOC activity and the SR Ca(2+) store in VSMCs. Based on these findings, and the fact that [Ca(2+)](i) signaling is essential to the regulation of contraction, production and secretion of extracellular matrix, cellular proliferation and apoptosis, we propose that the abnormal intracellular Ca(2+) regulation associated with Pkd2 haploinsufficiency is directly related to the vascular phenotype.     

Differential STIM1 expression in T and B cell subsets suggests a role in determining antigen receptor signal amplitude

Ca(2+) acts ubiquitously as a second messenger in transmembrane signal transduction. In lymphocytes, calcium mobilization is triggered by antigen and chemokine receptors, among others, and controls cell functions ranging from proliferation to migration. The primary mechanism of extracellular Ca(2+) entry in lymphocytes is the CRAC influx. STIM1 is a crucial component of the CRAC influx mechanism in lymphocytes, acting as a sensor of low Ca(2+) concentration in the ER and an activator of the Ca(2+) selective channel ORAI1 in the plasma membrane. While STIM1 function has been studied extensively, little is known regarding whether it is differentially expressed and thereby affects the magnitude of calcium mobilization responses. We report here that STIM1 expression differs in murine T and B lymphocytes, and in respective subsets. For example, mature T cells express ～4 times more STIM1 than mature B cells. Furthermore, we show that through the physiologic range of expression, STIM1 levels determine the magnitude of Ca(2+) influx responses that follow BCR-induced intracellular store depletion. Considered in view of previous reports that differences in amplitude of lymphocyte Ca(2+) mobilization determine alternate biological responses, these findings suggest that differential STIM1 expression may be important determinant of biological responses.