大鼠心肌细胞核钙调素入核转运与核钙调节关系的探讨

最近发现,钙调素作为细胞内钙受体,除了调节胞浆的多种功能之外,可能还参与胞浆信号向核内快速传递。本研究观察大鼠心肌细胞核对钙调素的入核转运与钙浓度的关系,并初步探讨其调节机制。大鼠心肌细胞核采用差速离心和密度梯度离心分离提纯。用荧光分光光度计测定荧光标记钙调素向细胞核转入量发现,大鼠心肌细胞核对核外的CaM向核孔转运量具有[Ca~(2+)]浓度依赖性,随核外[Ca~(2+)]浓度的增加而增加(P<0.001),在[Ca~(2+)]浓度为10~(-3)mol/L时,ryanodine受体的拮抗剂rutheniumred和cADP ribose受体拮抗剂8-Br cADP ribose显著抑制CaM的细胞核孔转运(分别降低20％和18％,P<0.05),而IP_3受体拮抗剂heparin和Ca~(2+)-ATPase抑制剂thapsigargin抑制CaM的细胞核孔转运更显著(分别降低90％和89％,P<0.001)。上述结果表明心肌细胞核对CaM的向核转运,受核外[Ca~(2+)]和核钙摄取、释放所调节。     

血管紧张素Ⅱ增加新生鼠脑细胞胞浆Ca~(2+)浓度

本文应用荧光钙测定技术观察了血管紧张素Ⅱ(AⅡ)对新生Wistar鼠脑细胞胞浆Ca~(2+)浓度([Ca~(2+)]_i)的影响。结果表明:血管紧张素Ⅱ在1nmol/L—1μmol/L浓度下可诱导新生鼠脑细胞[Ca~(2+)]_i增加,具量效关系。在无外Ca~(2+)存在对,其增加幅度有所减少。上述效应可被血管紧张素Ⅱ拮抗剂Saralasin所阻断,并呈剂量依赖关系。上述结果提示,血管紧张素Ⅱ可激活血管紧张素AⅡ受体,增加脑细胞[Ca~(2+)]_i,该效应通过细胞内Ca~(2+)释放和细胞外Ca~(2+)内流两条适径实现,前者的作用是主要的。     

用Fura-2测定缺氧时海马细胞内游离钙离子浓度的变化

本文用Fura-2荧光测定技术直接监测了缺氧时大鼠海马细胞内游离钙离子浓度[Ca~(2+)]_1的变化。实验发现,缺氧可使海马细胞[Ca~(2+)]_1显著增高,并且在缺氧过程中其增高呈现明显的时相性变化。在去除细胞外钙的情况下,缺氧仍能使[Ca~(2+)]_1增高,仅增高幅度有所降低;另外[Ca~(2+)]_1不再出现时相性变化特征。结果提示,胞外Ca~(2+)的内流以及内源Ca~(2+)的释放均参与了缺氧所致海马细胞[Ca~(2+)]_1的增高过程,缺氧时[Ca~(2+)]_1升高的时相性变化为胞外Ca~(2+)内流引起。     

Con A刺激致T淋巴细胞胞浆游离Ca~(2+)浓度升高

本文分别应用荧光Ca~(2+)指示剂Quin2和Indo-1研究了Con A刺激的T淋巴细胞[Ca~(2+)]i升高过程及其发生机制.结果表明Con A与T淋巴细胞作用可导致细胞[Ca~(2+)]i的迅速升高.这种增加的胞内游离Ca~(2+)不仅来自胞外Ca~(2+)的内流,也来源于胞内钙库的释放.其中Ca~(2+)内流与T细胞钙通道的开放有关.可被钙通道抑制剂戊脉胺抑制,细胞的去极化及钾通道阻断剂四乙胺均不能阻断Ca~(2+)的内流,提示Ca~(2+)内流不是通过电位操纵的钙通道实现的,也与拥通道的开闭无关.Ca~(2+)内流可能是通过Con A受体活化的受体操纵的钙通道而实现的.     

压力超负荷性心肌肥厚大鼠心肌细胞核钙转运的改变

通过腹主动脉缩窄(abdominalaorticcoarctation ,AAC)心肌肥厚大鼠模型制备、差速离心提纯心肌细胞核、酶学方法测定Ca2 +-ATPase活性、45Ca2 +同位素法测定核钙摄取和荧光分光光度计测定细胞核内自由钙浓度 ,初步揭示压力超负荷心肌肥厚大鼠心肌细胞核钙转导异常的环节。结果发现 :心肌细胞核上存在具有[Ca2 +]和ATP依赖性的高亲和力Ca2 +-ATPase ,以[Ca2 +]依赖的方式摄取45Ca2 +,并呈先升高后降低趋势。AAC术后4周大鼠心肌显著肥厚 ,伴有明显的血流动力学异常 ,与对照组比较 ,AAC大鼠心肌细胞核Ca2 +-ATPase活性减少51.93 %(p<0.001) ,但核45Ca2 +摄入量(核外[Ca2 +]浓度为800 -1600nmol/L时)和核内[Ca2 +](核外[Ca2 +]浓度为0 -1000nmol/L时)均明显增加(p<0.05) ;正常组离体心肌细胞核Ca2 +摄取受PKA刺激(p<0.05) ,而被PKC抑制剂和CaM抑制剂显著抑制(p<0.05) ,AAC大鼠心肌细胞核Ca2 +摄取仅受CaM抑制剂抑制(p<0.01) ,而PKA和PKC抑制剂对其无明显影响(p>0.05)。结论为心肌肥厚时 ,心肌细胞核Ca2 +转运系统及其磷酸化调节可能发生改变。     

钙离子研究进展

一、钙离子振荡的时间空间分布通常有两个平行的系统调节着细胞内Ca~(2+)的释放。在大多数可兴奋细胞,细胞外钙由电压敏感钙通道进入细胞,使胞内游离钙[Ca~(2+)]_i有一个初期上升,后者使内质网上的ryanodine敏感钙通道开放,将Ca~(2+)释放到胞浆内。而在许多非可兴奋细胞,细胞表面受体由G蛋白介导     

在ts-RSV LA90细胞转化过程中钙流变化的研究

本文以ts-RSV LA90细胞为模型,用放射性同位素示踪技术测定了通过细胞质膜的~(45)Ca~(2+)流水平;同时用钙指示剂Indo-1 AM和光学多道分析仪测定了胞内[Ca~(2+)]_i,初步研究了Ca~(2+)流和[Ca~(2+)]_i在v-src基因引起细胞转化过程中的动态变化。结果表明LA90细胞质膜上~(45)Ca~(2+)流的改变是细胞转化过程中可以检测到的早期事件之一,转化状态(33℃)细胞的~(45)Ca~(2+)流大于正常状态(40℃)的,细胞从正常到转化(40℃→33℃)的25分钟内~(45)Ca~(2+)流就有明显增大。TMB-8可以抑制转化引起的~(45)Ca~(2+)流出的增大,小牛血清可以刺激正常状态细胞的~(45)Ca~(2+)流出增大,~(45)Ca~(2+)流出与温度有一定依赖关系;细胞转化引起的~(45)Ca~(2+)流入增大,可被异博定抑制,~(45)Ca~(2+)流入不受温度的影响。LA90细胞[Ca~(2+)]_i在转化早期有明显升高,并维持在较正常细胞高2—3倍的水平,A23187-Br可提高正常LA90细胞[Ca~(2+)]_i,[Ca~(2+)]_i不受温度的影响。从质膜上~(45)Ca~(2+)流和[Ca~(2+)]_i的增大说明转化细胞虽然对胞外Ca~(2+)浓度依赖性下降,但维持增殖及转化状态仍然需要一定的胞外Ca~(2+),并通过提高质膜Ca~(2+)流入和释放内源性Ca~(2+),使转化细胞[Ca~(2+)]_i维持在较高水平上。LA90细膜质膜上~(45)Ca~(2+)流和[Ca~(2+)]_i的增大在细胞转化中起着重大作用。     

L-型钙通道自身调控异常参与缺血再灌注心肌钙超载的形成

钙超载作为心肌缺血再灌注损伤的重要机制之一,其形成原因与治疗策略一直是研究的热点。心肌遭受缺血再灌注后,参与细胞内钙循环的L-型电压依赖钙通道(L-type voltage-dependent calcium channel,L-VDCC)、肌浆网钙ATP酶2a(sarco/endoplasmic reticulum ATPase 2a,SERCA2a)和受磷蛋白(phospholamban,PLB)、Ryanodine受体2(RyR2)、Na~+/Ca~(2+)交换体、Na~+/H~+交换体等多种蛋白功能异常,导致舒张期[Ca~(2+)]_i上升,钙瞬变幅度降低,细胞出现钙超载。[Ca~(2+)]_i升高的过程大致可分为两个阶段:早期的[Ca~(2+)]_i升高过程(部分由钙通道介导)和晚期的[Ca~(2+)]_i升高过程(主要由Na~+/Ca~(2+)交换体介导)。L-VDCC活性增加参与钙超载的形成,但是L-VDCC蛋白在缺血再灌注过程中的分子变化机制尚不清楚。L-VDCC通道调控方式包括两类:自身调节和外源性调节,其中外源性调节蛋白PKG和PKA的调控不能解释细胞水平的L-VDCC活性增加现象,而在缺血再灌注过程中,钙依赖的失活(calcium-dependent inactivation,CDI)效应减弱、钙依赖的易化(calcium-dependent facilitation,CDF)效应增强、羧基远端部分肽链(distal carboxy terminus,DCT)的抑制效应减弱,这三种自身调节机制的改变引起L-VDCC活性的增加。因此,可以认为L-VDCC通道自身调控异常参与缺血再灌注损伤中心肌细胞钙超载的形成。     

硫化氢减少缺氧时人脐静脉内皮细胞的凋亡

目的探讨硫化氢对血管内皮细胞缺氧性损伤的保护作用及机制。方法将人脐静脉内皮细胞于2%O_2、5%CO_2、93%N_2培养箱中培养24h,复制缺氧模型。以硫氢化钠(NaHS)作为硫化氢供体,采用DCFH-DA和激光共聚焦系统检测细胞内活性氧含量;用Fura-2/AM和显微荧光成像系统检测细胞质内钙离子([Ca~(2+)]_i)浓度;使用Rh123和激光共聚焦系统检测细胞线粒体膜电位(mitochondria membrane potential,MMP);用Hoechst33342染色观察细胞核形态,计算细胞凋亡率。结果缺氧使细胞ROS含量显著升高,硫氢化钠预处理显著降低缺氧所致ROS含量的升高;缺氧使细胞[Ca~(2+)]_i显著升高,而NaHS、活性氧清除剂NAC、IP_3受体抑制剂Xesto C均抑制缺氧诱导[Ca~(2+)]_i的升高;缺氧显著降低细胞MMP,NaHS、NAC预处理缺氧细胞则抑制。MMP降低;NaHS、Xesto C预处理可显著降低缺氧所致细胞凋亡。结论硫化氢可能一方面通过减轻缺氧时ROS增多引起的线粒体膜电位下降,保护线粒体功能;另一方面抑制缺氧时ROS增多诱导的钙超载,从而减少缺氧时人脐静脉内皮细胞的凋亡。     

钙通道与钙释放通道

1.Ca~(2+)的重要生理作用胞内游离钙浓度([Ca~(2+)])的变化调节着细胞的代谢、基因表达等细胞共有的活动,以及始动兴奋、收缩或出胞分泌以及激活和失活离子通道等细胞不同的反应。[Ca~(2+)]的升高主要依赖于胞外钙经质膜上的钙通道内流或/和胞内储存钙的释放。释放的内钙也是藉细胞器膜的钙释放通道进入胞浆。可见通道启闭活动的正常是维持[Ca~(2+)]正常的一个重要保证。2.离子通道及其分类离子通道是贯穿于质膜或细胞器膜的大分子蛋白质,其中央形成能通过离子的亲水性孔道(pores)。离子的跨膜转运是通过膜上通道蛋白的功能来完     

心肌细胞核Ca^2＋库特点及其调节的离体研究

To investigate the regulation of Ca2+ in the isolated cardiac nuclei from rats which may illuminated the mechanism of nuclear calcium transport system. Elocity and isopyknic gradient centrifugation were employed to fractionate rat cardiac nuclei. Then fluo-4 confocal microscopy techniques was used to verify the changes of nuclear Ca2+. There are calcium-dependent Ca2+ uptake in the cardiac nuclear obtained from normal rats. The accumulation Ca2+ of cardiac nuclei in vitro from the incubating medium were not consistent with free [Ca2+] in incubating medium. The nuclear envelope was initially loaded with Ca2+ (1 mmol/L ATP and approximately 100 nmol/L Ca2+), Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope and nucleoplasm. Exposure of Ca2+ -loaded nuclei to IP3, ryanodine or ryanodine + thapsigargin, respectively, resulted in a rapid and transient elevation of nucleoplasmic Ca2+ free concentration, this effects were abolished by pretreatment of cardiac nuclei with Ca2+ -ATPase inhibitor thapsigargin. Thapsigargin and IP3 receptor antagonist heparin induced nucleoplasmic Ca2+ free concentration decrease. Fluorescence experiments indicated that both ryanodine receptors and Ca2+ -ATPase were distributed in the outer layer of nuclear envelope, and inositol 1,4,5-trisphosphate receptors mainly dispersively localized at inner layer of nuclear envelope. The present study demonstrates that nuclear calcium were regulated by free Ca2+, IP3 and ryanodine. The results suggested calcium transport system might be present in the myocardial nuclei, the myocardial nuclei might served as one of calcium pools in myocardial cell.     

NAADP mobilizes Ca2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors

Ca2+ release from the envelope of isolated pancreatic acinar nuclei could be activated by nicotinic acid adenine dinucleotide phosphate (NAADP) as well as by inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR). Each of these agents reduced the Ca2+ concentration inside the nuclear envelope, and this was associated with a transient rise in the nucleoplasmic Ca2+ concentration. NAADP released Ca2+ from the same thapsigargin-sensitive pool as IP3. The NAADP action was specific because, for example, nicotineamide adenine dinucleotide phosphate was ineffective. The Ca2+ release was unaffected by procedures interfering with acidic organelles (bafilomycin, brefeldin, and nigericin). Ryanodine blocked the Ca2+-releasing effects of NAADP, cADPR, and caffeine, but not IP3. Ruthenium red also blocked the NAADP-elicited Ca2+ release. IP3 receptor blockade did not inhibit the Ca2+ release elicited by NAADP or cADPR. The nuclear envelope contains ryanodine and IP3 receptors that can be activated separately and independently; the ryanodine receptors by either NAADP or cADPR, and the IP3 receptors by IP3.     

Calcium sensing receptor regulates cardiomyocyte function through nuclear calcium

Nuclear Ca2+ plays a pivotal role in the regulation of gene expression. IP3 (inositol-1,4,5-trisphosphate) is an important regulator of nuclear Ca2+. We hypothesized that the CaR (calcium sensing receptor) stimulates nuclear Ca2+ release through IICR (IP3-induced calcium release) from perinuclear stores. Spontaneous Ca2+ oscillations and the spark frequency of nuclear Ca2+ were measured simultaneously in NRVMs (neonatal rat ventricular myocytes) using confocal imaging. CaR-induced nuclear Ca2+ release through IICR was abolished by inhibition of CaR and IP3Rs (IP3 receptors). However, no effect on the inhibition of RyRs (ryanodine receptors) was detected. The results suggest that CaR specifically modulates nuclear Ca2+ signalling through the IP3R pathway. Interestingly, nuclear Ca2+ was released from perinuclear stores by CaR activator-induced cardiomyocyte hypertrophy through the Ca2+-dependent phosphatase CaN (calcineurin)/NFAT (nuclear factor of activated T-cells) pathway. We have also demonstrated that the activation of the CaR increased the NRVM protein content, enlarged cell size and stimulated CaN expression and NFAT nuclear translocation in NRVMs. Thus, CaR enhances the nuclear Ca2+ transient in NRVMs by increasing fractional Ca2+ release from perinuclear stores, which is involved in cardiac hypertrophy through the CaN/NFAT pathway.     

心肌肥厚大鼠心肌细胞核三磷酸肌醇受体的特征

为了研究细胞核三磷酸肌醇受体在心肌肥厚中的作用,制备了腹主动脉缩窄大鼠心肌肥厚模型、用差速离心和密度梯度离心提纯心肌细胞核,以［3H］IP3为配基,采用放射受体分析心肌细胞核膜IP3R与其配体的最大结合容量(Bmax)和解离常数(Kd)。大鼠心肌细胞核上存在IP3R、CaM和PKC激动剂PMA,能显著抑制该受体与IP3的结合(P<0.05);核外［Ca2+］也能剂量依赖的抑制细胞核IP3R与IP3的结合。腹主动脉缩窄术后4周,大鼠心肌显著肥大,伴有明显的血流动力学异常,其心肌细胞核IP3R的Bmax和Kd与对照组比较分别增加1.217和2.149倍(P<0.01)。心肌细胞核上存在IP3R,并受CaM和PMA及核外［Ca     

Extracellular ATP-induced nuclear Ca transient is mediated by inositol 1,4,5-trisphosphate receptors in mouse pancreatic β-cells

Extracellular ATP (eATP) induces an intracellular Ca²⁺ transient by activating phospholipase C (PLC)-associated P2X4 purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent Ca²⁺ release from intracellular stores in mouse pancreatic β-cells. Using laser scanning confocal microscopy, Ca²⁺ indicator fluo-4 AM, and the cell permeable nuclear indicator Hoechst 33342, we examined the properties of eATP-induced Ca²⁺ release in pancreatic β-cell nuclei. eATP induced a higher nuclear Ca²⁺ transient in pancreatic β-cell nuclei than in the cytosol. After pretreatment with thapsigargin (TG), an inhibitor of sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pumps, the amplitude of eATP-induced Ca²⁺ transients in the nucleus was still much higher than those in the cytosol. This effect of eATP was not altered by inhibition of either the plasma membrane Ca²⁺-ATPase (PMCA) or the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) by LaCl₃ or by replacement of Na⁺ with N-Methyl-Glucosamine. eATP-induced nuclear Ca²⁺ transients were abolished by a cell-permeable IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), but were not blocked by the ryanodine receptor (RyR) antagonist ryanodine. Immunofluorescence studies showed that IP3Rs are expressed on the nuclear envelope of pancreatic β-cells. These results indicate that eATP triggers nuclear Ca²⁺ transients by mobilizing a nuclear Ca²⁺ store via nuclear IP3Rs.     

Nucleoplasmic Ca(2+)loading is regulated by mobilization of perinuclear Ca(2+)

Regulation of nucleoplasmic calcium (Ca(2+)) concentration may occur by the mobilization of perinuclear luminal Ca(2+)pools involving specific Ca(2+)pumps and channels of both inner and outer perinuclear membranes. To determine the role of perinuclear luminal Ca(2+), we examined freshly cultured 10 day-old embryonic chick ventricular cardiomyocytes. We obtained evidence suggesting the existence of the molecular machinery required for the bi-directional Ca(2+)fluxes using confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific for ryanodine-sensitive Ca(2+)channels (RyR2), sarco/endoplasmic reticulum Ca(2+)ATPase (SERCA2)-pumps, and fluorescent BODIPY derivatives of ryanodine and thapsigargin. Using immunocytochemistry techniques, confocal imaging showed the presence of RyR2 Ca(2+)channels and SERCA2-pumps highly localized to regions surrounding the nucleus, referable to the nuclear envelope. Results obtained from Fluo-3, AM loaded ionomycin-perforated embryonic cardiomyocytes demonstrated that gradual increases of extranuclear Ca(2+)from 100 to 1600 nM Ca(2+)was localized to the nucleus. SERCA2-pump inhibitors thapsigargin and cyclopiazonic acid showed a concentration-dependent inhibition of nuclear Ca(2+)loading. Furthermore, ryanodine demonstrated a biphasic concentration-dependence upon active nuclear Ca(2+)loading. The concomitant addition of thapsigargin or cyclopiazonic acid with ryanodine at inhibitory concentrations caused an significant increase in nuclear Ca(2+)loading at low concentrations of extranuclear added Ca(2+). Our results show that the perinuclear lumen in embryonic chick ventricular cardiomyocytes is capable of autonomously regulating nucleoplasmic Ca(2+)fluxes.     

Structural Association of Endoplasmic Reticulum with Other Membrane Systems in Populus deltoides Apical Bud Cells and Its Alterations During the Short Day-induced Dormancy

A comparative study was carried out on the EM-cytochemical localization of calcium and Ca2+-ATPase activity in the suspension-cultured cells between the chilling-sensitive maize (Zea mays L. cv. Black Mexican Sweet) and chilling-insensitive Trititrigia (Triticum sect. Trititrigia mackey) at 4 ℃ chilling. When maize and Tyititrigia cells were cultured at 26 ℃, electron microscopic observations revealed that the electron-dense calcium antimonate deposits, an indication of the calcium localization, were localized mainly in the vacuoles, and few was found in the cytosol and nuclei. The electron-dense cerium phosphate deposits, an indication of Ca2+-ATPase activity, were abundantly distributed on the plasma membrane (PM). When the cells from both species were cultured at 4 ℃ for 1 and 3 h, an elevation of Ca2+ level in the cytosol and nuclei was observed, whereas the cerium phosphate deposits on the PM showed no quantitative difference from those of the 26 ℃-cultured cells, indicating that the enzymatic activities were not altered during these chilling periods. However, there was a distinct difference in the dynamics of the Ca2+ distribution and the PM Ca2+-ATPase activity between maize and Trititrigia when chilled at 4 ℃ for 12, 24 and 72 h. In maize cells, a large number of Ca2+ deposits still existed in the cytosol and nuclei, and the PM Ca2+-ATPase became less and less active, and even inactive at all. In Trititrigia cells, the increased cytosolic and nuclear Ca2+ ions decreased after 12 h chilling. By chilling up to 24 and 72 h, the intracellular Ca2+ concentration had been restored to a similar low level as those of the warm temperature-cultured cells, while the activity of the PM Ca2+-ATPase maintained high. The transient cytosolic and nuclear Ca2+ increase and the activities of PM Ca2+-ATPase during chilling are discussed in relation to plant cold hardiness.