金雀异黄酮对骨质疏松大鼠体内Sost表达的影响

目的:观察金雀异黄酮对去卵巢大鼠骨质疏松发生的影响并探讨其分子机制.方法:将60只雌性SD大鼠随机分为:空白对照组、模型对照组、尼尔雌醇组、金雀异黄酮高(18 mg/kg)、中(9 mg/kg)、低(4.5 mg/kg)剂量组.除空白对照组外,其余大鼠切除双侧卵巢诱发骨质疏松.模型复制成功后,给予含有不同剂量金雀异黄酮的饲料喂食骨质疏松大鼠.12周后行股骨干骨密度测定并行RT-PCR和Western blot检测股骨组织中sost基因表达水平的变化.结果:和模型对照组相比,中剂量金雀异黄酮组大鼠骨密度显著增加(P<0.01)股骨组织中sost表达明显下调.结论:金雀异黄酮对骨质疏松大鼠有治疗作用,该作用可能与下调sost表达有关.     

植物中CBL-CIPK途径转导特异Ca~(2+)信号的分子机制

Ca2+作为植物中普遍存在的第二信使,其信号传感器CBL(calcineurin B-like protein)及与CBL互作的蛋白CIPK(CBL-interacting protein kinase)所构成的CBL-CIPK信号转导途径在转导特异Ca2+信号过程中起重要的作用。由于植物同时受多种复杂环境因素影响,因此必须同时对各种复杂因素作出响应并转导并存的信号。CBL和CIPK在结构、表达以及功能上的特异性构成了CBL-CIPK途径能够转导特异Ca2+信号的分子基础。本文在介绍CBL、CIPK的基础上,着重对其组合成的CBL-CIPK途径转导特异Ca2+信号的分子机制进行综述。     

去极化时Ca~(2+) 内流引起蛋白激酶C_α浆膜转位

 为观察胞外Ca2 + 内流和肌浆网Ca2 + 释放两种来源的Ca2 + 对cPKCα转位激活的影响 ,揭示PKC在去极化 nAChR转录偶联中的作用 ,构建了pPKCα EGFP N1融合蛋白真核基因表达载体 .转染C2C1 2肌细胞后 ,采用激光共聚焦显微镜记录了KC1或咖啡因处理所引起的细胞Ca2 + 波变化及PKCα GFP融合蛋白在细胞内的分布 .结果提示 ,只有用KC1处理引起细胞膜去极化时 ,伴随Ca2 +内流 ,才能观察到PKCα GFP绿色荧光在细胞内发生的细胞浆至细胞膜分布变化 .然而 ,采用肌浆网Ca2 + 通道激动剂咖啡因刺激肌细胞 ,使肌浆网中Ca2 + 释放 ,未见PKCα GFP绿色荧光在浆、膜分布发生任何变化 .结果提示 ,去极化时外Ca2 + 内流可引起PKCα转位激活 ,肌浆网Ca2 + 释放对PKCα的转位激活没有影响 .     

介质中不同Ca~(2+)浓度对五种榕树幼苗钙含量的影响

选择高榕(Ficusaltissima)、木瓜榕(F.auriculata)、聚果榕(F.racemosa)、鸡嗉子榕(F.semicorda ta)以及对叶榕(F.hispida)5种榕树,对其幼苗进行不同Ca2+(CaCl2)浓度下的水培实验,结果表明,5种幼苗钙含量随介质Ca2+浓度的增加而增加,增加趋势大致相同。高Ca2+浓度下(10mmol/L)幼苗地上部的钙含量为低Ca2+浓度下(0.05mmol/L)的1.51～1.63倍。5种榕树幼苗钙含量随培养液Ca2+浓度变化的Yadj值的多重比较结果显示,对叶榕钙含量显著高于其他四种(P<0 01),高榕则显著低于其他四种(P<0 01),木瓜榕、聚果榕、鸡嗉子榕之间的差异不显著。不同榕树幼苗地上部分钙含量受介质钙浓度影响,但可能主要决定于基因型的差异,表明榕属植物富钙基因的存在,这为高钙榕树植物资源开发提供初步的理论依据。     

Ca~(2+)信号调控细胞死亡的亚细胞和分子机制

Ca2+在细胞死亡过程中起至关重要的作用。胞浆和/或线粒体(MT)内Ca2+超载不但是多种原因、多种方式细胞死亡的起始因素,也可通过激活蛋白酶、核酸酶、磷脂酶等酶,直接或间接通过与Bcl-2家族蛋白等重要调节因素的相互作用,全程参与死亡生化反应。内质网(ER)释放Ca2+与MT、溶酶体(LS)、细胞核相互作用,激活多种细胞死亡信号转导通路,导致细胞凋亡、坏死和自噬性细胞死亡。本文重点介绍Ca2+信号在亚细胞和分子水平调控细胞死亡的机制研究方面的最新进展。     

外源Ca~(2+)对高温强光下西葫芦幼苗形态特征、光合特性及荧光参数的影响

选用西葫芦(Cucurbita pepo)品种"阿兰"一代为试验材料,研究了外源Ca2+处理对高温强光交叉胁迫下西葫芦幼苗生长特征、光合特性及叶绿素荧光参数的影响.结果表明:高温强光胁迫下,5～20mmol·L-1Ca2+处理的西葫芦幼苗具有较高的株高和较大的叶面积,其叶绿素、类胡萝卜素含量及光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)、PSⅡ最大光化学效率(Fv/Fm)、PSⅡ实际光化学效率(ΦPSⅡ)和光化学猝灭系数(qP)均较高,而胞间CO2浓度(Ci)和非光化学猝灭系数(NPQ)较低,其中以10mmol·L-1Ca2+处理效果最好.说明5～20mmol·L-1Ca2+处理能有效缓解高温强光对西葫芦光合机构的不可逆伤害,使其保持较快的光合电子传递速率和较高的PSⅡ电子传递活性.Ca2+处理浓度超过40mmol·L-1时对高温强光胁迫没有缓解效应.     

TRPC6介导肺动脉高压大鼠肺动脉张力和肺动脉平滑肌细胞Ca~(2+)浓度的升高

经典瞬时感受器电位通道6(transient receptor potential channel6,TRPC6)蛋白是受体操纵性Ca2+通道(ROCC)的分子基础。本文旨在研究TRPC6/ROCC在野百合碱(monocrotaline,MCT)诱发的肺动脉高压大鼠模型中的作用。Sprague-Dawley大鼠随机分为正常对照组(CON组)和MCT组,CON组正常饲养三周,而MCT组按60mg/kg剂量一次性腹腔注射2%MCT,建立MCT诱导的慢性肺动脉高压大鼠模型。通过测定右心室收缩压(RVSP)和右心室重量指数(RVMI)、HE染色观察肺动脉血管形态,分析肺动脉结构重建。半定量RT-PCR和Western blot检测大鼠肺动脉TRPC6 mRNA和蛋白表达水平。血管张力实验中用可特异性激活ROCC、可透膜的DAG拟似物1-oleoyl-2-acetyl-sn-glycerol(OAG)检测大鼠离体肺动脉环的收缩效应。用荧光探针Fluo3-AM测定OAG诱导大鼠肺动脉平滑肌细胞(PASMCs)胞浆游离Ca2+浓度([Ca2+]i)。结果显示,与CON组相比,MCT组的RVSP、RVMI均明显增高(P0.01);形态学观察可见肺小动脉平滑肌层明显增厚,管腔减小;TRPC6的mRNA和蛋白质表达无明显变化。在CON组,OAG几乎不引起肺动脉环收缩,而在MCT组,肺动脉环的收缩反应显著增强,差别有显著性意义(P0.01)。相比较于CON组,MCT也可使OAG触发的PASMCs[Ca2+]i增量值显著升高(P0.05)。上述结果提示,MCT预处理对肺动脉TRPC6mRNA和蛋白质水平的表达无显著增强效应,但可促进TRPC6/ROCC介导的PASMCsCa2+内流和肺动脉张力升高,诱导大鼠产生肺动脉高压,并进一步诱发肺血管及右心室重构。     

Ca~(2+)参与NO对蚕豆气孔运动的调控

观察了Ca2 + 、Ca2 + 的螯合剂和Ca2 + 通道抑制剂对NO调控的蚕豆气孔运动的影响。结果表明 ,NO的供体 1～ 10 0 μmol/LSNP (sodiumnitroprusside ,硝普纳 )可诱导气孔关闭 ;除去表皮条缓冲液中的Ca2 + 后 ,NO不再影响气孔的运动 ;Ca2 + 的螯合剂EGTA和BAPTA几乎可以完全抑制NO诱导的气孔关闭作用 ;胞内钙通道抑制剂钌红 (rutheniumred)和L型Ca2 + 通道阻断剂硝苯吡啶 (nifedipine)能够减弱SNP诱导气孔运动的关闭趋势 ;加入Ca2 + 通道抑制剂LaCl3 ,则外源NO失去其诱导气孔关闭的作用。说明在NO调控的气孔运动中 ,在NO信号途径的下游可能涉及来自胞内和胞外Ca2 + 的参与 ,并且胞外Ca2 + 更为重要。     

大黄素影响巨噬细胞升高[Ca~(2+)]_i和释放TNF-a的作用特征

为了研究大黄素(emodin)对正常的和细菌脂多糖(LPS)刺激的大鼠腹腔巨噬细胞(PM准)释放肿瘤坏死因子琢(TNF-琢)和升高[Ca2+]i的影响,应用L929细胞系和MTT法检测TNF-琢量,同时用激光共焦扫描显微术检测单细胞[Ca2+]i变化动力学。结果显示大黄素能轻度促进正常PM准释放TNF-琢,并发现大黄素诱发PM准[Ca2+]i变化呈振荡波模式。大黄素显著抑制LPS刺激PM准过度释放TNF-琢和升高[Ca2+]i,10-5mol/L大黄素抑制了10mg/LLPS刺激的TNF-琢峰值的50%和[Ca2+]i峰值的68%。LPS诱发PM准[Ca2+]i变化呈现高幅值的“平台期”,大黄素使之转变为低幅值的波动变化。以上结果说明,大黄素对PM准释放TNF-琢和升高[Ca2+]i表现出的双向调节作用之间有一定的相关性,大黄素对LPS诱发的[Ca2+]i升高的调制,可能是抑制LPS刺激PM准释放TNF-琢的信号传导通路中的重要环节。     

大麦根细胞质膜Ca~(2+)-ATP酶和Ca~(2+)转运系统的特性

用大麦质膜微囊研究细胞质膜 Ca~(2+)转运过程,发现质膜 Ca~(2+)—ATP酶在反应系统中不存在Mg~(2+)时可正常表现活性。跨膜Ca~(2+)转运按其对Mg~(2+)的需求可分为两个过程,一个是不需Mg~(2+)的、具高Ca~(2+)亲和力和较低的转运能力;另一个则是需Mg~(2+)的、具低Ca~(2+)亲和力和较高的转运能力。前者的动力学特征与Ca~(2+)—ATP酶相近,而后者则相差很大。据此推测,大麦根细胞质膜上除Ca~(2+)—ATP酶外,还存在另一个不同的Ca~(2+)转运系统。由两者分别承担的Ca~(2+)转运过程在细胞钙信使系统中可能起着不同的作用。     

The Ca 2+ pumps and the Na + / Ca 2+ exchangers

The Ca 2+ ATPases or Ca 2+ pumps transport Ca 2+ ions out of the cytosol, by using the energy stored in ATP. The Na + / Ca 2+ exchanger uses the chemical energy of the Na + gradient (the Na + concentration is much higher outside than inside the cell) to remove Ca 2+ from the cytosol. Ca 2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na + / Ca 2+ exchangers are located on the plasma membrane. A Na + / Ca 2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca 2+ concentration in the cytosol and are vital to prevent Ca 2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.© Kluwer Academic Publishers     

Kinetic Characterization of Ca2+ Transport in Synaptic Membranes

Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane.     

NaHCO3胁迫下星星草根中Ca2＋与Ca2＋-ATPase的超微细胞化学定位

利用焦锑酸盐和磷酸铅沉淀技术分别对NaHCO3胁迫条件下星星草（Puccinellia tenuiflora）根中Ca2＋和Ca2＋-ATPase进行超微细胞化学定位研究,旨在进一步探讨Ca2＋在NaHCO3胁迫诱导胞内信号转导过程中的作用,以及Ca2＋-ATPase活性定位变化与NaHCO3胁迫下星星草抗盐碱能力的关系。结果表明：在正常状态下,根毛区细胞质内Ca2＋较少,主要位于质膜附近和液泡中,Ca2＋-ATPase主要定位于质膜和液泡膜,有一定活性。在0.448%NaHCO3胁迫下,根毛区细胞质中Ca2＋增多,液泡中Ca2＋减少,且主要集中于液泡膜附近,质膜和液泡膜Ca2＋-ATPase活性明显升高。在1.054%NaHCO3胁迫下,细胞质中分布的Ca2＋增多,而液泡中Ca2＋极少,Ca2＋-ATPase活性也降低。以上结果表明,Ca2＋亚细胞定位和Ca2＋-ATPase活性变化在星星草响应NaHCO3胁迫的信号传递过程中具有重要作用。     

Active calcium transport via coupling between the enzymatic and the ionophoric sites of Ca2+ + Mg2+-ATPase

The 20K dalton fragment of Ca²⁺ + Mg²⁺-ATPase obtained from the tryptically digested sarcoplasmic reticulum has been further purified using Bio-Gel P-100. This removed low-molecular-weight UV-absorbing and positive Lowry-reacting contaminants. The ionophoric activity of the 20K fragment in both oxidized cholesterol and phosphatidylcholine: cholesterol membranes is unaltered by this further purification. The 20K selectivity sequence in phosphatidylcholine: cholesterol membranes is Ba²⁺ > Ca²⁺ > Sr²⁺ > Mn²⁺ Mg²⁺. Digestion of intact sarcoplasmic reticulum vesicles with trypsin, which results in the dissection of the hydrolytic site (30K) from the ionophoric site (20K), is shown to disrupt energy transduction between ATP hydrolysis and calcium transport. This further implicates the 20K dalton fragment as a calcium transport site. These data and previous evidence are discussed in terms of a proposed model for the ATPase molecular structure and the mechanism of cation transport in sarcoplasmic reticulum.     

The role of the Na+/Ca2+ exchangers in Ca2+ dynamics in ventricular myocytes

The role of the Na⁺/Ca²⁺ exchanger (NCX) as the main pathway for Ca²⁺ extrusion from ventricular myocytes is well established. However, both the role of the Ca²⁺ entry mode of NCX in regulating local Ca²⁺ dynamics and the role of the Ca²⁺ exit mode during the majority of the physiological action potential (AP) are subjects of controversy. The functional significance of NCXs location in T-tubules and potential co-localization with ryanodine receptors was examined using a local Ca²⁺ control model of low computational cost. Our simulations demonstrate that under physiological conditions local Ca²⁺ and Na⁺ gradients are critical in calculating the driving force for NCX and hence in predicting the effect of NCX on AP. Under physiological conditions when 60% of NCXs are located on T-tubules, NCX may be transiently inward within the first 100 ms of an AP and then transiently outward during the AP plateau phase. Thus, during an AP NCX current (I_NCX) has three reversal points rather than just one. This provides a resolution to experimental observations where Ca²⁺ entry via NCX during an AP is inconsistent with the time at which I_NCX is thought to become inward. A more complex than previously believed dynamic regulation of I_NCX during AP under physiological conditions allows us to interpret apparently contradictory experimental data in a consistent conceptual framework. Our modelling results support the claim that NCX regulates the local control of Ca²⁺ and provide a powerful tool for future investigations of the control of sarcoplasmic reticulum (SR) Ca²⁺ release under pathological conditions.     

Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction

The correct spatial and temporal control of Ca²⁺ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long-standing interest in the role of caveolae in regulating intracellular Ca²⁺ concentration. In this review we provide an updated view of how caveolae may regulate both Ca²⁺ entry into cells and Ca²⁺-dependent signal transduction     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.     

Effect of phorbol 12-myristate 13-acetate on Ca2+-ATPase activity in rat liver nuclei

The effect of phorbol 12-myristate 13-acetate (PMA) on Ca²⁺-ATPase activity in rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺-Mg²⁺)-ATPase activity. The nuclear Ca²⁺-ATPase activity was significantly increased by the presence of PMA (2–20 μM) in the enzyme reaction mixture; the maximum effect was seen at 10 μM. The PMA (10 μM)-increased Ca²⁺-ATPase activity was not blocked by the presence of staurosporine (2 μM) or dibucaine (2 and 10 μM), an inhibitor of protein kinase. Meanwhile, vanadate (20 and 100 μM) caused a significant reduction in the nuclear Ca²⁺-ATPase activity increased by PMA (10 μM). The present finding suggests that PMA has an activating effect on liver nuclear Ca²⁺-ATPase independent of protein kinase. © 1994 Wiley-Liss, Inc.     

The Intracellular Loop of Orai1 Plays a Central Role in Fast Inactivation of Ca2+ Release-activated Ca2+ Channels

Store-operated Ca²⁺ entry (SOCE) due to activation of Ca²⁺ release-activated Ca²⁺ (CRAC) channels leads to sustained elevation of cytoplasmic Ca²⁺ and activation of lymphocytes. CRAC channels consisting of four pore-forming Orai1 subunits are activated by STIM1, an endoplasmic reticulum Ca²⁺ sensor that senses intracellular store depletion and migrates to plasma membrane proximal regions to mediate SOCE. One of the fundamental properties of CRAC channels is their Ca²⁺-dependent fast inactivation. To identify the domains of Orai1 involved in fast inactivation, we have mutated residues in the Orai1 intracellular loop linking transmembrane segment II to III. Mutation of four residues, V¹⁵¹SNV¹⁵⁴, at the center of the loop (MutA) abrogated fast inactivation, leading to increased SOCE as well as higher CRAC currents. Point mutation analysis identified five key amino acids, N¹⁵³VHNL¹⁵⁷, that increased SOCE in Orai1 null murine embryonic fibroblasts. Expression or direct application of a peptide comprising the entire intracellular loop or the sequence N¹⁵³VHNL¹⁵⁷ blocked CRAC currents from both wild type (WT) and MutA Orai1. A peptide incorporating the MutA mutations had no blocking effect. Concatenated Orai1 constructs with four MutA monomers exhibited high CRAC currents lacking fast inactivation. Reintroduction of a single WT monomer (MutA-MutA-MutA-WT) was sufficient to fully restore fast inactivation, suggesting that only a single intracellular loop can block the channel. These data suggest that the intracellular loop of Orai1 acts as an inactivation particle, which is stabilized in the ion permeation pathway by the N¹⁵³VHNL¹⁵⁷ residues. These results along with recent reports support a model in which the N terminus and the selectivity filter of Orai1 as well as STIM1 act in concert to regulate the movement of the intracellular loop and evoke fast inactivation.