Ca^(2+)-ATPase参与植物耐盐性调控的研究进展

盐胁迫下细胞质Ca^(2+)浓度升高,细胞会激活Ca^(2+)调节的靶酶或者与Ca^(2+)高度亲和的受体蛋白,其中,与Ca^(2+)高度亲和的受体蛋白中,植物钙泵(Ca^(2+)-ATPase)是P型ATP酶,包含内质网Ca^(2+)-ATPases与质膜Ca^(2+)-ATPas‐es,通过主动运输将Ca^(2+)从细胞质转移到质外体或细胞器。大量研究表明,植物的耐盐性在很大程度上与其维持钙泵即Ca^(2+)-ATPase活性的能力有关。多种植物Ca^(2+)-ATPase对盐胁迫表现出敏感性,并受到外源Ca^(2+)的保护,表明外源钙处理与Ca^(2+)-ATPase活性可能在盐胁迫下的细胞内钙稳态和信号转导中起重要作用。该研究概述了植物Ca^(2+)-ATPase类型、结构与性质,亚细胞定位Ca^(2+)-ATPase及外源钙与亚细胞定位Ca^(2+)-ATPase参与植物耐盐调控研究进展,重点对质膜、液泡膜、核膜、内质网及高尔基体Ca^(2+)-ATPases参与植物耐盐调控的研究进展进行了综述,并提出展望。该研究为了解植物耐盐性生理及分子机制提供帮助,同时为作物耐盐栽培提供新思路。

Role of platelet plasma membrane Ca^(2+)-ATPase in health and disease

Platelets have essential roles in both health and disease. Normal platelet function is required for hemostasis.Inhibition of platelet function in disease or by pharmacological treatment results in bleeding disorders.On the other hand,hyperactive platelets lead to heart attack and stroke.Calcium is a major second messenger in platelet activation,and elevated intracellular calcium leads to hyperactive platelets.Elevated platelet calcium has been documented in hypertension and diabetes;both conditions increase the likelihood of heart attack and stroke. Thus,proper regulation of calcium metabolism in the platelet is extremely important.Plasma membrane Ca2+-ATPase(PMCA)is a major player in platelet calcium metabolism since it provides the only significant route for calcium efflux.In keeping with the important role of calcium in platelet function,PMCA is a highly regulated transporter.In human platelets,PMCA is activated by Ca2+/calmodulin,by cAMP-dependent phosphorylation and by calpain-dependent removal of the inhibitory peptide.It is inhibited by tyrosine phosphorylation and calpain-dependent proteolysis.In addition,the cellular location of PMCA is regulated by a PDZ-domain-dependent interaction with the cytoskeleton during platelet activation.Rapid regulation by phosphorylation results in changes in the rate of platelet activation,whereas calpain-dependent proteolysis and interaction with the cytoskeleton appears to regulate later events such as clot retraction.In hypertension and diabetes,PMCA expression is upregulated while activity is decreased, presumably due to tyrosine phosphorylation.Clearly,a more complete understanding of PMCA function in human platelets could result in the identification of new ways to control platelet function in disease states.

Plasma membrane Ca^(2+)-ATPases:Targets of oxidative stress in brain aging and neurodegeneration

The plasma membrane Ca2+-ATPase(PMCA)pumps play an important role in the maintenance of precise levels of intracellular Ca2+[Ca2+]i,essential to the functioning of neurons.In this article,we review evidence showing age-related changes of the PMCAs in synaptic plasma membranes(SPMs).PMCA activity and protein levels in SPMs diminish progressively with increasing age. The PMCAs are very sensitive to oxidative stress and undergo functional and structural changes when exposed to oxidants of physiological relevance.The major signatures of oxidative modification in the PMCAs are rapid inactivation,conformational changes,aggregation, internalization from the plasma membrane and proteolytic degradation.PMCA proteolysis appears to be mediated by both calpains and caspases.The predominance of one proteolytic pathway vs the other,the ensuing pattern of PMCA degradation and its consequence on pump activity depends largely on the type of insult,its intensity and duration.Experimental reduction of PMCA expression not only alters the dynamics of cellular Ca2+ handling but also has a myriad of downstream conse-quences on various aspects of cell function,indicating a broad role of these pumps.Age-and oxidation-related down-regulation of the PMCAs may play an important role in compromised neuronal function in the aging brain and its several-fold increased susceptibility to neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease,and stroke.Therapeutic approaches that protect the PMCAs and stabilize[Ca2+]i homeostasis may be capable of slowing and/or preventing neuronal degeneration.The PMCAs are therefore emerging as a new class of drug targets for therapeutic interventions in various chronic degenerative disorders.

Effect of Huoxuequyu Recipe on Erythrocyte Membrane Ca^(2+)-ATPase Activity in Pregnant Rats with Asymmetrical Intrauterine Growth Retardation Induced by Passive Smoking

The effect of Huoxuequyu recipe on erythrocyte membrane Ca2+-ATPase activity in pregnant rats with passive smoking induced asymmetrical intrauterine growth retardation(IUGR)was observed.The rats were divided into three groups:control group,model group and treated group.The fetal mean birth weight and erythrocyte membrane Ca2+-ATPase activity were found to be decreased in the model group as compared with the control and treated groups.There was a significant difference between the model group and the control group(P<0.01),while no significant difference existed between the treated group and the control group(P>0.05).Furthermore,the findings indicated that the erythrocyte membrane Ca2+-ATPase activity seem to be positively correlated with the birth weight(P<0.05).On the basis of our observation,it is believed that the Huoxuequyu decoction could increase erythrocyte membrane Ca2+-ATPase activity and promote blood circulation, thereby exerting a beneficial effect on fetal development.

Effects of Calcium on the GABA_A-Coupled CI^-/HCO₃^--ATPase from Plasma Membrane of Rat Brain

The work is a study of the influence of Ca2+ (0.01 - 1 mM) on neuronal CI-, HCO3-, -ATPase complex: an enzyme that is a CI--pump which is functionally and structurally coupled to GABAA-receptors. It is found that influence of Ca2+ on the multifunctional complex starts at concentration of 50·M and at concentration of 0.1 mM, it reduces the “basal” one and increases the CI-, HCO3-, -stimulated Mg2+-ATPase activities. GABA (0.1 - 100μM) activates the “basal” Mg2+-ATPase activity in the ab-sence of calcium. The effect of GABA on the enzyme in the presence of 0.01 ·M Ca2+ does not change. At the same time, 1 mM Ca2+eliminates the GABA effect on the “basal” Mg2+-ATPase activity. Competitive blocker of GABAA-receptors bicuculline (5 - 20 μM) in the absence of Ca2+ ions elimi-nates the stimulation of the “basal” Mg2+-ATPase by anions. When 0.25 mM Ca2+ is added to the in-cubation medium the inhibitory bicuculline effect on the enzyme does not appear. We found that 0.1 mM o-vanadate (protein tyrosine phosphatase blocker) reduces the GABA-activated ATPase activity. At the same time, 0.1 mM genistein (a protein tyrosine kinase blocker) has no effect on enzyme activity. In the presence of Ca2+ (0.25 mM), the effect of o-vanadate on the “basal” and CI-, HCO3-, -ATPase activities does not appear. It is shown for the first time that high concentrations of Ca2+prevent the action of GABAA-ergic ligands on the study ATPase. It is assumed that there is the involvement of protein kinases and protein phosphatases in the modulation of the enzyme activity by calcium. The observed effect of calcium on the ATPase may play an important role in the study of the mechanisms of epileptogenesis and seizure activity.

Involvement of Plasma Membrane Ca^(2+)/H^+ Antiporter in Cd^(2+) Tolerance

Cation exchangers （CAXs） belong to the cation/Ca2＋exchanger superfamily which have been extensively investigated in plant tonoplasts over the last decade. Recently, the roles of CAXs involved in heavy metal accumulation and tolerance in plants have been studied for phytoremediation and food security. In this mini review, we summarize the roles of the Ca2＋/H＋ antiporter in Ca2＋ signal transduction, maintaining ion homeostasis and sequestering heavy metals into the vacuole. Moreover, we present a possible role of the plasma membrane Ca2＋/H＋ antiporter in heavy metal detoxification.

Contribution of plasma membrane Ca^(2+) ATPase to cerebellar synapse function

The cerebellum expresses one of the highest levels of the plasma membrane Ca2+ATPase,isoform 2 in the mammalian brain.This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex;i.e. the Purkinje neurons(PNs) .Here we review recent evidence,including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2(PMCA2) knockout mouse,to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour.These studies have also revealed that deletionof PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development,they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.

Allosteric inhibitors of plasma membrane Ca^(2+) pumps: Invention and applications of caloxins

Plasma membrane Ca2+pumps(PMCA)play a major role in Ca2+homeostasis and signaling by extruding cellular Ca2+with high affinity.PMCA isoforms are encoded by four genes which are expressed differentially in various cell types in normal and disease states.Therefore, PMCA isoform selective inhibitors would aid in delineating their role in physiology and pathophysiology.We are testing the hypothesis that extracellular domains of PMCA can be used as allosteric targets to obtain a novel class of PMCA-specific inhibitors termed caloxins. This review presents the concepts behind the invention of caloxins and our progress in this area.A section is also devoted to the applications of caloxins in literature. We anticipate that isoform-selective caloxins will aid in understanding PMCA physiology in health and disease. With strategies to develop therapeutics from bioactive peptides,caloxins may become clinically useful in car diovascular diseases,neurological disorders,retinopathy,cancer and contraception.

人晶状体上皮细胞系细胞膜Ca^(2+)-ATPase同工异构体2的表达

目的 检测人晶状体上皮细胞系B 3(HLEB 3)中,细胞膜钙ATP酶(PMCA)的同工异构体之一,PMCA2的表达情况。 方法 细胞培养HLEB 3,当细胞培养达 90%融合时,提取其总mRNA,用PMCA2特异性引物,用反向聚合酶链反应(RT PCR)法检测PMCA2在mRNA水平上的表达。提取细胞膜蛋白,经SDS PAGE电泳以及蛋白免疫印记法(Westernblot),用抗 PMCA2的特异抗体检测其在蛋白质水平上的表达。 结果 RT PCR显示PMCA2特异性引物在HLEB 3细胞扩增出唯一 550bp的片段。蛋白免疫印记法证实PMCA2的表达,相对分子质量约为126 000。 结论 HLEB 3细胞中,在mRNA水平以及蛋白质水平均有PMCA2的表达。并有可能在HLEB 3细胞的钙离子平衡调节中发挥重要作用。

质膜Ca^2＋-ATPase的结构与功能

质膜Ca2+-ATPase(PMCA)是P型ATPase家族的一员,在真核细胞中主要负责信号刺激后胞内高浓度Ca2+的清除扫尾工作,并对维持静息状态下较低Ca2+浓度起着重要的调节作用.PMCA的一级结构已被确定,拓扑学结构显示,它有10个跨膜区和3个胞浆功能区.它的4个编码基因可产生4种亚型(PMCA 1~4),这些亚型在功能与分布上存在差异.PMCA的活性可被钙调蛋白等多种因素调节,这与其结构特征息息相关.近年来,PMCA已被证实与脂筏结构有一定关联,它在信号传导和细胞凋亡中的作用也成为目前科学研究的焦点.本文主要对PMCA的结构、亚型和功能的研究现状进行综述.

烟草叶和根细胞质膜Ca^(2+)-ATPase特性的比较研究

测定了烟草叶和根细胞质膜Ca2+-ATPase的活性并对它们的特性进行了比较.结果表明,二者在许多方面都不尽相同.前者的最适pH为7.0,后者则为6.0,而且在一个相对较宽的pH范围内有高的活性.叶细胞质膜Ca2+-ATPase的最适反应温度为45℃,[ATP]0.5为2.2mmol/L,根细胞质膜Ca2+-ATPase则分别为40℃和1.9mmol/L.叶和根的细胞质膜Ca2+-ATPase均受Ca2+激活.曙红B(EB)对烟草叶和根细胞质膜Ca2+-ATPase有明显的抑制作用,当EB浓度达到50μmol/L后,抑制作用才不再有显著的增加.本文讨论了烟草叶和根细胞质膜Ca2+-ATPase特性出现差异的原因及其与环境的关系.

质膜Ca^(2+)-ATPase和钙调节异常在恶性肿瘤发生发展中的作用及研究进展

钙离子(Ca^(2+))作为细胞内普遍存在的第二信使在细胞的许多生理病理过程中发挥了重要作用。而质膜Ca^(2+)-ATPase(PMCA)是一种依赖ATP的驱动将细胞内的Ca^(2+)排出胞外的钙泵。它的表达异常将会导致细胞内Ca^(2+)平衡的紊乱以及各种疾病的发生。由于Ca^(2+)和神经传递、肌肉收缩密切相关,早期的研究大多集中在中枢神经系统、心血管系统。近年来,随着研究的进展。很多学者逐渐发现钙信号在一些恶性肿瘤的进程中发挥了重要作用,比如增殖、迁移等。很多研究也发现了在各种恶性肿瘤中PMCA表达的改变,包括乳腺癌,结直肠癌、胰腺癌等。同时,在一些恶性肿瘤细胞系中发现并证实了PMCA各种亚型的存在及其差异性的表达。一些研究者还在一些临床肿瘤标本中检测了PMCA各种亚型的表达水平,而且对其表达水平改变的机制进行了初步的研究并提出在某些特异性的恶性肿瘤中PMCA有可能成为潜在的治疗靶点。因此,该文将对PMCA和钙调节异常在各种恶性肿瘤发生发展中的作用及研究进展进行综述。

Plasma membrane calcium pump regulation by metabolic stress

The plasma membrane Ca2+-ATPase(PMCA)is an ATPdriven pump that is critical for the maintenance of low resting[Ca2+]i in all eukaryotic cells.Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism,has the capacity to cause ATP depletion and thus inhibit PMCA activity.This has potentially fatal consequences,particularly for non-excitable cells in which the PMCA is the major Ca2+efflux pathway.This is because inhibition of the PMCA inevitably leads to cytosolic Ca2+ overload and the consequent cell death.However,the relationship between metabolic stress,ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted.There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion.In particular,there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function.Moreover, membrane phospholipids,mitochondrial membrane potential,caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA.The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca2+ overload and cytotoxicity.

ACTIVE CALCIUM TRANSPORT IN PLASMA MEMBRANE VESICLES FROM DEVELOPING COTYLEDONS OF COMMON BEAN

Plasma membrane vesicles were prepared from the developing cotyledons of common beau (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes.The putative plasma membrane fraction was minimally contaminated by membranes Other than plasma membrane and hence was of high purity. It exhibited a Ca2+dependent ATPase activity, which was inhibited by 1μ mol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATPdependent 45Ca2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6μmol/L CaM and 20μmol/L IAA but inhibited by 2μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca2+ in mediating phytohormones activity is discussed.

PMCA1-4及其A端和C端剪接变异体在成年大鼠前庭组织中的表达

目的研究质膜钙ATP酶异构体1-4(plasma membrane Ca^(2+)-ATPase 1-4,PMCA1-4)在成年大鼠前庭组织的分布部位,及其A端和C端剪接变异体的表达类型。方法选择8周龄健康SD大鼠,解剖出内耳器官行前庭切片,同时取前庭椭圆囊斑和球囊斑提取总RNA。通过免疫荧光方法检测PMCA1-4在成年大鼠内耳前庭组织的表达部位,通过逆转录聚合酶链反应(RT-PCR)法检测PMCA1-4的A端和C端剪接变异体在成年大鼠前庭组织的表达类型。结果球囊和椭圆囊荧光染色切片中,于毛细胞和支持细胞的胞体外侧膜可见PMCA1表达,毛细胞纤毛中可见PMCA2强表达,毛细胞和支持细胞的胞体外侧膜可见PMCA3弱表达,PMCA4几乎不表达。4种PMCA亚型在球囊表达的剪接体分别为PMCA1x/b、PMCA2w/b、PMCA3z/(a,b)和PMCA4x/b,它们在椭圆囊的表达类型与球囊相同。结论PMCA1-4在成年大鼠前庭器官的表达部位存在差异,这4种PMCA亚型的剪接体类型也各不相同。这种差异性可能是为了满足前庭组织和亚细胞结构域对Ca2+调节的特殊需求。

质膜Ca^(2+)-ATP酶异构体2基因多态性与噪声性听力损失易感性的关系

目的 探讨质膜Ca2 + - ATP酶异构体 2 (PMCA2 )基因多态性与噪声性听力损失的关系。方法 采用横断面流行病学研究方法 ,对 194名噪声暴露作业工人进行调查和听力测试 ,按听力学评价的结果将其分为听力损失组和听力正常组 ;用多聚酶链反应 限制性片断长度多态性 (PCR -RFLP)方法和等位基因特异扩增法 (ASA)检测其PMCA2基因上rs2 2 892 74和rs6790 64 0两个单核苷酸位点的多态性。结果 在 93名噪声性听力损失的工人中 ,rs2 2 892 74位点AA、AG和GG基因型的频率分别为16. 1%、40. 9%和 43. 0 % ,等位基因A和G的频率为 3 6 6%和 63 . 4% ;在 10 1名听力正常的工人中 ,其基因型频率分别为 15 8%(AA)、3 2 7% (AG)和 5 .1 5 % (GG) ,等位基因频率为 3 2. 2 % (A)和 67. 8% (G)。rs6790 64 0位点在噪声性听力损失组CC、CT和TT基因型的频率分别为 0、 82 8%和 17. 2 % ,等位基因C和T的频率为 41. 4%和 5 8 .6% ;在听力正常组的基因型频率分别为 1 0 %(CC)、 76. 2 % (CT)和 2 2 . 8% (TT) ;等位基因频率为 3 9. 1% (C)和 60. 9% (T)。两位点的基因型分布及其等位基因频率在噪声性听力损失组与听力正常组之间差异均无显著性 (P >0 . 0 5 )。采用多元Logistic回归对两组间年龄、性别、吸烟状况。

绿豆下胚轴质膜微囊的提取和H^+-ATPase活性研究

以两相法提取纯化绿豆下胚轴质膜微囊,材料与两相体系重量之比为32∶8时,一次洗膜就可以得到纯度较高的质膜微囊。提取缓冲液中牛血清白蛋白的浓度对质膜H+-ATPase的潜在活性有影响。质膜H+-ATPase水解活性依赖于Mg2+,Ca2+对酶活性有明显的促进作用。壳梭孢素(fusicoccin, FC)对酶有明显的刺激作用,活体条件最大刺激达到72%,而离体条件下刺激为30%。

中华大蟾蜍小肠黏膜上皮斯钙素-1基因表达与质膜Ca^(2+)-ATP酶活性分析

目的探讨小肠黏膜上皮斯钙素-1(STC1)基因表达与质膜Ca2+-ATP酶活性的关系。方法基于耐受实验结果,将72只成体中华大蟾蜍随机分为高钙环境组(加0.1 mol/L CaCl2的自来水)、低钙环境组[加0.03mol/L乙二胺四乙酸(EDTA)的自来水]及正常对照组(自来水),分别于暴露前及暴露后12h、24h、48h、72h、96h、120h、144h取血浆和小肠黏膜,利用比色法和半定量RT-PCR法分别检测血浆钙水平、质膜Ca2+-ATP酶活性和STC1 mRNA表达水平。结果 0.1mol/L氯化钙暴露致中华大蟾蜍血浆钙水平在12h和24h时显著高于对照组水平(1.9mmol/L)(P<0.05),48h时回复至正常,72h后持续升高;而12～48h内小肠黏膜上皮质膜Ca2+-ATP酶活性增强和STC1 mRNA水平上调,72h后回复至正常水平。0.03mol/L EDTA暴露则使中华大蟾蜍血浆钙水平下降,但质膜Ca2+-ATP酶活性和STC1 mRNA水平与对照组相比未出现明显变化。结论血浆钙水平升高致小肠黏膜上皮质膜Ca2+-ATP酶活性升高,进而增强STC1表达,而STC1表达上调又以负反馈方式抑制质膜Ca2+-ATP酶的活性。

渗透胁迫下小麦根质膜Ca^(2+)-ATP_(ase)活性及动力学

在渗透势为－０．５和－１．０ＭＰａ的ＰＥＧ溶液胁迫下，小麦抗旱品种陕合６号根质膜（ＰＭ）Ｃａ２＋－ＡＴＰａｓｅ活性分别增加５９％和２５％，水分敏感品种郑引１号该酶活性是先略降５％而后增加４３％。８ｍｍｏｌ／ＬＥＧＴＡ能明显抑制ＰＭＣａ２＋－ＡＴＰａｓｅ活性，陕合６号抑制率为４３％～７７％，郑引１号为４６％～５６％，其中两品种在ＰＥＧ胁迫以后，ＥＧＴＡ的抑制率减小。经对Ｃａ２＋－ＡＴＰａｓｅ的动力学分析表明：随着胁迫强度的增加，陕合６号Ｋｍ值分别下降４６％和２２％，Ｖｍａｘ虽略有增加，但变化不大。郑引１号Ｋｍ值分别下降７５％和８２％，Ｖｍａｘ约降低到对照的１／３．

细胞内Ca^(2+)在HMG-CoA还原酶抑制剂抑制MCF-7细胞增殖中的作用

目的 探讨HMG CoA还原酶抑制剂洛伐他汀 (LOV)对人乳腺癌MCF 7细胞增殖功能和细胞内游离Ca2 + 浓度([Ca2 + ]i)变化的影响。方法 4、8、16μmol LLOV处理MCF 7细胞 1～ 3d后 ,MTT比色法检测细胞增殖功能 ,流式细胞仪测定细胞周期相的分布及凋亡率 ,激光共聚焦显微技术观察细胞 [Ca2 + ]i变化以及RT PCR方法分析LOV对MCF 7细胞质膜钙泵PMCA1mRNA表达的影响。结果 LOV对MCF 7细胞增殖有明显的抑制作用 ,并使细胞的生长阻滞于G0 G1 期 ,该作用存在一定的剂量和时间关系 ,但其诱导MCF 7细胞凋亡的作用不明显 ;同时LOV可持续升高MCF 7细胞 [Ca2 + ]i,但对MCF 7细胞质膜钙泵PMCA1mRNA表达无明显影响。结论 LOV导致的 [Ca2 + ]i持续升高可能与LOV影响MCF 7质膜PMCA1功能有关 ;[Ca2 + ]i的升高及相关信号通路的变化可能参与了LOV对MCF