高温胁迫对花生幼苗光合速率、叶绿素含量、叶绿体Ca2+-ATPase、Mg2+-ATPase及Ca2+分布的影响

将水培后盆栽的花生幼苗,置于培养箱42℃高温培养,定时测定幼苗叶光合速率、叶绿素含量和叶绿体Ca²⁺-ATPase、Mg²⁺-ATPase的相对活性,并观察幼叶细胞内Ca²⁺分布的变化。试验结果表明：高温胁迫过程中,光合速率及叶绿素含量都随处理时间的延伸而下降,并呈显著正相关;叶绿体Ca²⁺-ATPase和Mg²⁺-ATPase高温胁迫过程中相对活性呈先升后降趋势,Ca²⁺-ATPase热敏性高于Mg²⁺-ATPase;高温胁迫过程中,Ca²⁺具有从胞外转运到胞质内和叶绿体中的趋势,Ca²⁺能够稳定高温胁迫下叶肉细胞膜和叶绿体的超微结构。     

植物胞外钙调素与信号转导

植物体需要构建复杂的信号转导体系以调节自身的生长发育过程并适应外界环境的变化,这种功能的实现需要胞内和胞外诸多信号分子的参与,胞外钙调素的发现使人们开始相信植物细胞外多肽信使的存在。胞外钙调素的生物学功能极其广泛,几乎涉及到植物生长发育的各个阶段,其信号转导途径是目前研究得最多也是最为清楚的方面,异三聚体G蛋白、磷脂酶C(PLC)-肌醇三磷酸(IP3)-肌醇三磷酸受体(IP3R)信号通路、活性氧和Ca2 通道之间直接或间接的相互作用是胞外钙调素信号转导的核心。     

骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca²⁺泵(Ca²⁺-ATPase)是调节细胞内Ca²⁺浓度的重要蛋白质之一. Ca²⁺泵在转运Ca²⁺的过程中经历一系列构象变化. 其中,E1状态为外向的Ca²⁺高亲和状态,E2状态则为内向的Ca²⁺低亲和状态. 目前,骨骼肌内质网Ca²⁺泵转运Ca²⁺过程中的几个中间状态,包括E1-2Ca²⁺,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析. 介绍这几种状态的晶体结构,并分析Ca²⁺泵在执行功能过程中结构与功能的关系.     

人白细胞抗原-G1对NK92效靶识别过程的影响

人白细胞抗原G(human leukocyte antigen-G,HLA-G)属非经典的主要组织相容性复合体(major histocompatibility complex, MHC)Ⅰ类分子,为自然杀伤细胞（natural killer,NK）抑制性受体的识别配体,可以向NK细胞传递抑制性信号,从而抑制NK细胞的细胞毒作用.为了研究HLA-G对NK与靶细胞识别作用的影响机制,采用激光扫描共聚焦显微镜和流式细胞术对全长HLA-G1的表达和功能进行了探讨,并对效靶识别过程中靶细胞［Ca²⁺］_i（胞内自由钙离子浓度）进行了实时检测.结果发现,全长的HLA-G1表达于K562、JAR和CHO细胞的胞浆和胞膜上,它能够部分地抑制NK92的细胞毒作用. NK92与CHO和GFP-CHO细胞作用后,靶细胞［Ca²⁺］_i出现了明显的升高,HLA-G1的表达则抑制了靶细胞［Ca²⁺］_i的升高.结果提示：靶细胞［Ca²⁺］_i的升高是有效的细胞杀伤作用的必要条件,HLA-G的免疫抑制功能可能与靶细胞胞内钙离子浓度升高的抑制作用有密切关系.     

信息跨膜传递的分子机制

胞外信息作用于质膜表面的受体,转变为胞内信使分子完成信息的跨膜传递。β受体结合配体后活化G蛋白(通过α亚基与β、γ的解离)影响环化酶的活性。而钙联受体则通过触发肌醇磷脂的代谢,生成胞内信使甘油二酯(DG)和三磷酸肌醇酯(IP₃),胞内游离Ca²⁺浓度瞬间增高(Ca²⁺动员过程),通过不同的蛋白激酶引起特定的生理效应。DG活化蛋白激酶-C,IP₃动员胞内Ca²⁺,它们通过二个相互独立而协同的过程调节细胞的代谢。     

胞外ATP对AlCl3诱导的细胞死亡过程中胞内H2O2和Ca2+水平的影响及其生理机制分析

该实验以烟草悬浮细胞 BY 2 为材料,在烟草悬浮细胞中分别加入0.05、0.10、0.15、0.20 mmol·L^-1AlCl₃,以等体积去离子水处理的悬浮细胞液为对照,并依据前述实验结果选择0.15 mmol·L^-1 AlCl₃,分别添加5 mmol·L^-1 DMTU(H₂O₂ 抑制剂)、20 μmol·L^-1CaCl₂、15 μmol·L^-1 LaCl₃(Ca²⁺通道抑制剂)和50 μmol·L^-1 ATP设计多项处理,分析胞外ATP(eATP)对铝离子(Al³⁺)胁迫引起的植物细胞死亡及其胞内H₂O₂、Ca²⁺的影响,以揭示Al³⁺胁迫下植物调节细胞死亡的可能机制,进一步扩展对eATP功能的认知。结果显示：(1)随着 AlCl₃ 胁迫浓度的提高,细胞死亡水平和胞内H₂O₂水平上升,而胞内Ca²⁺和eATP水平则逐渐降低。(2)外援施加H₂O₂抑制剂 DMTU(二甲基硫脲)和Ca²⁺能够有效缓解AlCl₃诱导的细胞死亡水平的上升;而Ca²⁺通道抑制剂LaCl₃(三氯化镧)则加剧了AlCl₃胁迫下的细胞死亡。(3)在AlCl₃胁迫下对细胞添加外源ATP,能够缓解AlCl₃胁迫下胞内H₂O₂水平上升和Ca²⁺水平下降的同时,并显著降低AlCl₃胁迫导致的细胞死亡。研究表明, Al³⁺以剂量依赖的模式提升细胞死亡和细胞内H₂O₂的水平并降低胞内Ca²⁺和eATP水平,AlCl₃诱导的细胞死亡受到H₂O₂和Ca²⁺水平变化的调节,eATP可以通过调节H₂O₂与Ca²⁺水平缓解AlCl₃诱导的细胞死亡。推测Al³⁺胁迫可能通过抑制钙离子通道而破坏了细胞内H₂O₂和Ca²⁺之间的协同关系,外源ATP对Al³⁺诱导H₂O₂上升的缓解作用可能是由于其提升了细胞的抗氧化能力。     

拟南芥保卫细胞微丝骨架的解聚可能参与了细胞外钙调素诱导的气孔关闭

细胞外钙调素(CaM)在植物的许多生理活动中都执行着重要功能, 但它对气孔运动的作用及其调控机制, 人们了解的很少. 以模式植物拟南芥为材料, 研究了细胞外CaM在保卫细胞壁上的存在及其对气孔运动的调控机制. 结果表明, 拟南芥保卫细胞壁中存在有分子量为17 kD的CaM, 并应用W7-琼脂糖和CaM抗血清初步证明了保卫细胞壁中存在的CaM可能具有促进气孔关闭和抑制气孔开放的作用. 在应用外源CaM诱导气孔关闭的实验中, 保卫细胞微丝骨架由长而呈辐射状分布的聚合态逐步解聚, 气孔开度也随着降低. 药理学实验结果表明, 保卫细胞微丝骨架的解聚能明显地促进外源CaM诱导的气孔关闭, 而微丝骨架的聚合则抑制这一过程. 研究结果还表明, 外源CaM能诱导保卫细胞[Ca²⁺]_cyt升高; 当使用Ca²⁺螯合剂EGTA时, 外源CaM诱导的[Ca²⁺]_cyt升高和气孔关闭运动均受到抑制. 为此推测细胞外CaM可能是通过诱导保卫细胞[Ca²⁺]_cyt升高, 导致微丝骨架的解聚, 进而促进气孔的关闭运动.     

肺腺癌A549/DDP细胞周期变化及其多药耐药性

用Fura-2/AM标记药物敏感的肺腺癌细胞A₅₄₉和抗顺铂药物的肺腺癌细胞A₅₄₉/DDP两种细胞胞内游离Ca²⁺,用碘化丙锭(PI)标记细胞DNA,检测其胞内Ca²⁺的变化及两种细胞增殖能力和细胞周期.实验结果表明,抗药性细胞株A₅₄₉/DDP胞浆内游离Ca²⁺的浓度仅为药物敏感细胞株A₅₄₉的1/3左右,同时前者的细胞增殖能力较后者明显增强,而且细胞周期也明显缩短.当用BAPTA-AM和EGTA或A₂₃₁₈₇和Thapsigargin处理细胞以降低或升高其胞内自由Ca²⁺浓度时可改变细胞的生长周期,二者也呈现明显差别.这些结果表明,对顺铂产生耐药性的人肺腺癌A₅₄₉/DDP细胞胞内Ca²⁺浓度的降低,可能影响细胞的增殖,缩短细胞的生长周期,特别是影响起决定作用的G1期,从而有利于肿瘤细胞多药耐药特性的维持.     

甘草次酸与AT1受体结合的研究

运用受体放射配基结合法, 激光共聚焦显微镜技术, Northern blot, ³H-TdR掺入DNA等技术, 研究甘草次酸(glycyrrhetic acid, GA)与AT₁受体的结合以及此作用的可能机制. 研究表明, GA对血管平滑肌细胞(VSMC) AT₁受体有较好的结合作用(IC₅₀为35.0 μmol/L), 它能引起细胞内[Ca²⁺]_i增加, 激活转录因子c-myc, 从而产生对VSMC的增殖作用, 因此, GA具有AT₁受体激动剂样作用, 这一结果为中药甘草的药理作用提供一种新机制.     

多形核白细胞上嘌呤受体的初步研究

ATP和ADP能激活多型核白细胞引起细胞内［Ca²⁺］_i的明显升高,AMP则无此作用.多型核白细胞对ATP和ADP具有不同的浓度依赖性.当细胞外的钙离子被螯合后,ATP和ADP仍能引起细胞内游离钙浓度的升高.结果表明多形核白细胞存在着对ATP和ADP敏感的P2型嘌呤受体,并且属于P2型受体中的P2Y亚类.     

Assessment of frequency-dependent alterations in the level of extracellular Ca2+ in the synaptic cleft.

The synaptic cleft may be represented as a very thin disk of extracellular fluid. It is possible that at high stimulation frequencies the interval between pulses would be insufficient for diffusion of Ca2+ from the periphery of the cleft to replace extracellular Ca2+ depleted at the center of the cleft as a result of activation of postsynaptic, Ca2(+)-permeable channels. Computer modeling was employed to assess the impact of activation of glutamate receptor channels (GRCs) in the postsynaptic membrane on the level of extracellular Ca2+ within the synaptic cleft. The model includes calcium influx from the synaptic cleft into the postsynaptic compartment through GRC and calcium efflux through calcium pumps and Na/Ca exchangers. Concentrations of extracellular Ca2+ inside the cleft are estimated by using a compartmental model incorporating flux across the postsynaptic membrane and radial diffusion from the edges of the cleft. The simulations suggest that substantial extracellular Ca2+ depletion can occur in the clefts during activation of GRCs, particularly at high stimulation frequencies used to induce long-term potentiation (LTP). Only minimal transitory changes in extracellular Ca2+ are observed at low frequencies. These frequency-dependent alterations in extracellular Ca2+ dynamics are a direct reflection of the activity of GRCs and could be involved in the modulation of presynaptic function via a retrograde messenger mechanism, if there are extracellular Ca2+ sensors on the presynaptic membranes. The recently cloned extracellular Ca2(+)-sensing receptors that are known to be present in nerve terminals in hippocampus and other areas of the brain could potentially play such a role.     

Extracellular calcium sensing and signalling

Ca2+ is well established as an intracellular second messenger. However, the molecular identification of a detector for extracellular Ca2+--the extracellular calcium-sensing receptor--has opened up the possibility that Ca2+ might also function as a messenger outside cells. Information about the local extracellular Ca2+ concentration is conveyed to the interior of many cell types through this unique G-protein-coupled receptor. Here, we describe new emerging concepts concerning the signalling function of extracellular Ca2+, with particular emphasis on the extracellular calcium-sensing receptor.     

Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor.

Stimulation of B-cell antigen receptor (BCR) induces a rapid increase in cytoplasmic free calcium due to its release from intracellular stores and influx from the extracellular environment. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ligand-gated channels that release intracellular calcium stores in response to the second messenger, inositol 1,4,5-trisphosphate. Most hematopoietic cells, including B cells, express at least two of the three different types of IP3R. We demonstrate here that B cells in which a single type of IP3R has been deleted still mobilize calcium in response to BCR stimulation, whereas this calcium mobilization is abrogated in B cells lacking all three types of IP3R. Calcium mobilization by a transfected G protein-coupled receptor (muscarinic M1 receptor) was also abolished in only triple-deficient cells. Capacitative Ca2+ entry, stimulated by thapsigargin, remains unaffected by loss of all three types of IP3R. These data establish that IP3Rs are essential and functionally redundant mediators for both BCR- and muscarinic receptor-induced calcium mobilization, but not for thapsigargin-induced Ca2+ influx. We further show that the BCR-induced apoptosis is significantly inhibited by loss of all three types of IP3R, suggesting an important role for Ca2+ in the process of apoptosis.     

Characterization of the Ca2+ influx into embryonic cells after stimulation of the embryonic muscarinic receptor.

Embryonic cells transiently express an embryonic muscarinic system during morphogenesis. Stimulation of the embryonic muscarinic receptor results in biphasic intracellular Ca2+ mobilization: an initial "peak" due to Ca2+ release from intracellular stores is followed by a sustained "plateau" of enhanced cytoplasmic Ca2+ due to influx of extracellular Ca2+. In the present investigation, we characterized the Ca2+ influx by measuring the cytoplasmic free Ca2+ concentration [Ca2+]i using the Ca2+ indicator fura-2: 1. The increase of [Ca2+]i during the plateau depended linearly on the logarithm of the extracellular calcium concentration whereas the initial peak was almost independent from extracellular calcium. 2. The organic Ca2+ entry blockers verapamil, gallopamil, nifedipine, nitrendipine and the inorganic blockers Mn2+, Mg2+ and La3+ were without effect on both phases of Ca2+ mobilization. Only Ni2+ at concentrations above 1 mM was able to reduce the influx without affecting the intracellular Ca2+ release. 3. Substitution of extracellular Na+ by guanidine+, choline+ or tris+ and membrane depolarisation by increasing the extracellular K+ concentration had no effect on either phase of Ca2+ mobilization. We conclude that a non-voltage dependent, receptor-operated influx mechanism, probably a "second messenger operated Ca2+ channel", is responsible for the Ca2+ influx after stimulation of the embryonic muscarinic receptor.     

PPADS is a reversible competitive antagonist of the NAADP receptor

NAADP has been shown to act as a second messenger in a wide range of systems from plants to mammalian cells. Although it had always been considered as a canonical second messenger, recent work has shown that it is also active when applied extracellularly. It has also been suggested that NAADP might have a direct action on P2 receptors, based on the action of a pharmacological agent, PPADS, on Ca2+ signals in response to extracellular NAADP. We have therefore investigated whether PPADS can act directly on the intracellular NAADP-induced Ca2+-release system in the well characterised sea urchin egg homogenate system. Indeed, PPADS, and its structural analogue PPNDS were able to compete with [32P]NAADP for the binding site and binding curves revealed that both compounds display affinities in the low micromolar range. The binding of PPADS was reversible in contrast to that of NAADP. In fluorimetric Ca2+-release experiments, PPADS was able to competitively antagonise NAADP-induced Ca2+-release with an IC50 of 20 microM, while it did not affect the other Ca2+-release channels. This is the first report of a reversible, competitive antagonist of the sea urchin NAADP receptor. Furthermore, PPADS might reveal itself as an invaluable tool to investigate NAADP signalling and is a lead compound for the synthesis of potent and specific antagonists.     

The regulation of platelet-activating factor production in endothelial cells. The role of calcium and protein kinase C

Endothelial cells (EC) synthesize platelet-activating factor (PAF) when stimulated with agonists that bind to cell-surface receptors. We examined events that link receptor binding to synthesis of PAF by EC. Bovine EC stimulated with agonists that interact with specific cell-surface receptors accumulated PAF only in the presence of extracellular calcium. Hormonal stimulation of EC resulted in Ca2+ entry characteristic of that seen with receptor-operated calcium channels; Indo-1 measurements demonstrated that this inward flux of Ca2+ caused prolonged elevated levels of intracellular Ca2+. EC were exposed to melittin or theta toxin from Clostridium perfringens (pore-forming peptides that increase the permeability of the plasma membrane for small molecules) resulting in an inward flux of Ca2+ and accumulation of PAF. Ca2+ appears to be regulatory for PAF production at the level of phospholipase A2-mediated production of the PAF precursor 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine, as Ca2+ was required for the stimulated hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine. PAF accumulation in EC is also regulated by protein kinase C. Pretreatment of EC with phorbol esters that activate protein kinase C or with dioctanoylglycerol, followed by stimulation, resulted in a 2-fold increase in stimulated PAF production. The regulatory effect of protein kinase C also appears to be at a phospholipase A2-mediated hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine.     

G protein-coupled, extracellular Ca2+ (Ca o 2+ )-sensing receptor enables Ca o 2+ to function as a versatile extracellular first messenger

The cloning of a G protein-coupled, extracellular Ca²⁺ (Ca _o ²⁺ )-sensing receptor (CaR) has afforded a molecular basis for a number of the known effects of Ca _o ²⁺ on tissues involved in maintaining systemic calcium homeostasis, especially parathyroid gland and kidney. In addition to providing molecular tools for showing that CaR messenger RNA and protein are present within these tissues, the cloned CaR has permitted documentation that several human diseases are the result of inactivating or activating mutations of this receptor as well as generation of mice that have targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in these patients as well as in the CaR “knockout” mice have elucidated considerably the CaR’s physiological roles in mineral ion homeostasis. Nevertheless, a great deal remains to be learned about how this receptor regulates the functioning of other tissues involved in Ca _o ²⁺ metabolism, such as bone and intestine. Further study of these human diseases and of the mouse models will doubtless be useful in gaining additional understanding of the CaR’s roles in these latter tissues. Furthermore, we understand little of the CaR’s functions in tissues that are not directly involved in systemic mineral ion metabolism, where the receptor probably serves diverse other roles. Some of these functions may be related to the control of intra- and local extracellular concentrations of Ca²⁺, while others may be unrelated to either systemic or local ionic homeostasis. In any case, the CaR and conceivably additional receptors/sensors for Ca²⁺ or other extracellular ions represent versatile regulators of a wide variety of cellular functions and represent important targets for novel classes of therapeutics.     

The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation.

The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.     

Properties of receptor-controlled inositol trisphosphate formation in parotid acinar cells.

Activation of muscarinic receptors in rat parotid cells results in breakdown of polyphosphoinositides liberating inositol phosphates, including inositol trisphosphate. Formation of inositol trisphosphate appears independent of agonist-induced Ca2+ mobilization, since neither formation nor degradation of inositol trisphosphate are appreciably altered in low-calcium media, and elevation of cytosolic Ca2+ with a calcium ionophore does not cause an increase in cellular inositol trisphosphate. Further, activation of substance P receptors and alpha 1-adrenoreceptors, but not beta-adrenoreceptors, increases inositol trisphosphate formation. The dose-response curve for methacholine activation of inositol trisphosphate formation more closely approximates the curve for receptor occupancy than for Ca2+-activated K+ release. These results are all consistent with the suggestion that inositol trisphosphate could function as a second messenger linking receptor occupation to cellular Ca2+ mobilization.