细胞松弛素B和鬼笔环肽对梨花粉管钙离子浓度和尖端钙离子通道的影响

应用激光共聚焦显微镜和全细胞膜片钳技术研究了微丝骨架解聚剂细胞松弛素B(CB)和稳定剂鬼笔环肽(PD)对梨花粉管细胞内钙离子浓度动态变化和尖端质膜上钙离子通道的影响。结果显示:CB处理能促进花粉管内胞质钙离子[Ca2+]i浓度增加,同时还能激活质膜上的钙离子通道;而PD处理对花粉管内[Ca2+]i浓度及钙离子通道几乎没有影响。研究表明,微丝骨架的解聚激活了花粉管质膜上的钙离子通道,使得胞外钙离子大量流入,胞内钙离子浓度升高,从而抑制花粉管生长。     

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.     

模拟微重力诱导的细胞微丝变化影响COL1A1启动子活性

细胞骨架系统是细胞内的重力感受系统。已知微重力导致的细胞形态、功能、信号传导等多种变化均与细胞骨架系统变化有关,但微重力对相关基因调控的影响知之甚少。本研究以构建的基因工程细胞株（EGFP-ROS）为对象,以回转器模拟微重力效应,利用增强型绿色荧光蛋白（enhanced green fluorescence protein,EGFP）荧光半定量和细胞微丝荧光染色分析技术,探讨回转模拟微重力条件下,细胞微丝系统对Ⅰ型胶原α1链基因（collagen type Ialpha chain 1 gene,COL1A1）启动子活性的影响。空间飞行和回转模拟微重力后,细胞微丝解聚、张力纤维减少,表明微重力可降低细胞微丝结构的有序性,诱导细胞骨架重排。适合剂量的细胞松弛素B处理EGFP-ROS细胞诱导微丝骨架解聚,同时导致COL1A1启动子活性增加,细胞荧光强度增强,并呈现剂量依赖性。因此,一定程度的细胞微丝系统破坏将导致COL1A1启动子活性的增强,证明细胞微丝骨架系统参与了微重力对COL1A1启动子活性调节,且在微重力信号传导中起重要作用。     

蚕豆水孔蛋白可能参与微丝骨架对气孔运动的调节

水孔蛋白的抑制剂HgCl2可明显抑制壳梭孢菌素（FC）和微丝骨架的解聚剂细胞松弛素D（CD）对蚕豆保卫细胞原生质体膨胀的诱导作用,而对微丝骨架的稳定剂鬼笔环肽（phalloidin）的抑制作用影响不明显。这表明水孔蛋白可能介导了FC和微丝骨架对气孔运动的调节。     

模拟微重力效应通过微丝骨架调控成骨细胞BMP2-Smad信号

细胞微丝骨架在力信号传导和基因表达调控中起重要作用。为了研究微丝骨架在模拟微重力效应调控成骨细胞BMP2-Smad信号中的作用,作者通过构建反映Smad活性的报告基因载体转染MC3T3-E1细胞,并通过报告基因活性分析、Western blot等方法检测了微丝骨架解聚剂和回转模拟微重力效应对BMP2诱导Samd磷酸化、核质分布和转录活性的作用。结果显示,构建的报告基因载体在成骨细胞中正确表达并响应BMP2;破坏微丝骨架会抑制BMP2诱导的Smad1/5/8蛋白磷酸化、入核及转录活性;回转抑制Smad1/5/8磷酸化、入核及其转录活性,而微丝骨架稳定剂可对抗回转的抑制作用。因此,认为回转模拟微重力效应可通过解聚微丝骨架抑制BMP2-Smad信号传导。     

线粒体PT孔参与甘草诱导MGC-803细胞凋亡的调控

不久前我们从中药中首次筛选发现了甘草能显著诱导胃癌MGC-803细胞凋亡,本文进一步研究甘草诱导MGC-803细胞凋亡过程中凋亡百分率、线粒体膜电位、胞内游离钙、DNA电泳和细胞膜通透性以及染色质DNA凝聚的时相变化,并研究了线粒体PT孔专一抑制剂环孢菌素A(CsA)对凋亡过程的影响.我们观察到,细胞膜通透性增强、胞内游离钙升高和线粒体膜电位下降为细胞凋亡的早期事件,先于凋亡峰出现、染色质凝聚和DNA电泳梯状条带出现,CsA明显抑制线粒体膜电位下降,细胞膜通透性增强和胞内游离钙变化,并极大程度地延迟细胞凋亡过程.结果提示,钙和CsA敏感性的线粒体PT孔开放参与甘草提取物诱导MGC-803细胞凋亡的调控.     

建立稳定敲减TNIK表达的A549细胞系及对其初步分析

TNIK(TRAF2 and NCK interacting kinase)属丝氨酸/苏氨酸激酶家族成员,在多种生理及病理过程中起关键作用。研究发现,TNIK在肺鳞癌组织中高表达且可驱动癌细胞增殖等恶性表型,但其在肺腺癌中的作用仍未知。该研究在构建稳定敲减TNIK表达的肺腺癌A549细胞后,流式细胞术检测发现稳定敲减TNIK阻滞细胞周期进程并诱发凋亡,细胞增殖及运动实验证明肺癌细胞增殖与迁移能力被显著抑制。进一步通过免疫荧光染色分析发现,稳定敲减TNIK表达后会诱导细胞微丝骨架排列紊乱并抑制黏着斑动态周转(组装/解聚)。综上研究结果表明,肺腺癌细胞中TNIK可能通过调节微丝骨架排列,从而影响黏着斑动态周转最终控制细胞增殖及迁移运动;推测肺腺癌细胞中高表达的TNIK可能通过调控细胞微丝骨架系统以维持癌细胞恶性表型。     

fMLP诱导的中性粒细胞胞内游离钙离子浓度变化与细胞极性化过程

目的：观察不同浓度的fMLP诱导中性粒细胞的极性变化,并结合胞内游离钙离子浓度（[Ca^2＋]i）变化曲线分析不同时相细胞的极性化变化,探讨二者之间的关系。方法：使用激光共聚焦显微镜对胞内[Ca^2＋]．变化进行监测,细胞极性化情况通过倒置显微镜来分析。结果：胞内[Ca^2＋]i变化主要包括静息期（0s）、快速上升期（10s）、快速下降期（150s）、慢速下降期（250s）和终末期等5个阶段,在这5个阶段的10s时细胞膜开始皱缩,启动细胞极性化过程,之后呈现为不断的极性化和去极性化过程。结论：游离钙离子浓度升高可能启动了中性粒细胞的极性化,但对之后的极性化过程影响不明显。     

三尖杉酯碱诱导的HL-60细胞凋亡的钙调节

三尖杉酯碱 (harringtonine ,HT)是一种对急性粒细胞白血病、急性单核细胞白血病有良好疗效的抗癌药物 ,可在很宽的剂量范围内迅速诱导HL -6 0细胞凋亡 .细胞外钙离子螯合剂EGTA不抑制抗癌药物HT、喜树碱 (campothecin ,CAM )诱导的-细胞凋亡 ;而细胞内Ca ²⁺螯合剂BAPTA AM却可抑制该过程 .与此相一致 ,HT和CAM也不能诱导胞内Ca ²⁺已排空的HL -6 0细胞凋亡 ,说明HT ,CAM诱导的HL- 6 0细胞凋亡依赖于胞内Ca ²⁺但是HT ,CAM诱导HL -6 0细胞凋亡过程中胞内自由Ca ²⁺浓度变化不大 .利用视频反差增强显微术 (videoenhance mentcontrastmicroscopy ,VEC)研究了单个HL- 6 0细胞凋亡过程中胞内Ca ²⁺分布的动态变化 ,结果表明HT诱导HL -6 0细胞凋亡过程存在胞内Ca ²⁺由胞质向核的位移 .     

胞质微丝改变与鼻咽癌细胞增殖和凋亡的关系

胞质微丝改变与鼻咽癌细胞增殖和凋亡的关系廖新波,孙宁,唐慰萍（广东医院病理学教研室湛江５２４０２３）本文应用激光共聚焦显微镜（ＣＬＳＭ）技术,研究鼻咽癌细胞胞质微丝骨架改变与癌细胞增殖、凋亡的关系。鼻咽癌细胞（ＣＮＥ－ＺＺ）体外培养（分诱导凋亡组和对...     

FMLP诱导的嗜中性白细胞呼吸爆发与凋亡的关系研究

The relationship between apoptosis of neutrophils and the change of their intracellular free Ca2+ concentration [Ca2+]i was studied. FMLP and A23187 were used to elevate the [Ca2+]i while BAPTA was used to deplete it. Fluorescence microscope, flow cytometry and gel electrophoresis were used to study the percentage of cell apoptosis and the change of f-actin during apoptosis. The results showed that the apoptosis was obviously inhibited by fMLP and A23187, while accelerated by BAPTA. The detection of f-actin showed that the f-actin depolymerized obviously during apoptosis. The elevation of [Ca2+]i inhibit the actin depolymerization while depletion of [Ca2+]i accelerated it. This result indicated that the apoptosis of neutrophil was obviously inhibited by [Ca2+]i elevation but accelerated by [Ca2+]i depletion.     

Role of Ca2+ in H+ transport by rabbit gastric glands studied with A23187 and BAPTA, an incorporated Ca2+ chelator

The role of Ca2+ in stimulation of H+ gastric secretion by cAMP-dependent and -independent secretagogues was studied in isolated rabbit glands using Ca2+ ionophore, A23187, and an intracellular Ca2+ chelator (BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) incorporated as its acetoxymethyl ester (BAPTA-AM). Acetylcholine (ACh), tetragastrin (TG), histamine and forskolin induced a transitory increase of intracellular Ca2+ concentration, [Ca2+]i, measured in gastric glands loaded with Ca2+-sensitive dye fura-2, and provoked an acid secretory response evaluated with aminopyrine accumulation ratio (AP ratio). The Ca2+-ionophore A23187 also induced an increase in [Ca2+]i and in AP ratio. cAMP-dependent secretagogues were more potent stimulants of acid secretion than cAMP-independent secretagogues. cAMP analogue, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BR-cAMP) induced an increase in AP ratio without modifying [Ca2+]i. BAPTA-AM (5-25 microM) induced a transient decrease of resting [Ca2+]i which returned to basal level due to extracellular Ca2+ entry. Increases in [Ca2+]i produced by ACh and TG were abolished by BAPTA and those produced by Ca2+ ionophore A23187 were partially buffered. BAPTA inhibited in a dose-dependent manner H+ secretion induced by cholinergic and gastrinergic stimulants in the presence of cimetidine. A23187 increased the AP ratio to values similar to those obtained with ACh or TG and was not inhibited by BAPTA. BAPTA partially inhibited (40%) the increase in AP ratio induced by forskolin and histamine inspite of the complete inhibition of the Ca2+ response. BAPTA did not inhibit the response to 8-BR-cAMP. BAPTA inhibition of forskolin stimulation was reversed by A23187 and the response was potentiated. These results indicate that ACh and TG response are completely dependent on an increase of [Ca2+]i. The response to cAMP-dependent agonists histamine and forskolin depend both on Ca2+ and cAMP. For forskolin stimulation the response may be the result of a potentiation between Ca2+ and cAMP.     

Cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in hela cells

Results of recent studies using BAPTA/AM have raised a serious question on whether Ca(2+) signal is truly involved in regulating the progression of apoptosis. To resolve this question, we examined the differential effects of three different Ca(2+) signaling blockers (BAPTA/AM, membrane-impermeant BAPTA, and heparin) on UV-induced apoptosis in HeLa cells. We found that although the membrane-permeable form of BAPTA (i.e., BAPTA/AM) could not inhibit cell death, the membrane-impermeant form of BAPTA, loaded into the cytosol by electroporation, clearly protected cells from entering apoptosis. Furthermore, when we injected heparin to block Ca(2+) release from the endoplasmic reticulum (ER) to cytosol, apoptosis was greatly suppressed. These findings strongly suggest that elevation of cytosolic Ca(2+) is part of the signal that drives the progression of apoptosis. The negative result of BAPTA/AM is probably due to its dual effect on subcellular Ca(2+) distribution; besides suppressing the Ca(2+) elevation in cytosol, BAPTA/AM can also enter into the ER to reduce the free Ca(2+) level there. The depletion of Ca(2+) in ER is believed to stimulate apoptosis and thus would counterbalance the protection effect of BAPTA/AM in suppressing the cytosolic Ca(2+) elevation.     

Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.     

Stimulation of leucine transport by a mitogen through intracellular Ca2+ increase in human peripheral lymphocytes

The effect of concanavalin A and ionophore A23187 on leucine uptake by human peripheral lymphocytes has been examined. Preincubation of the cells with 32 micrograms/ml concanavalin A or 0.1 microM A23187 increased leucine uptake by 67% and 100%, respectively. Both concanavalin A and A23187 could, within 2 min, induce a more than 2-fold increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i). This increase by concanavalin A was completely blocked by the addition of 0.1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) to incubation medium; TMB-8 partially blocked the action of A23187. The stimulation of leucine uptake by concanavalin A and A23187 was strongly inhibited by the presence of TMB-8 in the medium, whereas the basal uptake was not affected by this intracellular Ca2+ antagonist. Amiloride did not inhibit the stimulation of leucine uptake by concanavalin A. The concanavalin A- and A23187-induced elevation of [Ca2+]i was accompanied by membrane hyperpolarization. Concanavalin A-stimulated leucine uptake was greatly inhibited by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid. These results indicate that the increase in [Ca2+]i may function as a signal of the stimulation by mitogen of leucine uptake mediated by system L, finally inducing membrane hyperpolarization in human lymphocyte.     

Both calcium and ROS as common signals mediate Na(2)SeO(3)-induced apoptosis in SW480 human colonic carcinoma cells

Recent studies have shown that reactive oxygen species (ROS) play a crucial role in Se-induced cell apoptosis. A number of studies have demonstrated that perturbed cellular calcium homeostasis has been implicated in apoptosis. The main objective of this study was to evaluate the role of Ca(2+) in Na(2)SeO(3)-induced apoptosis and the relationship between Ca(2+) and ROS in human colonic carcinoma cells SW480. When SW480 cells were exposed to 25-100 microM Na(2)SeO(3), both cell apoptosis and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Na(2)SeO(3) was able to induce increase of [Ca(2+)](i) and ROS production and disrupt mitochondrial membrane potential (Delta Psi m) in SW480 cells monitored by using a confocal laser scanning microscope. Ca(2+) channel inhibitor CoCl(2) and an intracellular Ca(2+) chelator o-phtalaldehyde, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA) completely inhibited [Ca(2+)](i) increase, but catalase had no effect on Na(2)SeO(3)-induced increase of [Ca(2+)](i). BAPTA-AM, CoCl(2), and mitochondrial Ca(2+) uptake inhibitor ruthenium red blocked Delta Psi m dissipation. The increase of ROS was also suppressed by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine and catalase, respectively. The mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) completely inhibited Na(2)SeO(3)-induced ROS increase. This showed that ROS increase is due to mitochondrial Ca(2+) overload. The Na(2)SeO(3)-induced apoptosis of SW480 cells was also inhibited by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine, and catalase, respectively. The results mentioned above imply that both calcium and Ca(2+)-dependent ROS as a signal molecule mediate apoptosis induced by Na(2)SeO(3) in SW480 cells.     

An intracellular calcium store regulates protein synthesis in HeLa cells, but it is not the hormone-sensitive store.

There is considerable evidence, reviewed by Brostrom and Brostrom [1], that Ca2+ stores are involved in the regulation of protein synthesis. We provide evidence in HeLa cells that is consistent with their findings that depletion of Ca2+ stores and not changes in cytosolic free Ca2+ ([Ca2+]i) inhibit protein synthesis, but we also show that the mechanism leading to depletion is critical. Specifically, depletion of stores by the Ca(2+)-mobilizing hormone histamine does not inhibit protein synthesis. In assessing the role of Ca2+ stores in protein synthesis, experiments in certain cell types have been complicated by the use of Ca2+ ionophores, which simultaneously elevate [Ca2+]i and deplete Ca2+ stores. We have measured total cell Ca2+, [Ca2+]i and protein synthesis in HeLa cells under conditions that allowed evaluation of the separate contributions of stores and [Ca2+]i. Using 1,2-bis(2-aminophenoxyethane)-N,N,N'N'-tetraacetic acid (BAPTA) as an intracellular Ca2+, chelator and thapsigargin, which inhibits the membrane Ca(2+)-ATPase of storage vesicles, total cell Ca2+ can be depleted and this depletion is enhanced by extracellular EGTA which blocks Ca2+ influx; [Ca2+]i is actually lowered by BAPTA under these conditions. Protein synthesis is inhibited by BAPTA in the presence of EGTA and by thapsigargin with or without EGTA. However, histamine which with EGTA, affects an equal degree of Ca2+ depletion does not inhibit protein synthesis. Thus, it is suggested that Ca2+ stores are not homogeneous, and that the hormone-sensitive store specifically does not play a role in the regulation of protein synthesis. In this respect, the hormone-sensitive and insensitive stores do not functionally communicate and may be separately regulated.     

Calcium signaling is involved in cadmium-induced neuronal apoptosis via induction of reactive oxygen species and activation of MAPK/mTOR network

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+](i)) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+](i) elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+](i) elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+](i) elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+](i), which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+](i) homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.     

CaV1.3 channels and intracellular calcium mediate osmotic stress-induced N-terminal c-Jun kinase activation and disruption of tight junctions in Caco-2 CELL MONOLAYERS

We investigated the role of a Ca(2+) channel and intracellular calcium concentration ([Ca(2+)](i)) in osmotic stress-induced JNK activation and tight junction disruption in Caco-2 cell monolayers. Osmotic stress-induced tight junction disruption was attenuated by 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-mediated intracellular Ca(2+) depletion. Depletion of extracellular Ca(2+) at the apical surface, but not basolateral surface, also prevented tight junction disruption. Similarly, thapsigargin-mediated endoplasmic reticulum (ER) Ca(2+) depletion attenuated tight junction disruption. Thapsigargin or extracellular Ca(2+) depletion partially reduced osmotic stress-induced rise in [Ca(2+)](i), whereas thapsigargin and extracellular Ca(2+) depletion together resulted in almost complete loss of rise in [Ca(2+)](i). L-type Ca(2+) channel blockers (isradipine and diltiazem) or knockdown of the Ca(V)1.3 channel abrogated [Ca(2+)](i) rise and disruption of tight junction. Osmotic stress-induced JNK2 activation was abolished by BAPTA and isradipine, and partially reduced by extracellular Ca(2+) depletion, thapsigargin, or Ca(V)1.3 knockdown. Osmotic stress rapidly induced c-Src activation, which was significantly attenuated by BAPTA, isradipine, or extracellular Ca(2+) depletion. Tight junction disruption by osmotic stress was blocked by tyrosine kinase inhibitors (genistein and PP2) or siRNA-mediated knockdown of c-Src. Osmotic stress induced a robust increase in tyrosine phosphorylation of occludin, which was attenuated by BAPTA, SP600125 (JNK inhibitor), or PP2. These results demonstrate that Ca(V)1.3 and rise in [Ca(2+)](i) play a role in the mechanism of osmotic stress-induced tight junction disruption in an intestinal epithelial monolayer. [Ca(2+)](i) mediate osmotic stress-induced JNK activation and subsequent c-Src activation and tyrosine phosphorylation of tight junction proteins. Additionally, inositol 1,4,5-trisphosphate receptor-mediated release of ER Ca(2+) also contributes to osmotic stress-induced tight junction disruption.     

Elevation of cytosolic calcium precedes anoxic gene expression in maize suspension-cultured cells.

Based on pharmacological evidence, we previously proposed that intracellular Ca2+ mediates the perception of O2 deprivation in maize seedlings. Herein, using fluorescence imaging and photometry of Ca2+ in maize suspension-cultured cells, the proposal was further investigated. Two complementary approaches were taken: (1) real time analysis of anoxia-induced changes in cytosolic Ca2+ concentration ([Ca]i) and (2) experimental manipulation of [Ca]i and then assay of the resultant anoxia-specific responses. O2 depletion caused an immediate increase in [Ca2+]i, and this was reversible within a few seconds of reoxygenation. The [Ca]i elevation proceeded independent of extracellular Ca2+. The kinetics of the Ca2+ response showed that it occurred much earlier than any detectable changes in gene expression. Ruthenium red blocked the anoxic [Ca]i elevation and also the induction of adh1 (encoding alcohol dehydrogenase) and sh1 (encoding sucrose synthase) mRNA. Ca2+, when added along with ruthenium red, prevented the effects of the antagonist on the anoxic responses. Verapamil and bepridil failed to block the [Ca]i rise induced by anoxia and were equally ineffective on anoxic gene expression. Caffeine induced an elevation of [Ca]i as well as ADH activity under normoxia. The data provide direct evidence for [Ca]i elevation in maize cells as a result of anoxia-induced mobilization of Ca2+ from intracellular stores. Furthermore, any manipulation that modified the [Ca]i rise brought about a parallel change in the expression of two anoxia-inducible genes. Thus, these results corroborate our proposal that [Ca]i is a physiological transducer of anoxia signals in plants.