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。     

The Nitric Oxide Prodrug JS‐K Induces Ca2+‐Mediated Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

Hepatocellular carcinoma is one of the most common and deadly forms of human malignancies. JS‐K, O²‐(2, 4‐dinitrophenyl) 1‐ [(4‐ethoxycarbonyl) piperazin‐1‐yl] diazen‐1‐ium‐1, 2‐diolate, has the ability to induce apoptosis of tumor cell lines. In the present study, JS‐K inhibited the proliferation of HepG2 cells in a time‐ and concentration‐dependent manner and significantly induced apoptosis. JS‐K enhanced the ratio of Bax‐to‐Bcl‐2, released of cytochrome c (Cyt c) from mitochondria and the activated caspase‐9/3. JS‐K caused an increasing cytosolic Ca²⁺ and the loss of mitochondrial membrane potential. Carboxy‐PTIO (a NO scavenger) and BAPTA‐AM (an intracellular Ca²⁺ chelator) significantly blocked an increasing cytosolic Ca²⁺ in JS‐K‐induced HepG2 cells apoptosis, especially Carboxy‐PTIO. Meanwhile, Carboxy‐PTIO and BAPTA‐AM treatment both attenuate JS‐K‐induced apoptosis through upregulation of Bcl‐2, downregulation of Bax, reduction of Cyt c release from mitochondria to cytoplasm and inactivation of caspase‐9/3. In summary, JS‐K induced HepG2 cells apoptosis via Ca²⁺/caspase‐3‐mediated mitochondrial pathway.     

烟草悬浮细胞在凋亡过程中Ca2+分布的共聚焦显微成像研究

以Fluo-3AM为游离Ca²⁺荧光探针,利用激光扫描共聚焦显微镜（Laser Scanning Confocal Microscope,LSCM）对烟草悬浮细胞在热激诱导的凋亡过程中Ca²⁺时空分布进行了研究。结果显示,在正常细胞中,Ca²⁺集中分布在细胞壁和细胞核中,细胞质中分布较少;在凋亡早期细胞中,细胞质中Ca²⁺的浓度增加;在凋亡晚期的细胞中,细胞核中的Ca²⁺浓度增加较为明显。结果提示,在凋亡过程中,Ca²⁺的定位分布存在一定的规律。     

Effect of tetramethylpyrazine (TMP) on Ca2+ signal transduction and cell viability in a model of renal tubular cells

Tetramethylpyrazine (TMP) is a compound purified from herb. Its effect on Ca²⁺ concentrations ([Ca²⁺]_i) in renal cells is unclear. This study examined whether TMP altered Ca²⁺ signaling in Madin‐Darby canine kidney (MDCK) cells. TMP at 100–800 μM induced [Ca²⁺]_i rises, which were reduced by Ca²⁺ removal. TMP induced Mn²⁺ influx implicating Ca²⁺ entry. TMP‐induced Ca²⁺ entry was inhibited by 30% by modulators of protein kinase C (PKC) and store‐operated Ca²⁺ channels. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 93% of TMP‐evoked [Ca²⁺]_i rises. Treatment with TMP abolished BHQ‐evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) abolished TMP‐induced responses. TMP at 200–1000 μM decreased viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester. Together, in MDCK cells, TMP induced [Ca²⁺]_i rises by evoking PLC‐dependent Ca²⁺ release from endoplasmic reticulum and Ca²⁺ entry via PKC‐sensitive store‐operated Ca²⁺ entry. TMP also caused Ca²⁺‐independent cell death.     

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability in OC2 human oral cancer cells. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The tamoxifen-induced Ca²⁺ influx was sensitive to blockade of L-type Ca²⁺ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), tamoxifen-induced [Ca²⁺]_i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca²⁺]_i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca²⁺]_i rises, in a nongenomic manner, by causing Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ influx from L-type Ca²⁺ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca²⁺]_i rise.     

钙与植物乙烯反应的关系研究

研究了Ca2+ 对番茄(Lycopersicon esculentum Mill cv. Lichun)黄化幼苗乙烯反应的影响.通过测定不同Ca2+ 浓度条件下番茄黄化幼苗的"三重反应"、内源乙烯释放量、乙烯受体基因NEVER-RIPE(NR)表达量及胞内CaM含量的变化,结果发现,随着培养基中Ca2+ 浓度从0 mmol/L增加到3.8 mmol/L,番茄黄化幼苗的"三重反应"表型明显增强,内源乙烯释放量、NR基因的表达量及胞内CaM的含量都有不同程度的增加;当Ca2+ 浓度由3.8 mmol/L进一步增加到10 mmol/L时,番茄黄化幼苗"三重反应"表型受到抑制,内源乙烯释放量、 NR基因的表达量及胞内CaM的含量都有所下降.因此,Ca2+ 对番茄黄化幼苗"三重反应"的影响与Ca2+ 调节内源乙烯合成和乙烯受体基因的表达有关,而且Ca2+ 可能是通过CaM含量的变化来调节乙烯作用的.     

细胞信号转导中Ca2+和微管骨架的关系

就钙离子和微管骨架在信号通路中的关系和二者可能作用方式的研究进展作了概述。     

Ca2+ regulation of heart contractility in Octopus

Isometric force development of electrically paced preparations isolated from the systemic heart of Octopus vulgaris were utilized to examine the regulation of contractility by Ca²⁺. Increases in extracellular Ca²⁺, to the physiological level, resulted in enhancement of twitch force. For instance, at 36 beats · min⁻¹ an increase in Ca²⁺ from 3 to 9 mmol · l⁻¹ resulted in a threefold increase in twitch force development. When steady-state contraction at 12 beats · min⁻¹ was followed by a rest period of either 5 or 10 min, the first contraction always exhibited either an increase in twitch force or stayed unchanged such that post-rest twitch force was about 133% of the last value in the steady-state train. Ryanodine (12.5 μmol · l⁻¹), which is considered to be a specific inhibitor of the Ca²⁺ storage and release capabilities of the sarcoplasmic reticulum (SR), was applied to further assess Ca²⁺ handling. Twitch force fell to about 22% of the preteatment level in preparations paced at either 12 or 36 beats · min⁻¹. In all preparations the frequency transition from 12 to 36 beats · min⁻¹ was associated with an increase in resting tension. The␣increase␣was 37 ± 14% prior to ryanodine treatment and was significantly elevated to 127 ± 33% following treatment. When steady-state contraction at 36 beats · min⁻¹ was followed by a rest period of 10 s, the first contraction was not significantly different from the last beat in the train prior to ryanodine; however, with ryanodine treatment, post-rest twitch force development significantly decreased. Twitch force development was regular at pacing rates of up to 300 beats · min⁻¹. Twitch force was maintained up to rates of 84 beats · min⁻¹ but␣decreased thereafter and reached a value of about 10% at 300 beats · min⁻¹. Resting tension increased substantially as frequency was elevated from 12 to 36 beats · min⁻¹ and then gradually increased as frequency was further elevated to 180 beats · min⁻¹. In conclusion, the Octopus ventricle is dependent upon extracellular Ca²⁺ for contraction. A post-rest potentiation of force development, the negative impact of ryanodine, and the ability to respond regularly at high pacing rates imply a strong reliance on the SR in Ca²⁺ cycling based on criteria established for vertebrate hearts. Accepted: 19 January 1997     

Antimicrobial and immunostimulatory peptide,KLK, induces an increase in cytosolic Ca2+ concentration by mobilizing Ca2+ from intracellular stores

The cationic antimicrobial immunomodulatory peptide, KLK (KLKL₅KLK), exerts profound membrane interacting properties, impacting on ultrastructure and fluidity. KLK–membrane interactions that lead to these alterations require the ability of the peptide to move into an α‐helical conformation. We show that KLK induces an increase of the intracellular Ca²⁺ concentration in human T24 cells. The effect of KLK is buffer‐sensitive, as it is detected when HBSS buffer is used, but not with PBS. This, together with the lack of effect of the middle leucine‐to‐proline‐substituted peptide derivative [KPK (KLKLLPLLKLK)], indicates that it is the conformational propensity rather than the net positive charge that contributes to the effect of KLK on intracellular Ca²⁺ level of T24 cells. We show that, although KLK slightly stimulates Ca²⁺ influx into the cell, the bulk increase of Ca²⁺ levels is due to KLK‐induced depletion of intracellular Ca²⁺ stores. Finally, we demonstrate a KLK‐induced switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet that contributes to the onset of early apoptotic changes in these cells.     

Intracellular Ca2+ Chelators Prevent Dna Damage And Protect Hepatoma 1Clc7 Cells From Quinone-Induced Cell Killing

Exposure of hepatoma lclc7 cells to 2,3-drniethoxy-1.4-naphthoquinone (DMNQ) resulted in a sustained elevation of cytosolic Ca²⁺. DNA single strand breaks and cell killing. DNA single strand break formation was prevented when cells were preloaded with either of the intracellular Ca²⁺ chelators. Quin 2 or BAPTA, to buffer the increase in cytosolic Ca²⁺ concentration induced by the quinone. DMNQ caused marked NAD⁺ depletion which was prevented when cells were preincubated with 3-aminobenzamide. an inhibitor of nuclear poly-(ADP-ribose)-synthetase activity. or with either of the two Ca²⁺ chelators. However. 3-aminobenzamide did not protect the hepatoma cells from loss of viability. Our results indicate that quinone-induced DNA damage. NAD⁺ depletion and cell killing are mediated by a sustained elevation of cytosolic Ca²⁺     

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.     

拟南芥叶细胞游离钙离子的测定

低温(4℃)条件下将钙离子荧光探针Fluo-3／AM导入拟南芥叶细胞,利用激光共聚焦显微技术检测了胞内钙离子荧光强度的分布。实验证明,低温导入Fluo-3／AM法测定拟南芥叶细胞中钙离子荧光强度的变化切实可行。茉莉酸(JA)处理能够诱导胞内游离钙离子浓度的升高。     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

Ca2+-Regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlortetracycline analysis

We have used chlortetracycline (CTC) analysis to investigate mechanisms that may play important roles during bull sperm capacitation in a culture medium (containing glucose, heparin, and caffeine) known to promote capacitation and fertilization in vitro. In initial experiments employing the Ca²⁺ ionophore A23187, we identified three discrete CTC patterns so similar to those described for mouse and human sperm that we have employed the same nomenclature: “F,” characteristic of uncapacitated, acrosome-intact cells; “B,” characteristic of capacitated, acrosome-intact, cells; “AR,” characteristic of capacitated, acrosome-reacted cells. Over a 60-min period, A23187 stimulated significant increases in B and AR pattern cells, with concomitant decreases in F pattern cells, suggesting a very rapid transition from the uncapacitated to the capacitated state and then on to exocytosis. Without ionophore, significant changes in the proportions of F and B pattern cells were also observed, but the maximum responses required 4 hr; the proportion of AR cells was consistently ～ 15% throughout, indicating a low incidence of spontaneous acrosome loss. Analysis of cells in media with altered composition indicated that the inclusion of either heparin or caffeine significantly promoted capacitation to about the same extent, but together, heparin plus caffeine had an even more stimulatory effect. Despite this, none of these treatments triggered acrosome loss above the levels seen in media lacking these constituents. In the presence of caffeine, with or without heparin, the inclusion of glucose had little effect on responses, but in the presence of heparin there were fewer B cells. In the presence of either quercetin, a Ca-ATPase inhibitor used at 50–200 μM, or W-7, a calmodulin antagonist used at 5–125 μM, capacitation per se was accelerated, as evidenced by significant decreases in F and significant increases in B pattern cells; only the highest concentration of each caused significant increases in AR cells. In addition, 25 and 125 μM W-7 markedly stimulated motility, both quantitatively and qualitatively. Finally the Na⁺ ionophore monensin at 500 μM significantly accelerated both capacitation and acrosomal exocytosis. The addition of the dihydropyridine calcium channel blocker nifedipine at 10 nM, just prior to monensin, did not inhibit capacitation (F to B transition) but blocked acrosomal exocytosis (B to AR transition). We suggest that Ca²⁺ is required for functional changes in bull sperm, with a Ca²⁺-ATPase modulating intracellular Ca²⁺ during capacitation and calcium channels controlling the Ca²⁺ influx required for acrosomal exocytosis. © 1995 Wiley-Liss, Inc.     

Intracellular Ca2+ stores regulate muscarinic receptor stimulation of phospholipase C in cerebellar granule cells

Muscarinic receptor activation of phosphoinositide phospholipase C (PLC) has been examined in rat cerebellar granule cells under conditions that modify intracellular Ca2+ stores. Exposure of cells to medium devoid of Ca2+ for various times reduced carbachol stimulation of PLC with a substantial loss (88%) seen at 30 min. A progressive recovery of responses was observed following the reexposure of cells to Ca2+-containing medium (1.3 mM). However, these changes did not appear to result exclusively from changes in the cytosolic Ca2+ concentration ([Ca2+]i), which decreased to a lower steady level (approximately 25 nM decrease in 1-3 min after extracellular omission) and rapidly returned (within 1 min) to control values when extracellular Ca2+ was restored. Only after loading of the intracellular Ca2+ stores through a transient 1-min depolarization of cerebellar granule cells with 40 mM KCl, followed by washing in nondepolarizing buffer, was carbachol able to mobilize intracellular Ca2+. However, the same treatment resulted in an 80% enhancement of carbachol activation of PLC. In other experiments, partial depletion of the Ca2+ stores by pretreatment of cells with thapsigargin and caffeine resulted in an inhibition (18 and 52%, respectively) of the PLC response. Furthermore, chelation of cytosolic Ca2+ with BAPTA/AM did not influence muscarinic activation of PLC in either the control or predepolarized cells. These conditions, however, inhibited both the increase in [Ca2+]i and the PLC activation elicited by 40 mM KCl and abolished carbachol-induced intracellular Ca2+ release in predepolarized cells. Overall, these results suggest that muscarinic receptor activation of PLC in cerebellar granule cells can be modulated by changes in the loading state of the Ca2+ stores.     

CuCl2胁迫对烟草BY-2悬浮细胞死亡的诱导

本文以不含叶绿体的烟草BY-2悬浮细胞系为实验材料,研究了CuCl2胁迫对BY-2细胞呼吸速率、呼吸途径以及细胞内H2O2和ATP含量的影响。结果表明：0.5mmol·L-1CuCl2胁迫明显导致了烟草BY-2细胞的死亡,引起了胞内H2O2爆发和ATP含量下降,严重抑制了BY-2细胞的总呼吸和交替氧化酶途径（alternative oxidase pathway,AOX）。此外,CuCl2对BY-2细胞的线粒体电子传递具有即时的抑制作用。加入外源腺苷（ATP合成的底物）显著抑制了CuCl2胁迫引起的ATP损耗,并阻止了细胞死亡。上述结果表明CuCl2胁迫导致的ATP损耗在CuCl2诱导烟草BY-2细胞死亡过程中起重要的作用。     

Effects of Al3+ on Ca2+-ATPase Activity on Chloroplasts of Rice and Relation to Calmodulin

The relationship between aluminium phytotoxicity and calmodulin has been studied with. calcium-dependent ATPase in chloroplasts of rice. This enzyme could be activated by extrinsic calmodulin. It showed that the activity of Ca2+-ATPase in chloroplasts was regulated by calmodulin. The activation of calmodulin to the enzyme might be inhibited by calmodulin antagonists, TFP and CPZ. The effects of A13+ on the activation of calmodulin was similar to that of the calmodulin antagonists. Calcium could reduce the inhibition of aluminiutn. It seems that there is a model of toxic responses in plants to aluminium: Al3+→calmodulin→target enzy mes→metabolism.     

Ca2+参与H2O2促进盐碱混合胁迫下燕麦种子的萌发和成苗

盐碱胁迫是制约作物高产优质的重要因素,Ca²⁺和H₂O₂作为信号分子参与作物逆境响应调节。为了解Ca²⁺是否参与H₂O₂对盐碱胁迫下植物种子萌发和成苗的调控,以燕麦（Avena nude）为试验材料,采用隶属函数分析方法,研究了胞外游离Ca²⁺螯合剂EGTA、质膜Ca²⁺通道阻断剂LaCl₃和液泡膜Ca²⁺释放抑制剂钌红（RR）与H₂O₂共处理对盐碱混合（NaCl：Na₂SO₄：NaHCO₃：Na₂CO₃=12：8：9：1）胁迫下种子萌发和成苗的影响。结果表明,25~200 mmol·L^-1盐碱混合胁迫显著抑制燕麦的种子萌发和成苗,抑制程度随浓度提高而增强;0.001~2 mmol·L^-1 H₂O₂能够促进燕麦种子的萌发和成苗,且0.5 mmol·L^-1 H₂O₂可以显著缓解75 mmol·L^-1盐碱混合胁迫对燕麦种子萌发和成苗的抑制作用;而EGTA、LaCl₃和RR均能消减H₂O₂对盐碱混合胁迫下燕麦种子萌发和成苗的促进作用。表明Ca²⁺参与H₂O₂促进盐碱混合胁迫下燕麦种子萌发和成苗的信号转导过程。