Perturbations of intracellular calcium distribution in kidney cells by nephrotoxic haloalkenyl cysteine S-conjugates

The Ca2(+)-sensitive dye fura-2 was used to investigate the disturbances in intracellular Ca2+ concentration [( Ca2+]i) and distribution induced by the nephrotoxic cysteine S-conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and its homocysteine analog S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) in LLC-PK1 cells. After 24-hr treatment with DCVC, the average [Ca2+]i increased from 88 +/- 23 nM to 415 +/- 92 nM. Digital image analysis revealed that the mitochondrial region, which was stained with rhodamine-123, contained lower Ca2+ concentration ([Ca2+]) than other cell areas. This distribution was different from the higher [Ca2+] in the nuclear and mitochondrial regions observed in control cells. In DCVHC-treated cells, there was also an increase in [Ca2+]i to 355 +/- 85 nM, but the increase in [Ca2+] was greater in the mitochondrial region, compared with the rest of the cell. After 72-hr treatment with DCVC or DCVHC, the average [Ca2+]i was 410 +/- 85 nM and 340 +/- 90 nM, respectively, and blebs with markedly higher [Ca2+] (600-1000 nM) than the rest of the cell appeared in both DCVC- and DCVHC-treated cells. Moreover, in DCVC-treated cells the mitochondria could not be stained with rhodamine-123, indicating severe mitochondrial damage and loss of membrane potential. All changes described above took place in viable (propidium iodide-negative) cells. The experiments demonstrate that severe perturbations of intracellular Ca2+ distribution, particularly in the mitochondrial region, precede bleb formation and cell death in the course of development of toxicity by DCVC and DCVHC.     

Cadmium induces Ca2+-dependent necrotic cell death through calpain-triggered mitochondrial depolarization and reactive oxygen species-mediated inhibition of nuclear factor-kappaB activity

This study investigates the mechanism of cell death induced by cadmium (Cd) in Chinese hamster ovary (CHO) cells. Cells exposed to 4 microM Cd for 24 h did not show signs of apoptosis, such as DNA fragmentation and caspase-3 activation. The pro-apoptotic (Bax) or anti-apoptotic (Bcl-2 and Bcl-xL) protein levels in the Bcl-2 family were not altered. However, an increase in propidium iodide uptake and depletion of ATP, characteristics of necrotic cell death, were observed. Cd treatment increased the intracellular calcium (Ca2+) level. Removal of the Ca2+ by a chelator, BAPTA-AM, efficiently inhibited Cd-induced necrosis. The increased Ca2+ subsequently mediated calpain activation and intracellular ROS production. Calpains then triggered mitochondrial depolarization resulting in cell necrosis. Cyclosporin A, an inhibitor of mitochondrial permeability transition, recovered the membrane potential and reduced the necrotic effect. The generated ROS reduced basal NF-kappaB activity and led cells to necrosis. An increase of NF-kappaB activity by its activator, PMA, attenuated Cd-induced necrosis. Calpains and ROS act cooperatively in this process. The calpain inhibitor and the ROS scavenger synergistically inhibited Cd-induced necrosis. Results in this study suggest that Cd stimulates Ca2+-dependent necrosis in CHO cells through two separate pathways. It reduces mitochondrial membrane potential by activating calpain and inhibits NF-kappaB activity by increasing the ROS level.     

Styrene 7,8-oxide induces mitochondrial damage and oxidative stress in neurons

Styrene 7,8-oxide (SO) is the main metabolite of styrene, a neurotoxic compound used industrially. Neurons exposed to SO undergo apoptosis with characteristic features including chromatin rearrangements and caspase activation. We report that the execution phase of apoptosis induced by SO (0.3 mM) in SK-N-MC neurons is triggered by translocation of apoptogenic factors (e.g., cytochrome c) into the cytosol. In addition, mitochondria exhibit lower Ca2+ capacity and loss of mitochondrial membrane potential (DeltaPsi). Lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), is increased after 12 h. Pre-treatment with the antioxidant MnTBAP (100 microM) prevents the decrease of Ca2+ capacity, cytochrome c release, activation of caspases, exposure of phosphatidylserine and cell death. Hence, the neurotoxic effects of SO are related to mitochondrial damage and oxidative stress.     

五甲基槲皮素预处理对大鼠心肌细胞缺氧复氧损伤保护作用的机制研究

目的 探讨五甲基槲皮素（PMQ）预处理对大鼠心肌细胞缺氧/复氧（A/R）损伤的保护作用及其线粒体功能的影响。方法 原代培养SD大鼠乳鼠心肌细胞,经终浓度分别为10,30,100 μmol·L-1 PMQ预处理24 h后,制作A/R损伤,检测培养液中乳酸脱氢酶（LDH） 活性、四唑盐（MTT）比色法检测细胞存活率、流式细胞法检测线粒体膜电位和细胞凋亡情况、线粒体肿胀法检测各组心肌细胞线粒体mPTP开放情况。结果 不同剂量PMQ（10, 30,100 μmol·L-1）预处理24 h后可剂量依赖性的降低LDH活性、增加细胞存活率、减少细胞凋亡（P 〈0.05或P 〈0.01）;30, 100 μmol·L-1 PMQ预处理24 h后,线粒体膜电位更为稳定、mPTP开放减少（P 〈0.05或P 〈0.01）。结论 PMQ预处理24 h后,可产生药理性延迟保护作用,机制与其稳定线粒体膜电位、抑制mPTP开放,进而减少细胞凋亡有关。     

Mechanism of apoptosis induced by a polysaccharide, from the loach Misgurnus anguillicaudatus (MAP) in human hepatocellular carcinoma cells

The biological activities of the polysaccharide have attracted more and more attention in the biochemical and medical areas due to their anti-cancer effects. To estimate the anti-tumor mechanism of MAP, a novel polysaccharide from the loach, Misgurnus anguillicaudatus, the apoptosis effects of the polysaccharide on the human hepatocellular carcinoma cells (SMMC-7721 cells) were studied. The present studies showed that MAP could induce cell apoptosis which was closely accompanied with an increase of intracellular-free calcium concentration ([Ca2+]i), the enhancement of reactive oxygen species (ROS) level, dissipation of mitochondria membrane potential (MMP), up-regulation of p53 mRNA, increase expression of Bax mRNA, and decrease expression of Bcl-2 mRNA. These results suggested that cell apoptosis induced by MAP mainly was mediated by mitochondrial pathways, not involved death receptors (DRs) pathways. The mechanism possibly is that MAP acts on mitochondria and boosts ROS, ROS mediates a release of Ca2+ from the intracellular Ca2+ pool, increasing [Ca2+]i targets the cells a start-up of the apoptosis program. However, further research on the molecular mechanisms of MAP effecting on the cells' mitochondria is necessary.     

Calcium plays a key role in the effects induced by a snake venom Lys49 phospholipase A2 homologue on a lymphoblastoid cell line.

A catalytically-inactive Lys49 phospholipase A2 homologue from the venom of the snake Bothrops asper induces diverse effects (necrosis, apoptosis and proliferation) in a lymphoblastoid cell line, depending on the toxin concentration. The increments in cytosolic Ca2+ levels induced by this toxin in this cell line were assessed. At high toxin concentration (100 microg/mL) the toxin induces drastic disruption of the plasma membrane, associated with a prominent Ca2+ influx and necrosis. Previous incubation of the cells with the chelating agent EGTA or with ruthenium red, an inhibitor of the uniporter mitochondrial Ca2+ transport, greatly reduced necrosis. At a toxin concentration of 12.5 microg/mL, apoptosis is the predominant response, being associated with lower increments in cytosolic Ca2+. This effect was inhibited by preincubation with ruthenium red and the cytosolic Ca2+ chelator BAPTA-AM. The proliferative response, which occurs at a low toxin concentration (0.5 microg/mL), is associated with a small and oscillatory increment in cytosolic Ca2+. It was inhibited by EGTA, ruthenium red and BAPTA-AM, by inhibitors of the endoplasmic reticulum Ca2+ -ATPase (SERCA) and by blockade of the ryanodine receptor. It is concluded that necrosis and apoptosis induced by this toxin are associated with increments in cytosolic Ca2+ levels following plasma membrane perturbation, together with the involvement of mitochondria. The cellular proliferative response depends on a limited Ca2+ influx through the plasma membrane, being associated with a concerted functional unit constituted by SERCA, the ryanodine receptor and mitochondria, which regulate the observed oscillations in cytosolic Ca2+ concentration.     

Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.     

右美托咪定对布比卡因所致神经细胞毒性的保护作用

摘要： 目的 探讨右美托咪定对布比卡因所致神经细胞毒性的保护作用。方法 体外培养小鼠神经母细胞瘤细胞株 N2a 细胞, 取对数期细胞分为 4 组： 对照组细胞培养液不加任何药物; 布比卡因组细胞中加入 1 000 µmol/L 布比卡因; 50、 200 µmol/L 右美托咪定浓度组细胞中加入 1 000 µmol/L 布比卡因后, 再分别加入 50、 200 µmol/L 右美托咪定。各组细胞加入药物后继续培养 24 h, 以 MTT 法检测细胞存活率; 流式细胞术检测细胞凋亡、 活性氧 （ROS）水平、 线粒体膜电位及 Caspase-3 的表达。结果 在 1 000 µmol/L 布比卡因作用下, 各药物组 N2a 细胞的存活率明显降低, 同时线粒体膜电位显著下降, 而细胞的凋亡率、 胞内 ROS 水平和 Caspase-3 表达则显著升高; 50、 200 µmol/L 右美托咪定可抑制布比卡因引起的 N2a 细胞毒性, 使细胞的存活率及线粒体膜电位均明显升高, 同时降低细胞的凋亡率、 胞内 ROS 水平和 Caspase-3 表达, 200 µmol/L 右美托咪定的变化较 50 µmol/L 更为明显。结论 右美托咪定可减轻布比卡因对 N2a 细胞的毒性作用, 其可能是通过抑制 ROS 的生成、 改变线粒体膜电位、 降低 Caspase-3 的表达, 从而抑制细胞的凋亡来实现的。     

Effects of antiarrhythmic drugs on apoptotic pathways in H9c2 cardiac cells

Antiarrhythmic drugs may induce cellular apoptosis in the heart. By using representatives of 5 different categories of antiarrhythmic drugs, that is, pilsicainide, propranolol, nifekalant, verapamil, and amiodarone, we investigated whether these ion channel blockers or beta-antagonists affect cardiac apoptosis in cell cultures. Cultured H9c2 cells were treated with the drugs at varying concentrations. To determine the degree of apoptosis, the percentage of hypodiploid cells, mitochondrial transmembrane potential (DeltaPsi(m)), and activities of caspases were measured quantitatively. At 24 h after administration, only amiodarone induced apoptosis in the H9c2 cells. Amiodarone at a concentration of 14.8 microM or higher decreased DeltaPsi(m) and activated caspase-2 within 3 h of administration, and it caused the appearance of hypodiploid cells and activation of caspases-3 and -9 at 6 h or later. Thus, amiodarone, but none of the other antiarrhythmic drugs tested, possesses a pro-apoptotic effect, mainly via the mitochondrial pathway, suggesting that this effect is distinct from the blocking action of Na+, K+, and Ca2+ channels or the beta-adrenergic receptor. Furthermore, induction of apoptosis in a dose-dependent manner by amiodarone indicates the importance of monitoring the serum concentration in order to avoid its adverse effects.     

BAPTA-AM对MDCK细胞的保护作用及其机制

目的观察BAPTA-AM抗D-氨基半乳糖(D-GalN)诱导MDCK细胞损伤作用,探讨其作用机制。方法采用MTT法,Annexin V-EGFP/PI与Hoechst33342/PI荧光染色,罗丹明123(Rhodamine 123)荧光染色,Caspase-8、Caspase-9活性测定及钙离子载体A23187诱导细胞内高钙等方法,分别测定D-GalN攻击下MDCK细胞活性,细胞凋亡状况,线粒体膜电位(△Ψm)、Caspase-8、Caspase-9活性和细胞内游离钙浓度([Ca2+]i)变化等。结果 BAPTA-AM能明显抑制D-GalN所致的细胞活力下降、减少细胞凋亡、维持△Ψm,抑制Caspase-8、Caspase-9的激活,减轻A23187所致细胞损伤,降低[Ca2+]i。结论 BAPTA-AM通过减轻钙超载,保护线粒体、抑制外源及内源性凋亡通路的激活,发挥抗肾细胞凋亡作用。     

Cadmium hepatotoxicity and alterations of the mitochondrial function

OBJECTIVE: To examine the effect of low concentrations of cadmium on isolated liver mitochondrial function as related to hepatotoxicity. METHODS: Tetraphenyl phosphonium ion uptake and retention, estimated with a tetraphenyl phosphonium-sensitive electrode, was used to monitor changes in liver inner mitochondrial membrane potential. Ca2+ efflux was measured spectrophotometrically with the Ca2+ indicator Arsenazo III. Mitochondrial swelling was measured spectrophotometrically at 540 nm. Oxygen consumption was measured with a Clark-type oxygen microelectrode. RESULTS: Incubation of isolated liver mitochondria with cadmium (5-30 microM) altered mitochondrial function as indicated by swelling, inhibition of respiration, loss of inner mitochondrial membrane potential, and loss of preaccumulated Ca2+. The presence of dithiothreitol (2 mM) in the incubation medium restored mitochondrial function to almost the control level. Cyclosporin A (1 microM), however, did not provide any protection against cadmium toxicity. CONCLUSIONS: The findings point to a direct effect of cadmium on liver mitochondrial function. Cadmium toxicity may be due to loss of reduced glutathione rather than to increased mitochondrial inner membrane permeability. The effect of cadmium on liver mitochondria seems to be an early event in cadmium-induced hepatotoxicity.     

Regulation of endoplasmic reticulum Ca2+ dynamics by proapoptotic BCL-2 family members

Uncontrolled cytosolic Ca(2+) overload is a common cause of cell death in several pathological conditions. Recent evidences reveal a more regulated role for intracellular Ca(2+) stores in controlling cell death. Proteins of the BCL-2 family include anti- and proapoptotic members that control the mitochondrial amplification loop of apoptosis. The antiapoptotic protein BCL-2 prevents this mitochondrial loop, while the "multidomain" proapoptotic proteins BAX and BAK are crucial to initiate it. BCL-2, BAX and BAK localize also to the endoplasmic reticulum (ER), the main intracellular Ca(2+) store. Overexpression of BCL-2 reduces resting ER Ca(2+) and death in response to apoptotic stimuli that mobilize Ca(2+). Our recent data indicate that multidomain proapoptotics also influence Ca(2+) metabolism. Cells deficient for Bax, Bak (DKO) display lowered steady state ER Ca(2+) concentrations ([Ca(2+)](er)) and secondarily decreased mitochondrial Ca(2+) uptake. Genetic and pharmacologic correction of [Ca(2+)](er) indicates that it controls death in response to Ca(2+)-dependent, mitochondria utilizing signals such as oxidative stress and lipid mediators; and that it participates in the regulation of the apoptotic response to most intrinsic stimuli, such as staurosporine. Thus, BAX and BAK control apoptosis not only at the mitochondria, but also at the ER, an obligate checkpoint for Ca(2+)-dependent apoptotic stimuli.     

On the role of mitochondria in cell injury caused by vanadate-induced Ca2+ overload

Incubation of isolated rat hepatocytes with vanadate (0.25, 0.5 and 1 mM) resulted in progressive accumulation of Ca2+ in the intracellular compartments. Vanadate- induced Ca2+ accumulation was related to inhibition of the plasma membrane Ca2+-extruding system, but did not involve either enhanced plasma membrane permeability to Ca2+ or the enhanced operation of a putative Na+/Ca2+ exchanger. After an initial rise in the cytosolic free Ca2+ concentration, as revealed by phosphorylase activation, Ca2+ was sequestered predominantly by the mitochondria with little contribution from the endoplasmic reticulum. As the amount of Ca2+ in the mitochondria increased, a progressive decrease in mitochondrial membrane potential occurred, together with an impairment of the ability of these organelles to further sequester Ca2+. Associated with this, there was a decrease in intracellular ATP level, formation of surface blebs and cytotoxicity. Addition of an uncoupler to vanadate-treated hepatocytes dramatically accelerated the appearance of plasma membrane blebs and toxicity. Our results demonstrate that under conditions in which the plasma membrane Ca2+ pump is inhibited, mitochondria play an important role in protecting hepatocytes against damage induced by Ca2+ overload.     

Involvement of enniatins-induced cytotoxicity in human HepG2 cells

Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, to characterize at which phase of the cell cycle progression the cells were blocked and to study the role of the mitochondrial in ENNs-induced apoptosis. In conclusion, apoptosis induction on HepG2 cells allowed to compare cytotoxic effects caused by both ENNs, A and B. It is reported the possible mechanism observed in MMP changes, cell cycle analysis and apoptosis/necrosis, identifying ENN B more toxic than ENN A.     

Schisandrin B decreases the sensitivity of mitochondria to calcium ion-induced permeability transition and protects against carbon tetrachloride toxicity in mouse livers

Schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, has been shown to protect against carbon tetrachloride (CCl4) hepatotoxicity in mice. In order to elucidate the molecular mechanism underlying the hepatoprotection afforded by Sch B, the effect of Sch B treatment on the sensitivity of mitochondria to Ca2+-stimulated permeability transition (PT) was investigated in mouse livers under normal and CCl4-intoxicated conditions. CCl4 hepatotoxicity caused an increase in the sensitivity of mitochondria to Ca2+-stimulated PT in vitro. The enhanced sensitivity to mitochondrial PT was associated with increases in mitochondrial Ca2+ content as well as the extent of reactive oxidant species (ROS) production and cytochrome c release. The hepatoprotection afforded by Sch B pretreatment against CCl4 toxicity was paralleled by the decrease in the sensitivity of hepatic mitochondria to Ca2+-stimulated PT as well as the attenuations of mitochondrial Ca2+ loading, ROS production and cytochrome c release under CCl4-intoxicated condition. In conclusion, the results suggest that the hepatoprotection afforded by Sch B pretreatment against CCl4 toxicity may be related to the increase in the resistance of hepatic mitochondria to Ca2+-stimulated PT.     

Silica nanoparticles induced intrinsic apoptosis in neuroblastoma SH-SY5Y cells via CytC/Apaf-1 pathway

The present study was to investigate effects of Silica nanoparticles (SiNPs) on nervous system and explore potential mechanisms in human neuroblastoma cells (SH-SY5Y). Cytotoxicity was detected by cell viability and Lactate dehydrogenase (LDH) release. Flow cytometry analysis was applied to assess mitochondrial membrane potential (MMP) loss, intracellular Ca²⁺ and apoptosis. To clarify the mechanism of SiNPs-induced apoptosis, intrinsic apoptosis-related proteins were detected. Our results showed that SiNPs caused cytotoxicity, cell membrane damage and Ca²⁺ increase in a dose-dependent manner in SH-SY5Y cells. Both the mitochondrial membrane potential (MMP) loss and potential mitochondria damage resulted in Cyt C release to the cytoplasm. The elevated Cyt C and Apaf1 further triggered intrinsic apoptosis via executive molecular caspase-9 and caspase-3. The present study confirmed that SiNPs induced intrinsic apoptosis in neuroblastoma SH-SY5Y cells via CytC/Apaf-1 pathway and provided a better understanding of the potential toxicity induced by SiNPs on human neurocyte.     

高能冲击波对肾脏钙离子腺苷三磷酸酶活性的影响及川芎嗪的保护作用

目的 观察高能冲击波对肾脏钙离子腺苷三磷酸酶(Ca2+-ATPase)活性的影响及川芎嗪的保护作用,探讨高能冲击波肾损伤机制.方法 30只健康家兔制成单肾动物模型,按完全随机设计法分为对照组10只、高能冲击波组10只和川芎嗪组10只,对照组、高能冲击波组分别静脉注射等量生理盐水,高能冲击波冲击肾脏前3天,川芎嗪组静脉注射川芎嗪;冲击肾脏24h后,光学法检测肾细胞膜和线粒体膜Ca2+-ATPase活性;采用原位缺口末端标记法和流式细胞术检测凋亡细胞;采用生化分析测定内生肌酐清除率.结果 与对照组比较,高能冲击波组肾细胞膜和线粒体膜Ca2+-ATPase活性均降低(P<0.05,P<0.01)、细胞凋亡率增加(P<0.01)、内生肌酐清除率降低(P<0.01);川芎嗪组肾细胞膜Ca2+-ATPase活性和内生肌酐清除率变化不明显(P＞0.05),线粒体膜Ca2+-ATPase活性降低(P<0.05)、细胞凋亡率增加(P<0.05),但幅度明显小于高能冲击波组.结论 高能冲击波冲击肾脏后肾脏Ca2+-ATPase活性降低是发生肾细胞凋亡导致肾功能损伤的重要机制,川芎嗪有改善肾功能的作用,与其阻制肾脏Ca2+-ATPase活性降低、抗细胞凋亡有关.