薯蓣皂苷对大鼠心肌收缩与钠-钙交换体的影响

目的：观察薯蓣皂苷（Dio）对大鼠心肌收缩作用以及胞内Ca²⁺浓度的影响,并初步探讨其作用机制与Na⁺-Ca²⁺交换体（NCX）的关系。方法：采用Langendorff逆行主动脉灌流法对大鼠离体心脏进行灌流,利用压力感受器插管法测定左心室相关心功能参数,记录及其在应用NCX选择性抑制剂SEA0400情况下对左心室收缩压（LVSP）、左心室舒张末期压（LVEDP）、左心室内压最大上升/下降速率（±dp/dt_max）以及心率（HR）的影响;利用激光共聚焦显微观察薯蓣皂苷及SEA0400对大鼠心肌细胞H9c2细胞内Ca²⁺浓度的影响。结果：离体心脏灌流结果显示,1 μmol/L Dio可显著增加LVSP,增加约19.7%（P<0.01）;增加左室内压最大上升速率（+dp/dt_max）,增加约9.6%;激光共聚焦测定Ca²⁺荧光强度实验结果显示：1 μmol/L Dio可使H9c2细胞中Ca²⁺相对荧光强度增加（P<0.01）;而在SEA0400存在的情况下,1 μmol/L的Dio使细胞内Ca²⁺相对荧光强度变为（17.09±0.63）,给予Dio后差异有显著性（P<0.01）。在细胞液中无Ca²⁺或无Na⁺时,给予1 μmol/L的Dio使Ca²⁺相对荧光强度减小,与给予1 μmol/L的Dio差异有显著性（P<0.01）。结论：Dio可增加左心室收缩压和最大上升速率,表现正性肌力作用;Dio可使细胞内Ca²⁺浓度增加,其作用机制与增加Na⁺内流,促进NCX反向转运有关。     

苜蓿体内H2S信号与Ca2+调节气孔运动的作用机制

为探究H₂S信号在苜蓿（Medicago sativa）体内调节气孔运动的作用,及在此过程中H₂S与Ca²⁺的关系,以蒺藜苜蓿（Medicago truncatula）的野生型和钙离子转运体突变体为试验材料,分别从转录水平、细胞水平和生理水平开展研究。采用qRT-PCR比较相关基因的表达量变化、荧光探针显示体内Ca²⁺含量、电极法测定H₂S含量、光学显微镜观察和测量气孔孔径等。结果表明：蒺藜苜蓿突变体NF3011和NF2734体内H₂S的含量与野生型相比极显著降低（P<0.01）;H₂S信号在一定程度上抑制钙离子转运体编码基因MTR_6g027580的表达;外源生理浓度H₂S熏蒸可诱导蒺藜苜蓿气孔关闭,与Ca²⁺通道阻断剂LaCl₃联合处理对野生型气孔运动未产生影响,而在突变体中的结果截然相反;利用荧光探针测定保卫细胞内的Ca²⁺含量,所得结果与气孔孔径的变化规律完全一致。综上所述,H₂S信号促进叶片保卫细胞内Ca²⁺的含量增加,最终表现为植物气孔孔径变小,在此过程中胞内Ca²⁺含量变化主要通过Ca²⁺转运体进行,少部分依赖Ca²⁺离子通道。该研究结果不仅在理论上丰富了H₂S信号的作用机制,更具应用于苜蓿生产实践并推广于其他作物的潜力。     

小麦悬浮细胞应答激发子刺激的过敏性反应中Ca2+和 NO的动态变化及其相互作用

以感染叶锈菌的小麦(Triticum aestivum)叶片细胞间隙液IWF-260作为激发子, 刺激小麦品种洛夫林10和郑州5389的悬浮细胞, 探讨由激发子引发悬浮细胞过敏性反应中Ca²⁺和NO的变化及相互作用。以荧光分子探针Fluo-3AM和DAF-FM DA分别对细胞内Ca²⁺和NO进行标记, 利用激光共聚焦扫描显微镜对其动态变化进行实时监测, 通过药物学实验对Ca²⁺和NO的产生机制及其可能存在的相互关系进行探讨。结果表明, 2个小麦品种悬浮细胞的[Ca²⁺]_cyt水平对激发子刺激的反应表现出明显的差异, 对叶锈菌小种表现不亲和的洛夫林10悬浮细胞分别在激发子刺激后330秒和700秒出现2个钙峰; 而对该小种表现亲和的郑州5389悬浮细胞在激发子刺激后[Ca²⁺]_cyt水平稍有波动但变化不明显。药物学实验证明, [Ca²⁺]_cyt的升高依赖于胞外钙离子内流, 钙离子与激发子刺激诱发的过敏性防卫反应紧密相关。同样, 在激发子刺激后, 洛夫林10悬浮细胞出现1个NO峰, 而郑州5389悬浮细胞胞质NO变化不明显。药物学实验初步证明, NO的产生与胞外钙离子内流密切相关。由此推测, 在小麦悬浮细胞应答激发子刺激诱发的过敏性反应中, NO可能在钙的下游发挥作用。     

外源Ca2+对高温强光下西葫芦幼苗形态特征、光合特性及荧光参数的影响

选用西葫芦(Cucurbita pepo)品种“阿兰”一代为试验材料,研究了外源Ca²⁺处理对高温强光交叉胁迫下西葫芦幼苗生长特征、光合特性及叶绿素荧光参数的影响.结果表明：高温强光胁迫下,5～20 mmol·L^-1 Ca²⁺处理的西葫芦幼苗具有较高的株高和较大的叶面积,其叶绿素、类胡萝卜素含量及光合速率（P_n）、气孔导度（G_s）、蒸腾速率（T_r）、PSⅡ最大光化学效率(F_v/F_m)、PSⅡ实际光化学效率(Φ_PSⅡ)和光化学猝灭系数(q_P)均较高,而胞间CO₂浓度（C_i）和非光化学猝灭系数（NPQ）较低,其中以10 mmol·L^-1Ca²⁺处理效果最好.说明5～20 mmol·L^-1Ca²⁺处理能有效缓解高温强光对西葫芦光合机构的不可逆伤害,使其保持较快的光合电子传递速率和较高的PSⅡ电子传递活性.Ca²⁺处理浓度超过40 mmol·L^-1时对高温强光胁迫没有缓解效应.     

NaHCO3胁迫下3种灌木Na+、K+、Ca2+的吸收及转运

通过盆栽试验,采用原子吸收分光光度法和非损伤微测技术,研究了NaHCO₃胁迫(300 mmol·L^-1)对大洋洲滨藜、四翅滨藜和宁夏枸杞3种灌木离子吸收及运转的影响.结果表明: 随着NaHCO₃浓度升高,两种滨藜和宁夏枸杞叶片中Na⁺含量升高,300 mmol·L^-1NaHCO₃胁迫下,宁夏枸杞叶肉细胞Na⁺的外排增加,两种滨藜净Na⁺外排降低;随着胁迫时间的延长,大洋洲滨藜和宁夏枸杞叶片的K⁺含量下降,Na⁺/K⁺升高,四翅滨藜叶片K⁺含量升高,Na⁺/K⁺降低;随着浓度的升高,宁夏枸杞叶片积累Ca²⁺减少,Na⁺/Ca²⁺高于对照,叶肉细胞Ca²⁺外排;两种滨藜叶Ca²⁺含量总体呈升高趋势,叶肉细胞Ca²⁺表现为内流.在NaHCO₃胁迫下,3种灌木通过不同的策略来消除Na⁺毒害.宁夏枸杞叶片Na⁺的积累抑制了对Ca²⁺的吸收;两种滨藜Ca²⁺的内流促使细胞质中游离Ca²⁺增加,增加的细胞质\[Ca²⁺\]cyt防治质膜H⁺ ATPase去极化,限制K⁺的外排,从而维持细胞内Na⁺/K⁺的平衡,其中四翅滨藜调控Na⁺/K⁺平衡的能力较强.     

油桃花芽破眠过程中H2O2代谢与Ca2+转运的关系

利用化学测定法分析高温、单氰胺和TDZ 3种破眠处理对“曙光”油桃休眠花芽H₂O₂代谢的主要影响,利用非损伤微测技术检测H₂O₂对休眠芽Ca²⁺转运的影响,研究H₂O₂在芽休眠解除过程中的调控作用.结果表明： 在深休眠时期,高温和单氰胺处理均能诱导芽内H₂O₂含量升高和过氧化氢酶(CAT)活性降低,并具有显著的破眠作用;TDZ对H₂O₂含量及CAT、过氧化物酶(POD)活性影响不大,破眠效果较差.休眠花芽原基组织钙通道活跃,对外源Ca²⁺呈吸收状态.外源H₂O₂可诱导休眠花芽原基组织Ca²⁺转运发生变化,低浓度H₂O₂降低Ca²⁺吸收速率,高浓度H₂O₂使组织对Ca²⁺的转运由吸收转变为释放.这表明休眠芽内H₂O₂信号和Ca²⁺信号相关联,通过诱导H₂O₂积累调控Ca²⁺信号可能在高温和单氰胺打破休眠的信号转导过程中起重要作用.     

胞质Ca2+参与外源H2S促进盐碱胁迫下裸燕麦种子萌发

硫化氢（Hydrogen sulfide,H₂S）是植物新型气体信号分子,钙离子（Calcium,Ca²⁺）为重要的第二信使,两者在植物逆境响应中分别发挥着重要作用。为明确胞质Ca²⁺在外源H₂S促进盐碱胁迫下作物种子萌发中的作用,以裸燕麦（Avena nude）为材料,采用培养皿培养,以混合盐碱（NaCl、Na₂SO₄、Na₂CO₃、NaHCO₃的摩尔比为12：8：1：9）模拟甘肃裸燕麦种植地盐碱环境,蒸馏水为对照,测定了胞外Ca²⁺螯合剂乙二醇-双-（2-氨基乙醚）四乙酸（EGTA）、质膜Ca²⁺通道阻断剂氯化镧（LaCl₃）、液泡Ca²⁺释放抑制剂钌红（RR）和内质网钙泵阻断剂毒胡萝卜素（Thaps）分别与H₂S供体硫氢化钠（NaHS）共处理下种子的发芽势、发芽率、发芽指数、活力指数、平均发芽速率、胚根长和胚芽长7个发芽指标,利用隶属函数分析方法综合评价胞质Ca²⁺对H₂S缓解盐碱胁迫抑制种子萌发的影响。结果表明,随着盐碱胁迫浓度增大,裸燕麦种子的发芽势、发芽率、发芽指数、活力指数、平均发芽速率、胚根长和胚芽长显著下降。与对照相比,15~75 mmol·L^-1盐碱胁迫导致裸燕麦种子萌发的隶属函数综合评价值（D）显著降低,30 mmol·L^-1盐碱胁迫下D值下降了73.1%;100~1 000 μmol·L^-1 NaHS不同程度提高了裸燕麦种子萌发的D值,且100 μmol·L^-1 NaHS缓解30 mmol·L^-1盐碱胁迫下D值下降的作用最大;EGTA、LaCl₃和RR均显著逆转了100 μmol·L^-1 NaHS对30 mmol·L^-1盐碱胁迫下D值下降的缓解作用,而Thaps对NaHS的作用无显著影响。表明胞质Ca²⁺参与外源H₂S促进盐碱胁迫下裸燕麦种子萌发的信号传导过程,且胞质Ca²⁺主要来源于胞外Ca²⁺的内流和液泡中Ca²⁺的释放。     

植物感受盐胁迫及相关钙信号的研究进展

盐胁迫对植物的生长和发育造成严重影响,其危害包括渗透胁迫、离子毒害等,严重损害了农业生产和粮食安全。在盐胁迫下,植物相关感受器接受刺激,使得Ca²⁺通过细胞膜以及细胞内钙库膜上打开的Ca²⁺通道进入细胞质基质,导致细胞质内Ca²⁺浓度升高,产生钙信号。钙离子作为重要的第二信使,在植物细胞内和细胞间传递信号,信号往下游传递,在不同生长和发育阶段引起植物一系列的生理响应来应对盐胁迫影响。钙信号主要通过钙调蛋白（CaM）、钙调素样蛋白（CML）、钙依赖性蛋白激酶（CDPK）、钙调磷酸酶B样蛋白（CBL）和CBL互作蛋白激酶（CIPK）感知并将特异的钙信号信息传递到下游;从而激活植物盐胁迫生理响应。本文主要综述植物如何感知盐胁迫刺激,以及钙信号产生与传导机制,并对该研究领域需解决的问题进行了展望。     

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。     

干旱条件下钙离子对一氧化氮诱导黄瓜不定根发生的影响

以黄瓜品种‘新春4号’为材料,研究干旱胁迫下一氧化氮(NO)和钙离子(Ca²⁺)处理下黄瓜的生根指标、内源Ca²⁺荧光强度以及抗氧化酶(超氧化物歧化酶SOD、过氧化氢酶CAT、抗坏血酸过氧化物酶APX)活性,分析干旱条件下黄瓜不定根发生过程中NO和Ca²⁺之间的关系.结果表明: 200 μmol·L^-1 氯化钙(CaCl₂)和0.05%聚乙二醇(PEG)共处理显著提高了干旱条件下黄瓜不定根的根长和根数;添加Ca²⁺螯合剂(EGTA)和通道抑制剂(BAPTA/AM)处理显著降低了干旱条件下NO诱导的不定根根数和根长.干旱条件下,NO和CaCl₂处理提高了黄瓜下胚轴内源Ca²⁺荧光强度;而NO清除剂(cPTIO)处理的Ca²⁺荧光强度显著低于NO处理.干旱条件下,NO和CaCl₂处理显著提高了黄瓜下胚轴抗氧化酶活性;而Ca²⁺抑制剂或螯合剂处理显著降低了NO诱导的抗氧化酶活性.由此可见,干旱条件下Ca²⁺参与了NO调控黄瓜抗氧化酶活性,缓解了干旱胁迫对不定根形成产生的伤害,进而促进了不定根的发生.     

Role of free cytosolic calcium in secretagogue-stimulated amylase release from dispersed acini from guinea pig pancreas

To determine the role of free cytosolic calcium ([Ca+2]i) in stimulated enzyme secretion from exocrine pancreas, we determined the effects of various pancreatic secretagogues on [Ca+2]i and amylase release in dispersed acini from the guinea pig pancreas. Cholecystokinin-octapeptide (CCK-OP), carbachol, and bombesin, but not vasoactive intestinal peptide, stimulated rapid increases in [Ca+2]i from 100 to 600-800 nM that were independent of extracellular calcium. The increases in [Ca+2]i were transient (lasting less than 5 min) and correlated with an initial rapid phase of amylase release. After 5 min, secretagogue-stimulated amylase release occurred at basal [Ca+2]i. Carbachol pretreatment of the acini abolished the effects of CCK-OP and bombesin on [Ca+2]i and the initial rapid phase of amylase release. 4 beta-phorbol 12-myristate 13-acetate (PMA) had no effect on [Ca+2]i but stimulated an increase in amylase release. The addition of CCK-OP or A23187 to PMA-stimulated acini caused an increase in [Ca+2]i and PMA-stimulated amylase release only during the first 5 min after addition of these agents. These results indicate that CCK-OP, carbachol, and bombesin release calcium from an intracellular pool, resulting in a transient increase in [Ca+2]i and that this increase in [Ca+2]i mediates enzyme secretion during the first few minutes of incubation. The results with PMA suggest that secretagogue-stimulated secretion not mediated by increased [Ca+2]i (sustained secretion) is mediated by 1,2-diacylglycerol.     

Neurotransmitter release from bradykinin-stimulated PC12 cells. Stimulation of cytosolic calcium and neurotransmitter release.

The effect of bradykinin on intracellular free Ca2+ and neurotransmitter secretion was investigated in the rat pheochromocytoma cell line PC12. Bradykinin was shown to induce a rapid, but transient, increase in intracellular free Ca2+ which could be separated into an intracellular Ca2+ release component and an extracellular Ca2+ influx component. The bradykinin-induced stimulation of intracellular free Ca2+ displayed a similar time course, concentration dependencies and extracellular Ca2+ dependence as that found for neurotransmitter release, indicating an association between intracellular free Ca2+ levels and neurotransmitter secretion. The selective BK1-receptor antagonist des-Arg9,[Leu8]BK (where BK is bradykinin) did not significantly affect the stimulation of intracellular free Ca2+ or neurotransmitter release. In contrast, these effects of bradykinin were effectively blocked by the selective BK2-receptor antagonist [Thi5,8,D-Phe7]BK, and mimicked by the BK2 partial agonist [D-Phe7]BK in a concentration-dependent manner. The stimulation of intracellular free Ca2+ and neurotransmitter release induced by bradykinin was shown not to involve voltage-sensitive Ca2+ channels, since calcium antagonists had no effect on either response at concentrations which effectively inhibit depolarization-induced responses. These results indicate that bradykinin, acting through the interaction with the BK2 receptor, stimulates an increase in intracellular free Ca2+ leading to neurotransmitter secretion. Furthermore, bradykinin-induced responses involve the release of intracellular Ca2+ and the influx of extracellular Ca2+ that is not associated with the activation of voltage-sensitive Ca2+ channels.     

Localization of intracellular calcium release in cells injured by venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) and dependence of calcium mobilization on G-protein activation

Venom from the ectoparasitic wasp Nasonia vitripennis induces cellular injury that appears to involve the release of intracellular calcium stores via the activation of phospholipase C, and culminates in oncotic death. A linkage between release of intracellular Ca2+ and oncosis has not been clearly established and was the focus of this study. When BTI-TN-5B1-4 cells were treated with suramin, an uncoupler of G-proteins, venom-induced swelling and oncotic death were inhibited in a dose-dependent manner for at least 24 h. Suramin also blocked increases in free cytosolic [Ca2+], arguing that venom induces calcium mobilization through G-protein signaling pathways. Endoplasmic reticulum (ER) was predicted to be the source of intracellular calcium release, but labeling with the fluorescent probe ER-tracker revealed no indication of organelle swelling or loss of membrane integrity as would be expected if the Ca(2+)-ATPase pump was disabled by crude venom. Incubation of cell monolayers with calmodulin or nitrendipine, modulators of ER calcium release channels, neither attenuated nor augmented the effects of wasp venom. These results suggest that wasp venom stimulates calcium release from ER compartments distinct from RyRs, L-type Ca2+ channels, and the Ca(2+)-ATPase pump, or calcium is released from some other intracellular store. A reduction of mitochondrial membrane potential delta psi(m) appeared to precede a rise in cytosolic free Ca2+ as evidenced by fluorescent microscopy using the calcium-sensitive probe fluo-4 AM. This argues that the initial insult to the cell resulting from venom elicits a rapid loss of (delta psi(m)), followed by unregulated calcium efflux from mitochondria into the cytosol. Mobilization of calcium in this fashion could stimulate cAMP formation, and subsequently promote calcium release from NAADP-sensitive stores.     

Characterization of the calcium response to thyrotropin-releasing hormone (TRH) in cells transfected with TRH receptor complementary DNA: importance of voltage-sensitive calcium channels.

TRH stimulates a biphasic increase in intracellular free calcium ion, [Ca2+]i. Cells stably transfected with TRH receptor cDNA were used to compare the response in lines with and without L type voltage-gated calcium channels. Rat pituitary GH-Y cells that do not normally express TRH receptors, rat glial C6 cells, and human epithelial Hela cells were transfected with mouse TRH receptor cDNA. All lines bound similar amounts of [3H][N3-Me-His2]TRH with identical affinities (dissociation constant = 1.5 nM). Both pituitary lines expressed L type voltage-gated calcium channels; depolarization with high K+ increased 45Ca2+ uptake 20- to 25-fold and [Ca2+]i 12- to 14-fold. C6 and Hela cells, in contrast, appeared to have no L channel activity. GH4C1 cells responded to TRH with a calcium spike (6-fold) followed by a sustained second phase. When TRH was added after 100 nM nimodipine, an L channel blocker, the initial calcium burst was unaffected but the second phase was abolished. GH-Y cells transfected with TRH receptor cDNA responded to TRH with a 6-fold [Ca2+]i spike followed by a plateau phase (>8 min) in which [Ca2+]i remained elevated or increased. Nimodipine did not alter the peak TRH response or resting [Ca2+]i but reduced the sustained phase, which was eliminated by chelation of extracellular Ca2+. In the transfected glial C6 and Hela cells without calcium channels, TRH evoked transient, monophasic 7- to 9-fold increases in [Ca2+]i, and [Ca2+]i returned to resting levels within 3 min. Thapsigargin stimulated a gradual, large increase in [Ca2+]i in transfected C6 cells, and subsequent addition of TRH caused no further rise. Removal of extracellular Ca2+ from transfected C6 cells shortened the [Ca2+]i responses to TRH, to endothelin 1, and to thapsigargin. The TRH responses were pertussis toxin-insensitive. In summary, TRH can generate a calcium spike in pituitary, C6, and Hela cells transfected with TRH receptor cDNA, but the plateau phase of the [Ca2+]i response is not observed when the receptor is expressed in a cell line without L channel activity.     

Blockage of amyloid beta peptide-induced cytosolic free calcium by fullerenol-1, carboxylate C60 in PC12 cells

Amyloid beta protein (Abeta) alters signal transduction systems, including increases in the cytosolic free calcium ([Ca2+]i) response which have pathophysiological significance in Alzheimer's disease (AD). The purposes of this study were to elucidate the mechanism involved in Abeta's effect on the Ca2+ signal and to evaluate the effect of fullerenol-1, a water-soluble hydroxyl and superoxide radical scavenger, on the Abeta-induced Ca2+ response. Both Abeta and bradykinin (BK) dose-dependently elevated [Ca2+]i in PC12 cells. Fullerenol-1, at a concentration range between 100 nM and 1 microM, dose-dependently reduced the Abeta-induced [Ca2+]i response, but did not alter the subsequent BK-mediated process. Thapsigargin, an inhibitor of Ca2+-ATPase, released Ca2+ from the internal store and diminished the BK-mediated calcium spike but did not affect the Abeta-induced Ca2+ response. In the absence of extracellular calcium, the Abeta-induced, but not BK-induced, calcium spike was completely abolished. The Ca induced by Abeta did not enter through the voltage-dependent calcium channels or ligand gated calcium channels, because the peak of Abeta-evoked Ca2+ was not significantly altered by various Ca2+ channel blockers or a NMDA receptor antagonist MK801. In addition, neither cholera toxin nor pertussis toxin altered the Abeta-induced Ca response. The results demonstrated that Abeta-stimulated [Ca2+]i increase is due to Ca influx from an extracellular source rather than from the intracellular store. Alteration of the membrane lipid structure and permeability by free radicals generated by Abeta may be a major cause of Ca -influx. Furthermore, fullerenol-1, a novel antioxidant, may provide therapeutic benefits in neurodegenerative diseases such as AD.     

EGF receptor—mediated intracellular calcium increase in human hepatoma BEL—7404 cells

Epidermal growth factor(EGF) induced intracellular free calcium ([Ca^2 ]i) response was studied in fura-2- or fluo-3-loaded human hepatoma cells of BEL-7404 cell line.Single cell[Ca^2 ]i analysis and [Ca^2 ]i measurement in cell populations revealed that EGF triggered a rapid[Ca^2 ]i increase in the dose-dependent and time-dependent manner.Pretreatment of cells with an endoplasmic reticulum(ER) Ca^2 -ATPase inhibitor,thapsigargin(TG) at 100nM concentration for 20 min,completely abolished EGF-induced [Ca^2 ]i increase,and chelating extracellular calcium by excess EGTA partially inhibited the increase.Furthermore,the expression of antisense EGF receptor sequence in BEL-7404 cells suppressed the [Ca^2 ]i response to EGF.The results suggest that EGF receptor-mediated [Ca^2 ]i increase in the human hepatoma cells is essentially dependent on the Ca^2 storage in ER.     

Characterization of calcium release-activated apoptosis of LNCaP prostate cancer cells

Apoptosis inhibition rather than enhanced cellular proliferation occurs in prostate cancer (CaP), the most commonly diagnosed malignancy in American men. Therefore, it is important to characterize residual apoptotic pathways in CaP cells. When intracellular Ca(2+) stores are released and plasma membrane "store-operated" Ca(2+) entry channels subsequently open, cytosolic [Ca(2+)] increases and is thought to induce apoptosis. However, cells incapable of releasing Ca(2+) stores are resistant to apoptotic stimuli, indicating that Ca(2+) store release is also important. We investigated whether release of intracellular Ca(2+) stores is sufficient to induce apoptosis of the CaP cell line LNCaP. We developed a method to release stored Ca(2+) without elevating cytosolic [Ca(2+)]; this stimulus induced LNCaP cell apoptosis. We compared the apoptotic pathways activated by intracellular Ca(2+) store release with the dual insults of store release and cytosolic [Ca(2+)] elevation. Earlier processing of caspases-3 and -7 occurred when intracellular store release was the sole Ca(2+) perturbation. Apoptosis was attenuated in both conditions in stable transfected cells expressing antiapoptotic proteins Bclx(L) and catalytically inactive caspase-9, and in both scenarios inactive caspase-9 became complexed with caspase-7. Thus, intracellular Ca(2+) store release initiates an apoptotic pathway similar to that elicited by the dual stimuli of cytosolic [Ca(2+)] elevation and intracellular store release.     

Sustained release of calcium elicited by membrane depolarization in ryanodine-injected mouse skeletal muscle fibers

The effect of micromolar intracellular levels of ryanodine was tested on the myoplasmic free calcium concentration ([Ca(2+)](i)) measured from a portion of isolated mouse skeletal muscle fibers voltage-clamped at -80 mV. When ryanodine-injected fibers were transiently depolarized to 0 mV, the early decay phase of [Ca(2+)](i) upon membrane repolarization was followed by a steady elevated [Ca(2+)](i) level. This effect could be qualitatively well simulated, assuming that ryanodine binds to release channels that open during depolarization and that ryanodine-bound channels do not close upon repolarization. The amplitude of the postpulse [Ca(2+)](i) elevation depended on the duration of the depolarization, being hardly detectable for pulses shorter than 100 ms, and very prominent for duration pulses of seconds. Within a series of consecutive pulses of the same duration, the effect of ryanodine produced a staircase increase in resting [Ca(2+)](i), the slope of which was approximately twice larger for depolarizations to 0 or +10 mV than to -30 or -20 mV. Overall results are consistent with the "open-locked" state because of ryanodine binding to calcium release channels that open during depolarization. Within the voltage-sensitive range of calcium release, increasing either the amplitude or the duration of the depolarization seems to enhance the fraction of release channels accessible to ryanodine.     

Mechanisms of calcium elevation in the micromeres of sea urchin embryos

The micromeres, the first cells to be specified in sea urchin embryos, are generated by unequal cleavage at the fourth cell division. The micromeres differentiate autonomously to form spicules and dispatch signals to induce endomesoderm in the neighbouring macromeres cells in the embryo. Using a calcium indicator Fura-2/AM and a mixture of dextran conjugated Oregon green-BAPTA 488 and Rhodamine red, the intracellular calcium ion concentration ([Ca2+]i) was studied in embryos at the 16-cell stage. [Ca2+]i was characteristically elevated in the micromeres during furrowing at the 4th cleavage. Subsequently, Ca2+ oscillated for about 10 min in the micromeres, resulting in episodic high levels of [Ca2+]i. High [Ca2+]i regions were associated with regional localizations of the endoplasmic reticulum (ER), though not with ER accumulated at the vegetal pole of the micromeres during the 4th division. Pharmacological studies, using a blocker of IP3-mediated Ca2+ release (Xestospongin), a store-operated Ca2+ entry inhibitor (2 aminoethoxydiphenyl borate (2-APB)) and an inhibitor of stretch-dependent ion channels (gadolinium), suggest that the high [Ca2+]i and oscillations in the micromeres are triggered by calcium influx caused by the activation of stretch-dependent calcium channels, followed by the release of calcium ions from the endoplasmic reticulum. On the basis of these new findings, a possible mechanism for autonomous formation of the micromeres is discussed.     

Biphasic activation of cytosolic free calcium and LH responses by gonadotropin-releasing hormone

Gonadotropin-releasing hormone (GnRH) stimulates rapid peak increases in [Ca2+]i and LH release, followed by lower but sustained elevations of both [Ca2+]i and hormone secretion. Omission of extracellular Ca2+ only slightly decreased the peak of [Ca2+]i, but reduced the peak LH response by 40% and prevented the prolonged increases in [Ca2+]i and LH release. Dihydropyridine calcium antagonists did not affect the peak [Ca2+]i and LH responses, but reduced the sustained increases by up to 50%. Whereas GnRH-induced mobilization of intracellular calcium initiates the LH peak, and Ca2+ entry through dihydropyridine-insensitive channels contributes to the peak and plateau phases of LH release, dihydropyridine-sensitive L-type Ca2+ channels participate only in the sustained phase of gonadotropin secretion.