羟墓自由基引起神经细胞［Ca~（2＋）］i增高的机制和Ebselen的抑制作用

用Ｆｕｒａ－２显微荧光测量技术研究了羟基自由基对单个皮层神经细胞内游离钙离子浓度（Ｃａ＾２＋）ｉ影响和硒化合物Ｅｂｅｌｅｎ对（Ｃａ＾２＋）ｉ的抑制作用,结果表明羟基自由基的作用首先引起胞内（Ｃａ＾２＋）ｉ以时间常数τ＝３８９５．４±５０７．２Ｓ速度缓慢增加,然后加入了以τ＝４２０．６＋１２２．０Ｓ的外钙大量涌入,钙通道阻断剂,疏基还原剂,疏基还原剂和自由基清除剂对羟基自由基损伤作用的影响提示外钙的     

羟墓自由基引起神经细胞［Ca~（2＋）］i增高的机制和Ebselen的抑制作用

用Ｆｕｒａ－２显微荧光测量技术研究了羟基自由基对单个皮层神经细胞内游离钙离子浓度［Ｃａ２＋］ｉ影响和硒化合物Ｅｂｅｌｅｎ对［Ｃａ２＋］ｉ的抑制作用。结果表明羟基自由基的作用首先引起胞内［Ｃａ２＋］ｉ以时间常数τ＝３８９５．４±５０７．２Ｓ速度缓慢增加,然后加入了以τ＝４２０．６±１２２．０Ｓ的外钙大量涌入。钙通道阻断剂、疏基还原剂、疏基还原制和自由基清除剂对羟基自由基损伤作用的影响提示外钙的大量涌入部分与通道的开放有关,疏基损伤在羟基自由基引起的［Ｃａ２＋］ｉ升高中起着重要的作用。具有类谷胱甘肽过氧化酶活性的小分子硒化合物Ｅｂｓｅｌｅｎ（１０－５ｍｏｌ／Ｌ和１０－６ｍｏｌ／Ｌ）抑制羟基自由基引起的［Ｃａ２＋］ｉ升高,推测它可以抑制钙库的释放或促进内钙的外排以及抑制外钙的流入。     

Fenton体系、H_2O_2损伤红细胞膜分子流动性的机理的比较研究

在系统中Ｈ２Ｏ２与所含有的亚铁离子通过Ｆｅｎｔｏｎ反应主要生成ＯＨ自由基损伤细胞,但Ｈ２Ｏ２也可损伤细胞膜或细胞。为区分Ｆｅｎｔｏｎ自由基体与Ｈ２Ｏ２分别对红细胞膜脂质及蛋白分子相对旋转时间影响机理的不同,分别对体外不同浓度Ｆｅｎｔｏｎ体系和不同浓度Ｈ２Ｏ２作用３０分钟后,红细胞膜剪切弹性模量μ、细胞表面指数Ｓｉ、膜蛋白分子τｐ及脂质分子τｌ相对旋转时间和它们的化学结构进行了比较分析。结果发现,（     

TFP刺激裂殖酵母细胞钙离子内流的质膜效应

ＴＦＰ（１０－１００μｍｏｌ／Ｌ）可引起裂殖酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅ）胞外Ｃａ２＋内流,ＴＦＰ浓度不同,促进Ｃａ２＋内流程度也不一样,５０μｍｏｌ／ＬＴＦＰ的促进作用最大。并且ＴＦＰ浓度越大,Ｃａ２＋内流出现峰值也越早,１０、２０、５０、１００μｍｏｌ／ＬＴＦＰ处理后,胞内总钙出现峰值时间分别为４５、４５、３０、１５分钟。胞外Ｈ＋浓度也会对ＴＦＰ引起的Ｃａ２＋内流产生不同影响,缓冲液的ｐＨ值为６．０时最有利于ＴＦＰ引起胞内Ｃａ２＋含量增加,碱性条件下ＴＦＰ的效果最不明显。由ＴＦＰ引起的Ｃａ２＋内流增加要比单一地增加外钙浓度效果好得多,ＴＦＰ在１０μｍｏｌ／Ｌ浓度的外钙条件下引起的胞内钙含量数值比１０００μｍｏｌ／Ｌ的外钙条件而无ＴＦＰＴ所引起的胞内钙含量还要高５３．９％。缓冲液中加入０．８％的钙离子通道阻断剂ＬａＣ１３或溶液中无葡萄糖的存在,ＴＦＰ的促进作用消失,说明ＴＦＰ促进Ｃａ２＋内流是通过钙离子通道来完成的并需要能量参与。     

TFP刺激裂殖酵母细胞钙离子内流的质膜效应*

ＴＦＰ（１０－１００μｍｏｌ／Ｌ）可引起裂殖酵母（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ　ｐｏｍｂｅ）胞外Ｃａ２＋内流,ＴＦＰ浓度不同,促进Ｃａ２＋内流程度也不一样,５０μｍｏｌ／ＬＴＦＰ的促进作用最大。并且ＴＦＰ浓度越大,Ｃａ２＋内流出现峰值也越早,１０、２０、５０、１００μｍｏｌ／ＬＴＦＰ处理后,胞内总钙出现峰值时间分别为４５、４５、３０、１５分钟。胞外Ｈ＋浓度也会对ＴＦＰ引起的Ｃａ２＋内流产生不同影响,缓冲液的ｐＨ值为６．０时最有利于ＴＦＰ引起胞内Ｃａ２＋含量增加,碱性条件下ＴＦＰ的效果最不明显。由ＴＦＰ引起的Ｃａ２＋内流增加要比单一地增加外钙浓度效果好得多,ＴＦＰ在１０μｍｏｌ／Ｌ浓度的外钙条件下引起的胞内钙含量数值比１０００μｍｏｌ／Ｌ的外钙条件而无ＴＦＰＴ所引起的胞内钙含量还要高５３．９％。缓冲液中加入０．８％的钙离子通道阻断剂ＬａＣ１３或溶液中无葡萄糖的存在,ＴＦＰ的促进作用消失,说明ＴＦＰ促进Ｃａ２＋内流是通过钙离子通道来完成的并需要能量参与。     

星形胶质细胞存在L-型钙通道的新证据

以纯化培养的小鼠星形胶质细胞(Astrocyte,AS)为实验材料,采用激光共聚焦钙成像和荧光分光光度计技术,探讨钙激动剂Bay k8644和钙拮抗剂nimodipine对星形胶质细胞胞内钙离子浓度的影响。结果显示,Bay k8644在0.0001、0.001和0.01mmol/L浓度下均可显著增加星形胶质细胞的细胞内钙水平,而nimodipine在0.001、0.01和0.1mmol/L浓度下均可显著降低星形胶质细胞胞内钙水平,并阻止KCl引起的细胞内钙升高。上述结果表明星形胶质细胞对L-型钙通道激动剂和拮抗剂的反应与神经元的反应相似,提示星形胶质细胞胞膜上也存在L-型钙通道。     

酿酒酵母细胞中钙离子信号传导途径的研究进展

酿酒酵母（SaccharomycescP增v括fdP）细胞可以通过ca2＋／钙调磷酸酶信号途径来应对许多外界环境胁迫。在交配信息素、盐或者其他环境压力存在的条件下,钙离子会通过细胞质膜上的未鉴定的钙转运蛋白x和M或者由Cchl和Midl组成的钙通道进入细胞质。胞质内钙离子浓度的增加会激活细胞质里的钙调磷酸酶（calcineurin）。钙调磷酸酶的一个非常重要的作用是去磷酸化细胞质内的转录因子Crzl,造成它快速地从细胞质转移到细胞核,从而诱导包括液泡膜上钙泵蛋白基因PMCl以及内质网膜和高尔基体膜上钙泵蛋白基因尸脚,在内的目标基因的表达。这两个钙泵蛋白和液泡膜上的Ca2＋／H＋交换蛋白Vcxl一起作用,将细胞质内的钙离子浓度控制在50～200nmol／L的正常生理浓度内．使细胞能够正常生长。该综述主要论述了酿酒酵母细胞内Ca2＋／钙调磷酸酶信号途径的最新研究进展。     

阿片类物质与Ca^2^＋的关系

本文介绍近来有关阿片类物质与Ｃａ＾２＾＋相互作用的研究进展。Ｃａ＾２＾＋作为第二信使参与信号转导作用。Ｃａ＾２＾＋可对抗阿片类物质的镇痛作用。Ｃａ＾２＾＋对阿片类物质的其他作用也表现出类似的作用。阿片类物质可抑制钙电流，降低胞内游离钙浓度。通过阻断钙通道，抑制久钙内和内钙释放柯能是阿片类物质产生作用的机制之一。     

猪冠状动脉平滑肌细胞的自发瞬时外向电流的特性

自发瞬时外向电流（spontaneous transient outward currents,STOCs）在小动脉的肌源性调节中起着非常重要的作用。本文应用穿孔膜片钳技术记录了猪冠状动脉平滑肌细胞上的STOCs,研究了其基本特性以及调节。结果显示：STOCs有明显的电压依赖性和钙依赖性,其频率和幅度具有变异性。STOCs可以随机叠加在阶跃刺激方案和斜坡刺激方案引出的全细胞钾电流上。STOCs可被大电导钙激活钾（large-conductance Ca^2＋-activated potassium,BKCa）通道的特异性阻断剂ChTX、螯合胞外钙离子和50μmol／L ryanodine完全抑制。钙离子载体A23187可以明显增加STOCs的幅度和频率;而L型钙通道阻断剂verapamil和CdCl2对STOCs的影响很小。咖啡因使STOCs瞬时爆发性增加,然后抑制。钠离子载体可明显增加STOCs的频率;钠钙交换体选择性抑制剂KB．R7943可明显抑制STOCs。由此可以认为STOCs是BKCa通道介导的。STOCs的产生和激活依赖于经钠钙交换的钙内流和经肌浆网ryanodine受体介导的钙释放,钠钙交换可能决定钙库重载,而细胞膜下肌浆网的胞内钙释放（钙火花）所致的局部钙浓度瞬时增加激活与其相邻的BKCa通道,产生STOCs。     

钙离子和蛋白激酶C对大鼠缺血海马脑片谷氨酸堆积的影响

采用大鼠海马脑片体外缺血模型观察钙离子和蛋白激酶Ｃ（ＰＫＣ）对神经元胞外谷氨酸（ＧＬＵ）堆积的影响,结果显示：海马脑片在体外“缺血”１０ｍｉｎ,ＧＬＵ在胞外的浓度增加４倍（从３２±４升高到１１３±１０ｐｍｏｌ／（ｍｉｎ．ｍｇＰｒ）．ｎ＝６）．Ｎ型钙通道拮抗剂蝙蝠葛苏林碱（ＤＳＬ）或无钙培养液均能有效抑制这种浓度的升高（Ｐ＜０．０１）．提示缺血１０ｍｉｎ引发的ＧＬＵ浓度升高是受Ｃａ２＋内流调控的．当脑片在缺血状况下孵育３０ｍｉｎ,ＤＳＬ只部分抑制这种ＧＬＵ堆积,而无钙培养液则无影响,但这额外的ＧＬＵ堆积可被ＰＫＣ抑制剂Ｈ－７完全阻断,而被ＰＫＣ激动剂ＰＤＢ所加强;且不受钙调蛋白抑制剂Ｃａｌｍｄａｚｏｌｉｕｍ和８－溴－ｃＡＭＰ影响．提示缺血３０ｍｉｎ,胞外ＧＬＵ的堆积受钙内流和ＰＫＣ双重调控。     

Ca2+- and voltage-dependent inactivation of the expressed L-type Ca(v)1.2 calcium channel

Ca2+-dependent regulation of the ion current through the alpha1Cbeta2aalpha2delta-1 (L-type) calcium channel transiently expressed in HEK 293 cells was investigated using whole cell patch clamp method. Ca2+ or Na+ ions were used as a charge carrier. Intracellular Ca2+ was either buffered by 10 mM EGTA or 200 microM Ca2+ was added into non-buffered intracellular solution. Free intracellular Ca2+ inactivated permanently about 80% of the L-type calcium current. The L-type calcium channel inactivated during a depolarizing pulse with two time constants, tau(fast) and tau(slow). Free intracellular calcium accelerated both time constants. Effect on the tau(slow) was more pronounced. About 80% of the channel inactivation during brief depolarizing pulse could be attributed to a Ca2+-dependent mechanism and 20% to a voltage-dependent mechanism. When Na+ ions were used as a charge carrier, the L-type current still inactivated with two time constants that were 10 times slower and were virtually voltage-independent. Ca2+ ions stabilized the inactivated state of the channel in a concentration-dependent manner.     

Mitochondrial calcium in relaxed and tetanized myocardium.

The elemental composition of rat cardiac muscle was determined with electron probe x-ray microanalysis (EPMA) of rapidly frozen papillary muscles and trabeculae incubated with ryanodine (1 microM) in either 1.2 or 10 mM [Ca2+]o-containing solutions, paced at 0.6 Hz or tetanized at 10 Hz. Total mitochondrial calcium increased significantly, by 4.2 mmol/kg dry weight during a 7 s tetanus, only in muscles tetanized in the presence of 10 mM [Ca2+]o when cytoplasmic Ca2+ is 1-4 microM (Backx, P. H., W.-D. Gao, M. D. Azan-Backx, and E. Marban. 1995. The relationship between contractile force and intracellular [Ca2+] in intact rat trabeculae. J. Gen. Physiol. 105:1-19). Comparison of total mitochondrial with free mitochondrial Ca2+ reported in the literature indicates that the total/free ratio is approximately 6000 at physiological or near-physiological levels of total mitochondrial calcium. Increases in free mitochondrial [Ca2+] consistent with regulation of mitochondrial enzymes should be associated with increases in total mitochondrial calcium detectable with EPMA. However, such increases in mitochondrial calcium occur only as the result of prolonged, unphysiological elevations of cytosolic [Ca2+].     

TRPC通道对PC12细胞缺氧缺糖再灌注后氧化损伤的保护作用

瞬时受体电位C通道(transient receptor potential canonical,TRPC)属于瞬时受体电位（TRP）离子通道家族成员之一,与Ca2+ 调控及氧化应激关系密切．然而,TRPC通道在缺血缺氧性脑损伤中的作用仍有争议．本实验采用MTT法、台盼蓝排斥实验、LDH漏出实验联合Annexin/PI染色流式分析等显示,当采用通道阻断剂-SKF96365特异阻断TRPC通道时,PC12细胞对缺氧缺糖再灌注（oxygen-glucose deprivation／reperfusion,OGD-R）引起的损伤变得更敏感,细胞凋亡增加．尽管同时采用SKF96365、NMDA受体、AMPA受体和L-型钙通道阻断剂可引起细胞损伤和死亡减弱,但仍呈现明显的SKF96365浓度依懒性,提示TRPC通道参与细胞对缺氧缺糖再灌注耐受的调节,具有保护作用．钙指示剂Fluo-3AM荧光标记结合激光共聚焦显微镜分析显示,SKF96365可明显降低缺氧缺糖再灌注后细胞内的Ca2+浓度．总之,实验结果提示,TRPC通道对缺氧缺糖再灌注引起的细胞损伤和凋亡具有保护作用,其机制可能涉及TRPC通道对细胞内钙浓度的调节,详尽机制有待进一步研究．     

An increase in free radical production by means of an anion channel blocker DIDS in mouse peritoneal neutrophils

DIDS (4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid) has been recognized as an anion channel blocker. In this study, we demonstrated that DIDS significantly enhanced the production of free radicals in mouse peritoneal neutrophils. By means of a luminol-chemiluminescence (LCL) monitoring system, DIDS markedly increased LCL which could be suppressed by SOD, sodium azide (NaN3), EGTA and BAPTA-AM and only slightly inhibited by staurosporine (STP). Depletion of the endoplasmic reticulum (ER)-Ca2+ store by means of thapsigargin (TG) had no effects on DIDS-enhanced LCL, but DIDS significantly increased the amount of intracellular free calcium as monitored by means of fura-2 staining. These results indicate that DIDS may enhance free radical production mediated by Ca2+ release from the mitochondria. Both phorbol-12-myristate-13-acetate (PMA) and DIDS can induce increased translocation of p47-phox of the neutrophil to the membrane fraction, which is inhibited by STP pretreatment. Since free radical generation could reduce the cytoplasmic pH (pHi), we further examined whether DIDS was capable of inducing intracellular acidification. The result indicated that DIDS certainly lowered the pHi which was also suppressed by pretreatment with either NaN3 or NaCN, but not by diphenyleneiodonium (DPI). These findings lead us to propose a working hypothesis that DIDS mainly induces superoxide production accompanied by decreasing pHi mediated through a Ca2+ -dependent effect on the mitochondria rather than on NADPH oxidase. Using the lipophilic fluorescent dye DiOC6(3), we showed that DIDS decreased the transitional mitochondrial membrane potential. NaN3, but not STP or pyrrolidine dithiocarbamate (PDTC), antagonized DIDS in the course of decreasing the mitochondrial membrane potential. Taken together, all of these findings imply a possible role of anion channels of the mitochondria in modulating free radical production and intracellular acidification of neutrophils through alteration of the mitochondrial transition membrane potential and Ca2+ -release.     

Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: evidence of its dependence on an initial calcium mobilization from intracellular stores

It has been repeatedly shown that stimulation of a human leukemic T-cell line, JURKAT, by lectins such as phytohaemagglutinin and anti-T3 antibody (OKT3) leads to an elevation in the concentration of cytosolic free Ca2. This Ca2+ transient results from both an intracellular mobilization and an influx of Ca2+ through specific membrane channels. The objective of this study was to investigate the mechanism by which receptor-mediated influx of Ca2+ is regulated in JURKAT cells, which demonstrably lack 'voltage-dependent calcium channels'. It was found that upon increased loading with quin2 or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) there was a pronounced decline of both phytohaemagglutinin-stimulated and OKT3-stimulated influx of 45Ca2+. Using 15 microM quin2/AM or 30 microM BAPTA/AM, agonist-stimulated 45Ca2+ influx was almost totally abolished. At these concentrations of both quin2/AM or BAPTA/AM, phytohaemagglutinin and OKT3 could still induce a rise of cytosolic free Ca2+ above 200 nM. In the presence of La3+ (200 microM), which completely inhibited the agonist-induced 45Ca2+ influx, both phytohaemagglutinin and OKT3 were able to raise the concentrations of cytosolic free Ca2+ to well above 200 nM by merely mobilizing Ca2+ from intracellular stores alone. The data suggest that an agonist-induced increase in the concentration of cytosolic free Ca2+, due to mobilization from intracellular stores, could either directly or indirectly, initiate receptor-mediated Ca2+ influx across the plasma membrane in JURKAT cells.     

The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes

We have examined the effects of extracellular and intracellular Ca2+ concentrations upon basal and insulin-stimulated 2-deoxyglucose uptake in isolated rat adipocytes. In the absence of extracellular Ca2+, both basal and insulin-stimulated glucose uptake were significantly reduced. Insulin-stimulated glucose transport was optimal at 1 and 2 mM Ca2+. Further increases in extracellular Ca2+ concentration (3 mM) significantly diminished insulin-stimulated glucose uptake. When intracellular Ca2+ concentrations were augmented by ionomycin (1 microM), insulin-stimulated glucose uptake was significantly reduced at extracellular Ca2+ concentrations of 2 and 3 mM. The levels of intracellular free Ca2+ concentrations were then measured with Ca2+ indicator fura-2. The correlation between the levels of intracellular free Ca2+ and the magnitude of insulin-stimulated glucose uptake revealed that the optimal effect of insulin is observed at Ca2+ levels between 140 and 370 nM. At both extremes outside of this window, both low and high levels of intracellular Ca2+ result in diminished cellular responsiveness to insulin. These data suggest that intracellular calcium concentrations may exert a dual role in the regulation of cellular sensitivity to insulin. First, there must exist a minimal concentration of intracellular calcium to promote insulin action. Second, increased levels of intracellular calcium may provide a critical signal for diminution of insulin action.     

Spontaneous and stimulated transients in cytoplasmic free Ca(2+) in normal human osteoblast-like cells: aspects of their regulation.

We characterize two patterns of transients in cytoplasmic free calcium ([Ca2+]i) in normal human osteoblast-like cells (hOB cells). Firstly, spontaneous oscillations in [Ca2+]i were found to be common. The [Ca2+]i oscillations were completely inhibited by thapsigargin, indicating that Ca2+ fluxes between intracellular Ca2+ pools and the cytosol contributed to the generation of the [Ca2+]i oscillations. Removing extracellular Ca2+ either attenuated or completely inhibited spontaneous [Ca2+]i oscillations. Gadolinium, an inhibitor of stretch activated cation channels (SA-cat channels), reduced the frequency of [Ca2+]i oscillations. Hence, entry of calcium from the extracellular space, possibly through SA-cat channels also seemed to be of importance in the regulation of these [Ca2+]i oscillations. The role of the observed spontaneous [Ca2+]i oscillations in hOB cell function is not clear. Secondly, a decrease in pericellular osmolality, which causes the plasma membrane to stretch, transiently increased [Ca2+]i in hOB cells. This effect was also observed in a Ca2+ free extracellular environment, suggesting that osmotic stimuli release Ca2+ from intracellular pools. This finding indicates a possible signaling pathway by which mechanical strain can promote anabolic effects on the human skeleton.     

Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.     

The role of cytoplasmic free calcium concentration in B-cell tolerance

Calcium is an important factor in the immune response. Extracellular calcium is required for antibody production by B lymphocytes. Several investigators have demonstrated that crosslinking of receptors on B lymphocytes by anti-mu antibody induces an increase in intracellular calcium. There are few data on the role of intracellular calcium mobilization or calcium influx in tolerance induction in B cells. We studied changes in free intracellular calcium concentration ([Ca+2]i) induced by exposure of dinitrophenyl (DNP)-specific B cells to the tolerance-inducing conjugate DNP-murine IgG2a (DNP-MGG). Splenic B cells enriched for DNP-specific cells and DNP-specific continuous B-cell lines were used for the studies. Exposure of B cells to the tolerogen DNP-MGG, the antigen DNP-keyhole limpet hemocyanin (DNP-KLH), or the antigen DNP-Ficoll induced an increase in free [Ca+2]i which was due to both mobilization of Ca+2 from endoplasmic reticulum (ER) and influx of extracellular Ca+2. This increase was DNP specific since no significant change was seen with carriers alone and no change was seen in cells that were not DNP specific. The DNP-MGG and DNP-Ficoll induced the same amount of Ca+2 release from ER but the release induced by DNP-KLH was higher. When B cells, which were made tolerant by in vitro incubation with DNP-MGG, were incubated with antigens, a mobilization of Ca+2 from endoplasmic reticulum occurred that was the same as that of nontolerant B cells. Since Ca+2 mobilization is associated with Ig receptor-dependent early B-cell activation, it is likely that the tolerant B cell can still receive an activation signal through the Ig receptors.     

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.