耳蜗外毛细胞两种胞内钙库的初步探讨

为探讨毛细胞胞内钙库的种类 ,本文观察了在无钠、无钙和含镧液体中 ,在不受胞外 Ca2 +内流和质膜上 Ca2 +转运机制影响的条件下 ,和在三磷酸肌醇敏感钙库的工具药 thapsigargin和 ryanodine敏感钙库的工具药 caffeine作用下 ,毛细胞胞内游离钙([Ca2 + ] i)的变化过程。分离的豚鼠耳蜗外毛细胞经钙敏荧光染料 5μmol/L fluo-3染色后 ,用激光扫描共聚焦显微镜监测 ,以 fluo-3荧光相对值指示毛细胞 [Ca2 + ] i的高低。 3 0 nmol/L thapsigargin使外毛细胞 [Ca2 + ] i由静态值 1.0增至 1.64± 0 .76,再加入 10mmol/L caffeine后更增至 2 .45± 1.5 9(x± s,n=11,F=7.90 ,P<0 .0 1)。Q值检验示两种试剂引起外毛细胞 [Ca2 + ] i增高程度的差别有极显著意义 (P<0 .0 1) ,表明毛细胞内有对三磷酸肌醇敏感和对 ryanodine敏感的两种钙库参与了胞内 Ca2 + 释放机制     

大鼠胃壁细胞GnRH受体的定位及GnRH类似物对壁细胞内钙的影响

目的　观察促性腺激素释放激素 (gonadotropin releasinghormone,GnRH)受体在培养的大鼠胃粘膜壁细胞中的定位 ,并探讨GnRH类似物阿拉瑞林 (alarelin)对壁细胞内游离钙动员的机制。　方法　采用免疫组织化学和原位杂交技术 ;应用Ca2 + 指示剂Fluo 3 AM作为细胞内钙离子的荧光探针 ,对负载培养的胃壁细胞 ,应用激光共聚焦显微镜技术检测单个细胞内钙荧光强度的变化。　结果　大鼠胃壁细胞呈GnRH受体免疫反应阳性 ,阳性物质位于细胞质 ,细胞核为阴性 ;同样壁细胞内可检测到GnRH受体mRNA杂交信号 ,阳性物质位于细胞质 ,细胞核为阴性 ;胞内Ca2 + 浓度变化为 :1 在Hank液中 ,GnRH类似物浓度为 10 - 8、10 - 7、10 - 6 mol L时 ,胃壁细胞内Ca2 + 浓度逐渐升高 ,其峰高 (峰值减去静息值 )分别为 7 1± 1 4、12 1± 1 7、16 8± 2 2。其达峰时间也逐渐增快 ,分别为 34 2± 6 4s、18 9± 1 2s、10 4± 2 3s。相邻两组间其峰高及达峰时间均存在显著性差异 (P <0 0 5 ) ,且呈明显剂量依赖性。 2 在D Hank液 (去除外钙 )中 ,阿拉瑞林可轻度短暂升高胞内Ca2 + ;用内罗啶孵育后再加入阿拉瑞林也可轻度短暂升高胞内Ca2 + ,二者无显著性差异。 3 当用拉西地平孵育后再加入阿拉瑞林 ,可明显抑制胞内Ca2 + 的增加     

前列腺凋亡反应基因4反义寡核苷酸抑制谷氨酸诱导的PC12细胞内钙离子浓度上调及其抗凋亡意义

目的　研究前列腺凋亡反应基因4(par -4)反义寡核苷酸拮抗谷氨酸对PC12细胞内游离钙离子浓度的上调作用及其抗凋亡意义。方法　脂质体介导转染par -4反义寡核苷酸。谷氨酸诱导PC12细胞凋亡。Hoechest33258 /碘化丙啶荧光染色观察细胞凋亡形态,流式细胞术分析凋亡百分率。Fura 2 /AM荧光染色结合激光共聚焦显微镜测定细胞内游离钙离子浓度。逆转录聚合酶链反应(RT- PCR)测定钙依赖性蛋白酶Calpain10的mRNA水平。Western印迹测定par -4蛋白表达量。结果与正常对照组(25 .6±4.1 )相比,谷氨酸诱导PC12细胞中par- 4蛋白表达上调( 90. 0±3 2,P<0. 01),par- 4反义寡核苷酸拮抗其上调( 52 .3±5 .0,P<0 .01 );谷氨酸诱导凋亡组凋亡百分率为53%, par -4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡(31%,P<0. 01);谷氨酸诱导PC12细胞内游离钙离子浓度上调,par- 4反义寡核苷酸拮抗其上调(荧光强度比值分别为167 .9±32 .4、228. 8±36. 8,P<0 .01);谷氨酸诱导PC12细胞内钙依赖性蛋白酶Calpain10mRNA水平上调( 46 .3±3 .7 ),par- 4反义寡核苷酸抑制其上调(34 8±2 1,P<0 01 )。结论　par -4反义寡核苷酸拮抗谷氨酸诱导的PC12细胞凋亡,其机制可能与抑制细胞内游离钙离子浓度上调和抑制Calpain10基因转录有关。     

HSPgp96诱导巨噬细胞呼吸爆发与细胞内外游离钙关系的研究

目的：探讨从小鼠H₂₂肝癌细胞中提纯的热休克蛋白gp96（HSPgp96）对小鼠腹腔巨噬细胞（PEMφ）呼吸爆发的影响及其与细胞内外游离钙的关系。方法：(1)用亲和层析和离子交换层析等方法从小鼠H₂₂肝癌细胞中获得纯化的HSPgp96。(2)用细胞内过氧化物荧光探针H₂DCF-DA监测HSPgp96作用于小鼠PEMφ过程中，单个细胞活性氧（ROS）信号的变化,反映PEMφ内呼吸爆发时的变化过程；用荧光探针Fluo-3/AM监测HSPgp96作用于小鼠PEMφ过程中，单个细胞内钙离子（［Ca2+］_i）信号的变化。(3)使用细胞膜和细胞内钙通道抑制剂及钙离子载体后，再观察HSPgp96作用后ROS信号的变化。结果：(1)加入HSPgp96后PEMφ内Fluo-3荧光强度立即上升，70s时增幅达161.05%±50.99%；当分别阻断细胞外钙内流、抑制细胞内钙库释放功能后110s增幅分别为84.81%±29.52%和46.21%±17.24%。同时阻断胞内外钙作用，HSPgp96这一诱发功能明显被抑制。加入钙离子载体后可见细胞内荧光强度迅速增强，于110 s时达156.98%±45.83%，随后迅速下降。(2)小鼠PEMφ受HSPgp96刺激后表征ROS的荧光强度立即上升，620 s达基础荧光值的636.78%±82.02%，随后始终维持在高水平。当分别阻断细胞外钙内流、抑制细胞内钙库释放功能后，再加入HSPgp96后细胞内ROS荧光值上升幅度较小。同时阻断了胞内外钙作用后，HSPgp96刺激后ROS荧光值上升没有明显高峰出现。结论：HSPgp96作用于小鼠PEMφ后，促进了细胞外钙内流并使细胞内钙库释钙，这是HSPgp96诱发细胞内活性氧增加的基本机制。     

锌对异丙肾上腺素致损豚鼠心室肌细胞钙通道电流的影响

本实验应用全细胞膜片钳技术从离子通道水平研究锌对心肌细胞保护作用机理。结果显示 (1)异丙肾上腺素 (ISO) 10 - 8mol L可使正常豚鼠心室肌细胞钙电流 (Ica)从 1383± 2 6 5pA增至 16 10± 2 70pA(P <0 0 5 ,n =6 )。 (2 )锌0 2mmol L可使正常豚鼠心室肌细胞Ica从 1383± 2 6 5pA减小到 70 7± 10 8pA(P <0 0 5 ,n =6 )。 (3)先加锌 0 2mmol L再加ISO 10 - 8mol L ,Ica幅值增加到 10 30± 2 5 0pA ,与ISO组相比差异显著 (P <0 0 5 ,n =6 )。提示微量元素锌可抑制因ISO引起的豚鼠心室肌Ica的增加。     

重组人内抑素对佐剂性关节炎大鼠成纤维样滑膜细胞内钙离子稳态的影响

目的 观察重组人内抑素(rhEndestatin)对佐剂性关节炎大鼠成纤维样滑膜细胞(AA FLSs)内钙离子(Ca2+)稳态的影响,探讨rhEndomafin促进AA FLSs凋亡的离子机制,为RA药物治疗及寻找其治疗新靶点提供实验依据.方法雄性SD大鼠12只,体质量140～160 g,分为正常组(n=3)和从模型组(n=9).模型组大鼠制备从模型,体外培养AA FLSs,应用Ca2+荧光指示剂Fluo-3/AM孵育培养的细胞,激光扫描共焦显微镜检测有、无细胞外Ca2+,而rhEndestatin作用所致AA FLSs胞内Ca2+荧光强度发生动态变化,可以判断rhEndostatin对AA FLSs胞内Ca2+浓度([Ca2+]I)的影响.结果在胞外有Ca2+的情况下,rhEndostatin可引起静态AA FLS[Ca2+];快速增加,rhEndostatin作用10s后,[Ca2+];急剧增加达峰值,继之随时间缓慢下降,停止加药50 s后,[Ca2+]I尚未回复到加药前基础水平;而在无细胞外钙环境中,rhEndostatin未引起AA FLSs[Ca2+]I变化.结论 rhEndestafin可促进AA FLSs胞外Ca2+内流,引起胞内Ca2+超载,从而促进从FLSs凋亡.     

异丙酚静脉全身麻醉对血糖的影响

1　资料与方法1.1　临床资料6 0例ASAI～II级中上腹部手术的病人 ,男 2 8例 ,女 32例 ,年龄 31～ 70岁 ,体重 45～ 74kg ,I组 (n =30 )为非糖尿病病人 ,II组 (n =30 )为糖尿病病人 (注 :术前血糖已控制在正常或接近正常范围 ) ,其中合并高血压的 15例 ,I组 7例 ,II组 8例 ,两组患者其它情况无统计学差异 ,详见表 1。表 1　两组病人一般情况比较 ( x±s)年龄 (岁 )体重 (kg)手术时间 (min)异丙酚用量 (mg)I组 (n =30 ) 52± 2 .51 53± 4 .5 30 0± 1 3 2 51 0± 35II组 (n =30 ) 53± 2 .35 54± 3 .0 2 78± 1 2 2 538± 40 　…     

粉防己碱(Tet)对MDCK细胞钙激活钾通道(KCa)的影响

目的MDCK细胞钙激活钾通道(KCa)的鉴定及探讨Tet对该通道的影响.方法内面向外式(inside-out)膜片钳技术.结果 (1)在对称性高钾溶液([K+].[K+]i=140mmolL1/140mmolL-1)中,主要记录到电导为50.475±0.600pS(n=5)的外向通道电流,随电压增加而增加.(2)浴液中钾离子浓度由140mmolL-1改变为45mmolL-1,在0mV记录到内向电流,从+30mv开始记录到记录到外向电流,电导为50.275±0.294pS(n=5).(3)在对称性高钾溶液、膜电位+60mV时.向浴液中加入不同浓度的Ca2+,从10-7molL-1开始,通道活动浓度依赖性增加.(4)对称性高钾溶液、膜电位为+60mV,向浴液中加入Tet,通道活动在1.5×10-5molL-1时最大,在6.0×10-5molL-1时明显受到抑制,几乎为0,洗脱后,部分恢复.结论(1)在MDCK细胞上记录到中电导钙激活钾通道(IKCa),该通道具有电压依赖性和胞内游离Ca2+浓度依赖性,无整流特性.(2)Tet在7.5×10-6molL-1时对MDCK细胞Ka没有显著影响;在1.5×10-5molL-1促进KCa开放,增加钾离子外流;在6.0×10-5molL-1时抑制MDCK细胞Kca开放,减少钾离子外流.     

新型阿片受体配基特异性结合μ受体并抑制cAMP生成

本文研究新合成阿片受体配基对稳定表达 μ阿片受体的CHO细胞的受体结合特性和对胞内cAMP的抑制作用。采用放射性配基结合的方法研究了阿片受体配基 [3H] diprenorphine(3H dip)在稳定表达 μ阿片受体的CHO细胞模型上 ,对 μ阿片受体的饱和性结合特征及和一系列新合成阿片配基A、B、C、D、E、F、G、及DAMGO([D Ala ,N Me Phe4 ,Gly ol5] enkephalin)和吗啡的竞争性结合特征。利用竞争性结合蛋白法测定阿片受体配基对胞内cAMP的抑制作用。 [3H] diprenorphine结合 μ阿片受体的Kd值为 1 0 6nmol L ;Bmax为 930fmol mg蛋白。结果表明新配基、DAMGO和吗啡竞争性结合 μ受体的IC50 值分别为 13 33± 3 73、14 36± 1 5 8、0 6 2± 0 0 3、5 6 38± 2 33、6 5 72±2 6 4 4、33 10± 11 33、0 5 5± 0 0 6、3 6 9± 1 5 9和 1 83± 0 5 0。其中C和G配基对 μ阿片受体的亲和力高于DAMGO和吗啡。B、D、E和F配基对μ受体的亲和力低于DAMGO和吗啡。新配基、DAMGO和吗啡抑制cAMP生成的IC50 值分别为 6 4 9± 1 5 9、1390± 6 1 10、0 84± 0 11、2 33± 1 2 4、2 5 0 0± 17 2 0、1 4 2± 1 2 1、0 0 1± 0 0 1、0 10± 0 0 5和0 0 4± 0 0 1。其中G配基的抑制胞内cAMP作用最强 ,强于吗啡 ,类     

顺铂(DDP)对MDCK细胞钙激活钾通道(KCa)的影响

目的探讨顺铂(DDP)对MDCK细胞KCa的影响.方法利用内面向外式(inside-out)膜片钳技术.结果(1)在对称性高钾溶液([K+].[K+]i=140mmolL-1/140mmolL-1)中,主要记录到电导为51.425±0.570pS(n=5)的外向通道电流,随电压增加而增加.(2)浴液中钾离子浓度由140mmolL-1改变为45mmolL-1,在0mV记录到内向电流,从+30mv开始记录到记录到外向电流,电导为50.385±0.307pS(n=5).(3)在对称性高钾溶液、膜电位+60mV时;向浴液中加入不同浓度的Ca2+,从10-7molL-1开始,通道活动浓度依赖性增加.(4)对称性高钾溶液、膜电位为+60mV,向浴液中加入DDP,从1.5×10-5molL-1开始,DDP使通道活性呈现浓度依赖性减少,洗脱后,通道活性部分恢复.结论(1)本实验在MDCK细胞上记录到中电导钙激活钾通道(IKCa),该通道具有电压依赖性和胞内游离Ca2+浓度依赖性,无整流特性.(2)DDP在7.5×10-6molL-1时对KCa没有显著影响;从1.5×10-5molL-1开始使通道活性呈现浓度依赖性减少,减少钾离子外流.     

儿童T细胞活化时细胞内钙离子的变化

本文用钙指示剂Ｆｌｕｏ－３标记正常儿童外周血单个核细胞，经流式细胞仪分析了几种激活剂对Ｔ细胞内钙离子浓度的影响。结果表明：（１）静止期Ｔ细胞内钙离子浓度为１６２±８７ｎＭ，抗ＣＤ３单克隆抗体交联ＧＡＭＩｇ刺激后，Ｔ细胞内钙离子浓度上升；（２）用抗ＣＤ３单克隆抗体和ＩＬ－２或ＰＭＡ和ｉｏｎｏｍｙｃｉｎ作用Ｔ细胞，细胞内钙离子浓度上升，但其浓度低于用抗ＣＤ３单克隆抗体交联ＧＡＭＩｇ刺激时的浓度；（３）去除抗ＣＤ３单克隆抗体和ＩＬ－２或ＰＭＡ和ｉｏｎｏｍｙｃｉｎ对Ｔ细胞的作用，培养后细胞内钙离子浓度又恢复到静止状态，再加入抗ＣＤ３单克隆抗体交联ＧＡＭＩｇ刺激发现细胞内钙离子显著地升高；（４）钙离子阻断测定提示早期钙离子升高是Ｔ细胞内钙离子池释放的，而细胞外钙离子的流入对于维持钙离子持续上升是必要的。     

Differential inhibition of interleukin 2- and interleukin 4-mediated human B cell proliferation by ionomycin: a possible regulatory role for apoptosis

Surface immunoglobulin (Ig) cross-linking by anti-IgM (mu) antibodies leads to B cell activation resulting in numerous early biochemical events including an increase in intracellular [Ca2+]. Furthermore, anti-mu-activated B cells become able to proliferate in response to interleukin (IL)2 and IL4. These studies examined the effect of the calcium ionophore ionomycin, an enhancer of cytoplasmic [Ca2+] levels, on IL2 and IL4-mediated proliferation of anti-mu-stimulated normal human B cells. Ionomycin inhibited the proliferative response of anti-mu-activated B cells to IL4. In contrast, IL2 and phorbol 12,13 dibutyrate (PBu2)-mediated B cell proliferation was refractory to the growth inhibitory effects of ionomycin. In an attempt to delineate a possible mechanism(s) for this differential growth effect of ionomycin, we first studied direct effects of ionomycin on activated B cells. Our data suggested that ionomycin induced DNA fragmentation in anti-mu-costimulated B cells. Interestingly, in contrast to PBu2, IL4 did not prevent ionomycin-dependent DNA fragmentation. Importantly, H7, an inhibitor of protein kinase C activation, down-regulated only the IL2 and PBu2-driven B cell proliferation but not B cell proliferative response to IL4. These results suggest that putative protein kinase C activation, either by direct treatment with phorbol ester or during IL2 signaling, counteracts the inhibitory effects of ionomycin. In contrast, IL4 signaling does not exhibit the same protective properties.     

Cytochrome P450 products and arachidonic acid-induced, non-store-operated, Ca2+ entry in cultured bovine endothelial cells.

Endothelial cells possess multiple mechanisms for the control of Ca2+ influx during agonist and mechanical stimulation. Increased intracellular Ca2+ during such events is important in the production of vasoactive substances including NO, prostacyclin, and, possibly, endothelium-derived hyperpolarizing factor(s). The present studies examined the effect of arachidonic acid on cellular Ca2+ entry and the underlying mechanisms by which this fatty acid regulates entry. Studies were conducted in cultured bovine aortic endothelial cells (passages 3 to 6) with changes in intracellular Ca2+ determined using the fluorescent Ca2+-sensitive indicator fura 2. Arachidonic acid (1 to 50 microM) stimulated Ca2+ entry from the superfusate without affecting Ca2+ release from intracellular stores. 2-aminoethoxydiphenyl borate (2APB) (100 microM) added at the peak of Ca2+ entry did not inhibit arachidonic acid-induced Ca2+ entry but, in contrast, significantly inhibited entry stimulated by ATP (1 microM). Arachidonic acid-induced Ca2+ entry was inhibited by econazole (1 microM), but not indomethacin (10 microM) or nordihydroguairetic acid (10 microM), suggesting the involvement of cytochrome P450 monooxygenase metabolite of arachidonic acid. Oleic acid (10 microM) was ineffective in inducing Ca2+ entry, whereas linoleic acid (10 microM) stimulated Ca2+ entry but by a mechanism insensitive to econazole. Collectively the data demonstrate that primary cultured aortic endothelial cells possess a Ca2+ entry mechanism modulated by arachidonic acid. This mode of Ca2+ entry appears to operate independently of store depletion-mediated mechanisms.     

β-淀粉样蛋白对培养神经细胞的毒性作用及神经节苷脂的保护作用

向培养液中加入15mmol／L的β-AP1-40，48h后，发现原代培养的海马细胞内Ca2+的浓度明显增高，对照组为23．9±3．6（n=51）而β-AP组为36．5±15．8（n=30），两组比较P＜0．001，有非常显著性的差别。对照组的乳酸脱氢酶没有明显变化，而β－AP组在加β－AP1－4048h后乳酸脱氢酶明显增加，从52iu／L增加到105iu／L，实增加了101％。实验结果表明，β－AP1-40对培养的神经细胞有明显的毒性作用，但没有发现形态学的变化和对细胞的特异性。如在培养液中加β－AP1-40的同时再加25mg／ml的神经节苷脂（GAs）可明显减弱β-AP1-40引发的胞内Ca2+浓度和培养液中LDH浓度的升高。β-AP＋GAs组胞内Ca2+浓度为27.7±5.58，与β-AP组比较，有显著性差异（P＜0．01）。β-AP组海马细胞培养液中乳酸脱氢酶的浓度增高为101％，而β-AP-GAs组培养液中乳酸脱氢酶的浓度增高仅为67％。在NG108－15细胞上也获得了相同的结果。单唾液酸神经节苷脂虽有与GAs类似的作用，但较弱。这些实验结果指出，神经节苷脂和单唾液酸神经节苷脂均能拮抗β-AP1-40的毒性作用，从而对细胞可起到保护作用。     

A simplified method for Ca2+ flux measurement on isolated human B cells that uses flow cytometry.

A method for Ca2+ flux measurement on isolated human peripheral B cells that uses flow cytometry is described. B cells were isolated by anti-CD19 magnetic bead sorting, and Ca2+ flux was measured with the fluo-3 reagent on a standard single-laser flow cytometer. The response of B-cell stimulation by anti-immunoglobulin B (anti-IgM), anti-IgD, protein A, concanavalin A, and ionomycin was determined. Percentage of responder B cells, the level of Ca2+, and the time of peak stimulation were measured. Bound anti-CD19 monoclonal antibody coupled with small paramagnetic particles did not affect Ca2+ flux. All the isolated B cells responded maximally at 10s with stimulation by 8 microg of ionomycin. The average isolated preparation contains 70% IgM+ and 85% IgD+ cells, all of which showed peak stimulation with 10 microg of anti-IgM and anti-IgD per ml, respectively, at 30s. Only at high concentrations of 80 microg/ml, concanavalin A produced a slower response, peaking at 90 s after stimulation. Stimulation with 20 microg of protein A per ml resulted in Ca2+ flux in only 40 to 60% of cells that had a rapid response and maximal stimulation resembling the pattern of activation of ionomycin. B cells from three patients with mixed cryoglobulinemia with high concentrations of monoclonal rheumatoid factors showed stimulation with aggregated IgG, whereas those from healthy control subjects did not, demonstrating the applicability of the methodology to detection of specific antigen stimulation of B cells. This methodology may be useful in testing the functional capacity of B cells in a variety of diseases. The methodology may also prove useful in studying antigen-specific B-cell responses when they involve a significant percentage of B cells.     

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.     

A transient acidification linked to intracellular Ca2+ in anti-mu-stimulated human B-lymphocytes.

Mitogen-induced cellular proliferation is in many cell types preceded by rapid changes in intracellular pH and free Ca2+ concentration. We studied the patterns of pH and Ca2+ changes in normal resting human B-lymphocytes after exposure to anti-mu antibodies and the monoclonal antibody IF5, reactive with the CD20 antigen, both able to activate resting B-lymphocytes to enter the G1 phase of the cell cycle. Monitoring intracellular pH with the pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein, we demonstrated that poly- and monoclonal anti-mu antibodies induced a rapid (maximum change within 2 min) intracellular acidification of 0.06 pH units followed by a slower (10-15 min) alkalinization towards, or slightly above, the resting pH of 6.88. The acidification response was amiloride-resistant, whereas the return to baseline was sensitive. Intracellular free Ca2+ was measured by using the fluorescent Ca2+ dye, indo-I. Exposure of cells to anti-mu resulted in a rapid increase (maximum change within 2 min) in cytoplasmic Ca2+ of 340 nM and a slower decline in fluorescence back to baseline of about 180 nM. In contrast to anti-mu, IF5 caused no change in cytoplasmic Ca2+ and pH. However, the Ca2+ ionophore ionomycin at low concentrations mimicked the Ca2+ response as well as the pH response to anti-mu. In Ca2(+)-free solutions the intracellular Ca2+ stores are usually rapidly depleted and, indeed, the Ca2+ and pH responses to anti-mu were reduced after 5 min and almost abolished after 35 min under such conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depolarization-induced increases in intracellular free calcium detected in single cultured neuronal cells

Microspectrofluorometry was used to examine intracellular free calcium changes in single NG108-15 neurons loaded with the Ca2+ sensitive probe, quin2. The changes in intracellular free Ca2+ were induced either by depolarizing cells with high K+ or veratridine or by the addition of ionomycin. Ca2+-free medium and various inorganic and organic calcium-channel blocking agents blocked changes in intracellular free Ca2+ levels, indicating that Ca2+ entry is most likely through voltage-sensitive calcium channels.