小牛血清和血小板源性生长因子对肺动脉平滑肌细胞Na^＋／H^＋交换器活性和细胞增殖的影响

目的：探讨小牛血清和血小板源性生长因子（PDGF）对大鼠肺动脉平滑肌细胞Na^ /H^ 交换器－1（NHE－1）表达和活性的影响，及其与细胞增殖的关系。方法：体外培养肺动脉平滑肌细胞，10％小牛血清、PDGF－BB作用24小时后检测细胞NHE－1mRNA表达、^22Na摄取量、细胞内pH(pHi)和^3H-TdR掺入量，观察不同浓度NHE－1抑制剂－乙基，异丙基氨氯吡咪（EIPA）对细胞凋亡率的影响。结果：10％小牛血清、PDGF作用后24小时，肺动脉平滑肌细胞中NHE－1mRNA表达、^22Na摄取量明显增加，同时伴有pHi增高和^3H-TdR掺入量增加，以10％小牛血清作用更为显著。随着EIPA浓度增加和作用时间延长，细胞凋亡率明显增高。结论：小牛血清、PDGF等生长因子参与调节肺动脉平滑肌细胞NHE－1表达和激活，使细胞内碱化，可能在细胞增殖中起重要作用。     

Na+/H+交换器-1反义RNA对大鼠肺动脉平滑肌细胞增殖的影响

目的 :探讨反义 RNA对大鼠肺动脉平滑肌细胞 Na / H 交换器 (NHE) 1表达和活性、细胞内 p H(p Hi)的影响 ,及其与细胞增殖的关系。方法 :构建含 NHE 1反义 RNA序列的 p L XSN反转录病毒重组载体 ,将其转染体外培养的大鼠肺动脉平滑肌细胞 ,G4 18筛选后获取含有重组载体的细胞克隆 ,检测细胞 NHE 1m RNA表达、2 2 Na摄取量、p Hi和 3H Td R掺入量。结果 :与转染 p L XSN空载体的细胞和正常对照组细胞比较 ,转染了重组载体的肺动脉平滑肌细胞中 NHE 1m RNA表达、2 2 Na摄取量明显减少 ,同时伴有 p Hi降低和 3H Td R掺入量减少 ,正常对照组和空载体组间无显著差异。结论 :NHE 1反义 RNA可以减少 NHE 1表达 ,从而诱导细胞酸化 ,抑制肺动脉平滑肌细胞增殖     

bcl-2、bax在NHE-1核酶基因转染所致缺氧大鼠肺动脉平滑肌细胞凋亡中的作用

目的　探讨 bcl 2、bax表达在 Na / H 交换器 1(NHE 1)抑制而诱导缺氧大鼠肺动脉平滑肌细胞 (PASMCs)凋亡中的作用。方法　将转染 NHE 1特异性核酶基因大鼠的 PASMCs置于缺氧条件下(O2 的体积分数 <1% )培养 ,分别在缺氧 0、2、6、12、2 4和 4 8h采用原位细胞凋亡检测法观察细胞凋亡情况 ,荧光指示剂 Fura 2 / AM测定法检测细胞内 Ca2 浓度 (〔 Ca2 〕i)变化 ,半定量逆转录聚合酶链反应方法检测细胞内 bcl 2 m RNA和 bax m RNA表达变化 ,免疫组化法检测细胞内 Bcl 2蛋白和 Bax蛋白表达的变化。结果　转染 NHE 1特异性核酶基因大鼠的 PASMCs在缺氧培养时 ,其细胞凋亡率随缺氧时间的延长而逐渐升高 ,但是〔 Ca2 〕i升高的幅度较小 ,从缺氧 6 h起就显著低于同时间点的对照组和转染逆转录病毒真核表达载体空载体组 (P均 <0 .0 1) ,bcl 2 m RNA及蛋白表达显著降低 ,bax m RNA和蛋白表达显著增加(P均 <0 .0 1)。结论　 NHE 1抑制可能通过阻止 PASMCs的〔 Ca2 〕i增加 ,并引起 bcl 2表达降低及 bax表达增加而诱导和促进缺氧培养的 PASMCs凋亡     

钠氢交换蛋白在依托泊苷诱导HL-60细胞凋亡中的作用及其机制的初步研究

本研究探讨钠氢交换蛋白(Na+/H+ exchanger-1,NHE1)在依托泊苷诱导HL-60细胞凋亡过程中的表达变化及其对凋亡过程的调控机制。应用DNA片段化检测和远末端缺口标记(TUNEL)法分析细胞凋亡;实时定量聚合酶链反应检测NHE1 mRNA表达量,Western blot法检测NHE1蛋白表达水平,并应用激光共聚焦显微镜分析细胞内pH值(pHi)的变化。同时应用NHE1特异抑制剂卡立泊来德(cariporide)作用于细胞观察凋亡及pHi的变化情况。结果表明:依托泊苷作用24小时后能有效诱导HL-60细胞凋亡,作用12小时后NHE1 mRNA表达增高了2.848±0.886倍(p0.01),NHE1蛋白表达水平也升高(p0.01)。依托泊苷作用24小时后细胞内pHi从7.11升高到7.46,而卡立泊来德预处理组的细胞在依托泊苷处理24小时后pHi没有明显升高并且凋亡率明显降低。结论:依托泊苷诱导的HL-60细胞凋亡过程中会出现NHE1表达上调,凋亡结果依赖于NHE1表达量升高导致的细胞内pH值升高。     

法舒地尔对人肺动脉平滑肌细胞增殖抑制作用研究

目的:探讨法舒地尔对血小板源性生长因子(platelet-derived growth factor,PDGF)诱导的人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cell,HPASMC)增殖的抑制作用及机制。方法:以体外培养的HPASMC为研究对象,PDGF-BB诱导增殖,法舒地尔进行预处理,MTT法检测细胞增殖;流式细胞仪检测细胞周期;Western blot检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)和p27kip1蛋白的表达。结果:细胞培养24 h后与空白组比较,PDGF组HPASMC细胞增殖比例,S期细胞比例和PCNA蛋白表达明显增加,p27kip1蛋白表达则明显降低;用法舒地尔预处理后,与PDGF组比较,细胞增殖比例,S期细胞比例和PCNA蛋白表达明显下降,p27kip1蛋白表达则明显增加。结论:法舒地尔可通过上调p27kip1蛋白表达来抑制PDGF诱导的HPASMC增殖和细胞周期进程。     

小牛与胎牛血清对体外培养骨髓基质细胞增殖与分化的影响

目的：观察小牛血清和胎牛血清对体外培养骨髓基质细胞增殖和分化的影响。方法：实验于2001-01／2004-12在吉林大学中日联谊医院骨科完成。①取四五月龄新西兰大白兔20只，体外培养骨髓基质细胞。②将传代培养的第2代骨髓间充质细胞按1&;#215;10^4／孔的细胞密度培养24h后，弃掉培养液，用磷酸盐缓冲液洗3次。加入含有不同浓度胎牛血清及新生牛血清的HamF12培养基继续培养24h，采用四甲基偶氮唑盐比色分析法观察两种血清对骨髓基质细胞增殖和分化的影响，用酶联免疫检测仪测定各孔在570nm波长处的光吸收值。结果：①小牛血清及胎牛血清均有明显促进骨髓间充质干细胞增殖的作用，随着血清浓度的增加，光吸收值也随之增大。②同一浓度下的胎牛血清比小牛血清具有更明显的促进骨髓间充质干细胞增殖的作用。③10％浓度的胎牛血清对骨髓间充质干细胞的增殖作用最为显著。结论：小牛血清及胎牛血清均有明显促进骨髓基质细胞增殖的作用．胎牛血清在骨髓间充质细胞的体外增殖过程中，与小牛血清相比较，可使细胞的增殖更加活跃。     

p27和p57蛋白在血小板源生长因子BB刺激下血管平滑肌细胞增殖过程中的表达

目的:观察不同剂量的血小板源生长因子BB刺激对血管平滑肌细胞生长率的影响及血管平滑肌细胞不同增殖程度下p27和p57蛋白表达量的变化。方法:实验于1998-09/2000-05在南方医科大学附属南方医院心内科实验室完成。①选用七八周雄性SD大鼠80只,体外分离SD大鼠主动脉中层平滑肌,贴壁法培养平滑肌细胞,选用2～4代培养的细胞。②胰酶消化的血管平滑肌细胞,浓度为1×108L-1,分别接种于6孔板上,无血清的培养基静止48h,分别加入血小板源生长因子BB10和20μg/L,无血清的培养基为对照组,分别在刺激1,3,5d后,计算不同剂量的血小板源生长因子BB刺激下的血管平滑肌细胞数量,每组重复4次,取平均值。③在用血小板源生长因子BB10和20μg/L刺激6,12,24h后收集细胞,用免疫印迹分析法检测细胞中p27和p57蛋白表达量。结果:①血管平滑肌细胞数量:血小板源生长因子BB刺激1和3及5d后血小板源生长因子BB10和20μg/L组明显高于对照组(t=4.06～21.76,P<0.05～0.01)。②血管平滑肌细胞中p27蛋白表达量(A值):血小板源生长因子BB刺激12和24h后血小板源生长因子BB10和20μg/L组明显低于对照组(P<0.05,0.01),血小板源生长因子BB20μg/L组明显低于血小板源生长因子BB10μg/L组(P<0.01)。p57蛋白表达量(A值):血小板源生长因子BB刺激24h后血小板源生长因子BB10和20μg/L组明显高于对照组(P<0.01),血小板源生长因子BB20μg/L组明显高于血小板源生长因子BB10μg/L组(P<0.01)。结论:①在血小板源生长因子BB刺激的血管平滑肌细胞增殖过程中,p27和p57蛋白表达量变化不同。②在增殖的血管平滑肌细胞中随刺激因子作用时间的延长,表达量逐渐下降,而p57蛋白表达水平随血管平滑肌细胞的增殖而增加,p27蛋白表达量的变化可能是决定血管平滑肌细胞增殖的关键因素。     

小牛与胎牛血清对体外培养骨髓基质细胞增殖与分化的影响

目的:观察小牛血清和胎牛血清对体外培养骨髓基质细胞增殖和分化的影响。方法:实验于2001-01/2004-12在吉林大学中日联谊医院骨科完成。①取四五月龄新西兰大白兔20只,体外培养骨髓基质细胞。②将传代培养的第2代骨髓间充质细胞按1×10/的细胞密度培养24h后,弃掉4孔培养液,用磷酸盐缓冲液洗3次。加入含有不同浓度胎牛血清及新生牛血清的HamF12培养基继续培养24h,采用四甲基偶氮唑盐比色分析法观察两种血清对骨髓基质细胞增殖和分化的影响,用酶联免疫检测仪测定各孔在570nm波长处的光吸收值。结果:①小牛血清及胎牛血清均有明显促进骨髓间充质干细胞增殖的作用,随着血清浓度的增加,光吸收值也随之增大。②同一浓度下的胎牛血清比小牛血清具有更明显的促进骨髓间充质干细胞增殖的作用。③10%浓度的胎牛血清对骨髓间充质干细胞的增殖作用最为显著。结论:小牛血清及胎牛血清均有明显促进骨髓基质细胞增殖的作用,胎牛血清在骨髓间充质细胞的体外增殖过程中,与小牛血清相比较,可使细胞的增殖更加活跃。     

p27和p57蛋白在血小板源生长因子BB刺激下血管平滑肌细胞增殖过程中的表达

目的：观察不同剂量的血小板源生长因子BB刺激对血管平滑肌细胞生长率的影响及血管平滑肌细胞不同增殖程度下p27和p57蛋白表达量的变化。方法：实验于1998-09／2000-05在南方医科大学附属南方医院心内科实验室完成。①选用七八周雄性SD大鼠80只，体外分离SD大鼠主动脉中层平滑肌，贴壁法培养平滑肌细胞，选用2～4代培养的细胞。②胰酶消化的血管平滑肌细胞，浓度为1&;#215;10^8L^-1，分别接种于6孔板上，无血清的培养基静止48h，分别加入血小板源生长因子BB10和20μg／L，无血清的培养基为对照组，分别在刺激1，3，5d后，计算不同剂量的血小板源生长因子BB刺激下的血管平滑肌细胞数量。每组重复4次，取平均值。③在用血小板源生长因子BB10和20μg/L刺激6，12，24h后收集细胞，用免疫印迹分析法检测细胞中p27和p57蛋宴表达量。结果：①血管平滑肌细胞数量：血小板源生长因子BB刺激1和3及5d后血小板源生长因子BB10和20μg／L组明显高于对照组（t=4．06-21．76，P〈0．05-0．01）。②血管平滑肌细胞中p27蛋白表达量（A值）：血小板源生长因子BB刺激12和24h后血小板源生长因子BB10和20μg/L．几组明显低于对照组（P〈0．05，0．01），血小板源生长因子BB20μg/L组明显低于血小板源生长因子BB10μg/L组（P〈0．01）。p57蛋白表达量（A值）：血小板源生长因子BB刺激24h后血小板源生长因子BB10和20μg/L组明显高于对照组（P〈0．01），血小板源生长因子BB20μg/L．几组明显高于血小板源生长因子BB10μg/L组（P〈0．01）。结论：①在血小板源生长因子BB刺激的血管平滑肌细胞增殖过程中，p27和p57蛋白表达量变化不同。②在增殖的血管平滑肌细胞中随刺激因子作用时间的延长，表达量逐渐下降，而p57蛋白表达水平随血管平滑肌细胞的增殖而增加，p27蛋白表达量的变化可能是决定血管平滑肌细胞增殖的关键因素。     

新霉素对脑胶质瘤细胞增殖及PDGF、VEGF和血管生成素表达的影响

目的：探讨新霉素对脑胶质瘤细胞增殖及PDGF、VEGF和血管生成素表达的影响。方法采用人脑胶质瘤细胞U251,DMEM培养基添加10％胎牛血清培养。MTT细胞活性实验检测细胞增殖,RealTimePCR检测mR-NA表达情况,酶联免疫吸附实验检测蛋白表达。结果MTT结果表明,新霉素对U251细胞增殖存在抑制作用,并以剂量依赖的方式进行,并发现新霉素的抑制作用随时间增强。RealTimePCR结果显示,新霉素作用后U251细胞中PDGF、VEGF和血管生成素mRNA的表达都受到不同程度的抑制,分别降低了26．75％、38．23％和43．87％。ELISA分析表明新霉素能够降低PDGF、VEGF和血管生成素蛋白水平的表达。结论新霉素能够抑制脑胶质瘤细胞U251中PDGF、VEGF和血管生成素的表达并抑制脑胶质瘤细胞增殖。     

Gallbladder Na+/H+ exchange activity is up-regulated prior to cholesterol crystal formation

BACKGROUND: Gallbladder Na+ and H2O absorption are increased prior to gallstone formation and may promote cholesterol nucleation. Na+/H+ exchange (NHE) isoforms NHE2 and NHE3 are involved in gallbladder Na+ transport in prairie dogs. We examined whether increased gallbladder Na+ absorption observed during early gallstone formation is the result of NHE up-regulation. MATERIALS AND METHODS: Native gallbladder and primary cultures of gallbladder epithelial cells (GBECs) harvested from prairie dogs fed nonlithogenic (CON) or 1.2% cholesterol diet for varying lengths of time to induce cholesterol-saturated bile (PreCRYS), cholesterol crystals (CRYS), or gallstones (GS) were used. NHE activity was assessed by measuring dimethylamiloride-inhibitable 22Na+ uptake under H+ gradient in primary GBECs. HOE-694 was used to determine NHE2 and NHE3 contributions. NHE protein and mRNA expression were examined by Western and Northern blots, respectively. RESULTS: Gallbladder total NHE activity was 25.1 +/- 1.3 nmol mg protein(-1) min(-1) in the control and increased during gallstone formation peaking at the PreCRYS stage (98.4 +/- 3.9 nmol mg protein(-1) min(-1)). There was a shift in NHE activity from NHE2 to NHE3 as the animals progressed from no stones through the PreCRYS and CRYS stages to gallstones. The increase in NHE activity was partly caused by an increased Vmax without any change in K(Na)m. Both NHE2 and NHE3 protein increased moderately during the PreCRYS stage without increases in mRNA expression. CONCLUSIONS: Increased gallbladder Na+ absorption observed prior to crystal formation is in part caused by an increase NHE activity which is not fully accounted for by an increase in NHE proteins and mRNA levels but may be explained by enhanced localization in the membranes and/or altered regulation of NHE.     

Fas和FasL在Na+/H+交换器-1抑制诱导缺氧大鼠肺动脉平滑肌细胞凋亡中的作用

目的探讨Fas、FasL在Na^＋／H^＋交换器-1（NHE-1）抑制所诱导的缺氧大鼠肺动脉平滑叽细胞（PASMCs）凋亡中的作用。方法将转染NHE-1特异性核酶基因的大鼠PASMCs置于缺氧条件下（O2的体积分数低下1％）培养。缺氧培养2、6、12、24和48h后用原位末端标记法（TUNEL）检测细胞凋亡情况；半定量逆转录-聚合酶链反应（sqRT—PCR）疗法检测细胞内fas和fasL mRNA表达变化；免疫细胞化学法检测细胞内Fas和FasL蛋白表达变化。结果转染NHE-1特异性核酶基因的大鼠PASMCs在缺氧培养时，其细胞凋亡率随缺氧时间的延长而逐渐升高，但细胞内fas、fasL mRNA及Fas、FasL蛋白表达与对照组细胞比较差异均无显著性。结论Fas／FasL死亡通路可能不参与NHE-1抑制而诱导缺氧大鼠PASMCs凋亡的调控。     

Na+/H+交换器-1抑制诱导大鼠肺动脉平滑肌细胞凋亡的可能机制

背景肺动脉平滑肌细胞( pulmonary artery smooth muscle cells,PASMC)的增殖在缺氧肺动脉高压肺血管重构中起重要作用.解放军第三军医大学附属新桥医院全军呼吸研究所在前期实验中通过构建 Na+ /H+交换器- 1( sodium/proton exchanger isoform-1, Na+ /H+ exchanger isoform-1, Na+ /H+ antiporter isoform-1, NHE-1)特异性核酶基因逆转录病毒载体,转染入体外培养的大鼠 PASMC内表达,发现 NHE-1抑制可诱导细胞内酸化而抑制 PASMC增殖,并促进其凋亡.但这种促凋亡作用是通过什么途径来完成,尚不清楚.目的探讨 Bcl-2、 Bax蛋白表达在 NHE-1抑制而诱导大鼠 PASMC凋亡中的作用.设计分组对照实验.地点和材料实验在解放军第三军医大学附属新桥医院全军呼吸内科研究所完成.转染表达 NHE-1特异性核酶基因的体外培养大鼠 PASMC( PRZ细胞)、转染 PLXSN空载体的大鼠 PASMC( PX细胞)、未处理大鼠 PASMC细胞( PA细胞)为前期实验所制备.干预已构建 NHE-1特异性核酶基因逆转录病毒载体,转染入体外培养的大鼠 PASMC内表达,同时转染 pLXSN空载体入另一组大鼠 PASMC内,后者与未转染的大鼠 PASMC细胞为对照组.用荧光指示剂( Fura-2/AM)测定法检测转染 NHE-1特异性核酶基因的大鼠 PASMC内 Ca2+( [Ca2+ ]i)变化; RT-PCR方法检测细胞内 Bcl-2和 Bax mRNA表达变化,免疫组化法检测细胞内 Bcl-2和 Bax蛋白表达变化.主要观察指标①转染 NHE-1特异性核酶基因的大鼠 PASMC内 Ca2+( [Ca2+ ]i)变化.②细胞内 Bcl-2和 Bax mRNA表达变化.③细胞内 Bcl-2和 Bax蛋白表达变化.结果转染 NHE-1特异性核酶基因后,大鼠 PASMC内 [Ca2+ ]i显著升高,细胞内 [Ca2+ ]i在 PA细胞 [(95.94± 6.39) nmol/L]与 PX细胞 [(98.08± 7.37) nmol/L]之间差异无显著性意义( P 》0.05),而 PRZ细胞 [(198.08± 16.59) nmol/L]显著高于 PA细胞及 PX细胞 , 差异有显著性意义( P《 0.001).Bcl-2 mRNA及蛋白表达显著降低( PA细胞、 PX细胞、 PRZ细胞的平均积分光密度分别为 2.21± 0.18, 2.09± 0.30, 1.45± 0.20), Bax mRNA和蛋白表达显著增加( PA细胞、 PX细胞、 PRZ细胞的 ABax/Aβ-actin分别为 0.17± 0.02, 0.23± 0.06, 0.59± 0.08).结论 NHE-1抑制诱导的 PASMC凋亡与 [Ca2+ ]i增加、 Bcl-2表达降低及 Bax表达增加有关.     

Inhibition of lipopolysaccharide-induced prostaglandin E2 production and inflammation by the Na+/H+ exchanger inhibitors

We analyzed the effects of the Na+/H+ exchanger (NHE) inhibitor 3,5-diamino-6-chloro-N-(diaminomethylidene)pyrazine-2-carboxamide hydrochloride (amiloride) and its analogs 5-(N,N-dimethyl)-amiloride (DMA) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on the lipopolysaccharide (LPS)-induced production of prostaglandin (PG) E2 in vitro and in vivo. In the mouse macrophage-like cell line RAW 264, these inhibitors suppressed the LPS (1 microg/ml)-induced production of PGE2 at 8 h in a concentration-dependent manner. They also reduced the LPS-induced release of arachidonic acid from membrane phospholipids at 4 h and the LPS-induced increase in the level of cyclooxygenase (COX)-2 protein at 6 h, but not the level of COX-2 mRNA at 3 h. The LPS-induced phosphorylation of mitogen-activated protein kinases and degradation of inhibitor of kappaB-alpha were not inhibited by these drugs. In an air pouch-type LPS-induced inflammation model in mice 30 mg/kg amiloride and 10 mg/kg EIPA as well as the COX inhibitor indomethacin (10 mg/kg), significantly reduced the level of PGE2 in the pouch fluid at 8 h and the vascular permeability from 4 to 8 h. The accumulation of pouch fluid and leukocytes in the pouch fluid at 8 h was significantly inhibited by amiloride and EIPA but not by indomethacin. These findings suggested that the NHE inhibitors suppress the production of PGE2 through inhibiting the release of arachidonic acid and the increase in COX-2 protein levels and thus induce anti-inflammatory activity.     

Protein kinase C-alpha regulation of gallbladder Na+ transport becomes progressively more dysfunctional during gallstone formation

Gallbladder Na+ absorption and biliary Ca2+ are both increased during gallstone formation and may promote cholesterol nucleation. Na+/H+ exchange (NHE) is a major pathway for gallbladder Na+ transport. Ca2+-dependent second messengers, including protein kinase C (PKC), inhibit basal gallbladder Na+ transport. Multiple PKC isoforms with species- and tissue-specific expression have been reported. In this study we sought to characterize Ca2+-dependent PKC isoforms in gallbladder and to examine their roles in Na+ transport during gallstone formation. Gallbladders were harvested from prairie dogs fed either nonlithogenic chow or 1.2% cholesterol-enriched diet for varying periods to induce various stages of gallstone formation. PKC was activated with the use of phorboldibutyrate, and we assessed gallbladder NHE regulation by measuring unidirectional Na+ flux and dimethylamiloride-inhibitable 22Na+ uptake. We measured gallbladder PKC activity with the use of histone III-S phosphorylation and used G? 6976 to determine PKC-alpha contributions. Gallbladder PKC isoform messenger RNA and protein expression were examined with the use of Northern- and Western-blot analysis, respectively. Prairie dog and human gallbladder expresses PKC-alpha, betaII, and delta isoforms. The PKC activation significantly decreased gallbladder J(Na)(ms) and reduced baseline 22Na+ uptake by inhibiting NHE. PKC-alpha mediated roughly 42% of total PKC activity under basal conditions. PKC-alpha regulates basal gallbladder Na+ transport by way of stimulation of NHE isoform NHE-2 and inhibition of isoform NHE-3. PKC-alpha blockade reversed PKC-induced inhibition of J(Na)(ms) and 22Na+ uptake by about 45% in controls but was progressively less effective during gallstone formation. PKC-alpha contribution to total PKC activity is progressively reduced, whereas expression of PKC-alpha mRNA, and protein increases significantly during gallstone formation. We conclude that PKC-alpha regulation of gallbladder NHE becomes progressively more dysfunctional and may in part account for the increased Na+ absorption observed during gallstone formation.     

Amiloride kills malignant glioma cells independent of its inhibition of the sodium-hydrogen exchanger

Previously, we demonstrated that malignant glioma cell lines have increased intracellular pH (pHi) as a result of increased activities of the type I sodium/hydrogen exchanger (NHE1). This alkalotic pHi of 7.2 to 7.4 is favorable for augmented glycolysis, DNA synthesis, and cell cycle progression. Conversely, reductions in pHi have been associated with reduced rates of proliferation in transformed cell types. The effects of reducing pHi directly and by NHE1 inhibition on human malignant glioma cells were systematically compared with those on primary rat astrocytes. Neither cariporide, nor direct acidification to pHi 6.9 altered the proliferative rates or viabilities of human U87 or U118 malignant glioma cell lines. However, amiloride significantly impaired glioma cell proliferation and viability while not affecting astrocytes at concentrations (500 microM) that exceeded its inhibition of NHE1 in glioma cells (IC50 = 17 microM). Preventing a reduction of pHi did not alter the drug's antiproliferative and cytotoxic effects on glioma cells. These findings indicated that amiloride's cytotoxic effects on glioma cells are independent of its ability to inhibit NHE1 or to reduce intracellular pHi. The amiloride derivative 2,4 dichlorobenzamil (DCB) inhibits the sodium-calcium exchanger (NCX) and was both antiproliferative and cytotoxic to glioma cells at low doses (20 microM). By contrast, KB-R7943 [(2-[2-[4-nitrobenzyloxy]phenyl]ethyl)-isothioureamethanesulfonate] preferentially blocks sodium-dependent calcium influx by NCX (reverse mode) and was nontoxic to glioma cells. It is proposed that DCB (20 microM) and amiloride (500 microM) impair calcium efflux by NCX, leading to elevations of intracellular calcium that initiate a morphologically necrotic, predominantly caspase-independent glioma cell death.