Nitschke R, Riedel A, Ricken S, Leipziger J, Benning N, Fischer K G, Greger R
Physiologisches Institut der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Pflugers Arch. 1996 Nov-Dec;433(1-2):98-108. doi: 10.1007/s004240050254.
The influence of intracellular pH (pHi) on intracellular Ca2+ activity ([Ca2+]i) in HT29 cells was examined microspectrofluorometrically. pHi was changed by replacing phosphate buffer by the diffusible buffers CO2/HCO3- or NH3/NH4+ (pH 7.4). CO2/HCO3- buffers at 2,5 or 10% acidified pHi by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca2+]i by 8-15 nmol/l. This effect was independent of the extracellular Ca2+ activity and the filling state of thapsigargin-sensitive Ca2+ stores. Removing the CO2/HCO3- buffer alkalinized pHi by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca2+]i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca2+-free solution and with thapsigargin showed that the [Ca2+]i transient was due to release from intracellular pools and stimulated Ca2+ entry. NH3/NH4+ (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca2+]i to a peak (Delta [Ca2+]i = 164 nmol/l). The peak [Ca2+]i increase was not influenced by removal of external Ca2+, but the decline to basal [Ca2+]i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP3) antagonist theophylline had any influence on the NH3/NH4+-stimulated [Ca2+]i increase, whereas carbachol-induced [Ca2+]i transients were reduced by more than 80% and 30%, respectively. InsP3 measurements showed no change of InsP3 during exposure to NH3/NH4+, whereas carbachol enhanced the InsP3 concentration, and this effect was abolished by U73122. The pHi influence on "capacitative" Ca2+ influx was also examined. An acid pHi attenuated, and an alkaline pHi enhanced, carbachol- and thapsigargin-induced [Ca2+]i influx. We conclude that: (1) an alkaline pHi releases Ca2+ from InsP3-dependent intracellular stores; (2) the store release is InsP3 independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca2+ influx; (4) the capacitative Ca2+ influx activated by InsP3 agonists is decreased by acidic and enhanced by alkaline pHi. The effects of pHi on [Ca2+]i should be of relevance under many physiological conditions.
采用显微分光荧光测定法研究了细胞内pH值（pHi）对HT29细胞内钙离子活性（[Ca2+]i）的影响。通过用可扩散缓冲液CO2/HCO3-或NH3/NH4+（pH 7.4）替代磷酸盐缓冲液来改变pHi。CO2/HCO3-缓冲液分别使pHi酸化2%、5%或10%，导致pHi分别降低0.1、0.32和0.38个pH单位，并使[Ca2+]i增加8 - 15 nmol/L。这种效应与细胞外钙离子活性以及毒胡萝卜素敏感的钙离子储存库的充盈状态无关。去除CO2/HCO3-缓冲液使pHi碱化0.14（2%）、0.27（5%）和0.38（10%）个单位，并分别使[Ca2+]i增强至峰值20、65和143 nmol/L。在无钙溶液和有毒胡萝卜素的情况下进行的实验表明，[Ca2+]i瞬变是由于细胞内储存库的释放以及刺激的钙离子内流所致。NH3/NH4+（20 mmol/L）使细胞内瞬间碱化0.6个pH单位，并使[Ca2+]i增加至峰值（Δ[Ca2+]i = 164 nmol/L）。[Ca2+]i增加的峰值不受去除细胞外钙离子的影响，但降至基础[Ca2+]i的速度更快。磷脂酶C抑制剂U73122和肌醇1,4,5 - 三磷酸（InsP3）拮抗剂茶碱对NH3/NH4+刺激的[Ca2+]i增加均无影响，而卡巴胆碱诱导的[Ca2+]i瞬变分别降低了80%以上和30%。InsP3测量显示，在暴露于NH3/NH4+期间InsP3无变化，而卡巴胆碱可提高InsP3浓度，且该效应被U73122消除。还研究了pHi对“容量性”钙离子内流的影响。酸性pHi减弱，碱性pHi增强卡巴胆碱和毒胡萝卜素诱导的[Ca2+]i内流。我们得出以下结论：（1）碱性pHi从依赖InsP3的细胞内储存库释放钙离子；（2）储存库释放不依赖InsP3，通过一种尚不清楚的机制发生；（3）储存库释放刺激容量性钙离子内流；（4）由InsP3激动剂激活的容量性钙离子内流在酸性条件下降低，在碱性条件下增强。pHi对[Ca2+]i的影响在许多生理条件下可能具有相关性。
