The effect of intracellular pH on cytosolic Ca2+ in HT29 cells

The influence of intracellular pH (pH(i)) on intracellular Ca2+ activity ([Ca2+](i)) in HT29 cells was examined microspectrofluorometrically. pH(i) 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 pH(i) 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 Ca'+ activity and the filling state of thapsigargin-sensitive Ca2+ stores. Removing the CO2/HCO3- buffer alkalinized pH(i) 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 (InsP(3)) 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. InsP(3) measurements showed no change of InsP(3) during exposure to NH3/NH4+, whereas carbachol enhanced the InsP(3) concentration, and this effect was abolished by U73122. The pH(i) influence on ''capacitative'' Ca2+ influx was also examined. An acid pH(i) attenuated, and an alkaline pH(i) enhanced, carbachol- and thapsigargin-induced [Ca2+](i) influx. We conclude that: (1) an alkaline pH(i) releases Ca2+ from InsP(3)-dependent intracellular stores; (2) the store release is InsP(3) independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca2+ influx; (4) the capacitative Ca2+ influx activated by InsP(3) agonists is decreased by acidic and enhanced by alkaline pH(i). The effects of pH, on [Ca2+](i) should be of relevance under many physiological conditions.