Effect of intracellular pH on agonist-induced [Ca2+](i) transients in HT29 cells

In this study we examined the influence of intracellular pH (pH(i)) on agonist-induced changes of intracellular Ca2+ activity ([Ca2+](i)) in HT29 cells. pH(i) and [Ca2+](i) were measured microspectrofluorimetrically using BCECF and fura-2. respectively. Buffers containing trimethylamine (TriMA), NH3/NH4+ and acetate were used to clamp pH(i) to defined values. The magnitudes of the peak and plateau of [Ca2+](i) transients induced by carbachol (CCH, 10(-6) mol/l) were greatly enhanced by an acidic pH(i) and nearly abolished by an alkaline pH(i). The relationship between pH(i) and the [Ca2+](i) peak was nearly linear from pH(i) 7.0 to 7.8. This effect of pH(i) was also observed at higher CCH concentrations (10(-4) and 10(-5) mol/l), at which the inhibitory effect of an alkaline pH(i) was more pronounced than the stimulatory effect of an acidic pH(i). An acidic pH(i) shifted the CCH concentration/response curve to the left, whereas an alkaline pH(i) led to a rightward shift. The influence of pH(i) on [Ca2+](i) transients induced by neurotensin (IO-S mol/l) or ATP (5 x 10(-7) mol/l) was similar to its influence on those induced by CCH, but generally not as pronounced. Measurements of cellular inositol 1,4,5-trisphosphate (InsP(3)) showed no changes in response to acidification with acetate (20 mmol/l) or alkalinization with TriMA (20 mmol/l). The InsP(3) increase induced by CCH was unaltered at an acidic pH(i), but was augmented at an alkaline pH(i). Confocal measurements of cell volume showed no significant changes induced by TriMA or acetate. Slow-whole-cell patch-clamp experiments showed no additional effect of CCH on the membrane voltage (V-m) measured after TriMA or acetate application. We conclude that pH(i) is a physiological modulator of hormonal effects in HT29 cells, as the [Ca2+](i) responses to agonists were significantly changed at already slightly altered pH(i). The measurements of InsP(3), cell volume and V-m show that pH(i) must act distally to the InsP(3) production, and not via changes of cell volume or V-m.