EFFECT OF INTRACELLULAR PH ON AGONIST-INDUCED [CA2+](I) TRANSIENTS INHT29 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 microspectrofluorimetri cally using BCECF and fura-2. respectively. Buffers containing trimeth ylamine (TriMA), NH3/NH4+ and acetate were used to clamp pH(i) to defi ned values. The magnitudes of the peak and plateau of [Ca2+](i) transi ents 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 relati onship 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 co ncentrations (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/resp onse curve to the left, whereas an alkaline pH(i) led to a rightward s hift. The influence of pH(i) on [Ca2+](i) transients induced by neurot ensin (IO-S mol/l) or ATP (5 x 10(-7) mol/l) was similar to its influe nce on those induced by CCH, but generally not as pronounced. Measurem ents of cellular inositol 1,4,5-trisphosphate (InsP(3)) showed no chan ges in response to acidification with acetate (20 mmol/l) or alkaliniz ation 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 ind uced by TriMA or acetate. Slow-whole-cell patch-clamp experiments show ed no additional effect of CCH on the membrane voltage (V-m) measured after TriMA or acetate application. We conclude that pH(i) is a physio logical modulator of hormonal effects in HT29 cells, as the [Ca2+](i) responses to agonists were significantly changed at already slightly a ltered pH(i). The measurements of InsP(3), cell volume and V-m show th at pH(i) must act distally to the InsP(3) production, and not via chan ges of cell volume or V-m.