Abstract

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 +/- 0.023 (n = 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 +/- 0.011 pH units/min in the presence of Naf, but in the absence of this ion or after treatment with 0.1 mM hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na+ independent H+ transport mechanisms in HEPES Ringer. In CO2/HCO3- Ringer, basal cell pH was 7.21 +/- 0.020 (n = 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO2, followed by return to the initial pH at a rate of -0.14 +/- 0.012 (n = 8) pH/min; this return was retarded or abolished in the absence of Cl- or after addition of 0.2 mM DIDS, suggesting extrusion of bicarbonate by Cl-/HCO3- exchange. This exchange was not Na+ dependent. When Na+ was added to cells incubated in 0 Na+ Ringer while blocking Na+/H+ exchange by HMA, cell alkalinization by 0.19 +/- 0.04 (n = 11) pH units was observed, suggesting the presence of Na+/HCO3- cotransport carrying HCO3- into these cells, which was abolished by DIDS. These experiments, thus, show that Na+/H+ and Cl-/HCO3- exchange and Na+/HCO3- cotransport participate in cell pH regulation in T84 cells.