Abstract
pHi recovery in acid-loaded Ehrlich ascites tumor cells and pHi maintenance at steady-state were studied using the fluorescent probe BCECF. Both in nominally HCO3(-)-free media and at 25 mM HCO3-, the measured pHi (7.26 and 7.82, respectively) was significantly more alkaline than the pHi value calculated assuming the transmembrane HCO3- gradient to be equal to the Cl- gradient. Thus, pHi in these cells is not determined by the Cl- gradient and by Cl-/HCO3- exchange. pHi recovery following acid loading by propionate exposure, NH4+ withdrawal, or CO2 exposure is mediated by amiloride-sensitive Na+/H+ exchange in HCO3(-)-free media, and in the presence of HCO3- (25 mM) by DIDS-sensitive, Na+)-dependent Cl-/HCO3- exchange. A significant residual pHi recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H+ pump in pHi regulation. pHi maintenance at steady-state involves both Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange. Acute removal of external Cl- induces a DIDS-sensitive, Na(+)-dependent alkalinization, taken to represent HCO3- influx in exchange for cellular Cl-. Measurements of 36Cl- efflux into Cl(-)-free gluconate media with and without Na+ and/or HCO3- (10 mM) directly demonstrate a DIDS-sensitive, Na(+)-dependent Cl-/HCO3- exchange operating at slightly acidic pHi (pHo 6.8), and a DIDS-sensitive, Na+)-independent Cl-/HCO3- exchange operating at alkaline pHi (pHo 8.2).
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