Bicarbonate-dependent proton extrusion in Chinese hamster ovary (CHO) cells adapted to growth at pH 6.7

A CHO cell model is described for in vitro studies of the mechanisms underlying heat resistance in cells adapted to growth in acidic environments. Adaptation is defined as a loss of pH 6.7-induced sensitization to 42.0 degrees C cytotoxicity and it is accompanied with an elevation of steady-state intracellular pH (pH(i)). CHO cells cultured between 75 and 100 days at pH 6.7 became fully adapted (6.7 G cells), and the adapted phenotype was maintained for at least 100 additional days of culture at pH 6.7. The surviving fraction (SF) of 6.7 G cells heated (42.0 degrees C) at pH 6.7 was comparable with that of cells cultured at pH 7.3 (7.3 G cells) and heated at pH 7.3, while the SF of 7.3 G cells acutely acidified to pH 6.7 and heated was an order of magnitude less. Although this resistance of 6.7 G cells to killing was observed at 42.0 degrees C, it was not observed at 43.0 and 45.0 degrees C. Both 6.7 G and 7.3 G cells were able to develop comparable levels of thermotolerance during 42.0 degrees C at their growth pHs. However, in agreement with the literature, development of thermotolerance was reduced in acutely acidified 7.3 G cells. An acute acidification of only 0.2 pH unit from pH 6.7 to 6.5 also reduced the ability of 6.7 G cells to develop thermotolerance during heating at 42.0 degrees C. The acquired 6.7 G phenotype reverted to the 7.3 G phenotype following 17 days of culture at pH 7.3. Amiloride (0.5 mM), an inhibitor of the Na+/H+ exchanger (NHE), did not sensitize 7.3 G and 6.7 G cells to 42.0 degrees at their growth pHs. However, sensitization was observed for acutely acidified 7.3 G cells. This is consistent with the hypothesis that extracellular acute acidification causes a decrease in pH(i), and that the recovery from that decrease is achieved in part by activation of the NHE. An elevation of steady-state pH(i), measured by analysing intracellular BCECF excitation spectra, was documented in a suspension assay for cells grown at pH 6.7 for 180 days. The elevation was bicarbonate-dependent (negligible in the absence of HCO3-, +0.17 pH units in the presence of HCO3-). These results suggest that the altered regulation of pH(i) in CHO cells adapted to pHe 6.7 is maintained by bicarbonate-dependent processes.