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

Abstract

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°C cytotoxicity and it is accompanied with an elevation of steady-state intracellular pH (pH). CHO cells cultured between 75 and 100 days at pH 6.7 became fully adapted (6.7G cells), and the adapted phenotype was maintained for at least 100 additional days of culture at pH 6.7. The surviving fraction (SF) of 6.7G cells heated (42.0°C) at pH 6.7 was comparable with that of cells cultured at pH 7.3 (7.3G cells) and heated at pH 7.3, while the SF of 7.3G cells acutely acidified to pH 6.7 and heated was an order of magnitude less. Although this resistance of 6.7G cells to killing was observed at 42.0°C, it was not observed at 43.0 and 45.0°C. Both 6.7G and 7.3G cells were able to develop comparable levels of thermotolerance during 42.0°C at their growth pHs. However, in agreement with the literature, development of thermotolerance was reduced in acutely acidified 7.3G cells. An acute acidification of only 0.2 pH unit from pH 6.7 to 6.5 also reduced the ability of 6.7G cells to develop thermotolerance during heating at 42.0°C. The acquired 6.7G phenotype reverted to the 7.3G 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 7.3G and 6.7G cells to 42.0° at their growth pHs. However, sensitization was observed for acutely acidified 7.3G 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 HCO⁻₃, +0.17 pH units in the presence of HCO⁻₃). These results suggest that the altered regulation of pH_i in CHO cells adapted to pHe 6.7 is maintained by bicarbonate-dependent processes.

Key Words:

• CHO
• Adaptation to low pH_e
• hyperthermia
• pH_i
• amiloride