Effects of Acute pH 6.6 and 42.0°C Heating on the Intracellular pH of Chinese Hamster Ovary Cells¹

Incubation of Chinese hamster ovary cells in pH 6.6 medium for 4 h prior to and during 42.0°C heating enhanced thermal cell killing compared to cells heated under normal pH 7.3 conditions. We examined the relationship between the extracellular pH and intracellular pH (pH_i) of Chinese hamster ovary cells using a flow cytometer with the pH-sensitive fluorescent molecule 2,3-dicyanohydroquinone. Using either normal (7.3) or low (6.6) pH conditions, the mean pH_i and population pH_i heterogeneity was studied as a function of time at 42.0°C. Cells incubated at pH 6.6 for 4 h had a resting pH_i 0.14 to 0.19 pH units lower than cells at normal pH 7.3, indicating the presence of an active pH_i regulatory system. Heating 1 h at 42.0°C at normal pH caused an increase in the pH_i of 0.14 pH units. With further heating the cells gradually returned to the unheated (7.3) control levels. Similar pH_i changes were observed with the cells incubated and heated at pH 6.6. However, the mean pH_i was always more acidic than cells heated at normal pH. Active pH_i regulation was still possible for a substantial (>30%) number of cells even after 10 h of heating under low pH conditions. These results suggest that a breakdown in pH_i regulation is not the mechanism of low pH-induced heat sensitization.