Modification of intracellular pH and thermotolerance development by amiloride

The intracellular pH (pHi) of cells heated at 45.0 degrees C in the presence or absence of amiloride and in choline chloride substituted sodium-free medium was measured with flow cytometry using the pH sensitive dye, carboxy-seminaphthorhodafluor (SNARF-1). Chinese hamster ovary (CHO) cells at pH 7.3 and low-pH-resistant (PHV2) cells at pH 6.6 were studied. Bimodal population distributions of pHi were obtained for both CHO and PHV2 cells following a treatment in which cells were heated 10 min at 45.0 degrees C, incubated 4 to 10 h at 37 degrees C, then reheated at 45 min at 45.0 degrees C. Amiloride or sodium-free medium modified the changes in pHi, but did not eliminate them entirely. Cells were sorted from the higher pHi and lower pHi subpopulations and plated for cell survival. The survival after both heat treatments was three to five-fold higher for cells sorted from the higher pHi subpopulation than cells sorted from the low pHi subpopulation. The development of thermotolerance was delayed in CHO cells but not in PHV2 cells when amiloride was present throughout the treatment regimen. Combining low pH with amiloride caused an even greater delay in thermotolerance development in CHO cells. However, the final fraction of thermotolerant cells after 14 h incubation was nearly identical, regardless of medium pH or the presence of amiloride.