MODIFICATION OF INTRACELLULAR PH AND THERMOTOLERANCE DEVELOPMENT BY AMILORIDE

The intracellular pH (pH(i)) of cells heated at 45.0 degrees C in the presence or absence of amiloride and in choline chloride substituted s odium-free medium was measured with flow cytometry using the pH sensit ive dye, carboxy-seminaphthorhodafluor (SNARF-1). Chinese hamster ovar y (CHO) cells at pH 7.3 and low-pH-resistant (PHV2) cells at pH 6.6 we re studied. Bimodal population distributions of pH(i) were obtained fo r both CHO and PHV2 cells following a treatment in which cells were he ated 10 min at 45.0 degrees C, incubated 4 to 10 h at 37 degrees C, th en reheated 45 min at 45.0 degrees C. Amiloride or sodium-free medium modified the changes in pH(i), but did not eliminate them entirely. Ce lls were sorted from the higher pH(i) and lower pH(i) subpopulations a nd plated for cell survival. The survival after both heat treatments w as three to five-fold higher for cells sorted from the higher pH(i) su bpopulation than cells sorted from the low pH(i) subpopulation. The de velopment of thermotolerance was delayed in CHO cells but not in PHV2 cells when amiloride was present throughout the treatment regimen. Com bining low pH with amiloride caused an even greater delay in thermotol erance development in CHO cells: However, the final fraction of thermo tolerant cells after 14 h incubation was nearly identical, regardless of medium pH or the presence of amiloride.