THE ROLE OF INTRACELLULAR PH AND ITS VARIANCE IN LOW PH SENSITIZATION OF KILLING BY HYPERTHERMIA

Average intracellular pH (pH(i)) was measured in Chinese hamster ovary (CHO) and murine NG108-15 neuroblastoma cells by the weak acid, [14C]5,5-dimethyl-2,4-oxazolidinedione-2 ([14C]DMO). Intercell variance in pH(i) in CHO cells was determined by flow cytometry (FCM) and automated image analysis using the pH-sensitive fluorescent dye, 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein. Our results are summarized as follows: (i) When extracellular pH (pH(e)) ranged from 6.1 to 7.9, the relationship between pH(e) and average pH(i) was similar for both cell lines, except that pH(i) of NG108 cells was 0.3 to 0.2 pH units higher than that of CHO cells. (ii) When both cell lines were heated at 43.5°C to give an isosurvival of 0.1 at pH(e) 7.2, lowering pH(e) (6.1-6.9) reduced survival more for CHO cells than for NG108-15 cells. However, plots of survival versus average pH(i) were identical for the two cell lines. (iii) Values of average pH(i) measured by [14C]DMO agree with those measured by FCM techniques. (iv) A distribution of pH(i) values was obtained for a population of cells. However, when the cells were sorted on the basis of low or high values of pH(i) and reanalyzed, the distributions of the sorted populations were almost identical to the distribution of the original population. These results indicated that the distribution of pH(i) for a cell population is much more homogeneous than that observed by FCM. (v) These observations indicate that pH(i) at the time of heating and not pH(e) is responsible for the low pH sensitization of heat killing.