Effects of hyperthermia on the intracellular pH and membrane potential of Chinese hamster ovary cells

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Abstract
The effects of hyperthermia (exposure to 41–47°C) on the intracellular pH and membrane potential have been studied using Chinese hamster ovary HA-1 cells. Our goal was to determine whether intracellular pH changes or changes in membrane potential correlated with cell killing. The intracellular pH (pHj) was measured using the DMO partitioning technique. A rapid acidification of the intracellular environment was observed at all the elevated temperatures studied. The pH; reached a plateau value of approximately 6.9, and started reversing towards normal values upon prolonged exposure to heat. Similar patterns were seen for ΔpH (pHi — pHo). The membrane potential difference (Δϕ) was measured using the fluorescence quenching of 3,3-dipropylthio-carbocyanine, and calibrated using a 86Rb+ diffusion potential. We found that Δϕ falls to zero only upon prolonged exposure to temperatures above 43°C. When the external pH was changed from normal values the drop in Δϕ occurred more readily. Development of thermo-tolerance resulted in an increase in the time required to make Δϕ change by half. The changes in Δϕ were shown to be irreversible. When the proton electrochemical gradient (ΔμH+) was calculated using the measured values of Δϕ and ΔpH, the trends observed were the same as those seen for Δϕ. The changes observed for pHj can be accounted for by the changes in the pK values of the components involved in the intracellular buffering. The changes in Δϕ and ΔμH+ may reflect the physical breakdown of the transmembrane H+ gradients, which may be the actual mechanical process of cell death. No correlation of cell survival with the measured parameters was observed.