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

Abstract

The effects of hyperthermia (exposure to 41-47 degrees 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 (pHi) was measured using the DMO partitioning technique. A rapid acidification of the intracellular environment was observed at all the elevated temperatures studied. The pHi reached a plateau value of approximately 6.9, and started reversing towards normal values upon prolonged exposure to heat. Similar patterns were seen for delta pH (pHi-pHo). The membrane potential difference (delta psi) was measured using the fluorescence quenching of 3,3-dipropylthio-carbocyanine, and calibrated using a 86Rb+ diffusion potential. We found that delta psi falls to zero only upon prolonged exposure to temperatures above 43 degrees C. When the external pH was changed from normal values the drop in delta psi occurred more readily. Development of thermotolerance resulted in an increase in the time required to make delta psi change by half. The changes in delta psi were shown to be irreversible. When the proton electrochemical gradient (delta mu H+) was calculated using the measured values of delta psi and delta pH, the trends observed were the same as those seen for delta psi. The changes observed for pHi can be accounted for by the changes in the pK values of the components involved in the intracellular buffering. The changes in delta psi and delta mu H+ 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.