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

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 (pH_j) 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 (pH_i — pH_o). 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 (Δμ_H⁺) 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 pH_j can be accounted for by the changes in the pK values of the components involved in the intracellular buffering. The changes in Δϕ and Δμ_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.

Key Words:

• hyperthermia
• intracellular pH
• membrane potential
• thermotolerance
• extracellular pH
• cell survival.