The effect of heat on [formula omitted] antiport function and survival in mammalian cells

Abstract

Purpose : Because intracellular pH (pH i) is a determinant of thermosensitivity, it is important to understand the relationship between the cytotoxicity and the mechanisms responsible for pH i regulation, such as the Na +/H + antiport. The objective of this study is to elucidate the relationship between heat damage and Na +/H + antiport activity. Methods and Materials : Various cell lines, EMT6, RIF-1, and its thermoresistant variant TR-4, and CCL39, and its variant that lacks the Na +/H + antiport (PS120), were all heated using a water bath. Parallel assessments of antiport function and pH i were made using the fluorescent dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Results : Exposure of EMT6 cells to 43–46°C for 30–60 min caused progressive decline in antiport activity, in parallel with cytotoxicity. When the same degree of cytotoxicity was induced by ionizing radiation, no alteration in Na +/H + antiport function was observed. Despite a 10-fold lower survival in RIF-1 compared to TR-4 cells after heating, there was no difference in the thermosensitivity of their antiports. Antiport activity in the TR-4 cells, however, was higher than that of RIF-1 cells both before and during heating. Intracellular pH for TR-4 cells decreased minimally during heating, in contrast to a decline of 1 pH unit in RIF-1 cells despite similar relative levels of antiport activity, suggesting that in this pair of cell lines, antiport activity does not play a major pH i regulatory role. PS120 and CCL39 cells had similar survival levels when heated at pH e 7.2 in the presence of NaHCO 3, which allows function of the other major regulator of pH i, the Na +-dependent HCO 3 −/Cl − exchanger. This occurred despite a drop in pH i in the PS120 cells during heating. A reduced survival was observed, however, in PS120 cells after 43°C for 30–60 min at either pH e 6.5 or pH e 7.2 in the absence of NaHCO 3. Intracellular pH for both lines decreased with increasing duration of heating under the various heating conditions, but the decline was consistently greater for PS120 than CCL39 cells. Conclusion : We demonstrated that damage to the Na +/H + antiport likely reflects early heat-induced change in membrane function, but is not a primary target for heat cytotoxicity. Although there is an association between survival, antiport function, and pH i level under most treatment conditions, the precise role of the Na +/H + antiport in mediating thermal cytotoxicity remains uncertain.