JOURNAL OF CELLULAR PHYSIOLOGY 1.14216-221 (1990)
Effects of Hyperthermia on the Membrane Potential and N a + Transport of V79 Fibroblasts ROSS B. MIKKELSEN* AND CRAIG R. ASHER Division of Radiobiology, Department of Radiation Oncology, New England Medical Center, Boston, Masbachusetts 02 1 I 7 The effects of hyperthermia (41-43°C) on the membrane potential (calculated from the transmembrane distribution of [3H]tetraphenylphosphonium) and Na+ transport of Chinese hamster V79 fibroblasts were studied. At 41 "C, hyperthermia induced a membrane hyperpolarization of log phase cells (5 to 26 mV) that was reversible upon returning to 37°C. The hyperpolarization was inhibited 50% by 1 m M ouabain or 0.25 m M amiloride, an inhibitor of Na+:H' exchange. Shifting temperature to 41 "C increased ouabain-sensitive Rb+ uptake indicating activation of the electrogenic Na+ pump. At 43°C for 60 min, the membrane potential of log phase cells depolarized (20-35 mV). Parallel studies demonstrated enhanced Na- uptake at 41°C only in the presence of ouabain. At 43"C, i%a+ uptake was increased relative to controls with or without ouabain present. At both 41 and 43"C, 0.25 m M amiloride inhibited heat-stimulated Na' uptake. Na ' efflux was enhanced at 41°C in a process inhibited by ouabain. Thus, one consequence of heat treatment at 41°C is activation of Na':H* exchange with the resultant increase in cytosolic "a'] activating the elcctrogenic N a + pump. At temperatures 243"C, the Na+ pump is inhibited.
One hypothesis for the cytotoxic action of hyperthermia invokes cellular membranes as heat targets (Hahn, 1982). Experimental evidence for this proposal includes the synergism of membrane active agents (e.g., amphotericin B) and heat (Hahn and Li, 19821, thermally induced changes in ion and solute transport (Kwock et al., 1978; Mikkelsen and Koch, 1981, 1982; Yi et al., 1983; Stevenson et al., 1983; Anderson and Hahn, 1985; Bates et al., 19851, and heat-induced changes in membrane structure (Verma and Wallach, 1974). Of related interest is the potential role of nontoxic hyperthermia clinically achievable during whole body hyperthermia to enhance tumor cell killing by increasing anti-tumor drug uptake or by modification of repair processes (Honess and Bleehen, 1988). Hahn et al. (1975) early on provided evidence for increased cytotoxic efficacy of some anti-tumor drugs in vitro by hyperthermia in part because of increased drug uptake. Previously, we (Mikkelsen and Wallach, 1982; Wallach et al., 1981; Mikkelsen et al., 1985) suggested that the therapeutic gain achieved by combining moderate hyperthermia (
Effects of Hyperthermia on the Membrane Potential and N a + Transport of V79 Fibroblasts ROSS B. MIKKELSEN* AND CRAIG R. ASHER Division of Radiobiology, Department of Radiation Oncology, New England Medical Center, Boston, Masbachusetts 02 1 I 7 The effects of hyperthermia (41-43°C) on the membrane potential (calculated from the transmembrane distribution of [3H]tetraphenylphosphonium) and Na+ transport of Chinese hamster V79 fibroblasts were studied. At 41 "C, hyperthermia induced a membrane hyperpolarization of log phase cells (5 to 26 mV) that was reversible upon returning to 37°C. The hyperpolarization was inhibited 50% by 1 m M ouabain or 0.25 m M amiloride, an inhibitor of Na+:H' exchange. Shifting temperature to 41 "C increased ouabain-sensitive Rb+ uptake indicating activation of the electrogenic Na+ pump. At 43°C for 60 min, the membrane potential of log phase cells depolarized (20-35 mV). Parallel studies demonstrated enhanced Na- uptake at 41°C only in the presence of ouabain. At 43"C, i%a+ uptake was increased relative to controls with or without ouabain present. At both 41 and 43"C, 0.25 m M amiloride inhibited heat-stimulated Na' uptake. Na ' efflux was enhanced at 41°C in a process inhibited by ouabain. Thus, one consequence of heat treatment at 41°C is activation of Na':H* exchange with the resultant increase in cytosolic "a'] activating the elcctrogenic N a + pump. At temperatures 243"C, the Na+ pump is inhibited.
One hypothesis for the cytotoxic action of hyperthermia invokes cellular membranes as heat targets (Hahn, 1982). Experimental evidence for this proposal includes the synergism of membrane active agents (e.g., amphotericin B) and heat (Hahn and Li, 19821, thermally induced changes in ion and solute transport (Kwock et al., 1978; Mikkelsen and Koch, 1981, 1982; Yi et al., 1983; Stevenson et al., 1983; Anderson and Hahn, 1985; Bates et al., 19851, and heat-induced changes in membrane structure (Verma and Wallach, 1974). Of related interest is the potential role of nontoxic hyperthermia clinically achievable during whole body hyperthermia to enhance tumor cell killing by increasing anti-tumor drug uptake or by modification of repair processes (Honess and Bleehen, 1988). Hahn et al. (1975) early on provided evidence for increased cytotoxic efficacy of some anti-tumor drugs in vitro by hyperthermia in part because of increased drug uptake. Previously, we (Mikkelsen and Wallach, 1982; Wallach et al., 1981; Mikkelsen et al., 1985) suggested that the therapeutic gain achieved by combining moderate hyperthermia (
