title: The effect of heat on Na+/H+ antiport function and survival in mammalian cells.
abstract: OBJECTIVE
  Because intracellular pH (pHi) is a determinate of thermosensitivity, it is important to understand the relationship between heat cytotoxicity and the mechanisms responsible for pHi 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
  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 pHi were made using the fluorescent dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF).
  
  
  RESULTS
  Exposure of EMT6 cells to 43-46 degrees 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 pHi regulatory role. PS120 and CCL39 cells and similar survival levels when heated at pHe 7.2 in the presence of NaHCO3, which allows function of the other major regulator of pHi, the Na+ -dependent HCO3-/Cl- exchanger. This occurred despite a drop in pHi in the PS120 cells during heating. A reduced survival was observed, however, in PS120 cells after 43 degrees C for 30-60 min at either pHe 6.5 or pHe 7.2 in the absence of NaHCO3. Intracellular pH was consistently greater for PS120 than CCL39 cells.
  
  
  CONCLUSIONS
  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 pHi level under most treatment conditions, the precise role of the Na+/H+ antiport in mediating thermal cytotoxicity remains uncertain.
authors.affiliation: Department of Radiation Oncology, University of Toronto, Princess Margaret Hospital/Ontario Cancer Institute, Canada.
authors.forename: F F
authors.initials: FF
authors.lastname: Liu
authors.forename: K
authors.initials: K
authors.lastname: Diep
authors.forename: I F
authors.initials: IF
authors.lastname: Tannock
authors.forename: R P
authors.initials: RP
authors.lastname: Hill
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000502:physiology / Q000528:radiation effects; D006224:Cricetinae; D003412:Cricetulus; D005354:Fibrosarcoma / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006979:Hyperthermia, Induced; D007424:Intracellular Fluid / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D051379:Mice; D009381:Neoplasms, Radiation-Induced / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D017923:Sodium-Hydrogen Exchangers / Q000502:physiology* / Q000528:radiation effects; D014407:Tumor Cells, Cultured / Q000528:radiation effects
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein