title: Thermotolerance attenuates heat-induced increases in [Ca2+]i and HSP-72 synthesis but not heat-induced intracellular acidification in human A-431 cells.
abstract: BACKGROUND
  Thermotolerance affects cell viability, retards translation of heat shock proteins, and protects RNA slicing mechanisms. We reported previously that heat shocking nonthermotolerant cells causes an intracellular acidification and an increase in cytosolic free Ca2+ ([Ca2+]i) in addition to an induction of heat shock protein 72kDa (HSP-72) production. This study characterized heat-induced changes in cytosolic Ca2+, H+, and HSP-72 synthesis in thermotolerant A-431 cells.
  
  
  METHODS
  We studied heat-induced changes in pH(i), [Ca2+]i, and HSP-72 using thermotolerant A-431 cell monolayers. pH(i) and [Ca2+]i were determined using fluorescence probes, and HSP-72 was measured by SDS-PAGE. The mRNA encoding HSP-72 was measured by Northern blots probed with a [32P]-labeled 2.3 kb fragment of an HSP-70 cDNA insert.
  
  
  RESULTS
  Heat shocking thermotolerant cells induced the same degree of intracellular acidification as that induced in nonthermotolerant cells, but the heat-induced increase in [Ca2+]i was less in thermotolerant cells. This diminished response was characterized by an increase in Km for external Ca2+ and was blocked by pretreatment with cycloheximide, indicating a newly synthesized protein is involved. Similar to what was seen in nonthermotolerant cells, the heat-induced increase in [Ca2+]i in thermotolerant cells depended on external Na+ concentration and was blocked by dichlorobenzamil, though thermotolerant cells were more sensitive to the inhibitor (IC50 = 0.21 mumol/L for nonthermotolerant, 0.025 mumol/Lm for thermotolerant). Thermotolerant cells contained high resting levels of HSP-72. Heat shocking these cells attenuated the HSF translocation from cytosol to nucleus and did not induce a further synthesis of HSP-72 mRNA and protein.
  
  
  CONCLUSIONS
  The results suggest that thermotolerance desensitizes the machinery required for Ca2+ entry. Low [Ca2+]i levels probably result in diminished HSP-72 mRNA production and less HSP-72 synthesis.
authors.affiliation: Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington DC 20307-5100, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Kiang
authors.forename: X Z
authors.initials: XZ
authors.lastname: Ding
authors.forename: D E
authors.initials: DE
authors.lastname: McClain
mesh_terms: D000064:Acclimatization / Q000502:physiology*; D000136:Acid-Base Equilibrium / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002294:Carcinoma, Squamous Cell; D002470:Cell Survival / Q000502:physiology*; D003600:Cytosol / Q000502:physiology; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D006358:Hot Temperature; D006801:Humans; D014407:Tumor Cells, Cultured
chemical_list: D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D002118:Calcium