title: Characterization of intracellular calcium pools and their desensitization in thermotolerant human A-431 cells.
abstract: BACKGROUND
  This study characterizes the intracellular Ca2+ pools in nonthermotolerant and thermotolerant human A-431 cells and the reduced cytotoxicity using the inhibitors of Ca2+ mobilizations.
  
  
  METHODS
  Nonthermotolerant and thermotolerant cells were treated with different Ca2+ mobilizers in the absence of external Ca2+. The cytosolic Ca2+ concentration using fura-2 fluorescence probe was measured to identify the presence of intracellular Ca2+ pools. The cytotoxicity of the increase in [Ca2+]i was studied using the colony forming efficiency assay.
  
  
  RESULTS
  The resting intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Ca2+ was 42 +/- 9 nm, determined by fura-2. Bradykinin (10 mumol/L), monensin (200 mumol/L), and ionomycin (1 mumol/L) sequentially treated to cells mobilized Ca2+ and increased [Ca2+]i by 64 +/- 23, 40 +/- 6, and 59 +/- 21 nm, respectively. The bradykinin effect was blocked by 5 mumol/L U-73122 (an inhibitor of inositol trisphosphate production); the ionomycin effect was inhibited by increasing intracellular pH (pHi) or treatment with 100 mumol/L ryanodine while the monensin effect was enhanced by increasing pHi, but was not inhibited by ryanodine. Cells that were made tolerant to lethal temperatures also responded to bradykinin, monensin, and ionomycin, but the magnitude of the response was diminished. Subsequent treatments with bradykinin, monensin, and ionomycin increased [Ca2+]i in thermotolerant cells to levels 68 +/- 8, 44 +/- 5, and 45 +/- 5%, respectively, of values found in nonthermotolerant cells. Higher concentrations of these agents did not further increase [Ca2+]i. The bradykinin-induced increase in inositol trisphosphates in thermotolerant cells was also reduced, which perhaps accounts for the attenuation in Ca2+ mobilization. Unlike nonthermotolerant cells, the monensin effect was not enhanced when pHi was increased. However, the ionomycin effect was still dependent on pHi and was blocked by ryanodine at a higher concentration.
  
  
  CONCLUSIONS
  These results show that there are bradykinin-, monensin-, and ryanodine-sensitive pools and that thermotolerance attenuates Ca2+ mobilization stimulated by these three agents. Ionomycin at 10 mumol/L or NaCN at 10 mM for 1 hour demonstrated cytotoxicity. Pretreatment with 100 mumol/L ryanodine and/or 5 mumol/L U-73122 reduced cytotoxicity produced by either NaCN or ionomycin. These results suggest that an attenuation of [Ca2+]i increases can diminish cytotoxicity.
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: M L
authors.initials: ML
authors.lastname: Koenig
mesh_terms: D000222:Adaptation, Physiological; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D014407:Tumor Cells, Cultured
chemical_list: D002118:Calcium