title: Potential interactions of calcium-sensitive reagents with zinc ion in different cultured cells.
abstract: BACKGROUND
  Several chemicals have been widely used to evaluate the involvement of free Ca(2+) in mechanisms underlying a variety of biological responses for decades. Here, we report high reactivity to zinc of well-known Ca(2+)-sensitive reagents in diverse cultured cells.
  
  
  RESULTS
  In rat astrocytic C6 glioma cells loaded with the fluorescent Ca(2+) dye Fluo-3, the addition of ZnCl2 gradually increased the fluorescence intensity in a manner sensitive to the Ca(2+) chelator EGTA irrespective of added CaCl2. The addition of the Ca(2+) ionophore A23187 drastically increased Fluo-3 fluorescence in the absence of ZnCl2, while the addition of the Zn(2+) ionophore pyrithione rapidly and additionally increased the fluorescence in the presence of ZnCl2, but not in its absence. In cells loaded with the zinc dye FluoZin-3 along with Fluo-3, a similarly gradual increase was seen in the fluorescence of Fluo-3, but not of FluoZin-3, in the presence of both CaCl2 and ZnCl2. Further addition of pyrithione drastically increased the fluorescence intensity of both dyes, while the addition of the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) rapidly and drastically decreased FluoZin-3 fluorescence. In cells loaded with FluoZin-3 alone, the addition of ZnCl2 induced a gradual increase in the fluorescence in a fashion independent of added CaCl2 but sensitive to EGTA. Significant inhibition was found in the vitality to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide in a manner sensitive to TPEN, EDTA and BAPTA in C6 glioma cells exposed to ZnCl2, with pyrithione accelerating the inhibition. Similar inhibition occurred in an EGTA-sensitive fashion after brief exposure to ZnCl2 in pluripotent P19 cells, neuronal Neuro2A cells and microglial BV2 cells, which all expressed mRNA for particular zinc transporters.
  
  
  CONCLUSIONS
  Taken together, comprehensive analysis is absolutely required for the demonstration of a variety of physiological and pathological responses mediated by Ca(2+) in diverse cells enriched of Zn(2+).
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Medical, Pharmaceutical and Health Sciences, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Fujikawa
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Medical, Pharmaceutical and Health Sciences, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Fukumori
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Medical, Pharmaceutical and Health Sciences, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Medical, Pharmaceutical and Health Sciences, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Takaya
authors.initials: T
authors.lastname: Kutsukake
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Medical, Pharmaceutical and Health Sciences, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Takarada
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Medical, Pharmaceutical and Health Sciences, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yoneda
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D000001:Calcimycin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured / Q000378:metabolism*; D002614:Chelating Agents / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D005021:Ethylamines / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism*; D051379:Mice; D011083:Polycyclic Compounds / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D051381:Rats; D013778:Tetrazolium Salts / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*; D017967:Zinc Compounds / Q000378:metabolism
chemical_list: C000598529:3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; D000814:Aniline Compounds; D002614:Chelating Agents; D002712:Chlorides; D005021:Ethylamines; C451182:FluoZin-3; D007202:Indicators and Reagents; C511824:N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine; D011083:Polycyclic Compounds; D011725:Pyridines; D013778:Tetrazolium Salts; D014966:Xanthenes; D017967:Zinc Compounds; C059715:Fluo-3; D000001:Calcimycin; C016837:zinc chloride; D015032:Zinc; D002118:Calcium