title: Mechanism of rise and decay of 2,5-di-tert-butylhydroquinone-induced Ca2+ signals in Madin Darby canine kidney cells.
abstract: We examined the effect of 2,5-di-tert-butylhydroquinone (BHQ) on intracellular Ca2+ concentrations ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. BHQ increased [Ca2+]i in a dose-dependent manner with an EC50 of 40 microM. The Ca2+ signal showed a slow onset, a gradual decay and a sustained plateau in normal Ca2+ medium. Depletion of the endoplasmic reticulum Ca2+ store by incubation with 0.1 mM BHQ for 6 min abolished the [Ca2+]i increase evoked by bradykinin or ATP, suggesting that BHQ depleted the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store. Removal of extracellular Ca2+ reduced the BHQ response by 50%. The Ca2+ signal was initiated by Ca2+ release from the internal store, followed by capacitative Ca2+ entry which was abolished by 100 microM La3+ or 50 microM Gd3+ and was partly inhibited by 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365). After depletion of the endoplasmic reticulum Ca2+ store, by incubation with another inhibitor of the endoplasmic reticulum Ca2+ pump, thapsigargin for 30 min, BHQ did not elevate [Ca2+]i, suggesting that the BHQ-induced Ca2+ influx was largely due to capacitative Ca2+ entry, and that BHQ released Ca2+ from the thapsigargin-sensitive endoplasmic reticulum store. We investigated the mechanism of decay of the BHQ response. Pretreatemt with La3+ (or Gd3+) or alkalization of the extracellular medium to pH 8 significantly potentiated the Ca2+ signal, whereas pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP) or oligomycin, or removal of extracellular Na+, had no effect. Collectively, our results suggest that BHQ increased [Ca2+]i in MDCK cells by depleting the endoplasmic reticulum Ca2+ store followed by capacitative Ca2+ entry, with both pathways contributing equally. The decay of the BHQ response is effected by Ca2+ efflux via the plasma membrane Ca2+ pump, but not by efflux via Na+/Ca2+ exchange or sequestration by the mitochondria.
authors.affiliation: Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, Taiwan. crjan@isca.vghks.gov.tw
authors.forename: C R
authors.initials: CR
authors.lastname: Jan
authors.forename: C M
authors.initials: CM
authors.lastname: Ho
authors.forename: S N
authors.initials: SN
authors.lastname: Wu
authors.forename: C J
authors.initials: CJ
authors.lastname: Tseng
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000493:pharmacokinetics; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005682:Gadolinium / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones / Q000494:pharmacology*; D017136:Ion Transport; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007811:Lanthanum / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology