Mechanism of rise and decay of 2,5-di-tert-butylhydroquinone-induced Ca2+ signals in Madin Darby canine kidney cells.

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.