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 μM. 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 μM La³⁺ or 50 μM Gd³⁺ and was partly inhibited by 1-[β-[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. Copyright (C) 1999 Elsevier Science B.V.