Mechanism of rise and decay of thapsigargin-evoked calcium signals in MDCK cells

We studied the effect of thapsigargin on intracellular calcium levels ([Ca²⁺](i)) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Thapsigargin elevated [Ca²⁺](i) dose dependently with an EC₅₀ of ~ 0.15 μM. The Ca²⁺ signal consisted of a slow rise, a gradual decay and a plateau. Depletion of the endoplasmic reticulum Ca²⁺ store with thapsigargin for 7 min abolished the [Ca²⁺](i) increases evoked by bradykinin. Removal of extracellular Ca²⁺ reduced the thapsigargin response by ~50%. The Ca²⁺ signal was initiated by Ca²⁺ release from the internal store followed by capacitative Ca²⁺ entry (CCE). The thapsigargin-evoked CCE was abolished by La³⁺ and Gd³⁺, and was partly inhibited by SKF 96365 and econazole. After depletion of the internal Ca²⁺ store for 30 min with another inhibitor of the internal Ca²⁺ pump, cyclopiazonic acid, thapsigargin failed to increase [Ca²⁺](i), thus suggesting that the thapsigargin-evoked Ca²⁺ influx was solely due to CCE. We investigated the mechanism of decay of the thapsigargin response. Pretreatment with La³⁺ (or Gd³⁺)) or alkalization of extracellular medium to pH 8 significantly potentiated the Ca²⁺ signal; whereas pretreatment with carbonylcyanide m- chlorophynylhydrozone (CCCP) or removal of extracellular Na⁺ had no effect. Collectively, our results imply that thapsigargin increased [Ca²⁺](i) in MDCK cells by depleting the internal Ca²⁺ store followed by CCE, with both pathways contributing equally. The decay of the thapsigargin response might be significantly governed by efflux via the plasmalemmal Ca²⁺ pump.