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

Chung Ren Jan, Chin Man Ho, Sheng Nan Wu, Ching Jiunn Tseng

Research output: Contribution to journalArticlepeer-review

62 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)259-267
Number of pages9
JournalLife Sciences
Volume64
Issue number4
DOIs
Publication statusPublished - 1998 Dec 18

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

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