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

• Published in: Life sciences (1973) 1998-12, Vol.64 (4), p.259-267
• Main Authors: Jan, Chung-Ren, Ho, Chin-Man, Wu, Sheng-Nan, Tseng, Ching-Jiunn
• Format: Article
• Language: English
• Subjects: capacitative Ca 2+ entry; fura-2; Gd 3; MDCK cells; thapsigargin
• ISSN: 0024-3205; 1879-0631
• DOI: 10.1016/S0024-3205(98)00561-X

We studied the effect of thapsigargin on intracellular calcium levels ([Ca 2+] i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Thapsigargin elevated [Ca 2+] i dose dependently with an EC 50 of ~ 0.15 μM. The Ca 2+ signal consisted of a slow rise, a gradual decay and a plateau. Depletion of the endoplasmic reticulum Ca 2+ store with thapsigargin for 7 min abolished the [Ca 2+] i increases evoked by bradykinin. Removal of extracellular Ca 2+ reduced the thapsigargin response by ~50%. The Ca 2+ signal was initiated by Ca 2+ release from the internal store followed by capacitative Ca 2+ entry (CCE). The thapsigargin-evoked CCE was abolished by La 3+ and Gd 3+, and was partly inhibited by SKF 96365 and econazole. After depletion of the internal Ca 2+ store for 30 min with another inhibitor of the internal Ca 2+ pump, cyclopiazonic acid, thapsigargin failed to increase [Ca 2+] i, thus suggesting that the thapsigargin-evoked Ca 2+ influx was solely due to CCE. We investigated the mechanism of decay of the thapsigargin response. Pretreatemt with La 3+ (or Gd 3+) or alkalization of extracellular medium to pH 8 significantly potentiated the Ca 2+ signal; whereas pretreatment with carbonylcyanide m-chlorophynylhydrozone (CCCP) or removal of extracellular Na + had no effect. Collectively, our results imply that thapsigargin increased [Ca 2+] i in MDCK cells by depleting the internal Ca 2+ store followed by CCE, with both pathways contributing equally. The decay of the thapsigargin response might be significantly governed by efflux via the plasmalemmal Ca 2+ pump.