Calcium signals induced by the endoplasmic reticulum ca2+ pump inhibitor: Cyclopiazonic acid in renal tubular cells

W. C. Chen, J. S. Chen, J. C.R. Tsai, C. R. Jan*

*Corresponding author for this work

Research output: Contribution to journalJournal Article peer-review

Abstract

Cyclopiazonic acid (CPA) is commonly used as a selective inhibitor of the endoplasmic reticulum (ER) Ca2+ pump, however, its effect on Ca+ signaling is unclear. The present study explored the effect of CPA on intracellular Ca2+ concentrations ([Ca2+]i) using Madin Darby canine kidney (MDCK) cells as a model. The Ca2+-sensitive fluorescent dye, fura 2, was used to measure [Ca2+]ichanges. CPA caused a dose-dependent rise in [Ca2+]i with an EC50 of 10 μM. The Ca2+ signal comprised an initial rise, a gradual decay and a plateau. The signal was due to external Ca2+ influx and internal Ca2+ release because removal of external Ca2+ reduced 50 μM CPA-induced response by 50%. Depletion of the ER Ca2+ store with 50 μM CPA for 5 rain abolished the [Ca2+]i rise induced by ATP, bradykinin, or thapsigargin. This suggests that CPA discharged IP3-sensitive ER Ca2+ stores. CPA induced a dose-dependent capacitative Ca2+ entry. CPA (50 μM) induced Mn2+ quench of fura-2 fluorescence, confirming that CPA induced Ca2+ influx. This Mn2+ quench was inhibited by 50 μM Gd3+ by 50%. Pretreatment with Gd3+ partly inhibited the rise of the CPA-induced [Ca2+]i signal but significantly slowed down the decay, while pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP) or removal of external Na+ had little effect. The findings suggest that CPA increases [Ca2+]i in MDCK cells by depleting the internal Ca2+ stores followed by capacitative Ca2+ entry, with both pathways contributing approximately equally. The decay of the CPA response might involve efflux via the plasmalemmal Ca2+ pump, but not efflux via Na+/Ca2+ exchange or sequestration by mitochondria or the ER.

Original languageEnglish
Pages (from-to)191-198
Number of pages8
JournalIn Vitro and Molecular Toxicology: Journal of Basic and Applied Research
Volume13
Issue number3
StatePublished - 2000
Externally publishedYes

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