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

Cyclopiazonic acid (CPA) is commonly used as a selective inhibitor of the endoplasmic reticulum (ER) Ca²⁺ pump, however, its effect on C^a+ 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 C^a2+ release because removal of external Ca²⁺ reduced 50 μM CPA-induced response by 50%. Depletion of the ER C^a2+ 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 C^a2+ 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.