Effect of betulinic acid on intracellular-free Ca²⁺ levels in Madin Darby canine kidney cells

The effect of betulinic acid, an anti-tumor and apoptosis-inducing natural product, on intracellular-free levels of Ca²⁺ ([Ca²⁺](i)) in Madin Darby canine kidney (MDCK) cells was examined by using fura-2 as a Ca²⁺ dye. Betulinic acid caused significant increases in [Ca²⁺](i) concentration dependently between 25 and 500 nM with an EC₅₀ of 100 nM. The [Ca²⁺](i) signal was composed of an initial gradual rise and a plateau. The response was decreased by removal of extracellular Ca²⁺ by 45±10%. In Ca²⁺-free medium, pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) abolished 250 μM betulinic acid-induced [Ca²⁺](i) increases. Conversely, pretreatment with betulinic acid only partly inhibited thapsigargin-induced [Ca²⁺](i) increases. Addition of 3 mM Ca²⁺ induced a [Ca²⁺](i) increase after pretreatment with 250 nM betulinic acid in Ca²⁺-free medium for 5 min. This [Ca²⁺](i) increase was not altered by the addition of 20 μM SKF96365 and 10 μM econazole. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 μM) abolished 250 nM betulinic acid-induced Ca²⁺ release. Pretreatment with 10 μM La³⁺ inhibited 250 nM betulinic acid-induced [Ca²⁺](i) increases by 85±3%; whereas 10 μM of verapamil, nifedipine and diltiazem had no effect. In Ca²⁺ medium, pretreatment with 2.5 nM betulinic aid for 260 s potentiated 10 μM ATP and 1 μM thapsigargin-induced [Ca²⁺](i) increases by 33±3% and 45±3%, respectively. Trypan blue exclusion revealed that acute exposure of 250 nM betulinic acid for 2-30 min decreased cell viability by 6±2%, which could be prevented by pretreatment with 2 μM U731222. Together, the results suggest that betulinic acid induced significant [Ca²⁺](i) increases in MDCK cells in a concentration-dependent manner, and also induced mild cell death. The [Ca²⁺](i) signal was contributed by an inositol 1,4,5-trisphosphate-dependent release of intracellular Ca²⁺ from thapsigargin-sensitive stores, and by inducing Ca²⁺ entry from extracellular medium in a La³⁺-sensitive manner. (C) 2000 Elsevier Science B.V.