Effect of betulinic acid on intracellular-free Ca 2+ levels in Madin Darby canine kidney cells

Abstract

The effect of betulinic acid, an anti-tumor and apoptosis-inducing natural product, on intracellular-free levels of Ca 2+ ([Ca 2+] i) in Madin Darby canine kidney (MDCK) cells was examined by using fura-2 as a Ca 2+ dye. Betulinic acid caused significant increases in [Ca 2+] i concentration dependently between 25 and 500 nM with an EC 50 of 100 nM. The [Ca 2+] i signal was composed of an initial gradual rise and a plateau. The response was decreased by removal of extracellular Ca 2+ by 45±10%. In Ca 2+-free medium, pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor) abolished 250 μM betulinic acid-induced [Ca 2+] i increases. Conversely, pretreatment with betulinic acid only partly inhibited thapsigargin-induced [Ca 2+] i increases. Addition of 3 mM Ca 2+ induced a [Ca 2+] i increase after pretreatment with 250 nM betulinic acid in Ca 2+-free medium for 5 min. This [Ca 2+] 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 Ca 2+ release. Pretreatment with 10 μM La 3+ inhibited 250 nM betulinic acid-induced [Ca 2+] i increases by 85±3%; whereas 10 μM of verapamil, nifedipine and diltiazem had no effect. In Ca 2+ medium, pretreatment with 2.5 nM betulinic aid for 260 s potentiated 10 μM ATP and 1 μM thapsigargin-induced [Ca 2+] 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 [Ca 2+] i increases in MDCK cells in a concentration-dependent manner, and also induced mild cell death. The [Ca 2+] i signal was contributed by an inositol 1,4,5-trisphosphate-dependent release of intracellular Ca 2+ from thapsigargin-sensitive stores, and by inducing Ca 2+ entry from extracellular medium in a La 3+-sensitive manner.