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 muM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) abolished 250 muM 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 muM SKF96365 and 10 muM econazole. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 muM) abolished 250 nM betulinic acid-induced Ca2+ release. Pretreatment with 10 muM La3+ inhibited 250 nM betulinic acid-induced [Ca2+](i) increases by 85 +/- 3%; whereas 10 muM of verapamil, nifedipine and diltiazem had no effect, In Ca2+ medium, pretreatment with 2.5 nM betulinic aid for 260 a potentiated 10 muM ATP and 1 muM 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 muM 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. All rights reserved.