Effect of betulinic acid on intracellular-free Ca(2+) 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 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 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) abolished 250 microM 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 microM SKF96365 and 10 microM econazole. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 microM) abolished 250 nM betulinic acid-induced Ca(2+) release. Pretreatment with 10 microM La(3+) inhibited 250 nM betulinic acid-induced [Ca(2+)](i) increases by 85+/-3%; whereas 10 microM of verapamil, nifedipine and diltiazem had no effect. In Ca(2+) medium, pretreatment with 2.5 nM betulinic aid for 260 s potentiated 10 microM ATP and 1 microM 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 microM 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.
Chou
K J
KJ
Department of Nephrology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
Fang
H C
HC
Chung
H M
HM
Cheng
J S
JS
Lee
K C
KC
Tseng
L L
LL
Tang
K Y
KY
Jan
C R
CR
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004285:Dogs; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D053978:Pentacyclic Triterpenes; D019284:Thapsigargin / Q000494:pharmacology; D014315:Triterpenes / Q000494:pharmacology*; D000094062:Betulinic Acid
D000972:Antineoplastic Agents, Phytogenic; D002273:Carcinogens; D005456:Fluorescent Dyes; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2; D000094062:Betulinic Acid