Mechanisms of AM404-induced [Ca(2+)](i) rise and death in human osteosarcoma cells.
The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca2+ levels ([Ca2+]i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. AM404 induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, Ni2+, nifedipine and verapamil. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), AM404-induced [Ca2+]i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca2+]i rise. At concentrations between 10 and 200 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM AM404 was partly reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.
Chang
Hong-Tai
HT
Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
Huang
Chorng-Chih
CC
Cheng
He-Hsiung
HH
Wang
Jue-Long
JL
Lin
Ko-Long
KL
Hsu
Pei-Te
PT
Tsai
Jeng-Yu
JY
Liao
Wei-Chuan
WC
Lu
Yih-Chau
YC
Huang
Jong-Khing
JK
Jan
Chung-Ren
CR
D017209:Apoptosis / Q000187:drug effects*; D001095:Arachidonic Acids / Q000633:toxicity*; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D012516:Osteosarcoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D013778:Tetrazolium Salts / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D013778:Tetrazolium Salts; D019284:Thapsigargin; D002118:Calcium; D016257:Fura-2; C107594:N-(4-hydroxyphenyl)arachidonylamide