Mechanisms of AM404-induced [Ca2+]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 >= 5 mu M increased [Ca2+](i) in a concentration-dependent manner with an EC50 value of 60 mu M. 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 mu M 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 mu M, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 mu M 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 encloplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis. (c) 2008 Published by Elsevier Ireland Ltd.