Mechanisms of AM404-induced [Ca 2+] i rise and death in human osteosarcoma cells

Abstract

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca 2+ levels ([Ca 2+] i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations ≥5 μM increased [Ca 2+] i in a concentration-dependent manner with an EC 50 value of 60 μM. The Ca 2+ signal was reduced partly by removing extracellular Ca 2+. AM404 induced Mn 2+ quench of fura-2 fluorescence implicating Ca 2+ influx. The Ca 2+ influx was sensitive to La 3+, Ni 2+, nifedipine and verapamil. In Ca 2+-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor), AM404-induced [Ca 2+] i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca 2+] i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca 2+] i rise. At concentrations between 10 and 200 μM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 μM AM404 was partly reversed by prechelating cytosolic Ca 2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca 2+] i rise by causing Ca 2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca 2+ influx via L-type Ca 2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.