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 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 60 μM. 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 μM 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 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 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.