AM-404 elevates renal intracellular Ca²⁺, questioning its selectivity as a pharmacological tool for investigating the anandamide transporter

The effect of N-(4-hydroxyphenyl)-arachidonamide (AM-404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca²⁺ levels ([Ca²⁺]_i) was studied in Madin Darby canine kidney (MDCK) cells. [Ca²⁺]_i was measured using fura-2 as a Ca²⁺ indicator. Between 2 and 40 μM, AM-404 increased [Ca²⁺]_i in a concentration-dependent fashion with an EC₅₀ value of 20 μM. Removal of extracellular Ca²⁺ abolished the [Ca²⁺]_i signals. The [Ca²⁺]_i increase was nearly abrogated by 10 μM La³⁺, but was insensitive to 50 μM Ni²⁺ and 10 μM of nifedipine, nimodipine, nicardipine, and verapamil. At a concentration that did not increase [Ca²⁺]_i, AM-404 (1 μM) did not alter the [Ca²⁺]_i increases induced by 10 μM ATP and 1 μM bradykinin. AM-404 (5 μM) also increased [Ca²⁺]_i in Chang liver cells, PC3 human prostate cancer cells, BFTC human bladder cancer cells, and MG63 human osteoblast-like cells. Together, this study shows for the first time that AM-404 at concentrations commonly used to inhibit the anandamide transporter in various systems induced an increase in [Ca²⁺]_i in different cell types. The [Ca²⁺]_i increase was solely due to extracellular Ca²⁺ influx. Thus caution must be exercised in using AM-404 as a selective inhibitor of the anandamide transporter.