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 muM, AM-404 increased [Ca2+](i) in a concentration-dependent fashion with an EC50 value of 20 muM. Removal of extracellular Ca2+ abolished the [Ca2+](i) signals. The [Ca2+](i) increase was nearly abrogated by 10 muM La3+, but was insensitive to 50 muM Ni2+ and 10 muM of nifedipine, nimodipine, nicardipine, and verapamil. At a concentration that did not increase [Ca2+](i), AM-404 (1 muM) did not alter the [Ca2+](i) increases induced by 10 muM ATP and 1 muM bradykinin. AM-404 (5 muM) 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. (C) 2002 Elsevier Science Inc. All rights reserved.