title: AM-404 elevates renal intracellular Ca(2+), questioning its selectivity as a pharmacological tool for investigating the anandamide transporter.
abstract: The effect of N-(4-hydroxyphenyl)-arachidonamide (AM-404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca(2+) levels ([Ca(2+)](i)) was studied in Madin Darby canine kidney (MDCK) cells. [Ca(2+)](i) was measured using fura-2 as a Ca(2+) indicator. Between 2 and 40 microM, AM-404 increased [Ca(2+)](i) in a concentration-dependent fashion with an EC(50) value of 20 microM. Removal of extracellular Ca(2+) abolished the [Ca(2+)](i) signals. The [Ca(2+)](i) increase was nearly abrogated by 10 microM La(3+), but was insensitive to 50 microM Ni(2+) and 10 microM of nifedipine, nimodipine, nicardipine, and verapamil. At a concentration that did not increase [Ca(2+)](i), AM-404 (1 microM) did not alter the [Ca(2+)](i) increases induced by 10 microM ATP and 1 microM bradykinin. AM-404 (5 microM) also increased [Ca(2+)](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 [Ca(2+)](i) in different cell types. The [Ca(2+)](i) increase was solely due to extracellular Ca(2+) influx. Thus caution must be exercised in using AM-404 as a selective inhibitor of the anandamide transporter.
authors.affiliation: Department of Surgery, Ping Tung Christian Hospital, Taipei 900, Taiwan.
authors.forename: W C
authors.initials: WC
authors.lastname: Chen
authors.forename: J K
authors.initials: JK
authors.lastname: Huang
authors.forename: J S
authors.initials: JS
authors.lastname: Cheng
authors.forename: J C
authors.initials: JC
authors.lastname: Tsai
authors.forename: A J
authors.initials: AJ
authors.lastname: Chiang
authors.forename: K J
authors.initials: KJ
authors.lastname: Chou
authors.forename: C P
authors.initials: CP
authors.lastname: Liu
authors.forename: C R
authors.initials: CR
authors.lastname: Jan
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology*; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D016257:Fura-2 / Q000493:pharmacokinetics; D006801:Humans; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D007811:Lanthanum / Q000494:pharmacology; D009529:Nicardipine / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D009553:Nimodipine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
chemical_list: D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D009553:Nimodipine; D007811:Lanthanum; D009532:Nickel; D000255:Adenosine Triphosphate; D014700:Verapamil; D009529:Nicardipine; D009543:Nifedipine; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2; C107594:N-(4-hydroxyphenyl)arachidonylamide