title: Synthesis and characterization of a fluorescent substrate for the N-arachidonoylethanolamine (anandamide) transmembrane carrier.
abstract: N-Arachidonoylethanolamine (AEA) is a proposed endogenous ligand of the central cannabinoid receptor (CB1). Previous studies indicate that AEA is translocated across membranes via a process that has the characteristics of carrier-mediated facilitated diffusion. To date, studies of this mechanism have relied on [(3)H]AEA as a substrate for the carrier. We have synthesized an analog of AEA, SKM 4-45-1, that is nonfluorescent in the extracellular environment. When SKM 4-45-1 is exposed to intracellular esterases, it is de-esterified and becomes fluorescent. We have carried out studies to demonstrate that SKM 4-45-1 accumulation in cells occurs via the AEA carrier. SKM 4-45-1 is accumulated by both cerebellar granule cells and C6 glioma cells. Uptake of SKM 4-45-1 into C6 glioma is inhibited by AEA (IC(50)=53.8 +/- 1.8 microM), arachidonoyl-3-aminopyridine amide (IC(50)=10.1 +/- 1.4 microM), and arachidonoyl-4-hydroxyanilineamide (IC(50)=6.1 +/- 1.3 microM), all of which also inhibit [(3)H]AEA accumulation. Conversely, [(3)H]AEA accumulation by cerebellar granule cells is inhibited by SKM 4-45-1 with an IC(50) of 7.8 +/- 1. 3 microM. SKM 4-45-1 is neither a substrate nor inhibitor of fatty acid amide hydrolase, an enzyme that catabolizes AEA. SKM 4-45-1 does not bind the CB1 cannabinoid receptor at concentrations <10 microM. In summary, the cellular accumulation of SKM 4-45-1 occurs via the same pathway as AEA uptake and provides an alternative substrate for the study of this important cellular process.
authors.affiliation: Department of Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
authors.forename: S
authors.initials: S
authors.lastname: Muthian
authors.forename: K
authors.initials: K
authors.lastname: Nithipatikom
authors.forename: W B
authors.initials: WB
authors.lastname: Campbell
authors.forename: C J
authors.initials: CJ
authors.lastname: Hillard
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D001932:Brain Neoplasms / Q000378:metabolism; D002186:Cannabinoids / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003511:Cyclohexanols / Q000378:metabolism; D063388:Endocannabinoids; D004950:Esterases / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism; D006801:Humans; D007783:Lactones / Q000138:chemical synthesis / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D053284:Polyunsaturated Alkamides; D051381:Rats; D017207:Rats, Sprague-Dawley; D014407:Tumor Cells, Cultured; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D001095:Arachidonic Acids; D002186:Cannabinoids; D002352:Carrier Proteins; D003511:Cyclohexanols; D063388:Endocannabinoids; D004950:Esterases; D005456:Fluorescent Dyes; D007783:Lactones; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C406526:SKM 4-45-1; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; C078814:anandamide