Carrier-Mediated Uptake of the Endogenous Cannabinoid Anandamide in RBL-2H3 Cells 1 FARIBORZ RAKHSHAN, THERESA A. DAY, RANDY D. BLAKELY, and ERIC L. BARKER Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy and Pharmacal Sciences, West Lafayette, Indiana (F.R., T.A.D., E.L.B.); Center for Molecular Neuroscience and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee (R.D.B.) Accepted for publication December 4, 1999 This paper is available online at http://www.jpet.org ABSTRACT Anandamide (N-arachidonylethanolamide) is an endogenous cannabinoid that mimics the pharmacologic effects of  9 -tet- rahydrocannabinol, the major bioactive substance in marijuana. Anandamide appears to be synthesized, released, and inacti- vated by mechanisms similar to those for other neurotransmit- ters. Of interest to the present studies are reports that anand- amide undergoes carrier-mediated uptake into neuronal or glial cells after release, followed by rapid intracellular degradation by the intracellular fatty acid amidohydrolase. In addition to effects in the brain, anandamide has multiple effects in the periphery, particularly on cells of the immune system that ex- press both a peripheral cannabinoid receptor and amidohydro- lase enzyme. We have performed a detailed characterization of anandamide uptake in the cognate mast cell line RBL-2H3 to test the hypothesis that the uptake system in peripheral cells is also carrier-mediated and functionally similar to that observed in the central nervous system. RBL-2H3 cells exhibited robust, saturable transport of [ 3 H]anandamide that was both time- and temperature-sensitive. This transport activity was not depen- dent on extracellular ion gradients for uptake and was inhibited selectively by other fatty acid-derived molecules, anandamide congeners, and the psychoactive cannabinoids such as  9 - tetrahydrocannabinol. We conclude that anandamide transport in the RBL-2H3 cells is carrier-mediated, and uptake in periph- eral cells is functionally and pharmacologically identical with that observed in neurons and astrocytes. Anandamide (N-arachidonylethanolamide), 2-arachidonyl- glycerol (2-AG), and a family of fatty acid ethanolamides have been identified as endogenous cannabinoids (Devane et al., 1992; Stella et al., 1997). Biochemical and pharmacologic evidence indicates that these fatty acid-derived neuromodu- lators act via the cloned cannabinoid receptors (CB1 and CB2) to elicit similar behavioral and physiologic effects to the psychoactive cannabinoids like  9 -tetrahydrocannabinol ( 9 - THC), thus supporting the assertion that anandamide is an endogenous cannabinoid (Crawley et al., 1993; Felder et al., 1993, 1995; Fride and Mechoulam, 1993; Smith et al., 1994). Although the precise physiologic role of the endogenous can- nabinoids has not been fully elucidated, anandamide and 2-AG have been implicated in modulation of memory, cogni- tion, blood pressure, pain, fever, and the immune system and as having potentially therapeutic effects in conditions such as convulsions, glaucoma, movement disorders, and multiple sclerosis (Hirst et al., 1998). As putative neuromodulators, mechanisms must exist for the synthesis, release, and termination of endocannabinoid signaling. Piomelli and coworkers (Di Marzo et al., 1994; Cadas et al., 1997) have performed a series of elegant studies implicating a calcium-dependent phosphodiesterase-medi- ated cleavage of a membrane phospholipid precursor, N- arachidonoyl-phosphatidylethanolamine, as the major route of fatty acid amide biosynthesis. Once formed and released, anandamide is rapidly transported into neurons and astro- cytes for subsequent hydrolytic degradation to ethanolamine and arachidonic acid (Deutsch and Chin, 1993; Di Marzo et al., 1994; Cravatt et al., 1996; Beltramo et al., 1997; Hillard et al., 1997). The fatty acid amidohydrolase (FAAH) respon- sible for anandamide metabolism has been cloned, and func- tional studies reveal that 2-AG and the sleep-inducing lipid, oleamide, also can serve as substrates for this enzyme (Cra- vatt et al., 1996; Goparaju et al., 1998). Thus, both synthesis and catalysis pathways exist for anandamide in the central nervous system. However, for degradation to occur, anand- amide first must be transported into cells possessing the FAAH activity, making the uptake process a critical and potentially rate-limiting step in the metabolism of anandam- ide. Received for publication September 2, 1999. 1 This work was supported in part by a research grant from Bristol-Myers Squibb. ABBREVIATIONS: 2-AG, 2-arachidonylglycerol; THC, tetrahydrocannabinol; 11-OH- 9 -THC, 11-hydroxy-nor- 9 -THC; FAAH, fatty acid amidohy- drolase; AM404, N-(4-hydroxyphenyl)-arachidonamide; KRH, Krebs-Ringer-HEPES; PMSF, phenylmethylsulfonyl fluoride; ATFK, arachidonyl trifluoromethyl ketone; MAFP, methyl arachidonyl fluorophosphonate. 0022-3565/00/2923-0960$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 292, No. 3 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 292:960–967, 2000 960 at ASPET Journals on May 23, 2016 jpet.aspetjournals.org Downloaded from