ACCELERATED COMMUNICATION Role of Fatty Acid Amide Hydrolase in the Transport of the Endogenous Cannabinoid Anandamide

A facilitated transport process that removes the endogenous cannabinoid anandamide from extracellular spaces has been identified. Once transported into the cytoplasm, fatty acid amide hydrolase (FAAH) is responsible for metabolizing the accumulated anandamide. We propose that FAAH contributes to anandamide uptake by creating and maintaining an inward concentration gradient for anandamide. To explore the role of FAAH in anandamide transport, we examined anandamide metabolism and uptake in RBL-2H3 cells, which natively express FAAH, as well as wild-type HeLa cells that lack FAAH. RBL-2H3 and FAAH-transfected HeLa cells demonstrated a robust ability to metabolize anandamide compared with vector-transfected HeLa cells. This activity was reduced to that observed in wildtype HeLa cells upon the addition of the FAAH inhibitor methyl arachidonyl fluorophosphonate. Anandamide uptake was reduced in a dose-dependent manner by various FAAH inhibitors in both RBL-2H3 cells and wild-type HeLa cells. Anandamide uptake studies in wild-type HeLa cells showed that only FAAH inhibitors structurally similar to anandamide decreased anandamide uptake. Because there is no detectable FAAH activity in wild-type HeLa cells, these FAAH inhibitors are probably blocking uptake via actions on a plasma membrane transport protein. Phenylmethylsulfonyl fluoride, a FAAH inhibitor that is structurally unrelated to anandamide, inhibited anandamide uptake in RBL-2H3 cells and FAAH-transfected HeLa cells, but not in wild-type HeLa cells. Furthermore, expression of FAAH in HeLa cells increased maximal anandamide transport 2-fold compared with wild-type HeLa cells. These results suggest that FAAH facilitates anandamide uptake but is not solely required for transport to occur. The discovery of the G protein-coupled CB1 and CB2 cannabinoid receptors (Matsuda et al., 1990; Munro et al., 1993) that are activated by D-tetrahydrocannabinol prompted the search for an endogenous agonist for these receptors. Several endocannabinoids were discovered in the early 1990s, the first being N-arachidonylethanolamide, or anandamide (Devane et al., 1992). Anandamide is a long-chain fatty acid amide that is believed to have therapeutic potential similar to marijuana (Devane et al., 1992). Some of these potential medicinal properties include: suppression of nausea and vomiting; appetite stimulation; alleviation of side effects associated with Parkinson’s disease and multiple sclerosis; reduction of intraocular pressure from glaucoma; treatment of pain, especially migraines; and regulation of memory, cognition, fever, blood pressure, and the immune system (Mechoulam et al., 1998; Mechoulam and Ben Shabat, 1999). Anandamide is considered a putative neurotransmitter, with its metabolism occurring intracellularly by a fatty acid amide hydrolase (FAAH) that cleaves anandamide into arachidonic acid and ethanolamine (Schmid et al., 1985; Deutsch and Chin, 1993; Desarnaud et al., 1995; Ueda et al., 1995; Cravatt et al., 1996). FAAH metabolizes several other fatty acid amides and esters, such as oleamide and the endocannabinoid 2-arachidonyl glycerol (Cravatt et al., 1996; Patterson et al., 1996). Although anandamide is capable of being synthesized by FAAH from its components arachidonic acid and ethanolamine in vitro (Devane and Axelrod, 1994; Ueda This work was supported in part by a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression (E.L.B.) and National Institutes of Health Grant R21-DA13268 (E.L.B.). This work appeared in abstract form in Day TA, Rakhshan F and Barker EL (2000) Role of fatty acid amide hydrolase in the uptake of the endogenous cannabinoid anandamide. Society for Neuroscience 30 Annual Meeting Abstract. ABBREVIATIONS: FAAH, fatty acid amide hydrolase; PMSF, phenylmethylsulfonyl fluoride; MAFP, methyl arachidonyl fluorophosphonate; ECL, enhanced chemiluminescence; KRH, Krebs-Ringer-HEPES; AM404, N-(4-hydroxyphenyl)-arachidonamide; ATFK, arachidonyl trifluoromethyl ketone; A.5-HT, arachidonoyl serotonin. 0026-895X/01/5906-1369–1375$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 6 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 861/908118 Mol Pharmacol 59:1369–1375, 2001 Printed in U.S.A. 1369 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from et al., 1995; Arreaza et al., 1997), the physiological concentrations of ethanolamine and arachidonic acid are not large enough to make this a plausible route in vivo (Schmid et al., 1990; Piomelli, 1994). Increases in intracellular calcium concentrations have been shown to stimulate the formation, cleavage, and release of anandamide from a membrane phospholipid precursor, N-arachidonoyl phosphatidylethanolamine (Di Marzo et al., 1994, 1996; Cadas et al., 1996, 1997). Once released from the membrane into the extracellular space, anandamide can activate CB1 receptors in the central nervous system, or CB2 receptors in the periphery (Devane et al., 1992; Felder et al., 1993; Pertwee et al., 1993; Mackie et al., 1993). Termination of anandamide signaling at the cannabinoid receptors occurs through an uptake mechanism that transports anandamide into the cell where it subsequently undergoes rapid degradation by FAAH (Ueda et al., 1995; Cravatt et al., 1996; Hillard et al., 1997; Beltramo et al., 1997; Piomelli et al., 1999). Current evidence suggests that anandamide uptake is a carrier-mediated process that is timeand temperature-dependent, saturable, and inhibited with a unique pharmacologic profile (Di Marzo et al., 1994; Beltramo et al., 1997; Rakhshan et al., 2000). Colocalization of FAAH and CB1 receptors in rat brain may indicate FAAH’s participation in anandamide signaling and uptake (Thomas et al., 1997; Egertova et al., 1998; Yazulla et al., 1999). The putative transmembrane domain and SH3-domain-binding sequence of FAAH suggests that FAAH may localize with the plasma membrane and associated proteins (Cravatt et al., 1996). We propose that FAAH may establish facilitated anandamide uptake by creating and maintaining an inward concentration gradient of anandamide. To determine the role of FAAH in anandamide uptake, we have studied anandamide uptake and FAAH enzymatic activity in RBL-2H3 cells that natively express FAAH and wild-type HeLa cells that lack FAAH. Although HeLa cells lack FAAH, we detected anandamide transport activity that had kinetics similar to RBL-2H3 cells. Additionally, our results revealed that FAAH inhibitors structurally similar to anandamide not only inhibited FAAH, but also decreased anandamide uptake, possibly by recognizing a distinct extracellularly accessible plasma membrane transporter. Phenylmethylsulfonyl fluoride (PMSF), a FAAH inhibitor that is structurally unrelated to anandamide, did not inhibit anandamide uptake in wild-type HeLa cells. However, FAAHtransfected HeLa cells and RBL-2H3 cells showed reduced anandamide uptake in the presence of PMSF, suggesting that FAAH inhibition may also reduce transport. Furthermore, expression of FAAH in HeLa cells increased maximal anandamide transport compared with wild-type HeLa cells, thus confirming a role for FAAH in facilitated anandamide uptake. Our data using wild-type and FAAH-transfected HeLa cells indicated that although FAAH is not required for anandamide uptake, FAAH may work in conjunction with other membrane proteins to facilitate anandamide transport. Materials and Methods FAAH Enzymatic Activity Assay. Experiments were performed on HeLa cells as described previously (Rakhshan et al., 2000). Wildtype HeLa cells, or HeLa cells transiently transfected with rat FAAH cDNA (generous gift from Dr. Ben Cravatt, Scripps Research Institute)/pBluescript II SK (Cravatt et al., 1996) using the vaccinia virus T7 expression system (Fuerst et al., 1986; Blakely et al., 1991), were washed with KRH buffer, scraped into 1.5-ml tubes with TrisEDTA buffer (20 mM Tris-HCl, 1 mM EDTA, pH 9.0, 0.7 mg/ml pepstatin A, and 0.5 mg/ml leupeptin), and homogenized. Lysed cell enzymatic assays were performed by a modification of a method published previously (Omeir et al., 1995). Membrane preparations were incubated with 5 nM anandamide (ethanolamine 1-H) (American Radiolabeled Chemicals, St. Louis, MO) for 5 min in the presence or absence of 500 nM methyl arachidonyl fluorophosphonate (MAFP). Reactions were terminated with the addition of 23 volume of chloroform/methanol (1:1, v/v). Production of [H]ethanolamine in the aqueous phase was compared with intact anandamide (ethanolamine 1-H) in the organic phase by liquid scintillation counting on a TopCount scintillation plate analyzer (Packard, Meriden, CT). Saturation kinetics were determined by using 125 mg of homogenized cell lysate from RBL-2H3 cells or FAAH-transfected HeLa cells and increasing concentrations of anandamide (ethanolamine 1-H) (American Radiolabeled Chemicals), with the specific activity diluted to ;0.9 Ci/mmol with nonisotopic anandamide. After a 5-min incubation at 37°C in the presence or absence of 10 mM MAFP, the reaction was terminated with the addition of 23 volume of chloroform/methanol (1:1, v/v). Production of [H]ethanolamine in the aqueous phase was compared with intact anandamide (ethanolamine 1-H) in the organic phase by liquid scintillation counting on a Packard TopCount scintillation plate analyzer. FAAH Vmax and Km values were derived by nonlinear least-square fits with Prism software (v. 3.0; GraphPad, San Diego, CA). Western Blot Analysis. Total postnuclear and plasma membrane enriched cell lysates were prepared using a method published previously (Stuhlsatz-Krouper et al., 1998). Briefly, HeLa or RBL2H3 cells were grown to confluence, washed with KRH buffer, and homogenized in 255 mM sucrose, 20 mM Tris, pH 7.4, 1 mM EDTA, and 1 mg/ml each of pepstatin A and leupeptin. For total postnuclear protein preparations, nuclei were removed by centrifugation at 1,000g for 10 min, followed by centrifugation of the supernatant at 356,000g for 30 min at 4°C. The pellet was resuspended in 1% Triton X-100, 50 mM Tris, pH 7.4, 2 mM EDTA, 150 mM NaCl with 1 mg/ml each of pepstatin and leupeptin. For plasma membrane enriched protein preparations, homogenized cell lysates were pelleted at 16,000g for 20 min, resuspended in the sucrose buffer mentioned above, placed on a 1.12 M sucrose layer, and centrifuged at 99,000g for 20 min, which resulted in an interfacial plasma membrane protein fraction. Protein samples were quantified by the Pierce bicinchoninic acid assay (Rockford, IL) and prepared for gel electrophoresis with the addition of 13 volume Laemmli buffer (62.5 mM TrisHCl, 20% glycerol, 2% SDS, 5% b-mercaptoethanol, and 5% bromphenol blue). Protein samples were loaded onto a 10% SDSpolyacrylamide gel electrophoresis Tris-HCl gel and electrophoresed at 150 V for ;1 h using the Mini-Protean 3 system (Bio-Rad, Hercules, CA). Resolved proteins were transferred to a polyvinylidene difluoride membrane using the Bio-Rad Mini Trans-Blot system. The membrane was blocked overnight in phosphate-buffered saline containing 0.1% Tween-20 and 5% (w/v) nonfat dry milk at 4°C. To determine the presence of FAAH, a-FAAH polyclonal 1° antibody (gift from Dr. Ben Cravatt, Scripps Research Institute), horseradish peroxidase-labeled goat-anti-rabbit 2° antibody (Bio-Rad), and ECL detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ) were used, followed by exposure to X-ray film (Amersham Pharmacia