Phytocannabinoids, such as the principal bioactive component of marijuana, ⌬ 9 -tetrahydrocannabinol, have been used for thousands of years for medical and recreational purposes. ⌬ 9 -Tetrahydrocannabinol and endogenous cannabinoids (e.g., anandamide) initiate their agonist properties by stimulating the cannabinoid family of G protein-coupled receptors (CB 1 and CB2). The biosynthesis and physiology of anandamide is well understood, but its mechanism of uptake (resulting in signal termination by fatty acid amide hydrolase) has been elusive. Mounting evidence points to the existence of a specific anandamide transport protein; however, no direct evidence for this protein has been provided. Here, we use a potent, competitive small molecule inhibitor of anandamide uptake (LY2318912, IC 50 7.27 ؎ 0.510 nM) to identify a high-affinity, saturable anandamide transporter binding site (LY2318912; K d ‫؍‬ 7.62 ؎ 1.18 nM, Bmax ‫؍‬ 31.6 ؎ 1.80 fmol͞mg protein) that is distinct from fatty acid amide hydrolase. Systemic administration of the inhibitor into rodents elevates anandamide levels 5-fold in the brain and demonstrates efficacy in the formalin paw-licking model of persistent pain with no obvious adverse effects on motor function. Identification of the anandamide transporter binding site resolves a missing mechanistic link in endocannabinoid signaling, and in vivo results suggest that endocannabinoid transporter antagonists may provide a strategy for positive modulation of cannabinoid receptors.anandamide ͉ fatty acid amide hydrolase ͉ cannabinoid ͉ marijuana ͉ transporter E ndocannabinoids are recognized as significant intracellular lipid signaling molecules in the central nervous system with extensive control of physiological and behavioral mood and affect. Increases in endocannabinoid neurotransmission have broad therapeutic potential, including reduction of nausea and emesis (1), appetite stimulation (2), analgesia (3), anxiolytic activity (4), antispasmodic activity (5), and lowering of intraocular pressure in glaucoma (6). Identification of a specific binding site for the phytocannabinoid, ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC) (7), cloning of the cannabinoid receptors (CB 1 and CB 2 ) (8, 9), and the identification of an endogenous ligand, anandamide (N-arachidonoylethanolamide) (10), provided evidence of an endogenous cannabinoid system. Anandamide represents a class of lipid neurotransmitters that stimulate not only presynaptic and postsynaptic CB 1 receptors but also TRPV1 ion channels (11, 12), 5-hydroxytryptamine receptors (13-16), and possibly other receptors, as well as CB 2 receptors in the periphery (10,(17)(18)(19). More recently, the enzymes that are responsible for anandamide synthesis (phospholipase D) and catabolism (fatty acid amide hydrolase, FAAH) have been identified and characterized (20,21). Unlike typical neurotransmitter molecules, anandamide is synthesized in the membrane bilayer, resulting in the phospholipid precursor of anandamide, Narachidonoylphosphatidylethanolamine (22-25). Calciumacti...

Cannabinoids, in the form of marijuana plant extracts, have been used for thousands of years for a wide variety of medical conditions, ranging from general malaise and mood disorders to more specific ailments, such as pain, nausea, and muscle spasms. The discovery of tetrahydrocannabinol, the active principal in marijuana, and the identification and cloning of two cannabinoid receptors (i.e., CB1 and CB2) has subsequently led to biomedical appreciation for a family of endocannabinoid lipid transmitters. The biosynthesis and catabolism of the endocannabinoids and growing knowledge of their broad physiological roles are providing insight into potentially novel therapeutic targets. Compounds directed at one or more of these targets may allow for cannabinoid-based therapeutics with limited side effects and abuse liability.

: 1. The mechanism of anandamide uptake and disposal has been an issue of considerable debate in the cannabinoid field. Several compounds have been reported to inhibit anandamide uptake or fatty acid amide hydrolase (FAAH; the primary catabolic enzyme of anandamide) activity with varying degrees of potency and selectivity. We recently reported the first evidence of a binding site involved in the uptake of endocannabinoids that is independent from FAAH. There are no direct comparisons of purported selective inhibitory compounds in common assay conditions measuring anandamide uptake, FAAH activity and binding activity. 2. A subset of compounds reported in the literature were tested in our laboratory under common assay conditions to measure their ability to (a) inhibit [(14)C]-anandamide uptake in cells containing (RBL-2H3) or cells lacking (HeLa) FAAH, (b) inhibit purified FAAH hydrolytic activity, and (c) inhibit binding to a putative binding site involved in endocannabinoid transport in both RBL and HeLa cell membranes. 3. Under these conditions, nearly all compounds tested inhibited (a) uptake of [(14)C]-anandamide, (b) enzyme activity in purified FAAH preparations, and (c) radioligand binding of [(3)H]-LY2183240 in RBL and HeLa plasma membrane preparations. General rank order potency was preserved within the three assays. However, concentration response curves were right-shifted for functional [(14)C]-anandamide uptake in HeLa (FAAH(-/-)) cells. 4. A more direct comparison of multiple inhibitors could be made in these three assay systems performed in the same laboratory, revealing more information about the selectivity of these compounds and the relationship between the putative endocannabinoid transport protein and FAAH. At least two separate proteins appear to be involved in uptake and degradation of anandamide. The most potent inhibitory compounds were right-shifted when transport was measured in HeLa (FAAH(-/-)) cells suggesting a requirement for a direct interaction with the FAAH protein to maintain high affinity binding of anandamide or inhibitors to the putative anandamide transport protein.