The endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) is an uncharged neuromodulatory lipid that, similar to many neurotransmitters, is inactivated through its cellular uptake and subsequent catabolism. AEA is hydrolyzed by fatty acid amide hydrolase (FAAH), an enzyme localized on the endoplasmic reticulum. In contrast to most neuromodulators, the hydrophilic cytosol poses a diffusional barrier for the efficient delivery of AEA to its site of catabolism. Therefore, AEA likely traverses the cytosol with the assistance of an intracellular carrier that increases its solubility and rate of diffusion. To study this process, AEA uptake and hydrolysis were examined in COS-7 cells expressing FAAH restricted to the endoplasmic reticulum, mitochondria, or the Golgi apparatus. AEA hydrolysis was detectable at the earliest measurable time point (

On the basis of temperature dependency, saturability, selective inhibition, and substrate specificity, it has been proposed that an anandamide transporter exists. However, all of these studies have examined anandamide accumulation at long time points when downstream effects such as metabolism and intracellular sequestration are operative. In the current study, we have investigated the initial rates (<1 min) of anandamide accumulation in neuroblastoma and astrocytoma cells in culture and have determined that uptake is not saturable with increasing concentrations of anandamide. However, anandamide hydrolysis, after uptake in neuroblastoma cells, was saturable at steady-state time points (5 min), suggesting that fatty acid amide hydrolase (FAAH) may be responsible for observed saturation of uptake at long time points. In general, arvanil, olvanil, and N-(4-hydroxyphenyl)arachidonylamide (AM404) have been characterized as transport inhibitors in studies using long incubations. However, we found these ''transport inhibitors'' did not inhibit anandamide uptake in neuroblastoma and astrocytoma cells at short time points (40 sec or less). Furthermore, we confirmed that these inhibitors in vitro were actually inhibitors of FAAH. Therefore, the likely mechanism by which the transport inhibitors raise anandamide levels to exert pharmacological effects is by inhibiting FAAH, and they should be reevaluated in this context. Immunofluorescence has indicated that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, and this finding is consistent with our observed kinetics of anandamide hydrolysis. In summary, these data suggest that anandamide uptake is a process of simple diffusion. This process is driven by metabolism and other downstream events, rather than by a specific membrane-associated anandamide carrier. T he endocannabinoids, including anandamide, are a class of neurotransmitters, similar to ⌬ 9 -tetrahydracannabinol, involved in multiple physiological events including nociception, memory, blood pressure, locomotion, and immunity (for review, see ref. 1). These compounds bind the CB1 and CB2 cannabinoid receptors, which are G i -coupled receptors that modulate ion channels and signal transduction pathways (2-4).Anandamide is readily taken up into cells. The first step of this process has been characterized by several laboratories as a process of facilitated diffusion (for reviews, see refs. 5 and 6). Although an anandamide transporter has never been isolated, its existence is based on an anandamide uptake process that is temperature-dependent, selective, and saturable. In addition, several studies identified compounds that inhibit anandamide accumulation, including N-(4-hydroxyphenyl)arachidonylamide (AM404) and the vanilloids arvanil and olvanil (7-16).After uptake, if fatty acid amide hydrolase (FAAH) is present, anandamide is hydrolyzed to arachidonic acid and ethanolamine (for review, see ref. 17). A recent report investigating the role of FAAH in anandamide metabolism showed that FAAH Ϫ...

Background: Transport inhibitors modulate endocannabinoid signaling by inhibiting their uptake through unknown mechanisms. Results: Effects of transport inhibitors upon endocannabinoid uptake and intracellular trafficking were lost in the absence of fatty acid-binding proteins. Conclusion: Fatty acid-binding proteins are physiological targets of transport inhibitors. Significance: These findings identify drug targets for modulating endocannabinoid signaling.

Anandamide is an endogenous compound that acts as an agonist at cannabinoid receptors. It is inactivated via intracellular degradation after its uptake into cells by a carrier-mediated process that depends upon a concentration gradient. The fate of anandamide in those cells containing an amidase called fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and ethanolamine. The active site nucleophilic serine of FAAH is inactivated by a variety of inhibitors including methylarachidonylfluorophosphonate (MAFP) and palmitylsulfonyl fluoride. In the current report, the net uptake of anandamide in cultured neuroblastoma (N18) and glioma (C6) cells, which contain FAAH, was decreased by nearly 50% after 6 min of incubation in the presence of MAFP. Uptake in laryngeal carcinoma (Hep2) cells, which lack FAAH, is not inhibited by MAFP. Free anandamide was found in all MAFP-treated cells and in control Hep2 cells, whereas phospholipid was the main product in N18 and C6 control cells when analyzed by TLC. The intracellular concentration of anandamide in N18, C6, and Hep2 cells was up to 18-fold greater than the extracellular concentration of 100 nM, which strongly suggests that it is sequestered within the cell by binding to membranes or proteins. The accumulation of anandamide and/or its breakdown products was found to vary among the different cell types, and this correlated approximately with the amount of FAAH activity, suggesting that the breakdown of anandamide is in part a driving force for uptake. This was shown most clearly in Hep2 cells transfected with FAAH. The uptake in these cells was 2-fold greater than in vector-transfected or untransfected Hep2 cells. Therefore, it appears that FAAH inhibitors reduce anandamide uptake by cells by shifting the anandamide concentration gradient in a direction that favors equilibrium. Because inhibition of FAAH increases the levels of extracellular anandamide, it may be a useful target for the design of therapeutic agents.Endocannabinoids, such as anandamide (arachidonyl ethanolamide) and 2-arachidonyl glycerol, are endogenous ligands that bind to the cannabinoid receptors (1-3). Emerging evidence suggests that they are involved in many physiological phenomena such as pain, locomotion, memory, learning, blood pressure, immunity, sleep, reproduction, mood, perception, response to stress, and so forth (for review see Ref. 4). ⌬ 9 -Tetrahydrocannabinol, the active component of marijuana, appears to mimic many of the physiological and pharmacological effects of the endogenous cannabinoids, in some cases to an extreme degree (e.g. a marijuana "high"). Anandamide is transported into the neuroblastoma, glioma, brain neuron, brain astrocyte, cerebellar granule cells, leukocyte, macrophage, leukemia, and lymphoma cells in culture (5-10). The driving force for uptake is substrate concentration (facilitated diffusion) rather than an active cotransport system (11,12). The transport appears to be carrier-mediated, and specific transport inhibitors have been described (9,11,(13...

The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors.Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics.