Anatomical studies of endocannabinoid transport are greatly limited by the lack of transporter-specific markers. Nevertheless, biochemical experiments have documented the existence of [3 H]anandamide uptake in primary cultures of rat cortical neurons and astrocytes , rat cerebellar granule cells , human neuroblastoma cells , and human astrocytoma cells . The CNS distribution of endocannabinoid transport was investigated by exposing metabolically active rat brain slices to [14C]anandamide and analyzing the distribution of radioactivity in the tissue by autoradiography . A receptor antagonist was included in the incubations to prevent the binding of [14C]anandamide to CB1 receptors, which are very numerous in certain brain regions , and AM404 was used to differentiate transportmediated [14C]anandamide reuptake from nonspecific binding . Substantial levels of AM404-sensitive [14C]anandamide reuptake were observed in the somatosensory, motor, and limbic areas of the cortex and in the striatum. Additional brain regions showing detectable [14C]anandamide accumulation included the hippocampus, thalamus, septum, substantia nigra, amygdala, and hypothalamus . Thus, endocannabinoid transport may be present in discrete regions of the rat brain that also express CB1 receptors . Distribution of Endocannabinoid Transport Outside the CNS. The endocannabinoid system is not confined to the brain, and it is reasonable to anticipate that mechanisms of endocannabinoid inactivation may also exist in peripheral tissues. In keeping with this expectation,industrial rolling racks carrier-mediated [ 3 H]anandamide transport was demonstrated in J774 macrophages , RBL-2H3 cells , and human endothelial cells .

Although the kinetic and pharmacological properties of endocannabinoid uptake in peripheral cells appear to be generally similar to those reported in the CNS, some important difference have been observed. For example, in contrast to neurons, [3 H]anandamide uptake in RBL-2H3 cells is inhibited by arachidonic acid . Such disparities might reflect the existence in non-neural tissues of mechanisms of endocannabinoid internalization that are distinct from those found in the CNS. Inhibition of Endocannabinoid Transport: Molecular Tools. A variety of compounds have been tested for their ability to interfere with [3 H]anandamide internalization . Amongthem, the anandamide analog AM404 stands out for its relatively high potency and its ability to block endocannabinoid transport both in vitro and in vivo. AM404 inhibits [ 3 H]anandamide uptake in rat brain neurons and astrocytes , human astrocytoma cells , rat brain slices , and RBL-2H3 cells . AM404 does not directly activate cannabinoid receptors in vitro , but it augments several CB1 receptor-mediated effects of anandamide. For example, AM404 enhances anandamideevoked inhibition of adenylyl cyclase activity in cortical neurons, an effect that is reversed by the CB1 antagonist SR141716A . Likewise, AM404 potentiates the inhibitory actions of anandamide on GABA-ergic neurotransmission in the periaqueductal gray matter . These findings are consistent with the hypothesis that AM404 protects anandamide from inactivation and, by doing so, magnifies the biological effects of this short-lived lipid mediator. It is important to point out, however, that AM404 is readily transported inside cells , where it can reach concentrations that may be sufficient to inhibit anandamide hydrolysis . To what extent this effect contributes to the ability of AM404 to prolong anandamide’s life span is at present unclear. An initial screening found that AM404 has no affinity for a panel of 36 different pharmacological targets, including G protein-coupled receptors and ligand-gated ion channels .

However, additional studies revealed that AM404 activates capsaicin receptor channels at concentrations similar to those necessary to inhibit endocannabinoid transport . The fact that AM404 can produce undesired effects underscores the need to introduce appropriate controls in the design of in vivo experiments with this compound. In particular, the effects of a cannabinoid receptor antagonist should be routinely tested to verify that endogenously produced anandamide and 2-AG are involved in the response to AM404 . Inhibition of Endocannabinoid Transport: Functional Studies. AM404 does not display a typical cannabimimetic profile when administered in vivo; this is consistent with its poor affinity for cannabinoid receptors. For example, AM404 has no antinociceptive effect in mice or rats and causes no hypotension in guinea pigs . Nevertheless, in the same models, AM404 increases the responses elicited by exogenous anandamide, and this potentiation is reversed by the CB1 antagonist SR141716A . Despite the absence of overt cannabimimetic properties, AM404 resembles anandamide and other cannabinoid receptor agonists in certain respects. For example, when administered alone, AM404 causes a reduction in motor activity, which is prevented by the CB1 antagonist SR141716A . Furthermore, AM404 reduces the yawning evoked by low doses of the mixed D1/D2 dopamine agonist apomorphine and inhibits the hyperactivity elicited by the selective D2 agonist quinpirole . AM404 also decreases the levels of circulating prolactin, but the role of CB1 receptors in this response is unknown . Can the effects of AM404 be explained by its in vitro affinity for vanilloid receptors ? The fact that SR141716A, a selective CB1 antagonist, blocks the motor inhibitory effects produced by AM404 argues against this possibility. Furthermore, vanilloid agonists such as capsaicin have very different, in some cases even opposite, effects. For example, capsaicin causes hyperkinesia and pain , whereas AM404 elicits hypokinesia and enhances anandamide’s analgesic properties .

Therefore, a more plausible interpretation of the available data is that, by inhibiting anandamide clearance, AM404 may cause this lipid to accumulate outside cells and activate local cannabinoid receptors. In further support of this possibility, the systemic administration of AM404 in rats was found to cause a time-dependent increase in circulating anandamide levels . Finally, it is important to point out that several anandamide responses are not affected by AM404. One example is the inhibition of intestinal motility, which anandamide may produce in rodents by activating CB1 receptors on the surface of enteric neurons . This effect is not enhanced by AM404, suggesting that the predominant pathway of endocannabinoid inactivation in the intestine may be through enzymatic hydrolysis, not transport . The fact that rat intestinal tissue contains high AAH levels is in agreement with this possibility . Alternatively, anandamide transport may occur in the intestine through transport mechanisms that are insensitive to AM404.Mechanisms and Kinetics. Long before the discovery of anandamide, Schmid and coworkers identified in rat liver an amidohydrolase activity, which catalyzes the hydrolysis of fatty acid ethanolamides to free fatty acid and ethanolamine . That anandamide may serve as a substrate for this activity was first suggested on the basis of biochemical evidence and then demonstrated by molecular cloning and heterologous expression of the enzyme involved . AAH is an intracellular membrane-bound protein whose primary structure displays significant similarities with a group of enzymes known as “amidase signature family” . AAH may act as a general hydrolytic enzyme not only for fatty acid ethanolamides but also primary amides  and even esters . Site-directed mutagenesis experiments indicate that this unusually wide substrate preference may be underpinned by a novel catalytic mechanism involving the amino acid residue lysine 142. This residue may act as a general acid catalyst, favoring the protonation and consequent detachment of reaction products from the enzyme’s active site . Three serine residues that are conserved in all amidase signature enzymes may also be essential for enzymatic activity: serine 241 may serve as the enzyme’s catalytic nucleophile, while serine 217 and 218 may modulate catalysis through an as-yet-unidentified mechanism . Like other hydrolase enzymes, AAH may act in reverse, catalyzing the synthesis of anandamide from free arachidonate and ethanolamine . The high KM values reported for anandamide synthase activity suggest, however, that under normal circumstances AAH acts predominantly as a hydrolase. One exception is represented by the rat uterus, where substrate concentrations in the micromolar range are required for the synthase reaction to occur, implying that in this tissue AAH could contribute to anandamide biosynthesis . In addition to AAH, other ill-characterized enzyme activities may participate in the breakdown of anandamide and 2-AG. A fatty acid ethanolamide-hydrolyzing activity catalytically distinct from AAH was described in rat brain membranes and human megakaryoblastic cells . Furthermore, evidence indicates that 2-AG degradation may be predominantly catalyzed by an enzyme different from AAH,marijuana drying rack possibly a monoacylglycerol lipase . Structure-Activity Relationship Studies. Modifications in three potential pharmacophores have helped define several general requisites for endocannabinoid hydrolysis by AAH. First, reducing the number of double bonds in the hydrophobic carbon chain causes a gradual increase in metabolic stability .

Thus, [3 H]anandamide hydrolysis is inhibited by fatty acid ethanolamides in the 20 carbon atom series with the following rank order of potency: 20:4  20:3 20:2 20:1 20:0  no effect . Second, replacing the ethanolamine moiety with a primary amide leads to good AAH substrates. For example, the rate of hydrolysis of arachidonylamide is approximately twice that of anandamide . Third, anandamide congeners containing a tertiary nitrogen in the ethanolamine moiety are poor AAH substrates . Fourth, introduction of a methyl group at the C2, C1, or C2 positions of anandamide yields analogs that are resistant to hydrolysis, likely as a result of increased steric hindrance around the carbonyl group . Fifth, substrate recognition at the AAH active site is stereoselective, at least with fatty acid ethanolamide congeners containing a methyl group in the C1_x0007_or C2 positions . Finally, as a result of AAH’s remarkable “directed nonspecificity” , fatty acid esters also serve as substrates for this enzyme. Thus, 2-AG is hydrolyzed by AAH at a rate that is about 4 times faster than anandamide is . AAH Distribution in the CNS. AAH is widely distributed in the brain, with particularly high levels in cortex, hippocampus, cerebellum, amygdala, thalamus, and pontine nuclei . Immunohistochemical studies suggest that neurons, not glia, are the predominant cell type expressing AAH , although astrocytes in primary culture have been shown to contain AHH activity . CB1 cannabinoid receptors are present in various brain regions that also express AAH, but there appears to be no direct correlation between the concentrations of these two proteins . This discrepancy may reflect the participation of AAH in the degradation of non-cannabinoid lipid amides, such as oleamide and OEA. AAH Distribution outside the CNS. AAH mRNA and enzyme activity have been measured in a variety of nonneural cells lines, including lung carcinoma , human breast carcinoma , leukemia basophils , human monocytic leukemia , rat renal endothelial and mesangial cells , rat macrophages , human platelets , and human lymphocytes . Furthermore, high AAH levels have been found in rat liver, testis, kidney, lung, spleen, uterus, small intestine, and stomach; whereas lower levels were observed in heart and skeletal muscle . The distribution of AAH in human tissues is somewhat different from the rat, with expression levels that are reportedly higher in pancreas, brain, kidney, and skeletal muscle than in liver . Inhibition of AAH Activity: Molecular Tools. The armamentarium of AAH inhibitors available to the experimentalist has been recently enriched by two important groups of molecules. The first are fatty acid sulfonyl fluorides, such as the compound AM374 . AM374 irreversibly inhibits AAH activity with an IC50 value in the low nanomolar range and displays a 50-fold preference for AAH inhibition versus CB1 cannabinoid receptor binding . In superfused hippocampal slices, AM374 augments anandamide’s ability to inhibit [3 H]acetylcholine release, although it does not affect release when it is applied alone . The second group of AAH inhibitors is represented by a series of substituted  -keto-oxazolopyridines , which are reversible and extremely potent . Little information is as yet available on the pharmacological selectivity and in vivo properties of these interesting compounds. AAH Inhibition: Functional Studies. Systemic administration of the potent AAH inhibitor AM374 does not produce clear cannabimimetic effects in rats but enhances the operant leverpressing response evoked by anandamide administration . These results suggest that AM374 protects exogenous anandamide from degradation but does not cause a significant accumulation of endogenously generated anandamide. This idea is consistent with the finding that, in contrast to the transport inhibitor AM404 , AM374 does not increase circulating anandamide levels in rats . Further studies will be required to fully evaluate the behavioral impact of AAH inhibitors and to assess the biological availability and pharmacokinetics of these molecules.In Search of a Role. What place will inhibitors of endocannabinoid clearance occupy in medicine, if any, will largely depend on the answers to two key questions.