Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) catalyse the hydrolysis of the endocannabinoids anandamide and 2-arachidonoyl glycerol. We investigated their ultrastructural distribution in brain areas where the localization and effects of cannabinoid receptor activation are known. In the hippocampus, FAAH was present in somata and dendrites of principal cells, but not in interneurons. It was located mostly on the membrane surface of intracellular organelles known to store Ca 2+ (e.g. mitochondria, smooth endoplasmic reticulum), less frequently on the somatic or dendritic plasma membrane. MGL immunoreactivity was found in axon terminals of granule cells, CA3 pyramidal cells and some interneurons. In the cerebellum, Purkinje cells and their dendrites are intensively immunoreactive for FAAH, together with a sparse axon plexus at the border of the Purkinje cell ⁄ granule cell layers. Immunostaining for MGL was complementary, the axons in the molecular layer were intensively labelled leaving the Purkinje cell dendrites blank. FAAH distribution in the amygdala was similar to that of the CB 1 cannabinoid receptor: evident signal in neuronal somata and proximal dendrites in the basolateral nucleus, and hardly any labelling in the central nucleus. MGL staining was restricted to axons in the neuropil, with similar relative signal intensities seen for FAAH in different nuclei. Thus, FAAH is primarily a postsynaptic enzyme, whereas MGL is presynaptic. FAAH is associated with membranes of cytoplasmic organelles. The differential compartmentalization of the two enzymes suggests that anandamide and 2-AG signalling may subserve functional roles that are spatially segregated at least at the stage of metabolism.

BACKGROUND AND PURPOSEGastrointestinal (GI) motility is regulated in part by fatty acid ethanolamides (FAEs), including the endocannabinoid (EC) anandamide (AEA). The actions of FAEs are terminated by fatty acid amide hydrolase (FAAH). We investigated the actions of the novel FAAH inhibitor AM3506 on normal and enhanced GI motility. EXPERIMENTAL APPROACHWe examined the effect of AM3506 on electrically-evoked contractility in vitro and GI transit and colonic faecal output in vivo, in normal and FAAH-deficient mice treated with saline or LPS (100 mg·kg KEY RESULTSFAAH was dominantly expressed in the enteric nervous system; its mRNA levels were higher in the ileum than the colon. LPS enhanced ileal contractility in the absence of overt inflammation. AM3506 reversed the enhanced electrically-evoked contractions of the ileum through CB1 and CB2 receptors. LPS increased the rate of upper GI transit and faecal output. AM3506 normalized the enhanced GI transit through CB1 and CB2 receptors and faecal output through CB1 receptors. LPS did not increase GI transit in FAAH-deficient mice. CONCLUSIONS AND IMPLICATIONSInhibiting FAAH normalizes various parameters of GI dysmotility in intestinal pathophysiology. Inhibition of FAAH represents a new approach to the treatment of disordered intestinal motility.

BACKGROUND AND PURPOSEDepolarization-induced suppression of inhibition (DSI) and excitation (DSE) are two forms of cannabinoid CB1 receptor-mediated inhibition of synaptic transmission, whose durations are regulated by endocannabinoid (eCB) degradation. We have recently shown that in cultured hippocampal neurons monoacylglycerol lipase (MGL) controls the duration of DSE, while DSI duration is determined by both MGL and COX-2. This latter result suggests that DSE might be attenuated, and excitatory transmission enhanced, during inflammation and in other settings where COX-2 expression is up-regulated. EXPERIMENTAL APPROACHTo investigate whether it is possible to control the duration of eCB-mediated synaptic plasticity by varied expression of eCB-degrading enzymes, we transfected excitatory autaptic hippocampal neurons with putative 2-AG metabolizing enzymes: COX-2, fatty acid amide hydrolase (FAAH), a/b hydrolase domain 6 (ABHD6), a/b hydrolase domain 12 (ABHD12) or MGL. KEY RESULTSWe found that overexpression of either COX-2 or FAAH shortens the duration of DSE while ABHD6 or ABHD12 do not. In contrast, genetic deletion (MGL -/-) and overexpression of MGL both radically altered eCB-mediated synaptic plasticity. CONCLUSIONS AND IMPLICATIONSWe conclude that both FAAH and COX-2 can be trafficked to neuronal sites where they are able to degrade eCBs to modulate DSE duration and, by extension, net endocannabinoid signalling at a given synapse. The results for COX-2, which is often up-regulated under pathological conditions, are of particular note in that they offer a mechanism by which up-regulated COX-2 may promote neuronal excitation by suppressing DSE while enhancing conversion of 2-AG to PGE2-glycerol ester under pathological conditions. Abbreviations 2-AG, 2-arachidonylglycerol; ABHD6, a/b hydrolase domain 6; ABHD12, a/b hydrolase domain 12; AEA, arachidonyl ethanolamide; DSE, depolarization-induced suppression of excitation; DSI, depolarization-induced suppression of inhibition; eCB, endocannabinoid; FAAH, fatty acid amide hydrolase; MGL, monoacylglycerol lipase

Background and purpose: Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH. Experimental approach: In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the 'tetrad' test for central CB receptor activation. Key results: Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 mM AEA by mouse, rat and human FAAH with IC50 values of 1.8, 1.4 and 2.4 mM respectively. The compound did not interact to any major extent with CB1 or CB2 receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 mg i.pl.) and biochanin A (100 mg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB1 receptor antagonist/inverse agonist AM251 (30 mg i.pl.). Biochanin A (15 mg·kg -1 i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg·kg -1 i.v. AEA in the tetrad test. Conclusions and implications: It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.

This study demonstrates definitively that FAAH inhibitors provide a new class of anti-spastic agents that may have utility in treating spasticity in MS and avoid the dose-limiting side effects associated with cannabis use.