Fatty Acid Amide Hydrolase Activity Research Articles

Closing the Gate to the Active Site: EFFECT OF THE INHIBITOR METHOXYARACHIDONYL FLUOROPHOSPHONATE ON THE CONFORMATION AND MEMBRANE BINDING OF FATTY ACID AMIDE HYDROLASE

Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of approximately 30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of approximately 11 kcal/mol that accounted for approximately 80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (DeltaG approximately 3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by approximately 2 kcal/mol, toward denaturant- and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.

A role for the FAAH‐interacting protein ERp57 in endogenous cannabinoid signaling

Cannabinoid receptor signaling is currently an active area of research due to therapeutic implications for analgesia, anxiety, appetite modulation, and regulation of immune responses. The long-chain fatty acid amide anandamide (AEA) is an endogenous agonist for the central nervous system and peripheral cannabinoid receptors CB1 and CB2, respectively. AEA is a putative neurotransmitter, and as such, a mechanism must exist for synaptic inactivation of endogenous cannabinoid signaling. The primary mechanism for AEA inactivation is thought to be transport into cells, followed by metabolism by fatty acid amide hydrolase (FAAH). We have shown that AEA reuptake occurs via a protein carrier-mediated caveolae-related endocytic process. The current objective of our research is to identify specific proteins that play a role in this process. We have discovered that ERp57 localizes to caveolin-rich membranes upon AEA treatment. Furthermore, we have identified ERp57 as a FAAH-interacting protein. Finally, RNAi strategies have been used to knockdown ERp57 expression in order to explore its putative function in AEA uptake and FAAH activity. This research supported by grant R21 DA018844 from NIH.

Development of highly sensitive fluorescent assays for fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is a pharmaceutical target whose inhibition may lead to valuable therapeutics. Sensitive substrates for high-throughput assays are crucial for the rapid-screening FAAH inhibitors. Here we describe the development of novel and highly sensitive fluorescent assays for FAAH based on substituted aminopyridines. Examining the relationship between the structure and the fluorescence of substituted aminopyridines suggested that a methoxy group in the para position relative to the amino group in aminopyridines greatly increased the fluorescence (i.e., quantum yields approach unity). These novel fluorescent reporters had a high Stokes’ shift of 94 nm, and their fluorescence in buffer systems increased with pH values from neutral to basic. Fluorescent substrates with these reporters displayed a very low fluorescent background and high aqueous solubility. Most importantly, fluorescent assays for FAAH based on these substrates were at least 25 times more sensitive than assays using related compounds with published colorimetric or fluorescent reporters. This property results in shorter assay times and decreased protein concentrations in the assays. Such sensitive assays will facilitate distinguishing the relative potency of powerful inhibitors of FAAH. When these fluorescent substrates were applied to human liver microsomes, results suggested that there was at least one amide hydrolase in addition to FAAH that could hydrolyze long-chain fatty acid amides. These results show that these fluorescent substrates are very valuable tools in FAAH activity assays including screening inhibitors by high-throughput assays instead of using the costly and labor-intensive radioactive ligands. Potential applications of novel fluorescent reporters are discussed.

Analgesic Effects of Fatty Acid Amide Hydrolase Inhibition in a Rat Model of Neuropathic Pain

Cannabinoid-based medicines have therapeutic potential for the treatment of pain. Augmentation of levels of endocannabinoids with inhibitors of fatty acid amide hydrolase (FAAH) is analgesic in models of acute and inflammatory pain states. The aim of this study was to determine whether local inhibition of FAAH alters nociceptive responses of spinal neurons in the spinal nerve ligation model of neuropathic pain. Electrophysiological studies were performed 14-18 d after spinal nerve ligation or sham surgery, and the effects of the FAAH inhibitor cyclohexylcarbamic acid 3-carbamoyl biphenyl-3-yl ester (URB597) on mechanically evoked responses of spinal neurons and levels of endocannabinoids were determined. Intraplantar URB597 (25 microg in 50 microl) significantly (p < 0.01) attenuated mechanically evoked responses of spinal neurons in sham-operated rats. Effects of URB597 were blocked by the cannabinoid 1 receptor (CB1) antagonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] (30 microg in 50 microl) and the opioid receptor antagonist naloxone. URB597 treatment increased levels of anandamide, 2-arachidonyl glycerol, and oleoyl ethanolamide in the ipsilateral hindpaw of sham-operated rats. Intraplantar URB597 (25 microg in 50 microl) did not, however, alter mechanically evoked responses of spinal neurons in spinal nerve ligated (SNL) rats or hindpaw levels of endocannabinoids. Intraplantar injection of a higher dose of URB597 (100 microg in 50 microl) significantly (p < 0.05) attenuated evoked responses of spinal neurons in SNL rats but did not alter hindpaw levels of endocannabinoids. Spinal administration of URB597 attenuated evoked responses of spinal neurons and elevated levels of endocannabinoids in sham-operated and SNL rats. These data suggest that peripheral FAAH activity may be altered or that alternative pathways of metabolism have greater importance in SNL rats.

Role of Endocannabinoids in Alcohol Consumption and Intoxication: Studies of Mice Lacking Fatty Acid Amide Hydrolase

Endocannabinoid signaling plays the important role in regulation of ethanol intake. Fatty acid amide hydrolase (FAAH) is a key membrane protein for metabolism of endocannabinoids, including anandamide, and blockade of FAAH increases the level of anandamide in the brain. To determine if FAAH regulates ethanol consumption, we studied mutant mice with deletion of the FAAH gene. Null mutant mice showed higher preference for alcohol and voluntarily consumed more alcohol than wild-type littermates. There was no significant difference in consumption of sweet or bitter solutions. To determine the specificity of FAAH for ethanol intake, we studied additional ethanol-related behaviors. There were no differences between null mutant and wild-type mice in severity of ethanol-induced acute withdrawal, conditioned taste aversion to alcohol, conditioned place preference, or sensitivity to hypnotic effect of ethanol. However, null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol. All three behavioral phenotypes (increased preference for ethanol, decreased sensitivity to ethanol-induced sedation, and faster recovery from ethanol-induced motor incoordination) seen in mutant mice were reproduced in wild-type mice by injection of a specific inhibitor of FAAH activity--URB597. These data suggest that increased endocannabinoid signaling increased ethanol consumption owing to decreased acute ethanol intoxication.

A Second Fatty Acid Amide Hydrolase with Variable Distribution among Placental Mammals

Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.

Follicle-Stimulating Hormone Activates Fatty Acid Amide Hydrolase by Protein Kinase A and Aromatase-Dependent Pathways in Mouse Primary Sertoli Cells

Among the biological activities of the endocannabinoid anandamide (N-arachidonoylethanolamine) (AEA), growing interest has been attracted by the regulation of mammalian fertility. Recently we have shown that treatment of mouse primary Sertoli cells with FSH enhances the activity of the AEA hydrolase [fatty acid amide hydrolase (FAAH)], though the molecular details were not elucidated. Here, we investigated whether FSH was also able to affect the enzymes that synthesize AEA (N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D), the endogenous content of this endocannabinoid, and the level of the AEA-binding vanilloid receptor 1 (transient receptor potential channel vanilloid receptor subunit 1). We show that FSH enhanced FAAH activity (up to approximately 500% of the controls) and expression (up to approximately 300%), leading to a marked reduction (down to approximately 15%) of AEA content. However N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D activity, and transient receptor potential channel vanilloid receptor subunit 1 binding were not affected. We also show that diacylglycerol lipase and monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoyl-glycerol, were not regulated by FSH, neither was the membrane transport of this endocannabinoid. In addition, we show that FAAH stimulation by FSH was abrogated by inhibitors of protein kinase A (PKA) and cytochrome-P(450) aromatase, and was conversely mimicked by N,O'-dibutyryl cAMP and estrogen. Finally, we demonstrate that FSH protects Sertoli cells against the pro-apoptotic activity of AEA, through PKA and aromatase-dependent activation of FAAH. Altogether these data suggest that FAAH is the only target of FSH among the elements of the endocannabinoid system, and that its regulation by PKA and aromatase-dependent pathways impacts Sertoli cell proliferation.

A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

Modulation of paraoxon toxicity by the cannabinoid receptor agonist WIN 55,212-2

Cannabinoids can reduce the pre-synaptic release of acetylcholine and other neurotransmitters in the mammalian brain through a retrograde signaling pathway. Organophosphorus insecticides elicit toxicity by inhibiting acetylcholinesterase and thereby increasing synaptic acetylcholine levels. Several studies suggest that some organophosphorus toxicants can potentially modify cannabinergic signaling by direct binding to cannabinoid receptors and inhibition of enzymes responsible for cannabinoid degradation (i.e., fatty acid amide hydrolase and monoacylglycerol lipase). We hypothesized that exposure to the cannabinoid receptor agonist WIN 55,212-2 (WIN) could alter the acute toxicity of the prototype anticholinesterase, paraoxon. In vitro, paraoxon inhibited hippocampal cholinesterase and fatty acid amide hydrolase activities, and displaced specific binding to the cannabinoid receptor ligand ([ 3H]CP 55,940) in a concentration-dependent manner. WIN (0.5, 1.5 or 5 mg/kg/day) had a complex dose-related effect on locomotor activity when evaluated for 2 h after either the first or last of seven daily exposures, and significantly decreased hippocampal CB1 binding following repeated dosing. Four hours after dosing, paraoxon (0.4 mg/kg, sc) elicited classical signs of cholinergic toxicity and significantly reduced hippocampal cholinesterase and fatty acid amide hydrolase activities as well as [ 3H]CP 55,940 binding. A single exposure to WIN (1.5 mg/kg) significantly reduced involuntary movements and SLUD signs following acute paraoxon exposure (0.4 and 0.6 mg/kg, sc). In contrast, when rats were challenged with paraoxon (0.4 mg/kg) after the seventh daily exposure to WIN (1.5 mg/kg/day), involuntary movements were significantly increased at later timepoints, while SLUD signs were unaffected. These results suggest that acute and repeated exposure to cannabinoid agonists may differentially modify acute cholinergic toxicity, possibly through modulation of acetylcholine release and adaptation in cannabinergic signaling associated with repeated cannabinoid exposures.

The fatty-acid amide hydrolase inhibitor URB597 does not affect triacylglycerol hydrolysis in rat tissues

The O-arylcarbamate URB597 (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester; also referred to as KDS-4103) is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the inactivation of the endogenous cannabinoid anandamide. URB597 demonstrates a remarkable degree of selectivity for FAAH over other serine hydrolases (e.g. cholinesterases) or other components of the endocannabinoid system (e.g. cannabinoid receptors). However, in a proteomic-based selectivity screen based on the displacement of fluorophosphonate-rhodamine (FPR) from mouse brain proteins, it was recently shown that URB597 prevents FPR binding to triacylglycerol hydrolase (TGH) with a median inhibitory concentration of 192 nM. To determine whether this effect correlates with inhibition of TGH activity, we investigated the ability of URB597 to inhibit triolein hydrolysis in rat liver and heart tissues, which are rich in TGH, as well as white adipose tissue (WAT), which is rich in adipose triacylglycerol lipase (TGL) and hormone-sensitive lipase. The results show that URB597 does not affect triolein hydrolysis in any of these tissues at concentrations as high as 10 μM, whereas it inhibits FAAH activity at low nanomolar concentrations. Moreover, intraperitoneal (i.p.) administration of URB597 at doses that maximally inhibit FAAH in vivo (0.3–3 mg kg −1) exerts no effect on triolein hydrolysis and tissue triacylglycerol (TAG) levels in rat liver, heart or WAT. The results indicate that URB597, while potent at inhibiting FAAH, does not affect TGH and TGL activities in rat tissues.

Pharmacological Characterization of Endocannabinoid Transport and Fatty Acid Amide Hydrolase Inhibitors

: 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.

Biochemical Changes in Endocannabinoid System are Expressed in Platelets of Female but not Male Migraineurs

The endogenous cannabinoid anandamide (AEA) plays important roles in modulating pain. Head pain is an almost universal human experience, yet primary headache disorders, such as migraine without aura (MoA) or episodic tension-type headache (ETTH), can represent a serious threat to well-being when frequent and disabling. We assessed the discriminating role of endocannabinoids among patients with ETTH or MoA, and control subjects. We measured the activity of AEA hydrolase and AEA transporter, and the level of cannabinoid receptors in peripheral platelets from MoA, ETTH and healthy controls. Sixty-nine headache patients and 36 controls were selected. Diagnosis of headache type was made according to the International Headache Society criteria. We observed significant sex differences concerning AEA membrane transporter and fatty acid amide hydrolase activity in all groups. An increase in the activity of AEA hydrolase and AEA transporter was found in female but not male migraineurs. Cannabinoid receptors were the same in all groups. Here we show that the endocannabinoid system in human platelets is altered in female but not male migraneurs. Our results suggest that in migraineur women an increased AEA degradation by platelets, and hence a reduced concentration of AEA in blood, might reduce the pain threshold and possibly explain the prevalence of migraine in women. The involvement of the endocannabinoid system in migraine is new and broadens our knowledge of this widespread and multifactorial disease.

Pharmacological Profile of the Selective FAAH Inhibitor KDS-4103 (URB597)

In the present article, we review the pharmacological properties of KDS-4103 (URB597), a highly potent and selective inhibitor of the enzyme fatty-acid amide hydrolase (FAAH), which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide. In vitro, KDS-4103 inhibits FAAH activity with median inhibitory concentrations (IC(50)) of 5 nM in rat brain membranes and 3 nM in human liver microsomes. In vivo, KDS-4103 inhibits rat brain FAAH activity after intraperitoneal (i.p.) administration with a median inhibitory dose (ID(50)) of 0.15 mg/kg. The compound does not significantly interact with other cannabinoid-related targets, including cannabinoid receptors and anandamide transport, or with a broad panel of receptors, ion channels, transporters and enzymes. By i.p. administration to rats and mice KDS-4103 elicits significant, anxiolytic-like, antidepressant-like and analgesic effects, which are prevented by treatment with CB1 receptor antagonists. By contrast, at doses that significantly inhibit FAAH activity and substantially raise brain anandamide levels, KDS-4103 does not evoke classical cannabinoid-like effects (e.g., catalepsy, hypothermia, hyperphagia), does not cause place preference, and does not produce generalization to the discriminative effects of the active ingredient of cannabis, Delta9-tetrahydrocannabinol (Delta9-THC). These findings suggest that KDS-4103 acts by enhancing the tonic actions of anandamide on a subset of CB(1) receptors, which may normally be engaged in controlling emotions and pain. KDS-4103 is orally available in rats and cynomolgus monkeys. Sub-chronic repeated dose studies (1,500 mg/kg, per os) in these two species have not demonstrated systemic toxicity. Likewise, no toxicity was noted in bacterial cytotoxicity tests in vitro and in the Ames test. Furthermore, no deficits were observed in rats on the rotarod test after acute i.p. treatment with KDS-4103 at doses up to 5 mg/kg or in a functional observation battery after oral doses up to 1,500 mg/kg. The results suggest that KDS-4103 will offer a novel approach with a favorable therapeutic window for the treatment of anxiety, depression and pain.

Endocannabinoid Degradation and Oxidative Defense Mechanisms Determine Anandamide-induced Cell Death in Liver Cell Populations

Background: We have shown that the endogenous cannabinoid anandamide (AEA) induces reactive oxygen species (ROS)-dependent cell death in primary hepatic stellate cells (HSCs), but not in primary hepatocytes. Aim: To determine cellular factors that control AEA-induced cell death in hepatic cell populations. Methods: Fatty acid amide hydrolase (FAAH) expression was analyzed by western blot and real time PCR in primary rat hepatocytes and primary rat HSCs. FAAH activity was determined colorimetrically. Cell death was analyzed by LDH release, propidium iodide uptake and western blot for caspase 3- and PARP cleavage. Glutathione (GSH) levels were measured by the DTNB method. mRNA expression of the endocannabinoid receptors CB1, CB2 and VR1 was determined by real time PCR. ROS production was monitored by DCFDA fluorescence. Results: FAAH mRNA, FAAH protein and FAAH activity were extremely high in primary rat hepatocytes, but virtually absent in primary rat HSCs. Hepatocytes from FAAH -/- mice, or wildtype hepatocytes which had been pretreated with the FAAH inhibitor URB597 were susceptible to AEA-mediated death (45% and 40% at 50 μM after 24h, respectively). Conversely, FAAH overexpression prevented AEA-induced death in HSCs. Anandamide induced an increase in ROS production in hepatocytes which had been pretreated with the FAAH inhibitor URB597 or depleted of GSH by BSO. GSH levels were 10-fold higher in hepatocytes than in HSCs. GSH depletion rendered hepatocytes susceptible to AEA-mediated death while GSH pretreatment prevented cell death in HSCs. The combination of FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24h at 50 μM AEA and lowered the threshold for AEA-induced cell death to 500 nM. AEA-induced hepatocyte death after BSO and URB597 pretreatment was purely necrotic and occurred independently of the endocannabinoid receptors CB1, CB2 and VR1 which were virtually absent (CB1, CB2) or expressed at low levels (VR1) in hepatocytes. Conclusion: FAAH and GSH determine endocannabinoid-induced necrosis in hepatocytes and HSCs, independently of expression of cannabinoid receptors. High expression of FAAH and high intracellular GSH levels efficiently prevent AEA-induced cell death in hepatocytes. FAAH-mediated endocannabinoid degradation in hepatocytes leads to a new physiological concept allowing the specific targeting of HSCs in liver fibrosis.

The Endocannabinoid Anandamide Selectively Induces Cell Death in Hepatoma Cell Lines

Background: Endocannabinoids mediate antineoplastic effects in several cancers including breast and prostate cancer. We have shown that the endocannabinoid anandamide (AEA) does not induce cell death in primary hepatocytes due to high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH). Aim: To determine whether the endocannabinoid AEA induces growth arrest and cell death in hepatoma cell lines. Methods: Rat and human hepatoma cell lines (FaO, McA-RH7777, HepG2, Hep3B, PCL/PRF/5) and primary rat hepatocytes were treated with varying concentrations of AEA. Cell death was determined by LDH release, western blot for caspase 3 and PARP cleavage and propidium iodide (PI) uptake. Proliferation was analyzed by 3H-thymidine uptake assay. Reactive oxygen species (ROS) formation was monitored by DCFDA fluorescence. FAAH expression was analyzed by western blot and real time PCR. FAAH overexpression was achieved by adenoviral infection. FAAH activity was measured colorimetrically and inhibited with the specific inhibitor URB597. Glutathione was depleted by BSO. Results: AEA induced dose-dependent cell death in all human and rat hepatoma cell lines, but not in primary rat hepatocytes. The rat hepatoma cell line FaO expressed almost no FAAH and was highly sensitive to AEA-induced cell death (60% at 50μM after 4h), whereas the rat hepatoma McA-RH cell line expressed higher levels and activity of FAAH and required longer AEA treatment to efficiently induce cell death (50% at 50μM after 24h). Overexpression of FAAH rendered FaO cells resistant to AEA whereas inhibition of FAAH activity strongly increased AEA sensitivity in McA-RH cells. Accordingly, FAAH overexpression inhibited, and FAAH inhibition increased AEA-induced cell death in human hepatoma cells. AEA markedly induced ROS formation in hepatoma cells. Blocking AEA-induced ROS formation by GSH pretreatment reduced AEA-induced cell death whereas GSH-depletion further increased AEA sensitivity of all hepatoma cells. Sublethal concentrations of AEA inhibited FCS-induced 3H-thymidine uptake (p<0.05). Conclusion: Anandamide induces ROS-dependent cell death in all investigated hepatoma cell lines, but not in primary hepatocytes. This higher sensitivity of hepatoma cells is due to the lower expression of FAAH. In addition, sublethal doses of AEA block proliferation in hepatoma cells. Modulation of the endocannabinoid system may represent a new option for the treatment of primary hepatocellular cancer.

Ex Vivo Imaging of Fatty Acid Amide Hydrolase Activity and Its Inhibition in the Mouse Brain

There is recent behavioral evidence that fatty acid amide hydrolase (FAAH) inhibitors produce a subset of cannabinoid receptor agonist effects, suggesting both anandamide-specific behavioral functions and possible regional differences in FAAH inhibitory effects. Here, we introduce a novel imaging method to quantify regional differences in brain FAAH activity. Upon intravenous [3H]anandamide administration, brain FAAH activity generates [3H]arachidonic acid, which is promptly trapped in membrane phospholipids. As a result, wild-type (WT) brains accumulate tritium in a regionally specific manner that is dependent upon regional FAAH activity, whereas brains from FAAH knockout (KO) mice show a uniform [3H]anandamide distribution. Increasing doses of anandamide + [3H]anandamide fail to alter regional tritium accumulation, suggesting insensitivity toward this process by anandamide-induced changes in regional cerebral blood flow. Regional tritiated metabolite levels in WT brains were highest in the somatosensory and visual cortices and the thalamus. Treatment with methylarachidonyl fluorophosphonate (MAFP) (1 mg/kg i.p.) reduced regional tritium accumulation in the somatosensory and visual cortices (p < 0.01), and at higher doses, the thalamus (p < 0.05). The selective FAAH inhibitor 1-oxazolo[4,5-b]pyridin-2-yl-1-dodecanone (CAY10435), although having similar efficacy as MAFP in reducing tritium in the thalamus and somatosensory and visual cortices, also reduces caudate putamen and cerebellum (p < 0.01) activity. These data indicate FAAH activity generates heterogeneous regional accumulation of [3H]anandamide and metabolites, and they suggest the modulation of endocannabinoid tone by FAAH inhibitors depends upon not only the dose and compound used but also on the degree of FAAH expression in the brain regions examined. This imaging method determines regionally specific FAAH inhibition and can elucidate the in vivo effects of pharmacological agents targeting anandamide inactivation.

Endocannabinoids in the intact retina:3H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide

There is much evidence for an endocannabinoid system in the retina. However, neither the distribution of endocannabinoid uptake, the regulation of endocannabinoid levels, nor the role of endocannabinoid metabolism have been investigated in the retina. Here we focused on one endocannabinoid, anandamide (AEA), and its major hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), in the goldfish retina. Immunoblots of FAAH immunoreactivity (IR) in goldfish retina, brain and rat retina, and brain homogenates showed a single band at 61 kDa that was blocked by preadsorption with peptide antigen. Specific FAAH IR (blocked by preadsorption) was most prominent over Müller cells and cone inner segments. Weaker label was observed over some amacrine cells, rare cell bodies in the ganglion cell layer, and in four lamina in the inner plexiform layer. FAAH activity assays showed that goldfish-retinal and brain homogenates hydrolyzed AEA at rates comparable to rat brain homogenate, and the hydrolysis was inhibited by methyl arachidonyl fluorophosphonate (MAFP) and N-(4 hydroxyphenyl)-arachidonamide (AM404), with IC(50)s of 21 nM and 1.5 microM, respectively. Cellular 3H-AEA uptake in the intact retina was determined by in vitro autoradiography. Silver-grain accumulation at 20 degrees C was most prominent over cone photoreceptors and Müller cells. Uptake was significantly reduced when retinas were incubated at 4 degrees C, or preincubated with 100 nM MAFP or 10 microM AM404. There was no differential effect of blocking conditions on the distribution of silver grains over cones or Müller cells. The codistribution of FAAH IR and 3H-AEA uptake in cones and Müller cells suggests that the bulk clearance of AEA in the retina occurs as a consequence of a concentration gradient created by FAAH activity. We conclude that endocannabinoids are present in the goldfish retina and underlay the electrophysiological effects of cannabinoid ligands previously shown on goldfish cones and bipolar cells.

Inhibitors of fatty acid amide hydrolase reduce carrageenan‐induced hind paw inflammation in pentobarbital‐treated mice: comparison with indomethacin and possible involvement of cannabinoid receptors

The in vivo effect of inhibitors of fatty acid amide hydrolase (FAAH) upon oedema volume and FAAH activity was evaluated in the carrageenan induced hind paw inflammation model in the mouse. Oedema was measured at two time points, 2 and 4 h, after intraplantar injection of carrageenan to anaesthetised mice. Intraperitoneal (i.p.) injections of the FAAH inhibitor URB597 (0.1, 0.3, 1 and 3 mg kg(-1)) 30 min prior to carrageenan administration, dose-dependently reduced oedema formation. At the 4 h time point, the ED(50) for URB597 was approximately 0.3 mg kg(-1). Indomethacin (5 mg kg(-1) i.p.) completely prevented the oedema response to carrageenan. The antioedema effects of indomethacin and URB597 were blocked by 3 mg kg(-1) i.p. of the CB(2) receptor antagonist SR144528. The effect of URB597 was not affected by pretreatment with the peroxisome proliferator-activated receptor gamma antagonist bisphenol A diglycidyl ether (30 mg kg(-1) i.p.) or the TRPV1 antagonist capsazepine (10 mg kg(-1) i.p.), when oedema was assessed 4 h after carrageenan administration. The CB(1) receptor antagonists AM251 (3 mg kg(-1) i.p.) and rimonabant (0.5 mg kg(-1) i.p.) gave inconsistent effects upon the antioedema effect of URB597. FAAH measurements were conducted ex vivo in the paws, spinal cords and brains of the mice. The activities of FAAH in the paws and spinal cords of the inflamed vehicle-treated mice were significantly lower than the corresponding activities in the noninflamed mice. PMSF treatment almost completely inhibited the FAAH activity in all three tissues, as did the highest dose of URB597 (3 mg kg(-1)) in spinal cord samples, whereas no obvious changes were seen ex vivo for the other treatments. In conclusion, the results show that in mice, treatment with indomethacin and URB597 produce SR144528-sensitive anti-inflammatory effects in the carrageenan model of acute inflammation.

Involvement of N-acylethanolamine-hydrolyzing acid amidase in the degradation of anandamide and other N-acylethanolamines in macrophages

Bioactive N-acylethanolamines including the endocannabinoid anandamide are known to be hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH). In addition, we recently cloned an isozyme termed “ N-acylethanolamine-hydrolyzing acid amidase (NAAA)”, which is active only at acidic pH [Tsuboi, Sun, Okamoto, Araki, Tonai, Ueda, J. Biol. Chem. 285 (2005) 11082–11092]. However, physiological roles of NAAA remained unclear. Here, we examined a possible contribution of NAAA to the degradation of various N-acylethanolamines in macrophage cells. NAAA mRNA as well as FAAH mRNA was detected in several macrophage-like cells, including RAW264.7, and mouse peritoneal macrophages. The homogenates of RAW264.7 cells showed both the NAAA and FAAH activities which were confirmed with the aid of their respective specific inhibitors, N-cyclohexanecarbonylpentadecylamine (CCP) and URB597. As analyzed with intact cells, RAW264.7 cells and peritoneal macrophages degraded anandamide, N-palmitoylethanolamine, N-oleoylethanolamine, and N-stearoylethanolamine. Pretreatment of the cells with CCP or URB597 partially inhibited the degradation, and a combination of the two compounds caused more profound inhibition. In contrast, the anandamide hydrolysis in mouse brain appeared to be principally attributable to FAAH despite the expression of NAAA in the brain. These results suggested that NAAA and FAAH cooperatively degraded various N-acylethanolamines in macrophages.

Inhibition of fatty-acid amide hydrolase enhances cannabinoid stress-induced analgesia: Sites of action in the dorsolateral periaqueductal gray and rostral ventromedial medulla

Recent research in our laboratory has demonstrated that stress activates an endogenous cannabinoid mechanism that suppresses sensitivity to pain [Nature 435 (2005) 1108]. In this work, CB 1 antagonists administered systemically blocked stress-induced analgesia induced by brief, continuous foot-shock. The present studies were conducted to examine the role of cannabinoid CB 1 receptors in the brainstem rostral ventromedial medulla (RVM) and midbrain dorsolateral periaqueductal gray (PAG) in cannabinoid stress-induced analgesia (SIA). Pharmacological blockade of vanilloid TRPV1 receptors with capsazepine, administered systemically, did not alter cannabinoid SIA, suggesting that cannabinoid SIA was not dependent upon TRPV1. Microinjection of the competitive CB 1 antagonist rimonabant (SR141716A) into either the RVM or dorsolateral PAG suppressed stress antinociception in this model. Rimonabant was maximally effective following microinjection into the dorsolateral PAG. The fatty-acid amide hydrolase (FAAH) inhibitor arachidonoyl serotonin (AA-5-HT) was subsequently used to block hydrolysis of endocannabinoids and enhance SIA. Systemic and site-specific injections of AA-5-HT into either the dorsolateral PAG or RVM induced CB 1-mediated enhancements of SIA. Palmitoyltrifluoromethylketone, a potent inhibitor of FAAH and phospholipase A2 activity, administered systemically, exerted similar effects. In all conditions, the antinociceptive effects of each FAAH inhibitor were completely blocked by coadministration of the CB 1 antagonist rimonabant. The present results provide evidence that a descending cannabinergic neural system is activated by environmental stressors to modulate pain sensitivity in a CB 1-dependent manner.