Inhibition of fatty acid amidohydrolase, the enzyme responsible for the metabolism of the endocannabinoid anandamide, by analogues of arachidonoyl-serotonin

Arachidonoyl-serotonin inhibits in a mixed-type manner the metabolism of the endocannabinoid anandamide by the enzyme fatty acid amidohydrolase. In the present study, compounds related to arachidonoyl-serotonin have been synthesised and investigated for their ability to inhibit anandamide hydrolysis by this enzyme in rat brain homogenates. Removal of the 5-hydroxy from the serotonin head group of arachidonoyl-serotonin produced a compound (N-arachidonoyltryptamine) that was a 2.3-fold weaker inhibitor of anandamide hydrolysis, but which also produced its inhibition by a mixed-type manner (Ki(slope) 1.3 microM; Ki(intercept) 44 microM). Replacement of the amide linkage in this compound by an ester group further reduced the potency. In contrast, replacement of the arachidonoyl side chain by a linolenoyl side chain did not affect the observed potency. N-(Fur-3-ylmethyl) arachidonamide (UCM707), N-(fur-3-ylmethyl)linolenamide and N-(fur-3-ylmethyl)oleamide inhibited anandamide hydrolysis with pI50 values of 4.53, 5.36 and 5.25, respectively. The linolenamide derivative was also found to be a mixed-type inhibitor. It is concluded that the 5-hydroxy group of arachidonoyl-serotonin contributes to, but is not essential for, inhibitory potency at fatty acid amidohydrolase.     

Coevolution between cannabinoid receptors and endocannabinoid ligands

Genes for receptors and ligands must coevolve to maintain coordinated gene expression and binding affinities. Researchers have debated whether anandamide or 2-arachidonyl glycerol (2-AG) is a more "intrinsic" ligand of cannabinoid receptors. We addressed this debate with a coevolutionary analysis, by examining genes for CB1, CB2, and ten genes that encode ligand metabolic enzymes: abhydrolase domain containing 4 protein, cyclooxygenase 2, diacylglycerol lipase paralogs (DAGLalpha, DAGLbeta), fatty acid amide hydrolase paralogs (FAAH1, FAAH2), monoglyceride lipase, N-acylethanolamine acid amidase, NAPE-selective phospholipase D, and protein tyrosine phosphatase non-receptor type 22. Gene trees (cladograms) of CB1, CB2, and ligand enzymes were obtained by searching for orthologs (tBLASTn) in the genomes of nine phylogenetically diverse species, aligning ortholog sequences with ClustalX, and applying Bayesian analysis (MrBayes). Mirrored cladograms provided evidence of coevolution (i.e., parallel cladogenesis). Next we constructed phylograms of CB1, CB2, and the ten enzymes. Phylogram branch lengths were proportional to three sets of maximum likelihood metrics: all-nucleotide-substitutions and NS/SS ratios (using PAUP()), and Ka/Ks ratios (using FUGE). Spurious correlations in all-nucleotide-substitutions trees (due to phylogenetic bias) and in Ka/Ks ratio trees (due to simplistic modeling) were parsed. Branch lengths from equivalent branches in paired trees were correlated by linear regression. Regression analyses, mirrored cladograms, and phylogenetic profiles produced the same results: close associations between cannabinoid receptors and DAGL enzymes. Therefore we propose that cannabinoid receptors initially coevolved with a fatty acid ester ligand (akin to 2-AG) in ancestral metazoans, and affinity for fatty acid ethanolamide ligands (e.g., AEA) evolved thereafter.     

N-Morpholino- and N-diethyl-analogues of palmitoylethanolamide increase the sensitivity of transfected human vanilloid receptors to activation by anandamide without affecting fatty acid amidohydrolase activity

The abilities of 19 analogues of palmitoylethanolamide and two analogues of oleoylethanolamide to affect the Ca(2+) influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) in response to anandamide (AEA) have been investigated using a FLIPR assay and a bovine serum albumin-containing assay medium. Only palmitoylethanolamide produced any effect in the absence of AEA. The ability of palmitoylethanolamide to potentiate the response to AEA was retained when the N-CH(2)CH(2)OH group was replaced by N-CH(2)CH(2)Cl,whereas replacement with N-alkyl substituents [from -H up to -(CH(2))(12)CH(3)] resulted either in a reduction or in a complete loss of this activity. The tertiary amide N-(CH(2)CH(3))(2) (19) and N-morpholino (20) analogues of palmitoylethanolamide potentiated the response to 1 microM AEA to a greater degree than the parent compound, whereas the N-(CH(3))(2) analogue was inactive. 19 and 20 produced leftward shifts in the dose-response curve for AEA activation of Ca(2+) influx into hVR1-HEK293 cells. EC(50) values for AEA to produce Ca(2+) influx into hVR1-HEK293 cells were 1.1, 1.1, 0.54 and 0.36 microM in the presence of 0, 1, 3 and 10 microM 19, respectively. The corresponding values for 20 were 1.5, 1.3, 0.77 and 0.17 microM, respectively. The compounds did not affect the dose-response curves to capsaicin. The ability of oleoylethanolamide to potentiate AEA is retained by the N-CH(2)CH(3) and N-CH(CH(3))(2) analogues (22 and 23, respectively). 22 and 23 produced a small ( approximately 25%) inhibition of the binding of [(3)H]-CP55,940 and [(3)H]-WIN 55,212-2 to CB(1) and CB(2) receptors, respectively, expressed in CHO cells. The compounds inhibited the metabolism of 2 microM [(3)H]-AEA by rat brain fatty acid amidohydrolase with IC(50) values of 5.6 and 11 microM, respectively. In contrast, 19 and 20 were without effect on either binding to CB receptors or fatty acid amidohydrolase activity. Minor reductions in the accumulation of 10 microM [(3)H]-AEA into C6 glioma cells were seen at 10 microM concentrations of 19 and 20. It is concluded that 19 and 20 selectively enhance AEA effects upon VR1 receptors without potentially confounding effects upon CB receptors or fatty acid amidohydrolase activity.     

Homologues and isomers of noladin ether,a putative novel endocannabinoid: interaction with rat cannabinoid CB(1) receptors

Two regioisomers and 13 analogues of the putative endocannabinoid noladin ether (2-arachidonyl glyceryl ether, 2-AGE, 1) were synthesized and tested for their interaction with CB(1) receptors in rat brain membranes. The results showed that a C-20 tetra-unsaturated moiety is necessary for high affinity, and that a series of alkyl glyceryl ethers of potential occurrence in brain tissues have less affinity than 2-AGE for CB(1) receptors.     

Amino acid determinants in cyclooxygenase-2 oxygenation of the endocannabinoid anandamide

The endocannabinoid arachidonylethanolamide (AEA, anandamide) is an endogenous ligand for the cannabinoid receptors and has been shown to be oxygenated by cyclooxygenase-2 (COX-2). We examined the structural requirements for COX-mediated, AEA oxygenation using a number of substrate analogues and site-directed mutants of COX-2. Fourteen AEA analogues were synthesized and tested as COX substrates. These studies identified the hydroxyl moiety of AEA as a critical determinant in the ability of COX enzymes to effect robust endocannabinoid oxygenation. In addition, these studies suggest that subtle structural modifications of AEA analogues near the ethanolamide moiety can result in pronounced changes in their ability to serve as COX-2 substrates. Site-directed mutagenesis studies have permitted the development of a model of AEA binding within the COX-2 active site. As with arachidonic acid, the omega-terminus of AEA binds in a hydrophobic alcove near the top of the COX-2 active site. The polar ethanolamide moiety of AEA, like the carboxylate of arachidonate, interacts with Arg-120 at the bottom of the COX-2 active site. Mutation of Tyr-385 prevents AEA oxygenation, suggesting that, as in the case of other COX substrates, AEA metabolism is initiated by Tyr-385-mediated hydrogen abstraction. Thus, AEA binds within the COX-2 active site in a conformation roughly similar to that of arachidonic acid. However, important differences have been identified that account for the isoform selectivity of AEA oxygenation. Importantly, the COX-2 side pocket and Arg-513 in particular are critical determinants of the ability of COX-2 to efficiently generate prostaglandin H(2) ethanolamide. The reduced efficiency of COX-1-mediated, AEA oxygenation can thus be explained by the absence of an arginine residue at position 513 in this isoform. Mutational analysis of Leu-531, an amino acid located directly across from the COX-2 side pocket, suggests that AEA is shifted away from this hydrophobic residue and toward Arg-513 relative to arachidonic acid. Coupled with earlier observations with the endocannabinoid 2-arachidonylglycerol, these results indicate that one possible function of the highly conserved COX-2 active site side pocket is to promote endocannabinoid oxygenation.     

Mechanisms underlying in vivo neuroprotection by the endocannabinoid anandamide and the cannabinoid/vanilloid receptor agonist,arvanil

The endocannabinoid anandamide (AEA) and some of its oxidative metabolites are putative ligands for the vanilloid receptor (VR₁). AEA affords protection against excitotoxicity induced in vivo by ouabain, a Na⁺/K⁺‐ATPase inhibitor. This effect is only partly dependent of the cannabinoid CB₁ receptor. Here, we assessed whether VR₁ is involved in neuroprotection by AEA and arvanil, which is a hydrolysis‐stable ligand for both VR₁ and CB₁. Using magnetic resonance imaging we show that: (i) modulation of VR₁, by the agonists arvanil and capsaicin and the antagonist capsazepine, leads to neuroprotective effects in the late but not acute phase after i.c. ouabain‐injection; (ii) arvanil is a potent neuroprotectant, acting at both CB₁ and VR₁; and (iii) the neuroprotective effects of AEA are mediated by CB₁ but not by lipoxygenase metabolites or VR₁.     

Endocannabinoid structure-activity relationships for interaction at the cannabinoid receptors

Anandamide (N -arachidonoylethanolamine) was the first ligand to be identified as an endogenous ligand of the G-protein coupled cannabinoid CB1 receptor. Subsequently, two other fatty acid ethanolamides, N -homo- gamma -linolenylethanolamine and N -7,10,13,16-docosatetraenylethanolamine were identified as endogenous cannabinoid ligands. A fatty acid ester, 2-arachidonoylglycerol (2-AG), and a fatty acid ether, 2-arachidonyl glyceryl ether also have been isolated and shown to be endogenous cannabinoid ligands. Recent studies have postulated the existence of carrier-mediated anandamide transport that is essential for termination of the biological effects of anandamide. A membrane bound amidohydrolase (fatty acid amide hydrolase, FAAH), located intracellularly, hydrolyzes and inactivates anandamide and other endogenous cannabinoids such as 2-AG. 2-AG has also been proposed to be an endogenous CB2 ligand. Structure-activity relationships (SARs) for endocannabinoid interaction with the CB receptors are currently emerging in the literature. This review considers cannabinoid receptor SAR developed to date for the endocannabinoids with emphasis upon the conformational implications for endocannabinoid recognition at the cannabinoid receptors.     

The endocannabinoid system,anandamide and the regulation of mammalian cell apoptosis

Endocannabinoids are a new class of lipid mediators, which include amides, esters and ethers of long-chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors able to mimic several pharmacological effects of Delta-9-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. The pathways leading to the synthesis and release of AEA and 2-AG from neuronal and non-neuronal cells are still rather uncertain. Instead, it is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Together with AEA and congeners these proteins form the 'endocannabinoid system'. Here, the involvement of AEA in apoptosis and the underlying signal transduction pathways will be reviewed, along with the metabolic routes and the molecular targets of this endocannabinoid. Also, recent findings on the apoptotic potential of AEA for neuronal cell differentiation and brain development will be discussed.     

Noladin ether, a putative endocannabinoid, inhibits mu-opioid receptor activation via CB2 cannabinoid receptors

We examined the occurrence of possible changes in mRNA expression and the functional activity of opioid receptors after acute in vivo and in vitro treatment with the putative endogenous cannabinoid noladin ether. While noladin ether (NE) demonstrates agonist activity at CB1 cannabinoid receptors, recent data indicate that NE acts as a full agonist at CB2 cannabinoid receptors too. Considering the functional interactions between opioids and cannabinoids, it is of interest to examine whether NE affects the opioid system. To that end, we studied the influence of NE on mu-opioid receptor (MOR) mRNA expression and MOR mediated G-protein signaling. We used real-time PCR and [35S]GTPgammaS binding assays to examine the changes of MOR mRNA levels and the capability of the mu-opioid agonist peptide ([D-Ala2,(NMe)Phe4,Gly5-ol]enkephalin (DAMGO) in activating regulatory G-proteins via MORs in forebrain membrane fractions of wild-type (w.t., CB1+/+) and CB1 receptor deficient transgenic mice (knockout, CB1-/-). We found, that the expression of MOR mRNAs significantly decreased both in CB1+/+ and CB1-/- forebrain after a single injection of NE at 1 mg/kg when compared to control. Consequently, MOR-mediated signaling is attenuated after acute in vivo treatment with NE in both CB1+/+ and CB1-/- mice. Inhibition on MOR mediated activation is observed after in vitro NE administration as well. Radioligand binding competition studies showed that the noticed effect of NE on MOR signaling is not mediated through MORs. Both in vivo and in vitro attenuations of NE can be antagonized by the CB2 selective antagonist SR144528. Taken together, our data suggest that the NE caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors.     

Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain

Anandamide is an endogenous signaling lipid that binds to and activates cannabinoid receptors in the brain and peripheral tissues. The endogenous precursors of anandamide, N-arachidonoyl phosphatidylethanolamines (NArPEs), are a family of complex glycerophospholipids that derive from the exchange reaction of an arachidonoyl group between the sn-1 position of phosphatidylcholine and the primary amine of phosphatidylethanolamine catalyzed by N-acyl transferase activity. A precise characterization of the molecular composition of NArPE species generating anandamide has not yet been reported. In the present study, using liquid chromatography coupled to electrospray ionization ion-trap mass spectrometry, we identified the major endogenous NArPE species, which mainly contained sn-1 alkenyl groups (C16:0, C18:0, C18:1) and monounsaturated (C18:1) or polyunsaturated (C20:4, C22:4, C22:6) acyl groups at the sn-2 position of the glycerol backbone. Using rat brain particulate fractions, we observed a calcium-dependent increase in both NArPEs and anandamide formation after incubation at 37 degrees C for 30 min. Furthermore, a targeted lipidomic analysis showed that Ca(2+) specifically stimulated the formation of PUFA-containing NArPE species. These results reveal a previously unrecognized preference of brain N-acyl transferase activity for polyunsaturated NArPE and provide new insights on the physiological regulation of anandamide biosynthesis.     

Arachidonic acid and anandamide have opposite modulatory actions at the glycine transporter,GLYT1a

The GLYT1 subtypes of glycine transporter are expressed in glia surrounding excitatory synapses in the mammalian CNS and may regulate synaptic glycine concentrations required for activation of the NMDA subtypes of glutamate receptor. In this report we demonstrate that the rate of glycine transport by GLYT1 is inhibited by arachidonic acid. The cyclo-oxygenase and lipoxygenase inhibitors indomethacin and nordihydroguaiaretic acid, and the protein kinase C inhibitor staurosporine, had no effect on the extent of arachidonic acid inhibition of transport, which suggests that the inhibitory effects of arachidonic acid result from a direct interaction with the transporter. In contrast to arachidonic acid, its amide derivative, anandamide, and the more stable analogue R1-methanandamide stimulate glycine transport. This stimulation is unlikely to be a secondary effect of cannabinoid receptor stimulation because the cannabinoid receptor agonist WIN 55 212-2 had no effect on transport. We suggest that the stimulatory effects of anandamide on GLYT1 are due to a direct interaction with the transporter.     

New metabolically stable fatty acid amide ligands of cannabinoid receptors: Synthesis and receptor affinity studies

We investigated the structure-activity relationships for the interactions of fatty acid amide analogs of the endocannabinoid anandamide with human recombinant cannabinoid receptors. Thirty-five novel fatty acid amides were synthesized using five different types of acyl chains and 11 different aromatic amine 'heads.' Although none of the new compounds was a more potent ligand than anandamide, we identified three amine groups capable of improving the metabolic stability of arachidonoylamides and their CB(1)/CB(2) selectivity ratio to over 20-fold, and several aromatic amines capable of improving the affinity of short chain or monosaturated fatty acids for cannabinoid CB(1) receptors. For the first time a tertiary amide of arachidonic acid was found to possess moderate affinity (K(i)=300 nM) for cannabinoid CB(1), but not CB(2), receptors.     

Conformational and amino acid residue requirements for the saposin C neuritogenic effect.

Prosaposin is the precursor of four activator proteins, termed saposins A, B, C, and D, that are required for much of glycosphingolipid hydrolysis. The intact precursor also has neurite outgrowth activity ex vivo and in vivo that is localized to amino acid residues 22-31 of saposin C. Across species, this saposin C region has a high degree of identity and similarity with amino acids in the analogous region of saposin A. Wild-type and mutant saposins C and A from human and mouse were expressed in E. coli. Pure proteins, synthetic peptide analogues, conformation-specific antibodies, and CD spectroscopy were used to evaluate the basis of the ex vivo neuritogenic effect. Wild-type saposin A had no neuritogenic activity whereas reduced and alkylated saposin A did. Introduction of the conserved saposin A Tyr 30 (Y30) into saposin C at the analogous position 31, a conserved Ala(A)/Gly(G)31, diminished neuritogenic activity by 50-60%. Nondenatured saposin A with an introduced A30 acquired substantial neuritogenic activity. Polyclonal antibodies directed against the NH2-terminus of saposin C cross-reacted well with reduced and alkylated saposins C and A, wild-type saposin C, and saposin A [Y30A], poorly with saposin C [A31Y], and not at all with wild-type saposin A. CD spectra of wild-type and mutant saposins C and A, the corresponding neuritogenic region of saposin C, and the analogous region of saposin A showed that more "saposin C-like" molecules had neuritogenic properties. Those with more "saposin A-like" spectra did not. These studies show that the neuritogenic activity of saposin C requires specific placement of amino acids, and that Y30 of saposin A significantly alters local conformation in this critical region and suppresses neuritogenic activity.     

Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells

Anandamide (AEA) is an endogenous agonist for the cannabinoid receptor 2 (CB2) which is expressed in osteoblasts. Arachidonic acid (AA) is the precursor for AEA and dietary n-3 polyunsaturated fatty acids (PUFA) are known to reduce the concentrations of AA in tissues and cells. Therefore, we hypothesized that n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which reduce AA in cells, could lower AEA in osteoblasts by altering enzyme expression of the endocannabinoid (EC) system. MC3T3-E1 osteoblast-like cells were grown for 6, 10, 15, 20, 25 or 30 days in osteogenic medium. Osteoblasts were treated with 10 μM of AA, EPA, DHA, oleic acid (OA) or EPA+DHA (5 μM each) for 72 h prior to their collection for measurement of mRNA and alkaline phosphatase (ALP) activity. Compared to vehicle control, osteoblasts treated with AA had higher levels of AA and n-6 PUFA while those treated with EPA and DHA had lower n-6 but higher n-3 PUFA. Independent of the fatty acid treatments, osteoblasts matured normally as evidenced by ALP activity. N-acyl phosphatidylethanolamine-selective phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH) and CB2 mRNA expression were higher at 20 days compared to 10 days. NAPE-PLD and CB2 mRNA was lower in osteoblasts treated with EPA compared to all other groups. Thus, mRNA expression for NAPE-PLD, FAAH, and CB2 increased during osteoblast maturation and EPA reduced mRNA for NAPE-PLD and CB2 receptor. In conclusion, EPA lowered mRNA levels for proteins of the EC system and mRNA for AEA synthesis/degradation is reported in osteoblasts.     

The conformation, location, and dynamic properties of the endocannabinoid ligand anandamide in a membrane bilayer

The endogenous cannabinoid ligand anandamide is biosynthesized from membrane phospholipid precursors and is believed to reach its sites of action on the CB1 and CB2 receptors through fast lateral diffusion within the cell membrane. To gain a better insight on the stereochemical features of its association with the cell membrane and its interaction with the cannabinoid receptors, we have studied its conformation, location, and dynamic properties in a dipalmitoylphosphatidylcholine multilamellar model membrane bilayer system. By exploiting the bilayer lattice as an internal three-dimensional reference grid, the conformation and location of anandamide were determined by measuring selected inter- and intramolecular distances between strategically introduced isotopic labels using the rotational echo double resonance (REDOR) NMR method. A molecular model was proposed to represent the structural features of our anandamide/lipid system and was subsequently used in calculating the multispin dephasing curves. Our results demonstrate that anandamide adopts an extended conformation within the membrane with its headgroup at the level of the phospholipid polar group and its terminal methyl group near the bilayer center. Parallel static (2)H NMR experiments further confirmed these findings and provided evidence that anandamide experiences dynamic properties similar to those of the membrane phospholipids and produces no perturbation to the bilayer. Our results are congruent with a hypothesis that anandamide approaches its binding site by laterally diffusing within one membrane leaflet in an extended conformation and interacts with a hydrophobic groove formed by helices 3 and 6 of CB1, where its terminal carbon is positioned close to a key cysteine residue in helix 6 leading to receptor activation.     

Molecular probes for the cannabinoid receptors

Cannabinoids produce most of their biochemical and pharmacological effects by interacting with CB1 and CB2 cannabinoid receptors, both of which are G-protein coupled membrane-bound functional proteins. CB1 is found in the central nervous system and in a variety of other organs including heart, vascular endothelium, uterus, vas deferens, testis and small intestine. Conversely, the CB2 receptor appears to be associated exclusively with the immune system and is found in the periphery of the spleen and other cells associated with immunochemical functions. Although both CB1 and CB2 have been cloned and the primary sequences are known, their three dimensional structures and the amino acid residues at the active site, critical for ligand recognition, binding and activation have not been characterized. In the absence of any X-ray crystallographic and NMR data, information on the structural requirements for ligand-receptor interactions is obtained with the help of suitably designed molecular probes. These ligands either interact with the receptor in a reversible fashion (reversible probes) or, alternatively, attach at or near the receptor active site with the formation of a covalent bond (irreversible probes). Subsequently, information related to ligand binding and receptor activation is further amplified with the help of receptor mutants and computer modeling. This review focuses on molecular probes related to the classical and non-classical cannabinoids that have been reported since the discovery of the first cannabinoid receptor over a decade ago.     

Human mast cells take up and hydrolyze anandamide under the control of 5-lipoxygenase and do not express cannabinoid receptors

Human mast cells (HMC-1) take up anandamide (arachidonoyl-ethanolamide, AEA) with a saturable process (K(m)=200+/-20 nM, V(max)=25+/-3 pmol min(-1) mg protein(-1)), enhanced two-fold over control by nitric oxide-donors. Internalized AEA was hydrolyzed by a fatty acid amide hydrolase (FAAH), whose activity became measurable only in the presence of 5-lipoxygenase, but not cyclooxygenase, inhibitors. FAAH (K(m)=5.0+/-0.5 microM, V(max)=160+/-15 pmol min(-1) mg protein(-1)) was competitively inhibited by palmitoylethanolamide. HMC-1 cells did not display a functional cannabinoid receptor on their surface and neither AEA nor palmitoylethanolamide affected tryptase release from these cells.     

Hemodynamic profile, responsiveness to anandamide, and baroreflex sensitivity of mice lacking fatty acid amide hydrolase

The endocannabinoid anandamide exerts neurobehavioral, cardiovascular, and immune-regulatory effects through cannabinoid receptors (CB). Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the in vivo degradation of anandamide. Recent experimental studies have suggested that targeting the endocannabinergic system by FAAH inhibitors is a promising novel approach for the treatment of anxiety, inflammation, and hypertension. In this study, we compared the cardiac performance of FAAH knockout (FAAH-/-) mice and their wild-type (FAAH+/+) littermates and analyzed the hemodynamic effects of anandamide using the Millar pressure-volume conductance catheter system. Baseline cardiovascular parameters, systolic and diastolic function at different preloads, and baroreflex sensitivity were similar in FAAH-/- and FAAH+/+ mice. FAAH-/- mice displayed increased sensitivity to anandamide-induced, CB1-mediated hypotension and decreased cardiac contractility compared with FAAH(+/+) littermates. In contrast, the hypotensive potency of synthetic CB1 agonist HU-210 and the level of expression of myocardial CB1 were similar in the two strains. The myocardial levels of anandamide and oleoylethanolamide, but not 2-arachidonylglycerol, were increased in FAAH-/- mice compared with FAAH+/+ mice. These results indicate that mice lacking FAAH have a normal hemodynamic profile, and their increased responsiveness to anandamide-induced hypotension and cardiodepression is due to the decreased degradation of anandamide rather than an increase in target organ sensitivity to CB1 agonists.     

Targeting the endocannabinoid system with cannabinoid receptor agonists: pharmacological strategies and therapeutic possibilities

Human tissues express cannabinoid CB₁ and CB₂ receptors that can be activated by endogenously released ‘endocannabinoids’ or exogenously administered compounds in a manner that reduces the symptoms or opposes the underlying causes of several disorders in need of effective therapy. Three medicines that activate cannabinoid CB₁/CB₂ receptors are now in the clinic: Cesamet (nabilone), Marinol (dronabinol; Δ⁹-tetrahydrocannabinol (Δ⁹-THC)) and Sativex (Δ⁹-THC with cannabidiol). These can be prescribed for the amelioration of chemotherapy-induced nausea and vomiting (Cesamet and Marinol), stimulation of appetite (Marinol) and symptomatic relief of cancer pain and/or management of neuropathic pain and spasticity in adults with multiple sclerosis (Sativex). This review mentions several possible additional therapeutic targets for cannabinoid receptor agonists. These include other kinds of pain, epilepsy, anxiety, depression, Parkinson''s and Huntington''s diseases, amyotrophic lateral sclerosis, stroke, cancer, drug dependence, glaucoma, autoimmune uveitis, osteoporosis, sepsis, and hepatic, renal, intestinal and cardiovascular disorders. It also describes potential strategies for improving the efficacy and/or benefit-to-risk ratio of these agonists in the clinic. These are strategies that involve (i) targeting cannabinoid receptors located outside the blood-brain barrier, (ii) targeting cannabinoid receptors expressed by a particular tissue, (iii) targeting upregulated cannabinoid receptors, (iv) selectively targeting cannabinoid CB₂ receptors, and/or (v) adjunctive ‘multi-targeting’.