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Vincenzo Di Marzo - One of the best experts on this subject based on the ideXlab platform.
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Anandamide acts as an intracellular messenger amplifying ca2 influx via trpv1 channels
The EMBO Journal, 2005Co-Authors: Mario Van Der Stelt, Luciano De Petrocellis, Aniello Schiano Moriello, Marcello Trevisani, Vittorio Vellani, Barbara Campi, Peter A Mcnaughton, P Geppetti, Vincenzo Di MarzoAbstract:The endocannabinoid Anandamide is able to interact with the transient receptor potential vanilloid 1 (TRPV1) channels at a molecular level. As yet, endogenously produced Anandamide has not been shown to activate TRPV1, but this is of importance to understand the physiological function of this interaction. Here, we show that intracellular Ca2+ mobilization via the purinergic receptor agonist ATP, the muscarinic receptor agonist carbachol or the Ca2+-ATPase inhibitor thapsigargin leads to formation of Anandamide, and subsequent TRPV1-dependent Ca2+ influx in transfected cells and sensory neurons of rat dorsal root ganglia (DRG). Anandamide metabolism and efflux from the cell tonically limit TRPV1-mediated Ca2+ entry. In DRG neurons, this mechanism was found to lead to TRPV1-mediated currents that were enhanced by selective blockade of Anandamide cellular efflux. Thus, endogenous Anandamide is formed on stimulation of metabotropic receptors coupled to the phospholipase C/inositol 1,4,5-triphosphate pathway and then signals to TRPV1 channels. This novel intracellular function of Anandamide may precede its action at cannabinoid receptors, and might be relevant to its control over neurotransmitter release.
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Anandamide as an intracellular messenger regulating ion channel activity.
Prostaglandins & other lipid mediators, 2005Co-Authors: Mario Van Der Stelt, Vincenzo Di MarzoAbstract:The endocannabinoid Anandamide (N-arachidonoylethanolamine) was proposed to be an extracellular retrograde messenger, which regulates excitability of neurons by cannabinoid CB1 receptor-dependent inhibition of neurotransmitter release. Recent findings indicate that the neuromodulatory actions of Anandamide might be more complex. Anandamide has been shown to directly modulate various ion channels, such as alpha7-nicotinic acetylcholine receptors, T-type Ca2+ channels, voltage-gated and background K+-channels and Transient Receptor Potential Vanilloid type 1 (TRPV1) channels. The binding site of Anandamide at some of these ion channels appears to be intracellular or at the bilayer interface. This rises the intriguing possibility that Anandamide, prior to its release into the synaptic cleft, may regulate ion homeostasis and excitability of neurons as an intracellular modulator of ion channels independent of its action at cannabinoid CB1 receptors. This possibility might extend the concept of Anandamide as an endocannabinoid retrograde messenger and may have profound implications for its role in neurotransmission and neuronal function. Here, we will review the evidence for this hypothesis.
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the activity of Anandamide at vanilloid vr1 receptors requires facilitated transport across the cell membrane and is limited by intracellular metabolism
Journal of Biological Chemistry, 2001Co-Authors: Luciano De Petrocellis, John B. Davis, Tiziana Bisogno, Mauro Maccarrone, Alessandro Finazziagro, Vincenzo Di MarzoAbstract:Abstract The endogenous ligand of CB1cannabinoid receptors, Anandamide, is also a full agonist at vanilloid VR1 receptors for capsaicin and resiniferatoxin, thereby causing an increase in cytosolic Ca2+ concentration in human VR1-overexpressing (hVR1-HEK) cells. Two selective inhibitors of Anandamide facilitated transport into cells, VDM11 and VDM13, and two inhibitors of Anandamide enzymatic hydrolysis, phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, inhibited and enhanced, respectively, the VR1-mediated effect of Anandamide, but not of resiniferatoxin or capsaicin. The nitric oxide donor, sodium nitroprusside, known to stimulate Anandamide transport, enhanced Anandamide effect on the cytosolic Ca2+ concentration. Accordingly, hVR1-HEK cells contain an Anandamide membrane transporter inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and an Anandamide hydrolase activity sensitive to phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, and a fatty acid amide hydrolase transcript. These findings suggest the following. (i) Anandamide activates VR1 receptors by acting at an intracellular site. (ii) Degradation by fatty acid amide hydrolase limits Anandamide activity on VR1; and (iii) the Anandamide membrane transporter inhibitors can be used to distinguish between CB1 or VR1 receptor-mediated actions of Anandamide. By contrast, the CB1 receptor antagonist SR141716A inhibited also the VR1-mediated effect of Anandamide and capsaicin on cytosolic Ca2+ concentration, although at concentrations higher than those required for CB1 antagonism.
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Vanilloid receptors on sensory nerves mediate the vasodilator action of Anandamide
Nature, 1999Co-Authors: Peter M. Zygmunt, Vincenzo Di Marzo, Jesper Petersson, David A. Andersson, Huai-hu Chuang, Morten Sørgård, David Julius, Edward D. HögestättAbstract:The endogenous cannabinoid receptor agonist Anandamide is a powerful vasodilator of isolated vascular preparations, but its mechanism of action is unclear. Here we show that the vasodilator response to Anandamide in isolated arteries is capsaicin-sensitive and accompanied by release of calcitonin-gene-related peptide (CGRP). The selective CGRP-receptor antagonist 8-37 CGRP, but not the cannabinoid CB1 receptor blocker SR141716A, inhibited the vasodilator effect of Anandamide. Other endogenous (2-arachidonylglycerol, palmitylethanolamide) and synthetic (HU 210, WIN 55,212-2, CP 55,940) CB1 and CB2 receptor agonists could not mimic the action of Anandamide. The selective 'vanilloid receptor' antagonist capsazepine inhibited Anandamide-induced vasodilation and release of CGRP. In patch-clamp experiments on cells expressing the cloned vanilloid receptor (VR1), Anandamide induced a capsazepine-sensitive current in whole cells and isolated membrane patches. Our results indicate that Anandamide induces vasodilation by activating vanilloid receptors on perivascular sensory nerves and causing release of CGRP. The vanilloid receptor may thus be another molecular target for endogenous Anandamide, besides cannabinoid receptors, in the nervous and cardiovascular systems.
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two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma Anandamide amidohydrolase
FEBS Letters, 1995Co-Authors: S Maurelli, Luciano De Petrocellis, Tiziana Bisogno, Aldo Di Luccia, Gennaro Marino, Vincenzo Di MarzoAbstract:The endogenous cannabimimetic substance, Anandamide (N-arachidonoyl-ethanolamine) and the recently isolated sleep-inducing factor, oleoyl-amide (cis-9,10-octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of 'Anandamide amidohydrolase' from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]Anandamide was found in both microsomal and 10,000 x g pellet fractions. The latter fractions, which displayed the highest Vmax for Anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl-amide, with an apparent Km of 9.0 +/- 2.2 microM. [14C]Anandamide- and [14C]oleoyl-amide-hydrolysing activities: (i) exhibited identical pH- and temperature-dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl-trifluoromethyl-ketone with the same IC50 (3 microM); (iv) were competitively inhibited by both Anandamide (or other polyunsaturated fatty acid-ethanolamides) and oleoyl-amide. Proteins solubilised from 10,000 x g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]Anandamide and [14C]oleoyl-amide. These data suggest that 'Anandamide amidohydrolase' enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep-inducing factor oleoyl-amide.
Daniele Piomelli - One of the best experts on this subject based on the ideXlab platform.
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the endogenous cannabinoid Anandamide has effects on motivation and anxiety that are revealed by fatty acid amide hydrolase faah inhibition
Neuropharmacology, 2008Co-Authors: Maria Scherma, Alexandros Makriyannis, Julie Medalie, Walter Fratta, Subramanian K Vadivel, Daniele Piomelli, Eva Mikics, Jozsef Haller, Sevil Yasar, Gianluigi TandaAbstract:Abstract Converging evidence suggests that the endocannabinoid system is an important constituent of neuronal substrates involved in brain reward processes and emotional responses to stress. Here, we evaluated motivational effects of intravenously administered Anandamide, an endogenous ligand for cannabinoid CB1-receptors, in Sprague–Dawley rats, using a place-conditioning procedure in which drugs abused by humans generally produce conditioned place preferences (reward). Anandamide (0.03–3 mg/kg intravenous) produced neither conditioned place preferences nor aversions. However, when rats were pre-treated with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (cyclohexyl carbamic acid 3′-carbamoyl-3-yl ester; 0.3 mg/kg intraperitoneal), which blocks Anandamide's metabolic degradation, Anandamide produced dose-related conditioned place aversions. In contrast, URB597 alone showed no motivational effects. Like URB597 plus Anandamide, the synthetic CB1-receptor ligand WIN 55,212-2 (50–300 μg/kg, intravenous) produced dose-related conditioned place aversions. When anxiety-related effects of Anandamide and URB597 were evaluated in a light/dark box, both a low Anandamide dose (0.3 mg/kg) and URB597 (0.1 and 0.3 mg/kg) produced anxiolytic effects when given alone, but produced anxiogenic effects when combined. A higher dose of Anandamide (3 mg/kg) produced anxiogenic effects and depressed locomotor activity when given alone and these effects were potentiated after URB597 treatment. Finally, anxiogenic effects of Anandamide plus URB597 and development of place aversions with URB597 plus Anandamide were prevented by the CB1-receptor antagonist AM251 (3 mg/kg intraperitoneal). Thus, additive interactions between the effects of Anandamide on brain reward processes and on anxiety may account for its aversive effects when intravenously administered during FAAH inhibition with URB597.
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the endogenous cannabinoid Anandamide produces δ 9 tetrahydrocannabinol like discriminative and neurochemical effects that are enhanced by inhibition of fatty acid amide hydrolase but not by inhibition of Anandamide transport
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: Marcello Solinas, Alexandros Makriyannis, Subramanian K Vadivel, Daniele Piomelli, Sevil Yasar, Gianluigi Tanda, Zuzana Justinova, Carrie E Wertheim, Steven R GoldbergAbstract:Anandamide is an endogenous ligand for brain cannabinoid CB(1) receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here we used a drug-discrimination procedure to test the hypothesis that Anandamide, given i.v. or i.p., would produce discriminative effects like those of delta-9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. We also used an in vivo microdialysis procedure to investigate the effects of Anandamide, given i.v. or i.p., on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methAnandamide (AM-356), a metabolically stable Anandamide analog, produced clear dose-related THC-like discriminative effects, but Anandamide produced THC-like discriminative effects only at a high 10-mg/kg dose that almost eliminated lever-press responding. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB-597), an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for metabolic inactivation of Anandamide, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of Anandamide, with 3 mg/kg Anandamide completely substituting for the THC training dose. URB-597 also potentiated the ability of Anandamide to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of Anandamide after URB-597 and methAnandamide were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the Anandamide transport inhibitors N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide (AM-404) and N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide (UCM-707) did not potentiate THC-like discriminative effects of Anandamide or its dopamine-elevating effects. Thus, Anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting brain area specificity for FAAH versus transport/FAAH inactivation of Anandamide.
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The endogenous cannabinoid Anandamide produces delta-9-tetrahydrocannabinol-like discriminative and neurochemical effects that are enhanced by inhibition of fatty acid amide hydrolase but not by inhibition of Anandamide transport.
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: Marcello Solinas, Alexandros Makriyannis, Subramanian K Vadivel, Daniele Piomelli, Sevil Yasar, Gianluigi Tanda, Zuzana Justinova, Carrie E Wertheim, Steven R GoldbergAbstract:Anandamide is an endogenous ligand for brain cannabinoid CB(1) receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here we used a drug-discrimination procedure to test the hypothesis that Anandamide, given i.v. or i.p., would produce discriminative effects like those of delta-9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. We also used an in vivo microdialysis procedure to investigate the effects of Anandamide, given i.v. or i.p., on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methAnandamide (AM-356), a metabolically stable Anandamide analog, produced clear dose-related THC-like discriminative effects, but Anandamide produced THC-like discriminative effects only at a high 10-mg/kg dose that almost eliminated lever-press responding. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB-597), an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for metabolic inactivation of Anandamide, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of Anandamide, with 3 mg/kg Anandamide completely substituting for the THC training dose. URB-597 also potentiated the ability of Anandamide to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of Anandamide after URB-597 and methAnandamide were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the Anandamide transport inhibitors N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide (AM-404) and N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide (UCM-707) did not potentiate THC-like discriminative effects of Anandamide or its dopamine-elevating effects. Thus, Anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting brain area specificity for FAAH versus transport/FAAH inactivation of Anandamide.
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characterization of the fatty acid amide hydrolase inhibitor cyclohexyl carbamic acid 3 carbamoyl biphenyl 3 yl ester urb597 effects on Anandamide and oleoylethanolamide deactivation
Journal of Pharmacology and Experimental Therapeutics, 2005Co-Authors: Darren Fegley, Andrea Duranti, Andrea Tontini, Giorgio Tarzia, Silvana Gaetani, Daniele PiomelliAbstract:Fatty acid amide hydrolase (FAAH) is an intracellular serine enzyme that catalyzes the hydrolysis of bioactive fatty acid ethanolamides such as Anandamide and oleoylethanolamide (OEA). Genetic deletion of the faah gene in mice elevates brain Anandamide levels and amplifies the effects of this endogenous cannabinoid agonist. Here, we show that systemic administration of the selective FAAH inhibitor URB597 (cyclohexyl carbamic acid 3′-carbamoyl-biphenyl-3-yl ester; 0.3 mg/kg i.p.) increases Anandamide levels in the brain of rats and wild-type mice but has no such effect in FAAH-null mutants. Moreover, URB597 enhances the hypothermic actions of Anandamide (5 mg/kg i.p.) in wild-type mice but not in FAAH-null mice. In contrast, the FAAH inhibitor does not affect Anandamide or OEA levels in the rat duodenum at doses that completely inhibit FAAH activity. In addition, URB597 does not alter the hypophagic response elicited by OEA (5 and 10 mg/kg i.p.), which is mediated by activation of peroxisome proliferator-activated receptor type-α. Finally, exogenously administered OEA (5 mg/kg i.p.) was eliminated at comparable rates in wild-type and FAAH -/- mice. Our results indicate that URB597 increases brain Anandamide levels and magnifies Anandamide responses by inhibiting intracellular FAAH activity. The results also suggest that an enzyme distinct from FAAH catalyzes OEA hydrolysis in the duodenum, where this lipid substance acts as a local satiety factor.
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Anandamide transport is independent of fatty acid amide hydrolase activity and is blocked by the hydrolysis resistant inhibitor am1172
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Darren Fegley, Alexandros Makriyannis, Andreas Goutopoulos, Satish Kathuria, R Mercier, Daniele PiomelliAbstract:The endogenous cannabinoid Anandamide is removed from the synaptic space by a high-affinity transport system present in neurons and astrocytes, which is inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). After internalization, Anandamide is hydrolyzed by fatty-acid amide hydrolase (FAAH), an intracellular membrane-bound enzyme that also cleaves AM404. Based on kinetic evidence, it has recently been suggested that Anandamide internalization may be mediated by passive diffusion driven by FAAH activity. To test this possibility, in the present study, we have investigated Anandamide internalization in wild-type and FAAH-deficient (FAAH(-/-)) mice. Cortical neurons from either mouse strain internalized [(3)H]Anandamide through a similar mechanism, i.e., via a rapid temperature-sensitive and saturable process, which was blocked by AM404. Moreover, systemic administration of AM404 to either wild-type or FAAH(-/-) mice enhanced the hypothermic effects of exogenous Anandamide, a response that was prevented by the CB(1) cannabinoid antagonist rimonabant (SR141716A). The results indicate that Anandamide internalization in mouse brain neurons is independent of FAAH activity. In further support of this conclusion, the compound N-(5Z, 8Z, 11Z, 14Z eicosatetraenyl)-4-hydroxybenzamide (AM1172) blocked [(3)H]Anandamide internalization in rodent cortical neurons and human astrocytoma cells without acting as a FAAH substrate or inhibitor. AM1172 may serve as a prototype for novel Anandamide transport inhibitors with increased metabolic stability.
Alexandros Makriyannis - One of the best experts on this subject based on the ideXlab platform.
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the endogenous cannabinoid Anandamide has effects on motivation and anxiety that are revealed by fatty acid amide hydrolase faah inhibition
Neuropharmacology, 2008Co-Authors: Maria Scherma, Alexandros Makriyannis, Julie Medalie, Walter Fratta, Subramanian K Vadivel, Daniele Piomelli, Eva Mikics, Jozsef Haller, Sevil Yasar, Gianluigi TandaAbstract:Abstract Converging evidence suggests that the endocannabinoid system is an important constituent of neuronal substrates involved in brain reward processes and emotional responses to stress. Here, we evaluated motivational effects of intravenously administered Anandamide, an endogenous ligand for cannabinoid CB1-receptors, in Sprague–Dawley rats, using a place-conditioning procedure in which drugs abused by humans generally produce conditioned place preferences (reward). Anandamide (0.03–3 mg/kg intravenous) produced neither conditioned place preferences nor aversions. However, when rats were pre-treated with the fatty acid amide hydrolase (FAAH) inhibitor URB597 (cyclohexyl carbamic acid 3′-carbamoyl-3-yl ester; 0.3 mg/kg intraperitoneal), which blocks Anandamide's metabolic degradation, Anandamide produced dose-related conditioned place aversions. In contrast, URB597 alone showed no motivational effects. Like URB597 plus Anandamide, the synthetic CB1-receptor ligand WIN 55,212-2 (50–300 μg/kg, intravenous) produced dose-related conditioned place aversions. When anxiety-related effects of Anandamide and URB597 were evaluated in a light/dark box, both a low Anandamide dose (0.3 mg/kg) and URB597 (0.1 and 0.3 mg/kg) produced anxiolytic effects when given alone, but produced anxiogenic effects when combined. A higher dose of Anandamide (3 mg/kg) produced anxiogenic effects and depressed locomotor activity when given alone and these effects were potentiated after URB597 treatment. Finally, anxiogenic effects of Anandamide plus URB597 and development of place aversions with URB597 plus Anandamide were prevented by the CB1-receptor antagonist AM251 (3 mg/kg intraperitoneal). Thus, additive interactions between the effects of Anandamide on brain reward processes and on anxiety may account for its aversive effects when intravenously administered during FAAH inhibition with URB597.
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the endogenous cannabinoid Anandamide produces δ 9 tetrahydrocannabinol like discriminative and neurochemical effects that are enhanced by inhibition of fatty acid amide hydrolase but not by inhibition of Anandamide transport
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: Marcello Solinas, Alexandros Makriyannis, Subramanian K Vadivel, Daniele Piomelli, Sevil Yasar, Gianluigi Tanda, Zuzana Justinova, Carrie E Wertheim, Steven R GoldbergAbstract:Anandamide is an endogenous ligand for brain cannabinoid CB(1) receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here we used a drug-discrimination procedure to test the hypothesis that Anandamide, given i.v. or i.p., would produce discriminative effects like those of delta-9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. We also used an in vivo microdialysis procedure to investigate the effects of Anandamide, given i.v. or i.p., on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methAnandamide (AM-356), a metabolically stable Anandamide analog, produced clear dose-related THC-like discriminative effects, but Anandamide produced THC-like discriminative effects only at a high 10-mg/kg dose that almost eliminated lever-press responding. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB-597), an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for metabolic inactivation of Anandamide, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of Anandamide, with 3 mg/kg Anandamide completely substituting for the THC training dose. URB-597 also potentiated the ability of Anandamide to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of Anandamide after URB-597 and methAnandamide were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the Anandamide transport inhibitors N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide (AM-404) and N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide (UCM-707) did not potentiate THC-like discriminative effects of Anandamide or its dopamine-elevating effects. Thus, Anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting brain area specificity for FAAH versus transport/FAAH inactivation of Anandamide.
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The endogenous cannabinoid Anandamide produces delta-9-tetrahydrocannabinol-like discriminative and neurochemical effects that are enhanced by inhibition of fatty acid amide hydrolase but not by inhibition of Anandamide transport.
Journal of Pharmacology and Experimental Therapeutics, 2007Co-Authors: Marcello Solinas, Alexandros Makriyannis, Subramanian K Vadivel, Daniele Piomelli, Sevil Yasar, Gianluigi Tanda, Zuzana Justinova, Carrie E Wertheim, Steven R GoldbergAbstract:Anandamide is an endogenous ligand for brain cannabinoid CB(1) receptors, but its behavioral effects are difficult to measure due to rapid inactivation. Here we used a drug-discrimination procedure to test the hypothesis that Anandamide, given i.v. or i.p., would produce discriminative effects like those of delta-9-tetrahydrocannabinol (THC) in rats when its metabolic inactivation was inhibited. We also used an in vivo microdialysis procedure to investigate the effects of Anandamide, given i.v. or i.p., on dopamine levels in the nucleus accumbens shell in rats. When injected i.v., methAnandamide (AM-356), a metabolically stable Anandamide analog, produced clear dose-related THC-like discriminative effects, but Anandamide produced THC-like discriminative effects only at a high 10-mg/kg dose that almost eliminated lever-press responding. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB-597), an inhibitor of fatty acid amide hydrolase (FAAH), the main enzyme responsible for metabolic inactivation of Anandamide, produced no THC-like discriminative effects alone but dramatically potentiated discriminative effects of Anandamide, with 3 mg/kg Anandamide completely substituting for the THC training dose. URB-597 also potentiated the ability of Anandamide to increase dopamine levels in the accumbens shell. The THC-like discriminative-stimulus effects of Anandamide after URB-597 and methAnandamide were blocked by the CB1 receptor antagonist rimonabant, but not the vanilloid VR1 receptor antagonist capsazepine. Surprisingly, the Anandamide transport inhibitors N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide (AM-404) and N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide (UCM-707) did not potentiate THC-like discriminative effects of Anandamide or its dopamine-elevating effects. Thus, Anandamide has THC-like discriminative and neurochemical effects that are enhanced after treatment with a FAAH inhibitor but not after treatment with transport inhibitors, suggesting brain area specificity for FAAH versus transport/FAAH inactivation of Anandamide.
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Anandamide transport is independent of fatty acid amide hydrolase activity and is blocked by the hydrolysis resistant inhibitor am1172
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Darren Fegley, Alexandros Makriyannis, Andreas Goutopoulos, Satish Kathuria, R Mercier, Daniele PiomelliAbstract:The endogenous cannabinoid Anandamide is removed from the synaptic space by a high-affinity transport system present in neurons and astrocytes, which is inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). After internalization, Anandamide is hydrolyzed by fatty-acid amide hydrolase (FAAH), an intracellular membrane-bound enzyme that also cleaves AM404. Based on kinetic evidence, it has recently been suggested that Anandamide internalization may be mediated by passive diffusion driven by FAAH activity. To test this possibility, in the present study, we have investigated Anandamide internalization in wild-type and FAAH-deficient (FAAH(-/-)) mice. Cortical neurons from either mouse strain internalized [(3)H]Anandamide through a similar mechanism, i.e., via a rapid temperature-sensitive and saturable process, which was blocked by AM404. Moreover, systemic administration of AM404 to either wild-type or FAAH(-/-) mice enhanced the hypothermic effects of exogenous Anandamide, a response that was prevented by the CB(1) cannabinoid antagonist rimonabant (SR141716A). The results indicate that Anandamide internalization in mouse brain neurons is independent of FAAH activity. In further support of this conclusion, the compound N-(5Z, 8Z, 11Z, 14Z eicosatetraenyl)-4-hydroxybenzamide (AM1172) blocked [(3)H]Anandamide internalization in rodent cortical neurons and human astrocytoma cells without acting as a FAAH substrate or inhibitor. AM1172 may serve as a prototype for novel Anandamide transport inhibitors with increased metabolic stability.
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Stereochemical selectivity of methAnandamides for the CB1 and CB2 cannabinoid receptors and their metabolic stability.
Bioorganic & Medicinal Chemistry, 2001Co-Authors: Andreas Goutopoulos, Pusheng Fan, Atmaram D. Khanolkar, Xian-qun Xie, Sonyuan Lin, Alexandros MakriyannisAbstract:Abstract Several chiral, analogues of the endogenous cannabinoid receptor ligand, arachidonylethanolamide (Anandamide), methylated at the 2,1′ and 2′ positions using asymmetric synthesis were evaluated in order to study (a) stereoselectivity of binding to CB1 and CB2 cannabinoid receptors; and (b) metabolic stability with regard to Anandamide amidase. Enantiomerically pure 2-methyl arachidonic acids were synthesized through diastereoselective methylation of the respective chiral 2-oxazolidinone enolate derivatives and CB1 and CB2 receptor affinities of the resulting chiral Anandamides were evaluated using a standard receptor binding assay. Introduction of a single 2-methyl group increased affinity for CB1, led to limited enantioselectivity and only modestly improved metabolic stability. However, a high degree of enantio- and diastereoselectivity was observed for the 2,1′-dimethyl analogues. ( R )- N -(1-methyl-2-hydroxyethyl)-2-( R )-methyl-arachidonamide ( 4 ) exhibited the highest CB1 receptor affinity in this series with a K i of 7.42 nM, an at least 10-fold improvement on Anandamide ( K i =78.2 nM). The introduction of two methyl groups at the 2-position of Anandamide led to no change in affinity for CB1 but somewhat enhanced metabolic stability. Conversely, chiral headgroup methylation in the 2- gem -dimethyl series led to chiral analogues possessing a wide range of CB1 affinities. Of these the ( S )-2,2,2′-trimethyl analogue ( 12 ) had the highest affinity for CB1 almost equal to that of Anandamide. In agreement with our previous Anandamide structure–activity relationship work, the analogues in this study showed high selectivity for the CB1 receptor over CB2. The results are evaluated in terms of stereochemical factors affecting the ligand's affinity for CB1 using receptor-essential volume mapping as an aid. Based on the results, a partial CB1 receptor site model is proposed, that bears two hydrophobic pockets capable of accommodating 1′- and 2-methyl groups
Shozo Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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an acid amidase hydrolyzing Anandamide as an endogenous ligand for cannabinoid receptors
FEBS Letters, 1999Co-Authors: Natsuo Ueda, Kenji Yamanaka, Yuka Terasawa, Shozo YamamotoAbstract:Abstract Anandamide loses its cannabimimetic activities upon hydrolysis to arachidonic acid and ethanolamine. So far the Anandamide hydrolyzing activity widely distributed in mammalian organs has been attributed exclusively to an enzyme referred to as Anandamide amidohydrolase with an optimum pH around 9. We found another enzyme hydrolyzing Anandamide in a human megakaryoblastic cell line (CMK). The enzyme present in the 12 000 ×g pellet of the cell homogenate was solubilized by freeze-thaw. The solubilized enzyme showed an optimal pH around 5, and was almost inactive at alkaline pH. The enzyme activity was increased by the addition of dithiothreitol. In contrast, Anandamide amidohydrolase of RBL-1 cells was mostly insoluble even after freeze-thaw, showed an optimal pH at 9, and was not affected by dithiothreitol. Furthermore, the enzyme of CMK cells was much less sensitive to phenylmethylsulfonyl fluoride and methyl arachidonoyl fluorophosphonate potently inhibiting Anandamide amidohydrolase, and effectively hydrolyzed palmitoylethanolamide, which was a poor substrate for Anandamide amidohydrolase. Thus, the enzyme of CMK cells is distinguishable from Anandamide amidohydrolase.
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Anandamide amidohydrolase reacting with 2‐arachidonoylglycerol, another cannabinoid receptor ligand
FEBS letters, 1998Co-Authors: Sravan Kumar Goparaju, Natsuo Ueda, Hiroko Yamaguchi, Shozo YamamotoAbstract:Two endogenous ligands for cannabinoid receptors, Anandamide (arachidonylethanolamide) and 2-arachidonoylglycerol, lose their biological activities by enzymatic hydrolysis. A cDNA for a rat liver enzyme hydrolyzing Anandamide as well as oleamide was overexpressed in COS-7 cells. When the particulate fraction was allowed to react with 2-arachidonoylglycerol, arachidonic acid was produced. In contrast, this hydrolytic reaction did not occur with the control cells. The hydrolysis of 2-arachidonoylglycerol proceeded about 4-fold faster than the Anandamide hydrolysis with a Km value as low as 6 μM and an optimal pH of 10. Phenylmethylsulfonyl fluoride and methyl arachidonyl fluorophosphonate inhibited the hydrolysis of both Anandamide and 2-arachidonoylglycerol in parallel. Furthermore, the hydrolysis of [14C]2-arachidonoylglycerol was inhibited by Anandamide dose-dependently. These results suggest that Anandamide and 2-arachidonoylglycerol can be inactivated by the same enzyme.
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Anandamide Amidohydrolase from Porcine Brain
Advances in experimental medicine and biology, 1997Co-Authors: Natsuo Ueda, Yuko Kurahashi, Kei Yamamoto, Shozo YamamotoAbstract:Ethanolamide of arachidonic acid was isolated from porcine brain as an endogenous ligand for cannabinoid receptors, and referred to as Anandamide.1 In consideration of various biological activities of Anandamide,2 it is very important to elucidate how the production and degradation of this new compound are regulated by enzymes within the cells. The enzyme activity hydrolyzing Anandamide to free arachidonic acid and ethanolamine has been considered physiologically important since Anandamide loses its biological activity at this step (Fig. 1). Several research groups have reported the Anandamide hydrolase activity in the particulate fractions of the brain and other tissues of various animal species.3–5 The specific enzyme activity in these crude preparations was in a range from 0.3 to 9 nmol/min/mg protein. It has also been reported that Anandamide is synthesized by the enzymatic condensation of arachidonic acid and ethanolamine. The Anandamide synthase activity was found in the brain with a specific activity of 2–3 nmol/min/mg protein.5,6,7 As reported recently, we found both the hydrolase and synthase activities in the microsome fraction of porcine brain,8 from which Anandamide was first isolated by Devane and others.1 In this paper we will discuss the partially purified Anandamide hydrolase of porcine brain with special reference to its identity with Anandamide synthase. We will also describe the inactivation of Anandamide by the catalysis of lipoxygenase enzymes.9
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Metabolism of Anandamide, an Endogenous Cannabinoid Receptor Ligand, in Porcine Ocular Tissues
Experimental eye research, 1997Co-Authors: Satoshi Matsuda, Natsuo Ueda, Yuko Kurahashi, Nobue Kanemitsu, Akiyo Nakamura, Yasuo Mimura, Shozo YamamotoAbstract:Anandamide (arachidonylethanolamide) is an endogenous ligand for cannabinoid receptors, and exerts various cannabimimetic activities. Since cannabinoids and Anandamide were pharmacologically active with the eye, we examined metabolism of Anandamide in a variety of porcine ocular tissues. In the presence of ethanolamine, [14C]arachidonic acid was converted to [14C]Anandamide by a homogenate of retina, choroid, iris, optic nerve and lacrimal gland with a specific enzyme activity of 1.9–4.2 nmol min−1mg−1protein at 37°C. On the other hand, [14C]Anandamide was hydrolysed to [14C]arachidonic acid by a homogenate of each tissue with a specific enzyme activity of 1.2–3.5 nmol min−1mg−1protein. Thus, both activities of Anandamide synthase and hydrolase were found in these ocular tissues. As for the subcellular distribution, the two enzyme activities were mostly recovered in particulate fractions rather than the cytosol. With the retina microsome palmitic acid was converted to its ethanolamide at a lower rate than arachidonic acid, and palmitoylethanolamide was less active than Anandamide as a substrate for the hydrolase.
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ENZYMES FOR Anandamide BIOSYNTHESIS AND METABOLISM
Journal of lipid mediators and cell signalling, 1996Co-Authors: Natsuo Ueda, Shozo Yamamoto, Yuko Kurahashi, Kei Yamamoto, Takashi TokunagaAbstract:Abstract Anandamide is an endogenous ligand for cannabinoid receptors. We tried to isolate and purify ‘Anandamide amidohydrolase’ which hydrolyzes Anandamide to arachidonic acid and ethanolamine. The enzyme activity was found in the microsomal fraction of procine brain homogenate. The enzyme was solubilized in 1% Triton X-100, and partially purified by hydrophobic chromatography to a specific activity of about 0.3 μmol/min per mg protein (37°C). Apparent Km for Anandamide was about 60 μM. The enzyme reacted also with also converted arachidonic acid to Anandamide in the presence of 250 mM concentration of ethanolamine. Several lines of evidence including experiments using various inhibitors suggested that the Anandamide synthase and amidohydrolase activities were derived from a single enzyme protein.
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Mechanisms for recycling and biosynthesis of endogenous cannabinoids Anandamide and 2‐arachidonylglycerol
Journal of neurochemistry, 2008Co-Authors: Ekaterina A. Placzek, Yasuo Okamoto, Natsuo Ueda, Eric L. BarkerAbstract:The mechanisms of endogenous cannabinoid biosynthesis are not completely understood. We hypothesized that Anandamide could be recycled by the cell to form new endocannabinoid molecules and released into the extracellular space. We determined that new endocannabinoids derived from exogenous Anandamide or arachidonic acid were synthesized and released from RBL-2H3 cells in response to ionomycin. Treatment of RBL-2H3 cells with nystatin and progesterone, agents that disrupt organization of lipid raft/caveolae, resulted in the attenuation of Anandamide and 2-arachidonyl glycerol synthesis and/or release in response to stimulation with ionomycin suggesting a role for these membrane microdomains in endocannabinoid biosynthesis. Furthermore, Anandamide synthesis may be independent of N-acyl phosphatidylethanolamine phospholipase D as expression of the enzyme was not detected in RBL-2H3 cells. We also established that extracellular calcium is necessary for endocannabinoid biosynthesis because release of intracellular calcium stores alone does not promote endocannabinoid biosynthesis. Next, we examined the role of calcium as a 'switch' to activate the synthesis of Anandamide and simultaneously reduce uptake. Indeed, [(3)H] Anandamide uptake was reduced in the presence of calcium. Our findings suggest a mechanism indicative of calcium-modulated activation of Anandamide synthesis and simultaneous termination of uptake.
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N-acylphosphatidylethanolamine-hydrolyzing phospholipase D is an important determinant of uterine Anandamide levels during implantation.
The Journal of biological chemistry, 2005Co-Authors: Yong Guo, Harald H.o. Schmid, Yasuo Okamoto, Natsuo Ueda, Haibin Wang, Philip J. Kingsley, Lawrence J. Marnett, Sanjoy K. Das, Sudhansu K. DeyAbstract:Implantation requires reciprocal interaction between blastocysts and a receptive uterus. In mice, one important player in this dialogue involves endocannabinoid signaling via cannabinoid receptor CB1. Anandamide is an endogenous cannabinoid ligand, and its levels are spatiotemporally regulated in the uterus during early pregnancy, showing lower levels in the receptive uterus and at the implantation site. However, the mechanism by which differential uterine Anandamide gradients are established under different pregnancy status is not clearly understood. Using multiple approaches, we show here that uterine Anandamide levels conducive to implantation are primarily regulated by spatiotemporal expression of Nape-Pld, the gene encoding N-acylphosphatidylethanolamine-hydrolyzing phospholipase D that generates Anandamide. The expression is well correlated with its activity and Anandamide levels. This study is clinically relevant, since elevated Anandamide levels in peripheral circulation are associated with spontaneous pregnancy failure in women.
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an acid amidase hydrolyzing Anandamide as an endogenous ligand for cannabinoid receptors
FEBS Letters, 1999Co-Authors: Natsuo Ueda, Kenji Yamanaka, Yuka Terasawa, Shozo YamamotoAbstract:Abstract Anandamide loses its cannabimimetic activities upon hydrolysis to arachidonic acid and ethanolamine. So far the Anandamide hydrolyzing activity widely distributed in mammalian organs has been attributed exclusively to an enzyme referred to as Anandamide amidohydrolase with an optimum pH around 9. We found another enzyme hydrolyzing Anandamide in a human megakaryoblastic cell line (CMK). The enzyme present in the 12 000 ×g pellet of the cell homogenate was solubilized by freeze-thaw. The solubilized enzyme showed an optimal pH around 5, and was almost inactive at alkaline pH. The enzyme activity was increased by the addition of dithiothreitol. In contrast, Anandamide amidohydrolase of RBL-1 cells was mostly insoluble even after freeze-thaw, showed an optimal pH at 9, and was not affected by dithiothreitol. Furthermore, the enzyme of CMK cells was much less sensitive to phenylmethylsulfonyl fluoride and methyl arachidonoyl fluorophosphonate potently inhibiting Anandamide amidohydrolase, and effectively hydrolyzed palmitoylethanolamide, which was a poor substrate for Anandamide amidohydrolase. Thus, the enzyme of CMK cells is distinguishable from Anandamide amidohydrolase.
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Anandamide amidohydrolase reacting with 2‐arachidonoylglycerol, another cannabinoid receptor ligand
FEBS letters, 1998Co-Authors: Sravan Kumar Goparaju, Natsuo Ueda, Hiroko Yamaguchi, Shozo YamamotoAbstract:Two endogenous ligands for cannabinoid receptors, Anandamide (arachidonylethanolamide) and 2-arachidonoylglycerol, lose their biological activities by enzymatic hydrolysis. A cDNA for a rat liver enzyme hydrolyzing Anandamide as well as oleamide was overexpressed in COS-7 cells. When the particulate fraction was allowed to react with 2-arachidonoylglycerol, arachidonic acid was produced. In contrast, this hydrolytic reaction did not occur with the control cells. The hydrolysis of 2-arachidonoylglycerol proceeded about 4-fold faster than the Anandamide hydrolysis with a Km value as low as 6 μM and an optimal pH of 10. Phenylmethylsulfonyl fluoride and methyl arachidonyl fluorophosphonate inhibited the hydrolysis of both Anandamide and 2-arachidonoylglycerol in parallel. Furthermore, the hydrolysis of [14C]2-arachidonoylglycerol was inhibited by Anandamide dose-dependently. These results suggest that Anandamide and 2-arachidonoylglycerol can be inactivated by the same enzyme.
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Anandamide Amidohydrolase from Porcine Brain
Advances in experimental medicine and biology, 1997Co-Authors: Natsuo Ueda, Yuko Kurahashi, Kei Yamamoto, Shozo YamamotoAbstract:Ethanolamide of arachidonic acid was isolated from porcine brain as an endogenous ligand for cannabinoid receptors, and referred to as Anandamide.1 In consideration of various biological activities of Anandamide,2 it is very important to elucidate how the production and degradation of this new compound are regulated by enzymes within the cells. The enzyme activity hydrolyzing Anandamide to free arachidonic acid and ethanolamine has been considered physiologically important since Anandamide loses its biological activity at this step (Fig. 1). Several research groups have reported the Anandamide hydrolase activity in the particulate fractions of the brain and other tissues of various animal species.3–5 The specific enzyme activity in these crude preparations was in a range from 0.3 to 9 nmol/min/mg protein. It has also been reported that Anandamide is synthesized by the enzymatic condensation of arachidonic acid and ethanolamine. The Anandamide synthase activity was found in the brain with a specific activity of 2–3 nmol/min/mg protein.5,6,7 As reported recently, we found both the hydrolase and synthase activities in the microsome fraction of porcine brain,8 from which Anandamide was first isolated by Devane and others.1 In this paper we will discuss the partially purified Anandamide hydrolase of porcine brain with special reference to its identity with Anandamide synthase. We will also describe the inactivation of Anandamide by the catalysis of lipoxygenase enzymes.9