8-11-14-eicosatrienoic-acid and anandamide

Anandamide is an arachidonic acid-derived endogenous cannabinoid that regulates normal physiological functions and pathophysiological responses within the central nervous system and in the periphery. Several cytochrome P450 (P450) isoforms metabolize anandamide to form hydroxylated and epoxygenated products. Human CYP2B6 and CYP2D6, which are expressed heterogeneously throughout the brain, exhibit clinically significant polymorphisms and are regulated by external factors, such as alcohol and smoking. Oxidative metabolism of anandamide by these two P450s may have important functional consequences for endocannabinoid system signaling. In this study, we investigated the metabolism of anandamide by wild-type CYP2B6 (2B6.1) and CYP2D6 (2D6.1) and by their common polymorphic mutants 2B6.4, 2B6.6, 2B6.9, and 2D6.34. Major differences in anandamide metabolism by the two isoforms and their mutants were found in vitro with respect to the formation of 20-hydroxyeicosatetraenoic acid ethanolamide (20-HETE-EA) and 14,15-epoxyeicosatetraenoic acid ethanolamide (14,15-EET-EA). Pharmacological studies showed that both 20-HETE-EA and 14,15-EET-EA bind to the rat brain cannabinoid CB1 receptor with lower affinities relative to that of anandamide. In addition, both products are degraded more rapidly than anandamide in rat brain homogenates. Their degradation occurs via different mechanisms involving either fatty acid amide hydrolase (FAAH), the major anandamide-degrading enzyme, or epoxide hydrolase (EH). Thus, the current findings provide potential new insights into the actions of inhibitors FAAH and EH, which are being developed as novel therapeutic agents, as well as a better understanding of the interactions between the cytochrome P450 monooxygenases and the endocannabinoid system.

Topics: 8,11,14-Eicosatrienoic Acid; Amidohydrolases; Animals; Arachidonic Acids; Aryl Hydrocarbon Hydroxylases; Brain; Cannabinoid Receptor Modulators; Cytochrome P-450 CYP2B6; Cytochrome P-450 CYP2D6; Endocannabinoids; Epoxide Hydrolases; Humans; Hydroxyeicosatetraenoic Acids; Hydroxylation; Male; Oxidation-Reduction; Oxidoreductases, N-Demethylating; Polymorphism, Genetic; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1

Oxidation of the endocannabinoid anandamide by cytochrome P450 (P450) enzymes has the potential to affect signaling pathways within the endocannabinoid system and pharmacological responses to novel drug candidates targeting this system. We previously reported that the human cytochromes P450 2D6, 3A4, and 4F2 are high-affinity, high-turnover anandamide oxygenases in vitro, forming the novel metabolites hydroxyeicosatetraenoic acid ethanolamides and epoxyeicosatrienoic acid ethanolamides. The objective of this study was to investigate the possible biological significance of these metabolic pathways. We report that the 5,6-epoxide of anandamide, 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA), is a potent and selective cannabinoid receptor 2 (CB2) agonist. The K(i) values for the binding of 5,6-EET-EA to membranes from Chinese hamster ovary (CHO) cells expressing either recombinant human CB1 or CB2 receptor were 11.4 microM and 8.9 nM, respectively. In addition, 5,6-EET-EA inhibited the forskolin-stimulated accumulation of cAMP in CHO cells stably expressing the CB2 receptor (IC(50) = 9.8 +/- 1.3 nM). Within the central nervous system, the CB2 receptor is expressed on activated microglia and is a potential therapeutic target for neuroinflammation. BV-2 microglial cells stimulated with low doses of interferon-gamma exhibited an increased capacity for converting anandamide to 5,6-EET-EA, which correlated with increased protein expression of microglial P450 4F and 3A isoforms. Finally, we demonstrate that 5,6-EET-EA is more stable than anandamide in mouse brain homogenates and is primarily metabolized by epoxide hydrolase. Combined, our results suggest that epoxidation of anandamide by P450s to form 5,6-EET-EA represents an endocannabinoid bioactivation pathway in the context of immune cell function.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Binding, Competitive; CHO Cells; Cricetinae; Cricetulus; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Endocannabinoids; Humans; Mice; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2

Members of the cytochrome P450 (P450) family of drug-metabolizing enzymes are present in the human brain, and they may have important roles in the oxidation of endogenous substrates. The polymorphic CYP2D6 is one of the major brain P450 isoforms and has been implicated in neurodegeneration, psychosis, schizophrenia, and personality traits. The objective of this study was to determine whether the endocannabinoid arachidonoylethanolamide (anandamide) is a substrate for CYP2D6. Anandamide is the endogenous ligand to the cannabinoid receptor CB1, which is also activated by the main psychoactive component in marijuana. Signaling via the CB1 receptor alters sensory and motor function, cognition, and emotion. Recombinant CYP2D6 converted anandamide to 20-hydroxyeicosatetraenoic acid ethanolamide and 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid ethanolamides (EET-EAs) with low micromolar K(m) values. CYP2D6 further metabolized the epoxides of anandamide to form novel dioxygenated derivatives. Human brain microsomal and mitochondrial preparations metabolized anandamide to form hydroxylated and epoxygenated products, respectively. An inhibitory antibody against CYP2D6 significantly decreased the mitochondrial formation of the EET-EAs. To our knowledge, anandamide and its epoxides are the first eicosanoid-like molecules to be identified as CYP2D6 substrates. Our study suggests that anandamide may be a physiological substrate for brain mitochondrial CYP2D6, implicating this polymorphic enzyme as a potential component of the endocannabinoid system in the brain. This study also offers support to the hypothesis that neuropsychiatric phenotype differences among individuals with genetic variations in CYP2D6 could be ascribable to interactions of this enzyme with endogenous substrates.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Arachidonic Acids; Brain; Cytochrome P-450 CYP2D6; Endocannabinoids; Epoxy Compounds; Humans; Hydroxylation; Microsomes; Mitochondria; Polyunsaturated Alkamides; Time Factors

The endocannabinoid anandamide is an arachidonic acid derivative that is found in most tissues where it acts as an important signaling mediator in neurological, immune, cardiovascular, and other functions. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to the physiologically active molecules hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), which play important roles in blood pressure regulation and inflammation. To determine whether anandamide can also be oxidized by P450s, its metabolism by human liver and kidney microsomes was investigated. The kidney microsomes metabolized anandamide to a single mono-oxygenated product, which was identified as 20-HETE-ethanolamide (EA). Human liver microsomal incubations with anandamide also produced 20-HETE-EA in addition to 5,6-, 8,9-, 11-12, and 14,15-EET-EA. The EET-EAs produced by the liver microsomal P450s were converted to their corresponding dihydroxy derivatives by microsomal epoxide hydrolase. P450 4F2 was identified as the isoform that is most probably responsible for the formation of 20-HETE-EA in both human kidney and human liver, with an apparent Km of 0.7 microM. The apparent Km values of the human liver microsomes for the formation of the EET-EAs were between 4 and 5 microM, and P450 3A4 was identified as the primary P450 in the liver responsible for epoxidation of anandamide. The in vivo formation and biological relevance of the P450-derived HETE and EET ethanolamides remains to be determined.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acids; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Endocannabinoids; Epoxy Compounds; Humans; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Kidney; Kinetics; Microsomes, Liver; Polyunsaturated Alkamides; Spectrometry, Mass, Electrospray Ionization

Linoleoylamide is physiological constituent of neurons. The effects of this agent, also a sleep-inducing agent, on ion currents in pituitary GH(3) cells were investigated. Hyperpolarization-elicited K(+) currents in GH(3) cells bathed in a high-K(+), Ca(2+)-free solution were studied to determine the effects of linoleoylamide and other related compounds on the I(K(IR)) that was sensitive to inhibition by E-4031 and identified as an erg (ether-à-go-go-related-gene) current. Linoleoylamide suppressed the amplitude of I(K(IR)) in a concentration-dependent manner with an IC(50) value of 5 microM. Oleamide (20 microM) inhibited the amplitude of I(K(IR)), while neither arachidonic acid (20 microM) nor 14,15-epoxyeicosatrienoic acid (20 microM) had an effect on it. In GH(3) cells incubated with anandamide (20 microM) or arachidonic acid (20 microM), the linoleoylamide-induced inhibition of I(K(IR)) remained unaltered. In inside-out patches, arachidonic acid (20 microM) and 14,15-epoxyeicosatrienoic acid (20 microM) stimulated large-conductance Ca(2+)-activated K(+) channels; however, linoleoylamide (20 microM) had little or no effect on them. Under current-clamp mode, linoleoylamide (20 microM) increased the firing rate. In IMR-32 neuroblastoma cells, linoleoylamide also suppressed I(K(IR)). This study provides the evidence that linoleoylamide has a depressant effect on the erg current, and suggests that this effect may affect hormonal secretion.

Topics: 8,11,14-Eicosatrienoic Acid; alpha-Linolenic Acid; Amides; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Calcium Channels, L-Type; Dose-Response Relationship, Drug; Endocannabinoids; Humans; Hydantoins; Imidazoles; Imidazolidines; Infant, Newborn; Linoleic Acids; Membrane Potentials; Neuroblastoma; Patch-Clamp Techniques; Piperazines; Piperidines; Pituitary Neoplasms; Polyunsaturated Alkamides; Potassium Channels; Pyridines; Tumor Cells, Cultured

TRPV4 is a widely expressed cation channel of the 'transient receptor potential' (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4alphaPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5',6'-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta; Arachidonic Acid; Arachidonic Acids; Calcium; Cannabinoid Receptor Modulators; Cation Transport Proteins; Cell Line; Electrophysiology; Endocannabinoids; Endothelium, Vascular; Humans; Ion Channel Gating; Ion Channels; Mice; Polyunsaturated Alkamides; TRPV Cation Channels

The recently discovered endogenous agonist for the cannabinoid receptor, anandamide (arachidonylethanolamide), can be formed enzymatically by the condensation of arachidonic acid with ethanolamine. 5Z,8Z,11Z-Eicosatrienoic acid (mead acid) has been found to substitute for arachidonic acid in the sn-2 position of phospholipids and accumulate during periods of dietary fatty acid deprivation in rats. In the present study, the chemically synthesized ethanolamide of mead acid was evaluated as a potential agonist at the two known subtypes of cannabinoid receptor: CB1 (central) and CB2 (peripheral). This compound was equipotent to anandamide in competing with [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the human CB1 receptor and from ATt-20 cells expressing the human CB2 receptor. Mead ethanolamide was also equipotent to anandamide in inhibiting forskolin-stimulated cAMP accumulation in cells expressing the CB1 receptor. It inhibited N-type calcium currents with a lower potency than anandamide. Mead and arachidonic acid were equally efficacious as substrates for the enzymatic synthesis of their respective ethanolamides in rat and adult human hippocampal P2 membranes. Palmitic acid was not an effective substrate for the enzymatic synthesis of palmitoyl ethanolamide. Mead ethanolamide exhibits several characteristics of a novel agonist to CB1 and CB2 receptors and may represent another candidate endogenous ligand for the CB1 receptor. Due to the anticonvulsant properties of GABA and the positional similarity of L-serine to ethanolamine in membrane phospholipids, these compounds were synthetically coupled to arachidonic acid, and their resulting arachidonamides were tested as potential cannabinoid agonists. The arachidonamides of GABA and L-serine were inactive in both binding and functional assays at the CB1 receptor.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cannabinoids; CHO Cells; Cricetinae; Cyclohexanols; Endocannabinoids; Humans; L Cells; Mice; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug