anandamide and 20-hydroxy-5-8-11-14-eicosatetraenoic-acid

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

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