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

Abstract

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°C for 30 min. Furthermore, a targeted lipidomic analysis showed that Ca2+ 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.—Astarita, G., F. Ahmed, and D. Piomelli. Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain. J. Lipid Res. 2008. 49: 48–57. 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°C for 30 min. Furthermore, a targeted lipidomic analysis showed that Ca2+ 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.—Astarita, G., F. Ahmed, and D. Piomelli. Identification of biosynthetic precursors for the endocannabinoid anandamide in the rat brain. J. Lipid Res. 2008. 49: 48–57. ERRATAJournal of Lipid ResearchVol. 53Issue 6PreviewAn affiliation for Dr. Daniele Piomelli, Italian Institute of Technology, was inadvertently omitted on the following papers Dr. Piomelli co-authored: Full-Text PDF Open Access N-Acyl-substituted derivatives of phosphatidylethanol­amines (NAPEs) constitute a family of unusual glycerophospholipids that occur in a wide range of vertebrate animals, including fish (1Chapman K.D. Emerging physiological roles for N-acylphos-phatidylethanolamine metabolism in plants: signal transduction and membrane protection.Chem. Phys. Lipids. 2000; 108: 221-229Crossref PubMed Scopus (91) Google Scholar), reptiles (2Astarita G. Rourke B.C. Andersen J.B. Fu J. Kim J.H. Bennett A.F. Hicks J.W. Piomelli D. Postprandial increase of oleoylethanolamide mobilization in small intestine of the Burmese python (Python molurus).Am. J. Physiol. Regul. Integr. Comp. Physiol. 2006; 290: R1407-R1412Crossref PubMed Scopus (56) Google Scholar), and mammals (3Schmid H.H. Schmid P.C. Natarajan V. N-Acylated glycerophospholipids and their derivatives.Prog. Lipid Res. 1990; 29: 1-43Crossref PubMed Scopus (423) Google Scholar). They are thought to serve as biological precursors for fatty acid ethanolamides (4Schmid H.H. Pathways and mechanisms of N-acylethanol-amine biosynthesis: can anandamide be generated selectively?.Chem. Phys. Lipids. 2000; 108: 71-87Crossref PubMed Scopus (142) Google Scholar), a class of signaling lipids involved in critical physiological processes such as pain, feeding, and inflammation (5Schmid H.H. Berdyshev E.V. Cannabinoid receptor-inactive N-acylethanolamines and other fatty acid amides: metab­olism and function.Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 363-376Abstract Full Text PDF PubMed Scopus (100) Google Scholar, 6Calignano A. La Rana G. Piomelli D. Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide.Eur. J. Pharmacol. 2001; 419: 191-198Crossref PubMed Scopus (222) Google Scholar, 7Rodríguez de Fonseca F. Navarro M. Gómez R. Escuredo L. Nava F. Fu J. Murillo-Rodríguez E. Giuffrida A. LoVerme J. Gaetani S. et al.An anorexic lipid mediator regulated by feeding.Nature. 2001; 414: 209-212Crossref PubMed Scopus (597) Google Scholar). 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Fatty acid amide hydrolase, the degradative enzyme for anandamide and oleamide, has selective distribution in neurons within the rat central nervous system.J. Neurosci. Res. 1997; 50: 1047-1052Crossref PubMed Scopus (152) Google Scholar). Anandamide is rapidly hydro-lyzed to arachidonic acid and ethanolamine by fatty acid amide hydrolases (14Cravatt B.F. Giang D.K. Mayfield S.P. Boger D.L. Lerner R.A. Gilula N.B. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.Nature. 1996; 384: 83-87Crossref PubMed Scopus (1806) Google Scholar), and selective fatty acid amide hydrolase inhibitors have been shown to alleviate pain, anxiety, and depression in rodent models (15Gaetani S. Cuomo V. Piomelli D. Anandamide hydrolysis: a new target for anti-anxiety drugs?.Trends Mol. Med. 2003; 9: 474-478Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 16Kathuria S. Gaetani S. Fegley D. Valino F. Duranti A. Tontini A. Mor M. Tarzia G. La Rana G. Calignano A. et al.Modu­lation of anxiety through blockade of anandamide hydrolysis.Nat. Med. 2003; 9: 76-81Crossref PubMed Scopus (1244) Google Scholar, 17Connell K. Bolton N. Olsen D. Piomelli D. Hohmann A.G. Role of the basolateral nucleus of the amygdala in endocannabinoid-mediated stress-induced analgesia.Neurosci. Lett. 2006; 397: 180-184Crossref PubMed Scopus (60) Google Scholar, 18Gobbi G. Bambico F.R. Mangieri R. Bortolato M. Campolongo P. Solinas M. Cassano T. Morgese M.G. Debonnel G. Duranti A. et al.Antidepressant-like activity and modulation of brain monoaminergic transmission by blockade of anandamide hydrolysis.Proc. Natl. Acad. Sci. USA. 2005; 102: 18620-18625Crossref PubMed Scopus (577) Google Scholar). However, although much attention has focused on the pharma­cological properties and physiological roles of anandamide, much less is known about the biochemical mechanisms responsible for the formation of this endogenous cannabinoid substance. An important step in understanding the regulation of anandamide biosynthesis in the central nervous system is the complete characterization of its precursors. The proposed mechanism for anandamide biosynthesis in neural cells involves two enzymatic steps (Scheme 1): 1) a calcium-dependent N-acyl transferase activity, which remains to be molecularly cloned, catalyzes the exchange of an arachidonoyl group between the sn-1 position of phosphatidylcholine and the primary amine of phospha­tidylethanolamine (PE), leading to the formation of N-arachidonoyl phosphatidylethanolamine (NArPE) (3Schmid H.H. Schmid P.C. Natarajan V. N-Acylated glycerophospholipids and their derivatives.Prog. Lipid Res. 1990; 29: 1-43Crossref PubMed Scopus (423) Google Scholar, 19Hansen H.S. Lauritzen L. Moesgaard B. Strand A.M. Hansen H.H. Formation of N-acyl-phosphatidylethanolamines and N-acetylethanolamines: proposed role in neurotoxicity.Bio­chem. 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Reddy P.V. Zuzarte-Augustin M.L. Schmid H.H. Biosynthesis of N-acylethanolamine phos-pholipids by dog brain preparations.J. Neurochem. 1983; 41: 1303-1312Crossref PubMed Scopus (85) Google Scholar, 24Cadas H. di Tomaso E. Piomelli D. Occurrence and biosynthesis of endogenous cannabinoid precursor, N-arachidonoyl phosphatidylethanolamine, in rat brain.J. Neurosci. 1997; PubMed Google Scholar). In NArPEs can anandamide through which at two other NArPE generated by the of a phospholipase J. L. J. D. R. Kim et biosynthetic pathway for Natl. Acad. Sci. USA. 2006; PubMed Scopus Google Scholar, J. L. J. Kim S. G. R. A. et involved in the biosynthesis of Scholar); and a from the of phospholipase A K. Y. T. M. N. Biosynthesis of anandamide and by of phospholipase and J. PubMed Scopus Google Scholar, D. A. Cravatt B.F. of N-acyl phosphatidylethanolamine phospholipase D mechanisms for the biosynthesis of 2006; PubMed Scopus Google Scholar, Cravatt B.F. through ethanolamine and a role for in this 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). However, although NArPE is a central of the biosynthetic pathway leading to anandamide a precise characterization of the composition of this phospholipid in the brain has not yet been reported. in this has been by the brain of NArPE, which a of the phospholipid (21Sugiura T. Kondo S. Sukagawa A. Tonegawa T. Nakane S. Yamashita A. Ishima Y. Waku K. Transacylase-mediated and phosphodiesterase-mediated synthesis of N-arachidonoyletha-nolamine, an endogenous cannabinoid-receptor ligand, in rat brain microsomes. Comparison with synthesis from free arachidonic acid and ethanolamine.Eur. J. Biochem. 1996; 240: 53-62Crossref PubMed Scopus (204) Google Scholar, 23Natarajan V. Schmid P.C. Reddy P.V. Zuzarte-Augustin M.L. Schmid H.H. 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Piomelli D. acid or as fatty acid amide hydrolase and Med. PubMed Scopus Google Scholar), was a from Dr. A. Duranti of anandamide by the reaction of arachidonoyl with a of ethanolamine or in at for with The with and They by and was by by the reaction of fatty acid with a of in at for using as a The with by chromatography and by was and in and as with and with and and in of for and anandamide by using an system coupled to a with an Anandamide was using a and with a of in to in at a of was at was in the ionization was at and was from to V. was as at a of and a of was at using an the of the of The of was identified and by using an system with an using a at A of in and acid to of in was at a of was in the was with at and at V. was as at a of with of and of was as the identified by of their and with those of as of by reaction with of and of V. was target was at and at range was with to by the in using as an The of was of as and and analysis by of and generated using the from in and at for and at for 30 min. The was in and for the particulate of at 37°C for in or was and lipids by and as Lipid and in for the of species of by the ion-trap using or was in the as the which to the of the of and showed ionization as by the signal of of known of an identified as sn-2 which derive from a (Scheme or a (Scheme mechanism of major was by the from position which be by a mechanism a of the on the sn-2 glycerol with of A was identified as N-acyl of species by in derivatives of Open in a new mechanisms for of using the ion-trap In both derivatives of and to the sn-2 fatty acyl group generated both N-acyl and the sn-1 fatty derivatives of the fatty acid at position we and the sn-2 from the and two of the sn-1 group and the other of the N-acyl such observed for that formation is by the of a group or bond to the at the sn-1 However, both and produced as major in the acid which have at the sn-2 and of the glycerol and a of acid K. A. Y. T. S. M. H. G. of a lipid Biophys. Acta. 2002; PubMed Scopus Google Scholar). was in the of the which derive from the of the N-acyl or from the of N-acyl J. of as a by mass with electrospray 2000; PubMed Google Scholar). this of to the species as the N-arachidonoyl not for precise of the fatty acyl groups present on the glycerol backbone. we the to the fatty acid composition of brain NArPE species. A to species by the mass was previously H.H. Hansen Hansen H.S. ionization mass for the of cannabinoid N-acylethanolamine PubMed Scopus Google Scholar). However, although this to the ionization processes and of is not for the of in a complex biological such as brain species are not by a mass for two 1) species have and 2) of the brain lipid to a in and to to the we using a of and a of a of on a of for liquid chromatography of A. 2000; PubMed Scopus Google Scholar, A of for the of in biological 2006; PubMed Scopus Google Scholar, B. R. analysis of and in human Sci. 2005; PubMed Scopus Google Scholar). These the for in and species both by and by of of their fatty acid species or acyl those with and we to by a acyl the of this lipid class a of the species. a complete we coupled with as of species a of on we of by from brain with acyl was identified by and of with as which to to species. In such we of fatty acyl glycerol which the and to their NArPEs from rat brain from the in a as a complex of and species. shown in a a of the NArPE was to of for by a of mass In mass with mass to the of to the of which have mass of from the species, as species. In we that constitute of the NArPE in the rat brain constitute the with of In we identified major endogenous which mainly contained sn-1 alkenyl groups (C16:0, C18:0, C18:1) and monounsaturated (C18:1) or (C20:4, C22:4, C22:6) sn-2 acyl groups the sn-1 was in species fatty at the sn-2 position acid brain and of the major NArPE species in rat N-arachidonoyl NArPE results are as of Open in a new NArPE, N-arachidonoyl NArPE results are as of in Ca2+ in neurons NArPE and anandamide biosynthesis V. Schmid P.C. Schmid H.H. N-Acylethanolamine phospholipid metabolism in normal and ische-mic rat brain.Biochim. Biophys. Acta. 1986; 878: 32-41Crossref PubMed Scopus (112) Google Scholar, 24Cadas H. di Tomaso E. Piomelli D. Occurrence and biosynthesis of endogenous cannabinoid precursor, N-arachidonoyl phosphatidylethanolamine, in rat brain.J. Neurosci. 1997; PubMed Google Scholar, H. S. M. L. Piomelli D. Biosynthesis of an endogenous cannabinoid in neurons and its by and Neurosci. 1996; PubMed Google Scholar). the NArPE species produced by Ca2+ we brain particulate with or and NArPE formation by observed a increase in NArPE species in with or with rat brain A targeted lipidomic analysis showed that Ca2+ incubation specifically stimulated the formation of the NArPE species polyunsaturated acyl groups at the sn-2 position of the glycerol backbone. In we identified an increase in NArPE species (C20:4, with a increase in NArPE species monounsaturated fatty Furthermore, after Ca2+ we to new NArPE species with at the sn-2 such as and which in we the of Ca2+ incubation on the of of the NArPEs and that the of both lipids by Ca2+ Furthermore, the in anandamide particulate in the of the fatty acid amide hydrolase increase in NArPE species after brain particulate with or not are as of Open in a new of on the of NArPE and anandamide in rat brain particulate at 37°C for 30 min. and are as by not are as of NArPEs are the biological precursors for anandamide, an endogenous signaling lipid that binds to and activates cannabinoid receptors in the brain and peripheral (8Devane W.A. Hanus L. Breuer A. Pertwee R.G. Stevenson L.A. Griffin G. Gibson D. Mandelbaum A. Etinger A. Mechoulam R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor.Science. 1992; 258: 1946-1949Crossref PubMed Scopus (4722) Google Scholar). In the present study, we an that 1) to the precise composition of NArPE species in the rat brain and 2) to that Ca2+ the formation of a of such species, through the of a selective N-acyl transferase activity. NArPEs a and in which was by the formation of a and sn-2 in and by sn-1 fatty acid and N-acyl derivatives in These results are in with that of sn-2 as the through a A. S. T. of using mass 2001; PubMed Scopus Google Scholar). 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NArPE species fatty acid species, although present in brain to be less to Ca2+ a previously unrecognized preference of brain N-acyl transferase activity for polyunsaturated a preference derive from a of the N-acyl transferase polyunsaturated species or from a of this enzyme in membrane in PUFA-containing E.A. membrane in the of the lipid signaling J. 2006; PubMed Scopus Google Scholar, Lipid are in arachidonic acid and and their composition is of a electrospray 2002; 41: PubMed Scopus Google Scholar). of the biosynthetic involved the that NArPE and increase in a between In the of NArPE species in the rat brain new insights the physiological regulation of anandamide The of the of for is was by from the and the of and to