Identification of a New Class of Molecules, the Arachidonyl Amino Acids, and Characterization of One Member That Inhibits Pain

Abstract

In mammals, specific lipids and amino acids serve as crucial signaling molecules. In bacteria, conjugates of lipids and amino acids (referred to as lipoamino acids) have been identified and found to possess biological activity. Here, we report that mammals also produce lipoamino acids, specifically the arachidonyl amino acids. We show that the conjugate of arachidonic acid and glycine (N-arachidonylglycine (NAGly)) is present in bovine and rat brain as well as other tissues and that it suppresses tonic inflammatory pain. The biosynthesis of NAGly and its degradation by the enzyme fatty acid amide hydrolase can be observed in rat brain tissue. In addition to NAGly, bovine brain produces at least two other arachidonyl amino acids: N-arachidonyl ␥-aminobutyric acid (NAGABA) and N-arachidonylalanine. Like NAGly, NAGABA inhibits pain. These findings open the door to the identification of other members of this new class of biomolecules, which may be integral to pain regulation and a variety of functions in mammals.Molecules found in bacteria that consist of a lipid moiety conjugated to an amino acid have been termed lipoamino acids (1-3). Burstein et al. (4) found that the lipoamino acid Narachidonylglycine (NAGly) 1 causes hot plate analgesia in mice, indicating its possible biological relevance in mammals. NAGly was first synthesized (5) as a structural analog of the endogenous cannabinoid anandamide (6), and it was found to lack affinity for the cannabinoid CB1 receptor. We hypothesized that NAGly may be produced by mammalian tissues because it is composed of the naturally occurring compounds glycine and arachidonic acid. Herein we show that at least three arachidonyl amino acids are natural constituents in mammalian brain: NAGly, N-arachidonyl ␥-aminobutyric acid (NAGABA), and N-arachidonylalanine (NAAla). One member of this group, NAGly, is characterized in detail here. It is synthesized in situ in rat brain tissue from the precursors arachidonic acid and glycine, and it is hydrolyzed by the enzyme fatty acid amide hydrolase (FAAH). NAGly is widely distributed among mammalian tissues, implying multiple functions. One possible physiological function of NAGly is pain suppression, indicated by its marked suppression of formalininduced pain behavior in rats, confirming a previous report of analgesic activity in mice (4). EXPERIMENTAL PROCEDURESTissue Extraction and Purification-The procedure comprised a liquidliquid extraction modified from that described by Folch et al. (7) followed by a series of solid-phase separations. Fresh bovine brain and rat organs were homogenized in the methanol fraction of 20 volumes of 2:1 chloroform:methanol and centrifuged for 15 min at 31,000 ϫ g at 4°C. Chloroform was then added to the supernatant. NaCl (0.2 volume, 0.73%) was mixed with the crude homogenate, and the solution was allowed to separate overnight at 4°C or centrifuged at 1,000 ϫ g for 15 min. The upper phase was discarded and the interphase washed twice. The lower phase was then applied to diethylaminopropyl silica-based...

The elmiric acids: Biologically active anandamide analogs

“…Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Prior reports (Burstein et al 1997;Huang et al, 2001), suggest that NAGly might have analgesic properties similar to those reported for anandamide (Pertwee, 2001;Walker et al, 1999) but would be inactive in assays for psychotropic action such as the "ring test (Pertwee, 1972). The latter was in agreement with a report showing a lack of affinity by NAGly for the cannabinoid receptor, CB1 (Sheskin et al, 1997).…”

“…Based on the report by Huang et al (Huang et al, 2001) showing that EMA-1 (20:4) is a potent inhibitor of FAAH, experiments were carried out in more complex models to see whether this property would result in a net increase in anandamide concentrations. An initial experiment using RAW cells indicated that this was, in fact, the case (Burstein et al, 2002).…”

“…The closely related analog of anandamide, N-arachidonylglycine (NAGly), is in a sense, a comparable acid analog to anandamide as the THC metabolite, THC-11-oic acid is to THC. NAGly is an endogenous substance found in rat brain and other sites that occurs in amounts greater than the closely related endocannabinoid, anandamide (Huang et al, 2001). However, the origin of NAGly in vivo is not completely understood.…”

“…Early observations raised the possibility that anandamide might be a precursor, which by an oxidative process could lead to EMA-1 (20:4) synthesis (Burstein et al, 2000). In a subsequent study, it was reported that deuterium labelled arachidonic acid and labelled glycine are converted to doubly labelled EMA-1 (20:4) by the P2 membrane fraction from rat brain (Huang et al, 2001). This suggests a direct condensation of glycine with arachidonic acid.…”

A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma

“…However, only limited information is available about the quantifiable amounts of these minor ECs in plasma and tissues. Another class of related compounds are amino acid conjugates (e.g., N-arachidonoyl-gamma-aminobutyric acid (A-GABA), Narachidonoyl-glycine (A-GLY) and N-arachidonoyl-serine (A-SER)), which have also been proposed to exert physiological effects [13,14]. Furthermore, N-acylethanolamines other than AEA, which exhibit distinct saturated or unsaturated fatty acids and show no (or little) direct effect on CB receptors (i.e., linoleoyl ethanolamide (LEA), palmitoyl ethanolamide (PEA), oleoyl ethanolamide (OEA), stearoyl ethanolamide (SEA)), have been shown to act as ECs entourage molecules, possibly acting in concert with ECs [15][16][17][18].…”