Short CommunicationRegulation of anandamide tissue levels by N-arachidonylglycine
Introduction
NAGly (Fig. 1), an endogenous substance found in rat brain and other sites [1], occurs in amounts greater than the closely related endocannabinoid anandamide (N-arachidonylethanolamide). Earlier reports [2] suggested that NAGly may have analgesic properties similar to those of anandamide [3], [4] but is inactive in assays for psychotropic action such as the “ring test” [5]. The latter finding was in agreement with a report showing a lack of binding by NAGly to the cannabinoid receptor CB1 [6]. This activity profile is reminiscent of that observed for the naturally occurring cannabinoid acids [7], which exhibit analgesic effects but are inactive in the ring test. NAGly also showed substantial potency ( μM) in reducing the in vitro activity of FAAH [1], [8], the enzyme primarily responsible for the degradation of anandamide to arachidonic acid and ethanolamine under physiological conditions [9], [10], [11]. However, it has little effect on anandamide transport or on the VR1 vanilloid receptor [1]. This suggests, as one of several possibilities, that NAGly may act as an endogenous regulator of tissue anandamide concentrations by virtue of its ability to inhibit FAAH and its presence in a number of tissue sites in vivo[1]. NAGly also showed in vivo anti-inflammatory activity in the mouse paw edema assay (Burstein SH and Pearson W, unpublished data).
The biological origin of NAGly is not well understood; however, two possible biosynthetic pathways have been investigated, and data supporting the existence of each have been reported [1], [12]. Burstein et al. [12], using anandamide radiolabeled in the ethanolamine moiety, showed that Chang hepatocytes incubated with this precursor produced a radiolabeled substance that chromatographically co-migrated with NAGly in several TLC systems. This suggested the possibility that NAGly may, under some conditions, be generated by an oxidative metabolism of anandamide. A second pathway involving the condensation of arachidonyl CoA with glycine was proposed by Huang et al. [1] in which the process is mediated by a subcellular rat brain preparation. To support this suggestion, they used deuterium-labeled precursors and demonstrated the synthesis of deuterated NAGly by mass spectrometric analysis. As is often the case in biosyntheses, each pathway may operate under a specific set of physiological circumstances.
We sought to determine in this study whether the in vivo administration of NAGly would increase circulating blood levels of anandamide that would, in turn, reflect an elevation of cellular concentrations of anandamide. This might help explain the analgesic [1] and anti-inflammatory actions of NAGly (Burstein SH and Pearson W, unpublished data) since anandamide is known to exhibit both actions in experimental models [3], [4], [13]. The results obtained from these studies are reported below.
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Materials
RAW 264.7 murine monocyte cells were prepared from stocks supplied and maintained by the University tissue culture facility. Minimum essential medium (MEM) was purchased from ICN. Fetal bovine serum and penicillin–streptomycin solution were obtained from GIBCO BRL. Deuterated arachidonic acid (d8-arachidonic acid) was obtained from the Cayman Chemical Co. Bovine serum albumin was obtained from Sigma, and Sep-Pak Plus C18 cartridges were purchased from the Waters Corp. TLC plates were obtained
Elevation of anandamide levels in RAW cells
In this study, evidence was sought to determine whether NAGly would cause a rise in anandamide levels in a physiologically relevant system, namely, an intact cell model such as the cultured macrophage RAW 264.7 cell line. In addition, experiments were also done in RAW cells to rule out the possibility that NAGly might serve as a metabolic precursor for anandamide, an effect that would result in an increase in anandamide concentration [1]. Treatment of RAW cells with 10 μM NAGly caused a 50%
Acknowledgements
The assistance of James E. Evans, Director, Mass Spectrometry Facility, University of Massachusetts Medical School, is gratefully acknowledged. This publication was made possible by Grants DA09439 (S.H.B.), DA09017 (S.H.B.), AR38501 (R.B.Z.), K02DA00375 (J.M.W.), NS33247 (J.M.W.), and DA13012 (J.M.W.) from The National Institute on Drug Abuse. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute on Drug Abuse.
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