Abstract
N-arachidonoyl glycine is an endogenous arachidonoyl amide that activates the orphan G protein-coupled receptor (GPCR) GPR18 in a pertussis toxin (PTX)-sensitive manner and produces antinociceptive and antiinflammatory effects. It is produced by direct conjugation of arachidonic acid to glycine and by oxidative metabolism of the endocannabinoid anandamide. Based on the presence of enzymes that conjugate fatty acids with glycine and the high abundance of palmitic acid in the brain, we hypothesized the endogenous formation of the saturated N-acyl amide N-palmitoyl glycine (PalGly). PalGly was partially purified from rat lipid extracts and identified using nano-high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry. Here, we show that PalGly is produced after cellular stimulation and that it occurs in high levels in rat skin and spinal cord. PalGly was up-regulated in fatty acid amide hydrolase knockout mice, suggesting a pathway for enzymatic regulation. PalGly potently inhibited heat-evoked firing of nociceptive neurons in rat dorsal horn. In addition, PalGly induced transient calcium influx in native adult dorsal root ganglion (DRG) cells and a DRG-like cell line (F-11). The effect of PalGly on the latter cells was characterized by strict structural requirements, PTX sensitivity, and dependence on the presence of extracellular calcium. PalGly-induced calcium influx was blocked by the nonselective calcium channel blockers ruthenium red, 1-(β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SK&F96365), and La3+. Furthermore, PalGly contributed to the production of NO through calcium-sensitive nitric-oxide synthase enzymes present in F-11 cells and was inhibited by the nitric-oxide synthase inhibitor 7-nitroindazole.
Footnotes
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This work was supported by National Institutes of Health/National Institute on Drug Abuse Grants DA16825 and DA018224, the Linda and Jack Gill Center for Biomolecular Science, Indiana University; a Faculty Research Support grant from Indiana University, Bloomington; and the MetaCyt Grant to Indiana University from the Lilly Foundation Inc., Indianapolis, IN. This work was part of the doctoral thesis of N.R. titled, The Signaling Properties in Sensory Neurons of the Novel Endogenous Lipid N-Palmitoyl Glycine (2007). This work was previously presented in the following symposia: Rimmerman N, Bradshaw HB, O'Dell DK, and Walker JM (2006) Cellular signaling properties of the newly identified fatty acid amide N-palmitoyl glycine found in rat central nervous system and skin in 16th Annual Symposium on the Cannabinoids, Burlington, VT, International Cannabinoid Research Society, p. 13; Hughes HV, Rimmerman N, Vasko MR, and Walker JM (2007) N-Palmitoyl glycine induces calcium mobilization in native dorsal root ganglion cells and nitric oxide production in F-11 cells expressing neuronal nitric oxide synthase. 17th Annual Symposium on the Cannabinoids, Burlington, VT, International Cannabinoid Research Society, p. 171; Bradshaw HB, Vefring E, Jahnsen JA, O'Dell KD, Burstein S, Walker JM (2005) Identification of novel brain-derived fatty acid amides in extracts of the rat brain. 15th Annual Symposium on the Cannabinoids, Burlington, VT, International Cannabinoid Research Society.
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N.R. and H.B.B. contributed equally to this work
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ABBREVIATIONS: AEA, N-arachidonoyl ethanolamine (anandamide); GPCR, G protein-coupled receptor; CB, cannabinoid; TRPV, transient receptor potential vanilloid; NAGly, N-arachidonoyl glycine; SAR, structure-activity relationship; PEA, N-palmitoyl ethanolamine; PalGly, N-palmitoyl glycine; HPLC, high-performance liquid chromatography; QqTOF, quadrupole time-of-flight; MS/MS, mass spectrometry; 2-AG, 2-arachidonoyl glycerol; FAAH, fatty acid amide hydrolase; KO, knockout; WT, wild type; DRG, dorsal root ganglion; WDR, wide dynamic range; LinGly, N-linoleoyl glycine; SK&F96365, 1-(β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole; PTX, pertussis toxin; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride; AM, acetoxymethyl ester; I-RTX, 5′-iodoresiniferatoxin; MK801, 5H-dibenzo[a,d]cyclohepten-5,10-imine; URB597, cyclohexyl carbamic acid 3′-carbamoylbiphenyl-3-yl ester; SR144528, N-(1,3,3-trimethylbicyclo(2.2.1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide; nNOS, neuronal nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; MAPK, mitogen-activated protein kinase; kb, kilobase(s); MRM, multiple reaction monitoring; PAM, peptidylglycine α-amidating monooxygenase; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; HEK, human embryonic kidney; YFP, yellow fluorescent protein; PBS, phosphate-buffered saline; 7-NI, 7-nitroindazole; SNAP, S-nitroso-N-acetyl-dl-penicillamine; ANOVA, analysis of variance; LSD, least significant difference; LC, liquid chromatography; AUC, area under the curve; NOS, nitric oxide synthase; TRP, transient receptor potential; DAF-2, 4,5-diaminofluorescein.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received February 5, 2008.
- Accepted April 17, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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