N-Palmitoyl Glycine, a Novel Endogenous Lipid That Acts As a Modulator of Calcium Influx and Nitric Oxide Production in Sensory Neurons

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-(beta-[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

No more pain upon G(q)-protein-coupled receptor activation: role of endocannabinoids (Retracted article. See vol. 43, pg. 991, 2016)

Marijuana has been used to relieve pain for centuries. The analgesic mechanism of its constituents, the cannabinoids, was only revealed after the discovery of cannabinoid receptors (CB1 and CB2) two decades ago. The subsequent identification of the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), and their biosynthetic and degradation enzymes discloses the therapeutic potential of compounds targeting the endocannabinoid system for pain control. Inhibitors of the anandamide and 2-AG degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase, respectively, may be superior to direct cannabinoid receptor ligands as endocannabinoids are synthesized on demand and rapidly degraded, focusing action at generating sites. Recently, a promising strategy for pain relief was revealed in the periaqueductal gray (PAG). It is initiated by G(q)-protein-coupled receptor (G(q)PCR) activation of the phospholipase C-diacylglycerol lipase enzymatic cascade, generating 2-AG that produces inhibition of GABAergic transmission (disinhibition) in the PAG, thereby leading to analgesia. Here, we introduce the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosynthesis and degradation processes, particularly in the PAG. We also review recent studies disclosing the G(q)PCR-phospholipase C-diacylglycerol lipase-2-AG retrograde disinhibition mechanism in the PAG, induced by activating several G(q)PCRs, including metabotropic glutamatergic (type 5 metabotropic glutamate receptor), muscarinic acetylcholine (M1/M3), and orexin 1 receptors. Disinhibition mediated by type 5 metabotropic glutamate receptor can be initiated by glutamate transporter inhibitors or indirectly by substance P, neurotensin, cholecystokinin and capsaicin. Finally, the putative role of 2-AG generated after activating the above neurotransmitter receptors in stress-induced analgesia is discussed

Nerve growth factor scales endocannabinoid signaling by regulating monoacylglycerol lipase turnover in developing cholinergic neurons

Endocannabinoid, particularly 2-arachidonoyl glycerol (2-AG), signaling has recently emerged as a molecular determinant of neuronal migration and synapse formation during cortical development. However, the cell type specificity and molecular regulation of spatially and temporally confined morphogenic 2-AG signals remain unexplored. Here, we demonstrate that genetic and pharmacological manipulation of CB(1) cannabinoid receptors permanently alters cholinergic projection neuron identity and hippocampal innervation. We show that nerve growth factor (NGF), implicated in the morphogenesis and survival of cholinergic projection neurons, dose-dependently and coordinately regulates the molecular machinery for 2-AG signaling via tropomyosine kinase A receptors in vitro. In doing so, NGF limits the sorting of monoacylglycerol lipase (MGL), rate limiting 2-AG bioavailability, to proximal neurites, allowing cell-autonomous 2-AG signaling at CB(1) cannabinoid receptors to persist at atypical locations to induce superfluous neurite extension. We find that NGF controls MGL degradation in vitro and in vivo and identify the E3 ubiquitin ligase activity of breast cancer type 1 susceptibility protein (BRCA1) as a candidate facilitating MGL’s elimination from motile neurite segments, including growth cones. BRCA1 inactivation by cisplatin or genetically can rescue and reposition MGL, arresting NGF-induced growth responses. These data indicate that NGF can orchestrate endocannabinoid signaling to promote cholinergic differentiation and implicate BRCA1 in determining neuronal morphology

Monoacylglycerol Lipase Limits the Duration of Endocannabinoid-Mediated Depolarization-Induced Suppression of Excitation in Autaptic Hippocampal Neurons

Depolarization-induced suppression of excitation (DSE) is a major form of cannabinoid-mediated short-term retrograde neuronal plasticity and is found in numerous brain regions. Autaptically cultured murine hippocampal neurons are an architecturally simple model for the study of cannabinoid signaling, including DSE. The transient nature of DSE—tens of seconds—is probably determined by the regulated hydrolysis of the endocannabinoid 2-arachidonoyl glycerol (2-AG). No less than five candidate enzymes have been considered to serve this role: fatty acid amide hydrolase (FAAH), cyclooxygenase-2 (COX-2), monoacylglycerol lipase (MGL), and α/β-hydrolase domain (ABHD) 6 and 12. We previously found that FAAH and COX-2 do not have a role in determining the duration of autaptic DSE. In the current study, we found that two structurally distinct inhibitors of MGL [N-arachidonoyl maleimide and 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184)] prolong DSE in autaptic hippocampal neurons, whereas inhibition of ABHD6 by N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4′-(aminocarbonyl)[1,1′-biphenyl]-4-yl ester, carbamic acid (WWL70) had no effect. In addition, we developed antibodies against MGL and ABHD6 and determined their expression in autaptic cultures. MGL is chiefly expressed at presynaptic terminals, optimally positioned to break down 2-AG that has engaged presynaptic CB1 receptors. ABHD6 is expressed in two distinct locations on autaptic islands, including a prominent localization in some dendrites. In summary, we provide strong pharmacological and anatomical evidence that MGL regulates DSE in autaptic hippocampal neurons and, taken together with other studies, emphasizes that endocannabinoid signaling is terminated in temporally diverse ways

Targeted lipidomics: Discovery of new fatty acyl amides

The discovery of endogenous fatty acyl amides such asN-arachidonoyl ethanolamide (anandamide),N-oleoyl ethanolamide (OEA), andN-arachidonoyl dopamine (NADA) as important signaling molecules in the central and peripheral nervous system has led us to pursue other unidentified signaling molecules. Until recently, technical challenges, particularly those associated with lipid purification and chemical analysis, have hindered the identification of low abundance signaling lipids. Improvements in chromatography and mass spectrometry (MS) such as miniaturization of high-performance liquid chromatography components, hybridization of multistage mass spectrometers and time-of-flight technology, the development of electrospray ionization (ESI) and of information-dependent acquisition, now permit rapid identification of novel, low abundance, signaling lipids