Compartmentalization of endocannabinoids into lipid rafts in a dorsal root ganglion cell line

Abstract

Background and purpose: N-arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids binding to the cannabinoid receptors CB 1 and CB 2 to modulate neuronal excitability and synaptic transmission in primary afferent neurons. To investigate the compartmentalization of the machinery for AEA and 2-AG signalling, we studied their partitioning into lipid raft fractions isolated from a dorsal root ganglion X neuroblastoma cell line (F-11). Experimental approach: F-11 cells were homogenized and fractionated using a detergent-free OptiPrep density gradient. All lipids were partially purified from methanolic extracts of the fractions on solid phase cartridges and quantified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Protein distribution was determined by Western blotting. Key results: Under basal conditions, the endogenous cannabinoid AEA was present in both lipid raft and specific non-lipid raft fractions as was one of its biosynthetic enzymes, NAPE-PLD. The 2-AG precursor 1-stearoyl-2-arachidonoyl-sn-glycerol (DAG), diacylglycerol lipase α (DAGLα), which cleaves DAG to form 2-AG, and 2-AG were all co-localized with lipid raft markers. CB 1 receptors, previously reported to partition into lipid raft fractions, were not detected in F-11 membranes, but CB 2 receptors were detected at high levels and partitioned into non-lipid raft fractions. Conclusions and implications: The biochemical machinery for the production of 2-AG via the putative diacylglycerol pathway is localized within lipid rafts, suggesting that 2-AG synthesis via DAG occurs within these microdomains. The observed co-localization of AEA, 2-AG, and their synthetic enzymes with the reported localization of CB 1 raises the possibility of intrinsic-autocrine signalling within lipid raft domains and/or retrograde-paracrine signalling. © 2008 Nature Publishing Group All rights reserved.