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* Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy;
Department of Biomedical Sciences, University of Teramo, Teramo, Italy; and
Instituto di Ricovero e Cura a Carattere Scientifico C. Mondino, Mondino-Tor Vergata Center for Experimental Neuropharmacology, Rome, Italy
Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-
-cyclodextrin (MCD) doubles the binding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylate cyclase and p42/p44 MAPK activity. In the present study, we investigated whether type-2 cannabinoid receptors (CB2R), widely expressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB2R, and that receptor activation triggers similar [35S]guanosine-5'-O-(3-thiotriphosphate) binding in MCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection against apoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetase N-acyl-phosphatidylethanolamine-phospholipase D, and the AEA hydrolase fatty acid amide hydrolase were not affected by MCD, whereas the AEA membrane transporter was inhibited (
55%) compared with controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD in DAUDI or C6 cells, whereas the transport of 2-arachidonoylglycerol was reduced to 50%. Instead, membrane cholesterol enrichment almost doubled the uptake of AEA and 2-arachidonoylglycerol in both cell types. Finally, transfection experiments with human U937 immune cells, and the use of primary cells expressing CB1R or CB2R, ruled out that the cellular environment could account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstrate that lipid rafts control CB1R, but not CB2R, and endocannabinoid transport in immune and neuronal cells.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was partly supported by Fondazione TERCAS (Finanziamento 2004; to M.M.), by Ministero della Salute (Progetto di Ricerca Finalizzata Fondazione Santa Lucia 2004 (to A.F.-A.), and by Ricerca Corrente 2005 (to M.M.).
2 Address correspondence and reprint requests to Professor Mauro Maccarrone, Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy. E-mail address: mmaccarrone{at}unite.it
3 Abbreviations used in this paper: AEA, anandamide or arachidonoylethanolamide; AC, adenylate cyclase; 2-AG, 2-arachidonoylglycerol; AMT, AEA membrane transporter; CPZ, capsazepine; CB1/2R, type 1/2 cannabinoid receptor; DAGL, diacylglycerol lipase; FAAH, fatty acid amide hydrolase; GPCR, G protein-coupled receptor; GTP
S, guanosine-5'-O-(3-thiotriphosphate); MAFP, methyl-arachidonoyl fluorophosphonate; MAGL, monoacylglycerol lipase, MCD, methyl--cyclodextrin; NAPE, N-acyl-phosphatidylethanolamine; NArPE, N-arachidonoyl-phosphatidyl ethanolamine; NMR, nuclear magnetic resonance; PLD, phospholipase D; PTX, pertussis toxin; RTX, resinferatoxin; TRPV1, transient receptor potential channel vanilloid receptor subunit 1.
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