Effect of Lipid Rafts on Cb2 Receptor Signaling and 2-Arachidonoyl-Glycerol Metabolism in Human Immune Cells

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Metabolism in Human Immune CellsSignaling and 2-Arachidonoyl-Glycerol Effect of Lipid Rafts on Cb2 Receptor

Mauro MaccarroneOddi, Nicoletta Pasquariello, Alessandro Finazzi-Agrò and Monica Bari, Paola Spagnuolo, Filomena Fezza, Sergio

http://www.jimmunol.org/content/177/8/4971doi: 10.4049/jimmunol.177.8.4971

2006; 177:4971-4980; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Effect of Lipid Rafts on Cb2 Receptor Signaling and2-Arachidonoyl-Glycerol Metabolism in Human Immune Cells1

Monica Bari,* Paola Spagnuolo,† Filomena Fezza,* Sergio Oddi,† Nicoletta Pasquariello,†

Alessandro Finazzi-Agro,* and Mauro Maccarrone2†‡

Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-�-cyclodextrin (MCD) doubles thebinding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylatecyclase and p42/p44 MAPK activity. In the present study, we investigated whether type-2 cannabinoid receptors (CB2R), widelyexpressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD doesnot affect AEA binding to CB2R, and that receptor activation triggers similar [35S]guanosine-5�-O-(3-thiotriphosphate) binding inMCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection againstapoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetaseN-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 diacylglycerollipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD inDAUDI or C6 cells, whereas the transport of 2-arachidonoylglycerol was reduced to �50%. Instead, membrane cholesterolenrichment almost doubled the uptake of AEA and 2-arachidonoylglycerol in both cell types. Finally, transfection experimentswith human U937 immune cells, and the use of primary cells expressing CB1R or CB2R, ruled out that the cellular environmentcould account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstratethat lipid rafts control CB1R, but not CB2R, and endocannabinoid transport in immune and neuronal cells. The Journal ofImmunology, 2006, 177: 4971–4980.

A nandamide (or arachidonoylethanolamide (AEA)3) andthe other endocannabinoid 2-arachidonoylglycerol(2-AG) bind to and activate two inhibitory G protein-

coupled receptors (GPCR), namely type-1 (CB1R) and type-2(CB2R) cannabinoid receptors (1–3). CB1R are localized mainlyin the CNS (4), but are also expressed in peripheral tissues likeimmune cells (5–7). Conversely, CB2R are predominantly ex-pressed peripherally, but they are also present in the brain (8, 9).Therefore, activation of CB1 or CB2 receptors by AEA or 2-AGhas many central (10) and peripheral (11) effects. These actions arecontrolled through not yet fully characterized cellular mechanisms,

which regulate the release of endocannabinoids from membraneprecursors, their uptake by cells, and finally their intracellular dis-posal. The key agent in AEA synthesis is the N-acyl-phosphati-dylethanolamines (NAPE)-hydrolyzing phospholipase D (NAPE-PLD) (12), whereas degradation occurs through a putative AEAmembrane transporter (AMT) (13–15) and fatty acid amide hy-drolase (FAAH) (16). Besides CB receptors, AEA binds also totype 1 vanilloid receptors (now called transient receptor potentialchannel vanilloid receptor subunit 1 (TRPV1)), and thus it can beconsidered a true “endovanilloid” (17). On the other hand, 2-AG isreleased from membrane lipids by means of an sn-1-specific diac-ylglycerol lipase (DAGL) (18), and is hydrolyzed by a specificmonoacylglycerol lipase (MAGL) (19). The transport of 2-AGthrough the cellular membrane has been shown to be saturable andenergy-independent, and might occur through the same AMT thattransports AEA (13, 20, 21). Altogether, AEA and 2-AG, with othercongeners like N-arachidonoyl-dopamine, noladin ether, and vi-rodhamine and the proteins that bind, transport, synthesize, and hy-drolyze these lipids form the “endocannabinoid system” (22, 23).

Lipid rafts are subdomains of the plasma membrane that containhigh concentrations of cholesterol and glycosphingolipids, and arewell-known modulators of the activity of a number of GPCR (24–26). In fact, they modulate signaling and membrane trafficking inmany cell types (27), including human immune cells (28–30). Notsurprisingly, lipid rafts have been proposed as a potential regulatorof CBR activity (3, 31, 32), and indeed, we have shown recentlythat methyl-�-cyclodextrin (MCD), a membrane cholesterol deple-tor (33) that is widely used to disrupt the integrity of lipid rafts(24–26), doubles the CB1R binding and signaling in rat C6 gliomacells (34). CB1R activation after MCD treatment could also ac-count for the ability of this raft disruptor to block apoptosis in-duced in vitro by AEA in the same C6 cells (34, 35). In addition,

*Department of Experimental Medicine and Biochemical Sciences, University ofRome Tor Vergata, Rome, Italy; †Department of Biomedical Sciences, University ofTeramo, Teramo, Italy; and ‡Instituto di Ricovero e Cura a Carattere Scientifico C.Mondino, Mondino-Tor Vergata Center for Experimental Neuropharmacology,Rome, Italy

Received for publication February 10, 2006. Accepted for publication July 14, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was partly supported by Fondazione TERCAS (Finanziamento 2004; toM.M.), by Ministero della Salute (Progetto di Ricerca Finalizzata Fondazione SantaLucia 2004 (to A.F.-A.), and by Ricerca Corrente 2005 (to M.M.).2 Address correspondence and reprint requests to Professor Mauro Maccarrone, De-partment of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100Teramo, Italy. E-mail address: [email protected] Abbreviations used in this paper: AEA, anandamide or arachidonoylethanolamide;AC, adenylate cyclase; 2-AG, 2-arachidonoylglycerol; AMT, AEA membrane trans-porter; CPZ, capsazepine; CB1/2R, type 1/2 cannabinoid receptor; DAGL, diacyl-glycerol lipase; FAAH, fatty acid amide hydrolase; GPCR, G protein-coupled recep-tor; GTP�S, guanosine-5�-O-(3-thiotriphosphate); MAFP, methyl-arachidonoylfluorophosphonate; MAGL, monoacylglycerol lipase, MCD, methyl-�-cyclodextrin;NAPE, N-acyl-phosphatidylethanolamine; NArPE, N-arachidonoyl-phosphatidyl eth-anolamine; NMR, nuclear magnetic resonance; PLD, phospholipase D; PTX, pertus-sis toxin; RTX, resinferatoxin; TRPV1, transient receptor potential channel vanilloidreceptor subunit 1.

The Journal of Immunology

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two parallel studies have shown that cholesterol depletion by MCDreduces also the activity of AMT (34, 36), possibly by promotinga faster endocytosis of the transporter molecules (37).

This growing evidence suggesting that lipid rafts might modu-late the endocannabinoid signaling prompted us to investigate alsothe possible effect of lipid rafts integrity on CB2 receptors, onAEA metabolism in immune cells, and on the proteins that syn-thesize, transport, and degrade 2-AG. We have chosen humanDAUDI leukemia cells, because they have active AMT and FAAH(38), express functional CB2R (39), and are protected by CB2Ractivation against AEA-induced apoptosis (38). Overall, DAUDIcells share several aspects of the endocannabinoid system and en-docannabinoid-induced apoptosis with C6 cells (38), which weused in our previous study on the effect of lipid rafts on CB1R(34). On the other hand, in DAUDI cells lipid rafts regulate im-portant functions like exosome secretion (40), or growth arrestinduced by antitumor drugs (41). We perturbed raft integrity alsoby means of membrane cholesterol enrichment, under the sameexperimental conditions already used for C6 cells (42). In addition,we checked for the first time the effect of membrane cholesteroldepletion or enrichment on 2-AG metabolism in C6 cells. Takentogether, this study and the two previous reports (34, 42) monitorthe effect of lipid rafts integrity on all the major proteins that bindand metabolize AEA and 2-AG, both in neuronal and immunecells. The results point out that CB1R and endocannabinoid trans-porters are probably localized within lipid rafts, at variance withCB2R and the other proteins of the endocannabinoid system.

Materials and MethodsMaterials

Chemicals were of the purest analytical grade. AEA, cholesterol, MCD,pertussis toxin (PTX), resinferatoxin (RTX), and guanosine-5�-O-(3-thiotriphosphate) (GTP�S) were obtained from Sigma-Aldrich. Methyl-arachidonoyl fluorophosphonate (MAFP) was purchased from CaymanChemical. Capsazepine (N-[2-(4-chlorophenyl) ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide, CPZ) was from Cal-biochem, VDM11 was from Tocris-Cookson, and 2�-amino-3�-methoxy-flavone (PD98059) was from Alexis Corporation. [3H]AEA (223 Ci/mmol), [3H]CP55.940 (126 Ci/mmol), [35S]GTP�S (1250 Ci/mmol),[3H]RTX (43 Ci/mmol), adenosine 5�-[�-32P]triphosphate (3000 Ci/mmol),and sn-1-stearoyl-2-[14C]arachidonoyl-glycerol (56 mCi/mmol) were pur-chased from PerkinElmer Life Sciences. N-[3H]Arachidonoyl-phosphati-dyl-ethanolamine ([3H]NArPE, 200 Ci/mmol) and 2-oleoyl-[3H]glycerol(20 Ci/mmol) were from ARC. 2-[3H]AG was synthesized from 1,3-diben-zyloxy-2-propanol and [3H]arachidonic acid (200 Ci/mmol; ARC), as reportedpreviously (43). N-[1(S)-endo-1,3,3-trimethyl-bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide(SR144528) and N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-3-pyrazole carboxamide (SR141716) were gifts from Sanofi-AventisRecherche (Montpellier, France). 6-Dodecanoyl-2-dimethylamino-naphtalene (laurdan) was obtained from Molecular Probes. The cDNAsencoding for the human cannabinoid receptors 1 and 2 were purchasedfrom the University of Missouri cDNA Resource Center. The pcDNA3.1and pcDNA3.1/CT-GFP expression vectors and LipofectAMINE 2000were from Invitrogen Life Technologies.

Cell culture and treatment and determination of apoptosis

Human DAUDI leukemia cells were cultured in RPMI 1640 medium (In-vitrogen Life Technologies), supplemented with 25 mM HEPES, 2.5 mMsodium pyruvate, 100 U/ml penicillin, 100 �g/ml streptomycin, and 10%heat-inactivated FCS as described previously (38). Cholesterol depletionwas performed by preincubating DAUDI cells for 30 min at 37°C with theindicated amounts of MCD, which removes cholesterol from the plasmamembranes (24, 44). After MCD pretreatment, cells were washed in PBSand then were treated with AEA (or vehicle in the controls) as detailedbelow. Cell viability was assessed by trypan blue dye-exclusion. Rat C6glioma cells were cultured and treated with MCD as described previously(34). Also primary Sertoli cells, isolated from 16-day-old mice and cul-tured as reported (45), and primary HUVEC (BioWhittaker), cultured aspreviously reported (46), were treated with 2.5 mM MCD for 30 min at

37°C. Then, Sertoli cells and HUVEC were subjected to CBR bindingassays as detailed below.

Cholesterol enrichment was performed by preincubating DAUDI cellsor C6 cells for 30 min at 37°C with a cholesterol-polyvinylpyrrolidone-BSA dispersion (or vehicle in the controls), as described previously (47).Briefly, 20 �l of a stock solution of cholesterol in methanol (250 mg/ml)was added to 5 ml of sterile PBS containing 3.5% polyvinylpyrrolidone and2% BSA. The dispersion was sonicated four times for 1 min, at 30-s in-tervals, using a Vibracell sonifier (Sonics & Materials), and was added tothe cells. After incubation for 30 min at 37°C, DAUDI cells or C6 cellswere washed twice in sterile PBS and were finally resuspended in culturemedium. Cell viability was assessed by trypan blue dye-exclusion and wasfound to be �95%, in keeping with previous studies (47).

Apoptosis was estimated 48 h after treatment with AEA (or vehicle inthe controls) (38), by using the cell-death detection ELISA kit (BoehringerMannheim), based on the evaluation of DNA fragmentation by an immu-noassay for histone-associated DNA fragments in the cell cytoplasm (38).This method has been validated recently for DAUDI cells by comparisonwith cytofluorimetric analysis performed in a FACSCalibur Flow Cytom-eter (BD Biosciences) (38). This latter technique quantifies apoptotic bodyformation in dead cells by staining with propidium iodide (50 �g/ml).Control DAUDI cells contained �4.0 � 1.0 apoptotic bodies for every 100cells analyzed (38).

Cholesterol quantitation and analysis of cell membrane fluidity

Membrane lipids were extracted from DAUDI cells (5 � 106/test) andcholesterol content was measured by means of cholesterol oxidase (kitfrom Biovision) (47). Membrane fluidity of the cells was determined bymeans of the fluorescent probe laurdan, as already described previously(47). Membrane fluidity is inversely proportional to the ratio of laurdanfluorescence at 440 nm vs that at 490 nm (F440/F490): the higher the ratio,the lower the fluidity (48).

Receptor binding assays and AEA-stimulated [35S]GTP�Sbinding

Binding of [3H]CP55.940 to DAUDI cells (200 � 106/test) was performedon membrane fractions by rapid filtration assays, as reported (38). Bindingdata were elaborated through nonlinear regression analysis, using the Prism4 program (GraphPad), to calculate maximum binding (Bmax) and disso-ciation constant (Kd) of [3H]CP55.940. Saturation binding of[3H]CP55.940 was further analyzed through Scatchard plots, generated byPrism 4. Also the binding of 200 pM [3H]RTX was evaluated by rapidfiltration assays, performed as reported previously (49). In all binding ex-periments, nonspecific binding was determined in the presence of 1 �M“cold” agonist (38, 49).

AEA-stimulated [35S]GTP�S binding was determined essentially as de-scribed previously (50, 51). Cells were homogenized in ice-cold assaybuffer (50 mM Tris-HCl (pH 7.4), 3 mM MgCl2, and 1 mM EGTA) andcentrifuged twice at 48,000 � g for 10 min at 4°C. Pellets were resus-pended and homogenized in membrane buffer (50 mM Tris-HCl, 3 mMMgCl2, 100 mM NaCl, 50 �M phenylmethanesulfonyl fluoride, and 0.2mM EGTA), and protein concentration was determined. Then, membranepreparations were preincubated in 4 mU/ml adenosine deaminase (183U/mg protein; Sigma-Aldrich) for 10 min at 30°C. The binding of[35S]GTP�S stimulated by 1 �M AEA was assayed in the presence of 30�M GDP, 0.1 nM [35S]GTP�S, 50 �g of protein, and assay buffer in a finalvolume of 1 ml. Nonspecific binding was determined in the absence ofagonist and in the presence of 10 �M unlabeled GTP�S (50, 51).

U937 cell culture and transfection

The human leukemic monocyte lymphoma U937 cells were cultured inRPMI 1640 medium, containing 10% FBS, 0.11 mg/ml sodium pyruvate,2 mM L-glutamine, 100 U/ml penicillin, 100 �g/ml streptomycin, and 10%heat-inactivated FCS as described (38). The pcDNA-CB1R and pcDNA-CB2R protein expression plasmids encoding for type-1 and type-2 canna-binoid receptors, respectively, were produced by inserting the full-lengthamino acid sequences of human CB1R and CB2R into pcDNA3.1 (Invitro-gen Life Technologies). These plasmids were transfected into U937 cells(75 � 106/test), using the LipofectAMINE 2000 reagent according to themanufacturer’s instructions. The transfection efficiency was found to be�25%, by counting GFP-positive cells in at least five fields of vision underthe fluorescent microscope (52). The expression of CB receptors was eval-uated 24 h after transfection by Western blotting of cell extracts, usingspecific anti-CB1R or anti-CB2R Abs (Cayman Chemical). Twenty-fourhours after transfection, cells were treated with 2.5 mM MCD for 30 min,and were subjected to receptor binding assays as reported above. U937

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cells transfected with the mock plasmid pcDNA3.1/CT-GFP were used ascontrol.

Determination of AEA uptake, synthesis, and hydrolysis

The activity of the AMT in DAUDI cells was measured as described pre-viously (46). Cells (2 � 106/test) were incubated for 15 min, at 37 or 4°C,with 400 nM [3H]AEA, then they were washed three times in 2 ml of PBScontaining 1% BSA and were finally resuspended in 200 �l of PBS. Mem-brane lipids were then extracted (46), resuspended in 0.5 ml methanol,mixed with 3.5 ml of Sigma-Fluor liquid scintillation mixture for nonaque-ous samples (Sigma-Aldrich), and radioactivity was measured in aLKB1214 Rackbeta scintillation counter (Amersham Biosciences). To dis-criminate noncarrier-mediated from carrier-mediated transport of AEAthrough cell membranes, [3H]AEA uptake at 4°C was subtracted from thatat 37°C (46). Apparent Michaelis-Menten constant (Km) and maximumvelocity (Vmax) of the uptake of [3H]AEA (0–800 nM range) by AMT weredetermined by nonlinear regression analysis through the Prism 4 software,as reported (46). AMT activity was expressed as pmol AEA taken up permin per mg protein. The synthesis of AEA through the activity of NAPE-PLD (EC 3.1.4.4) was assayed in DAUDI cell homogenates (50 �g/test),using 100 �M [3H]NArPE as reported previously (53). NAPE-PLD activ-ity was expressed as pmol [3H]AEA released per min per mg protein. Thehydrolysis of [3H]AEA by the fatty acid amide hydrolase (EC 3.5.1.4;FAAH) activity was assayed in DAUDI cell extracts (20 �g/test) by mea-suring the release of [3H]arachidonic acid from 10 �M [3H]AEA at pH 9.0,using reversed phase high performance liquid chromatography (38). FAAHactivity was expressed as pmoles of arachidonate released per minute permilligram of protein.

Determination of 2-AG uptake, synthesis, and hydrolysis.

The uptake of 2-AG by DAUDI cells or C6 cells (2 � 106/test) was assayedas described above for AMT, using 400 nM [3H]2-AG as substrate. Ap-parent Km and Vmax of the uptake of [3H]2-AG (0–800 nM range) weredetermined by nonlinear regression analysis through the Prism 4 program(46). The transport activity was expressed as pmol 2-AG taken up perminute per milligram of protein. The activity of DAGL was assayed with10 �M sn-1-stearoyl-2-[14C]arachidonoyl-glycerol as substrate (18), andthat of MAGL was determined using 10 �M 2-oleoyl-[3H]-glycerol assubstrate, as reported (19). Both DAGL and MAGL activities were ex-pressed as pmol product per minute per milligram of protein.

Other enzymatic assays

DAUDI cells (5 � 106/test) were incubated for 15 min at 37°C with AEAand related compounds, then they were washed, homogenized and sub-jected to enzymatic assays. Forskolin (1 �M)-stimulated adenylate cyclase(AC) (EC 4.6.1.1) activity was determined according to the amount ofcAMP (54), detected in cell extracts with the cAMP Enzyme Immunoassaykit (Cayman Chemical), as described previously (46). AC activity wasexpressed as pmol cAMP per min per mg protein. The activity of p42/p44MAPK (EC 2.7.1.37; MAPK) was assayed in cell extracts by the phos-phorylation of MAPK-specific peptide substrate at 30°C with adenosine5�-[�-32P]triphosphate (55), using the Biotrak MAPK Enzyme Assay sys-tem (Amersham Biosciences) as reported previously (56). MAPK activitywas expressed as pmol phosphate per min per mg protein. The effect ofpertussis toxin (PTX) on enzymatic activities was determined by preincu-bating DAUDI cells for 3 h at 37°C with 5 �g/ml PTX before addition ofAEA, or vehicle in control experiments (55).

Statistical analysis

Data reported in this article are the means � SD of at least three indepen-dent experiments, each performed in duplicate. Statistical analysis was per-formed by the nonparametric Mann-Whitney U test, elaborating experi-mental data by means of the InStat 3 program (GraphPad).

ResultsEffect of MCD on CB2 receptors

Human DAUDI leukemia cells were treated with MCD in a con-centration range (0.5–5 mM) widely used to disrupt lipid rafts(24–29). In particular, 2.5 mM MCD has been recently shown todouble the binding efficiency of CB1 receptors, defined as theBmax/Kd ratio by analogy with the catalytic efficiency (Vmax/Km)of enzymes, and to enhance �3-fold CB1R-dependent signaling innerve cells (34). Therefore, we used the same range of MCD con-centration to test the effect of lipid rafts on CB2R binding and

signaling in immune cells. Independently of the dose used, MCDdid not affect the binding of [3H]CP55.940, a CB1R and CB2Ragonist (57). DAUDI cells express type-2 CB receptors (38, 39),and consistently [3H]CP55.940 binding was fully displaced by 0.1�M SR144528 (Fig. 1A), a selective CB2R antagonist (57). Sat-uration curves like those shown in Fig. 1B allowed to calculate theconstants for [3H]CP55.940 binding, i.e., Bmax values of 301 �10 or 296 � 9 fmol/mg protein, and Kd values of 294 � 30 or306 � 28 pM, for untreated or MCD-treated cells, respectively.These data were further confirmed by Scatchard analysis of thesaturation binding of [3H]CP55.940 to control (Fig. 1C) or MCD-treated cells (Fig. 1D), which yielded Bmax values of 306 or 299fmol/mg protein, and Kd values of 305 or 312 pM, for control orMCD-treated cells respectively. Thus, the binding efficiency ofCB2R was always �1. Incidentally, Bmax and Kd values of CB2Rin DAUDI cells are close to those reported for the same receptorfrom other sources (2, 57). These data show that, unlike CB1R innerve cells, CB2 receptors in leukemic cells are not affected bylipid rafts disruption. To ascertain whether the cellular environ-ment per se could be responsible for the different effect of MCD onCB1 and CB2 receptors, human lymphoma U937 cells were trans-fected with plasmids encoding for type-1 or type-2 cannabinoidreceptors. These cells are a widely used model of immune cells,and were chosen because they have a functional endocannabinoidsystem (38). We found that U937 cells are devoid of CB receptors,based on Western blot (data not shown) and binding data (Table I).These observations extend previous findings from our group (38),yet conflicting reports have shown that the same cell line expressesa “brain-type” (i.e., type-1) (58), a type-2 (59), or both type-1 andtype-2 (60) cannabinoid receptors. These discrepancies may be dueto different subclones of U937 cells, and underline the need tocharacterize the biochemical background of a cell type before run-ning biological assays (61). Twenty-four hours after transfection,U937 cells expressed CBR proteins (data not shown), and weretreated with 2.5 mM MCD for 30 min to perform receptor bindingassays. Table I shows that both CB receptors were functional intransfected cells, and that CB1R or CB2R binding was minimizedby the corresponding selective antagonists SR141716 orSR144528 (57), respectively. Yet, MCD enhanced CB1R bindingonly (Table I), ruling out any effect of the gross cellular environ-ment per se. In addition, we sought to ascertain whether the effectof MCD on CB receptors in immortalized cell lines was represen-tative also of normal cells. To this end, we chose two primary celltypes that are known to bind [3H]CP55.940 through CB2R orCB1R only: mouse Sertoli cells (45) and HUVEC (Ref. 46 andreferences therein), respectively. We could not use human periph-eral lymphocytes, because they express both CB receptors (5, 6),thus preventing to dissect the effect of MCD on CB1R vs CB2R.In keeping with the data on DAUDI cells (Fig. 1A) and on C6 cells(34), treatment of Sertoli cells with 2.5 mM MCD did not affect[3H]CP55.940 binding, which instead was increased up to �160%of the controls in HUVEC (Fig. 2). The selective antagonistsSR141716 and SR144528 minimized binding of [3H]CP55.940 toCB1R or CB2R, respectively (Fig. 2).

Effect of MCD on CB2-dependent signaling pathways

The main signaling pathways triggered by agonist binding to CB2receptors include inhibition of AC and stimulation of MAPK, bothmediated by Gi/o proteins (for review, see Refs. 1–3, 57). Here,pretreatment of DAUDI cells with MCD did not affect the bindingof [35S]GTP�S stimulated by various amounts of AEA, up to asaturating concentration of 1 �M (data not shown). The effect of 1�M AEA was fully prevented by 0.1 �M SR144528 at all con-centrations of MCD (data not shown). In addition, pretreatment

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with 2.5 mM MCD did not affect the basal activity of AC or thatof MAPK, nor did it significantly potentiate the effect of AEA upto 1 �M on these enzymes; at the latter saturating concentrationAEA inhibited AC down to �50% of the controls, and increasedMAPK up to �240% (Fig. 3, A and B, and data not shown). The

activity of AC and MAPK in AEA-treated control cells was �55and �225% of the basal levels, respectively (Fig. 3, A and B).Additionally, the effects of 1 �M AEA on AC and MAPK wereabolished by 0.1 �M SR144528, or by 5 �g/ml PTX, an inhibitorof Gi/o proteins (55, 56) (Fig. 3, A and B). These findings suggestthat MCD does not affect CB2R binding nor the agonist-inducedCB2R signaling through Gi/o proteins.

Effect of MCD on TRPV1 receptors, AEA synthesis, uptake, anddegradation

We further investigated the effect of MCD on the other proteins ofthe endocannabinoid system that bind and metabolize AEA. Thebinding of [3H]RTX, a selective TRPV1 agonist (49, 62), toDAUDI cell membranes was not affected by MCD, yet it was fullyinhibited by 1 �M CPZ, a selective TRPV1 antagonist (49, 62)(data not shown). Independently of the dose, MCD did not affectthe activity of NAPE-PLD, responsible for AEA synthesis (12),nor that of the AEA-hydrolase FAAH (data not shown), whereas itreduced the activity of the AEA transporter AMT down to �55%at 2.5 mM (Fig. 4A). As expected, the activities of AMT and of

Table 1. Effect of cellular environment on CBR modulation by MCDa

Treatment of U937 CellsCBR Binding

(fmol/mg protein)b

None NDc

�2.5 mM MCD ND�CB1R-plasmid 50 � 8 (100%)�CB1R-plasmid � 0.1 �M SR141716 10 � 3 (20%)*�CB1R-plasmid � 2.5 mM MCD 78 � 8 (156%)*�CB2R-plasmid 55 � 7 (100%)�CB2R-plasmid � 0.1 �M SR144528 11 � 3 (20%)*�CB2R-plasmid � 2.5 mM MCD 60 � 7 (109%)

a Values in parentheses represent percentage of the control, set to 100.b Substrate was 400 pM [3H]CP55.940.c ND, Not detectable.* Denotes p � 0.01 compared with CB1R- or CB2R-transfected cells, respectively

( p � 0.05 in all other cases).

FIGURE 1. Effect of MCD on CB2R binding in DAUDI cells. A, Binding of 400 pM [3H]CP55.940 to DAUDI cells pretreated for 30 min with variousamounts of MCD, in the absence or in the presence of the CB2R antagonist SR144528 (0.1 �M). Values were expressed as percentage of the untreatedcontrol (100% � 170 � 20 fmol/mg protein). B, Saturation curves of the binding of [3H]CP55.940 to DAUDI cells, untreated or pretreated with 2.5 mMMCD for 30 min. Scatchard analysis of the saturation binding of [3H]CP55.940 to control (C) or MCD-treated (D) cells shown in B. All the data pointsfitted a linear relationship (r � 0.996 or 0.997, for control or MCD-treated cells, respectively), from which Bmax and Kd values were calculated (see“Results”). In A, � denotes p � 0.01 compared with SR144528-untreated cells (p � 0.05 in all other cases).

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FAAH were fully inhibited by the corresponding selective inhib-itors VDM11 (10 �M) and MAFP (100 nM) (62). The lack ofspecific inhibitors of NAPE-PLD did not allow to test the inhibi-tion of this enzyme. The kinetic analysis of AMT in controlDAUDI cells yielded Km and Vmax values of 100 � 15 nM and145 � 4 pmol/min per mg protein (Table II), in keeping with aprevious report (38). In DAUDI cells treated with 2.5 mM MCDthe values of Km and Vmax were 92 � 3 nM and 81 � 1 pmol/minper mg protein, respectively (Table II). Overall, MCD reduced thecatalytic efficiency of AMT, defined as the Vmax/Km ratio, to�60% of the control. Furthermore, the uptake of 400 nM[3H]AEA was reduced to �45% by 400 nM 2-AG.

Effect of MCD on 2-AG synthesis, uptake, and degradation

MCD did not affect at any concentration the activity of DAGL,which is responsible for 2-AG synthesis (18), nor that of MAGL,the main responsible for 2-AG hydrolysis (19) (data not shown).Yet, it dose-dependently reduced the uptake of [3H]2-AG, down to�50% at 5 mM (Fig. 4B). Interestingly, the uptake of [3H]2-AGwas fully inhibited by the AMT inhibitor VDM11 (Fig. 4B). Spe-cific inhibitors of DAGL or MAGL are not yet commercially avail-able, thus further inhibition experiments were not feasible. Addi-tionally, kinetic analysis of the uptake of [3H]2-AG in control orMCD-treated DAUDI cells yielded Km and Vmax values of 134 �11 nM and 80 � 2 pmol/min per mg protein, or 111 � 33 nM and50 � 3 pmol/min per mg protein, respectively (Table II). There-fore, MCD reduced the catalytic efficiency of [3H]2-AG uptake to75% of the control. Finally, the uptake of 400 nM [3H]2-AG wasreduced to �55% by 400 nM AEA.

Effect of MCD on membrane properties

To confirm that MCD treatment affected the membrane propertiesof DAUDI cells as it does in C6 cells (34), we checked the cho-

lesterol content and membrane fluidity of DAUDI cells. MCD is acholesterol depletor (33), and indeed it produced a dose-dependentdecrease in membrane cholesterol content (Table III). This effectwas paralleled by a dose-dependent decrease in the fluorescenceratio of laurdan (Table III), which is an index of membrane flu-idity: the higher the ratio, the lower the fluidity (48). At the con-centration of 2.5 mM, MCD reduced cholesterol content and flu-orescence ratio of DAUDI cell membranes to 38 and 63% of thecontrol values, indicating that membranes of MCD-treated cellswere more fluid than those of controls. These changes in DAUDIcells were superimposable on those recently reported for C6 cells(34), suggesting that the different effect of MCD on CB1 and CB2receptors was not due to a different effect of this substance on cellmembranes.

FIGURE 2. Effect of MCD on CB1 and CB2 receptors in primary cells.Binding of 400 pM [3H]CP55.940 to CB2R in mouse Sertoli cells or toCB1R in HUVEC. Both cells were pretreated for 30 min with 2.5 mMMCD, and binding assays were performed in the absence or in the presenceof the CB2R or CB1R antagonists SR144528 or SR141716, respectively(each used at 0.1 �M). �, Denotes p � 0.01 compared with untreated(none) controls; #, denotes p � 0.01 compared with MCD-treated cells(p � 0.05 in all other cases).

FIGURE 3. Effect of MCD on CB2R signaling in DAUDI cells. Activ-ity of (A) AC and (B) MAPK, assayed in DAUDI cells pretreated for 30min with 2.5 mM MCD. The effect of 1 �M AEA was tested alone, in thepresence of SR144528 (0.1 �M), or after pretreatment with 5 �g/ml per-tussis toxin (PTX). Activity values were expressed as percentage of theuntreated control (100% � 55 � 6 pmol/min per mg protein, for AC; 2.2 �0.2 pmol/min per mg protein, for MAPK). In both panels, � denotes p �0.01 compared with AEA untreated (none) controls; # denotes p � 0.01compared with AEA-treated cells (p � 0.05 in all other cases).

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Effect of MCD on AEA-induced apoptosis in DAUDI cells

Treatment with AEA induces apoptosis via activation of TRPV1receptors, both in DAUDI cells and C6 cells (38). This AEA-

induced apoptosis is blocked by activation of CB2R in DAUDIcells, and of CB1R in C6 cells (38). We have already demonstratedthat in C6 cells MCD enhanced CB1R-mediated protection againstapoptosis induced by AEA, by reinforcing the CB1R signalingthrough MAPK (34). These data are in keeping with the conceptthat activation of CB receptors is antiapoptotic (38, 63, 64).

In the present study, we show that also in DAUDI cells 10 �MCPZ, a selective TRPV1 antagonist (49, 62), quenched the AEA-induced apoptosis down to �40% of controls, whereas 1 �MSR144528 or 20 �M PD98059, a MAPK inhibitor (55), furtherincreased it up to �170 or �150% of controls, respectively (Fig.5). However, treatment with MCD (2.5 or 5 mM) did not protectat all DAUDI cells against AEA-induced apoptosis, in much thesame way as it did not enhance CB2R binding and signaling inthese cells (compare Fig. 5 with Figs. 1 and 3).

Effect of cholesterol enrichment on the endocannabinoid systemof DAUDI cells

We sought to extend the study of the role of lipid rafts perturbationon the endocannabinoid system also in the opposite way, i.e., byincreasing membrane cholesterol (65, 66). Table III shows thatcholesterol treatment of DAUDI cells led to a �300% increase inmembrane cholesterol content and a �50% increase in fluores-cence ratio (i.e., decrease in membrane fluidity). These changes incell membranes were superimposable on those reported in C6 cellsunder the same experimental conditions (42). In parallel, in cho-lesterol-treated DAUDI cells AMT activity was enhanced up to�180% of untreated controls, whereas TRPV1 binding, NAPE-PLD activity, and FAAH activity were not affected (Table IV).Kinetic analysis of AMT in cholesterol-treated DAUDI cellsshowed apparent Km and Vmax values of 96 � 10 nM and 255 �5 pmol/min per mg protein, respectively, indicating an improved

Table II. Effect of MCD or cholesterol treatment on the kinetic properties of AMT or 2-AG uptake inDAUDI cellsa

Parameter Control �2.5 mM MCD �Cholesterol

AMTVmax (pmol/min per mg protein) 145 � 4 (100%) 81 � 1 (56%)* 255 � 5 (176%)*Km (nM) 100 � 15 (100%) 92 � 3 (92%) 96 � 10 (96%)Catalytic efficiency (Vmax/Km) 1.45 (100%) 0.88 (61%)* 2.65 (183%)*

[3H]2-AG uptakeVmax (pmol/min per mg protein) 80 � 2 (100%) 50 � 3 (62%)* 137 � 2 (171%)*Km (nM) 134 � 11 (100%) 111 � 33 (83%) 121 � 7 (90%)Catalytic efficiency (Vmax/Km) 0.60 (100%) 0.45 (75%)** 1.10 (183%)*

a Values in parentheses represent percentage of the control, set to 100.*, Denotes p � 0.01 compared with untreated control; ** denotes p � 0.05 compared with untreated control ( p � 0.05 in

all other cases).

FIGURE 4. Effect of MCD on the transport of AEA and 2-AG inDAUDI cells. Activity of the AEA transporter AMT (A) and uptake of2-AG (B), assayed in DAUDI cells pretreated for 30 min with variousamounts of MCD. AEA transport and 2-AG uptake were assayed also inthe presence of the AMT inhibitor VDM11 (10 �M), under the same ex-perimental conditions. Activity values were expressed as percentage of theuntreated control (100% � 120 � 10 pmol/min per mg protein, for AMT;60 � 5 pmol/min per mg protein, for 2-AG uptake). In both panels, �

denotes p � 0.01 compared with VDM11 untreated cells; �� denotes p �0.05 compared with MCD untreated cells; ��� denotes p � 0.01 comparedwith MCD-untreated cells (p � 0.05 in all other cases).

Table III. Effect of MCD or cholesterol treatment on cholesterolcontent and fluidity of DAUDI cell membranesa

SampleCholesterol Content

(nmol/106 cells)Fluorescence Ratio

(F440:F490)

Control 3.00 � 0.35 (100%) 1.90 � 0.20 (100%)� 0.5 mM MCD 2.49 � 0.25 (83%) 1.71 � 0.22 (90%)� 1 mM MCD 2.22 � 0.25 (74%)* 1.61 � 0.20 (85%)� 2.5 mM MCD 1.14 � 0.15 (38%)** 1.19 � 0.15 (63%)**� 5 mM MCD 0.90 � 0.11 (30%)** 1.05 � 0.12 (55%)**� Cholesterol 8.70 � 0.80 (290%)** 2.95 � 0.24 (155%)**

a Values in brackets represent percentage of the control, set to 100.*, Denotes p � 0.05 compared with control; ** denotes p � 0.01 compared with

control ( p � 0.05 in all other cases).

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catalytic efficiency (�180% of untreated controls) (Table II). Al-together, these data recall those obtained about the cholesterol-dependent modulation of the endocannabinoid system in C6 gli-oma cells (42). However, a major difference between these two celltypes is that cholesterol enrichment did not affect CB2R binding ofAEA in DAUDI cells (Table IV), whereas it halved the CB1Rbinding in C6 cells (42). Taken together with the effect of MCD,these data demonstrate that CB2R, unlike CB1R, is not modulatedby perturbation of lipid rafts integrity. In addition, cholesterol en-richment did not significantly affect DAGL or MAGL activity inDAUDI cells, whereas it increased [3H]2-AG uptake up to 175%of untreated controls (Table IV). Kinetic analysis demonstratedthat in cholesterol-treated DAUDI cells apparent Km and Vmaxvalues for [3H]2-AG uptake were 121 � 7 nM and 137 � 2 pmol/min per mg protein, respectively, yielding a catalytic efficiency�180% of that of controls (Table II).

Effect of MCD or cholesterol treatment on 2-AG metabolism inrat C6 glioma cells

To extend the analysis of the effect of cholesterol depletion orenrichment also to 2-AG metabolism in neuronal cells, we inves-

tigated the effect of MCD or cholesterol addition on the activity ofDAGL and MAGL, and on [3H]2-AG uptake in C6 glioma cells.Table V shows that 2.5 mM MCD or cholesterol treatment had noeffect on DAGL and MAGL activity, whereas they reduced to�60% or enhanced to �190% [3H]2-AG uptake, respectively.These effects of MCD and cholesterol on C6 cells mirrored thoseon DAUDI cells (Table V), suggesting that 2-AG synthesis orhydrolysis is not modulated by membrane cholesterol content ineither neuronal or immune cells.

DiscussionWe report here unprecedented evidence that the cholesterol deple-tor MCD does not affect the binding of endocannabinoids to CB2receptors, and subsequent G protein-dependent signaling throughAC and MAPK. This is at variance with CB1 receptors, whosebinding and signaling are almost doubled by MCD treatment undersimilar experimental conditions (34). In addition, we demonstratethat the different effect of MCD on CB1 or CB2 receptors does notdepend on the gross cellular environment, and is observed also innormal cells. Furthermore, we show that MCD treatment reducesthe uptake of 2-AG without affecting its metabolism via DAGLand MAGL activity, in much the same way as it reduces AMTactivity without affecting the other proteins that bind (TRPV1) ormetabolize (NAPE-PLD or FAAH) AEA. Conversely, 2-AG up-take and AMT activity of DAUDI cells are enhanced by choles-terol enrichment, which does not affect CB2R, TRPV1, or the en-zymes that metabolize AEA or 2-AG. Taken together, these datasuggest that type-1 cannabinoid receptors and endocannabinoidtransporters are localized within lipid rafts, both in neuronal andimmune cells. Incidentally, this is the first report showing MAGLand DAGL activity in cells of the immune system.

The cannabinoid receptor subtypes, CB1 and CB2, are encodedby different genes, exhibit 44% amino acid identity throughout thewhole protein, and have been classified into the class A rhodopsin-like family of GPCR (1–3, 57). In the absence of crystal structures,studies have been conducted to understand the three-dimensionalstructure of CB receptors and their mechanisms of action by usingcomputer molecular modeling and nuclear magnetic resonance(NMR) approaches. Central and peripheral CB1 and CB2 receptorsare both activated by AEA or 2-AG, and trigger common signalingpathways mainly based on AC inhibition and MAPK activation(1–3, 57). Therefore, it would be of utmost importance to identifya possible differential regulation of CB1R and CB2R, also in viewof the fact that these two receptor subtypes have been recognized

Table IV. Effect of cholesterol treatment on the endocannabinoid system of DAUDI cellsa

Parameter Control �Cholesterol

CB2R binding (fmol/mg protein)b 160 � 20 (100%) 150 � 20 (94%)TRPV1 binding (fmol/mg protein)c 80 � 10 (100%) 76 � 10 (95%)NAPE-PLD activity (pmol/min per mg protein)d 37 � 4 (100%) 40 � 4 (108%)AMT activity (pmol/min per mg protein)e 120 � 10 (100%) 215 � 20 (179%)*FAAH activity (pmol/min per mg protein)f 245 � 22 (100%) 230 � 24 (94%)DAGL activity (pmol/min per mg protein)g 22 � 3 (100%) 18 � 2 (82%)[3H]2-AG uptake (pmol/min per mg protein)h 60 � 5 (100%) 105 � 10 (175%)*MAGL activity (pmol/min per mg protein)i 82 � 9 (100%) 70 � 8 (85%)

a Values in parentheses represent percentage of the control, set to 100.b Substrate was 400 pM [3H]CP55.940.c Substrate was 200 pM [3H]resinferatoxin.d Substrate was 100 �M [3H]NArPE.e Substrate was 400 nM [3H]AEA.f Substrate was 10 �M [3H]AEA.g Substrate was 10 �M sn-1-stearoyl-2-[14C]arachidonoyl-glycerol.h Substrate was 400 nM [3H]2-AG.i Substrate was 10 �M 2-oleoyl-[3H]glycerol.* Denotes p � 0.01 compared with untreated control ( p � 0.05 in all other cases).

FIGURE 5. Effect of MCD on apoptosis of DAUDI cells. Apoptoticbodies formation induced after 48 h by 10 �M AEA in DAUDI cellspretreated for 30 min with various amounts of MCD. Apoptotic bodieswere induced in the absence (control) or in the presence of the CB2Rantagonist SR144528 (1 �M), the TRPV1 antagonist capsazepine (CPZ, 10�M), or the MAPK inhibitor PD98059 (20 �M); 100% � 0.40 � 0.05absorbance units at 405 nm. � denotes p � 0.01 compared with corre-sponding control (p � 0.05 in all other cases).

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as distinct drug discovery targets for numerous potential therapeu-tic applications. These include food intake, cancer, and immunesuppression (22, 67, 68). Recently, we have shown that in rat C6glioma cells CB1R are regulated by lipid rafts, so that raft pertur-bation by cholesterol depletion enhanced CB1R binding and sig-naling (34), whereas raft perturbation by cholesterol enrichmenthad the opposite effect (42). Shortly afterward, an independentreport has shown that CB1R are localized within lipid rafts also inhuman MDA-MB231 cells, a breast cancer cell line (69). Morenotably, a solid-state NMR study has shown that AEA undergoesa fast lateral diffusion within the bilayer outer leaflet before mak-ing a productive interaction with CB1R (70), giving ground to theconcept that the membrane environment is critical for CB1R bind-ing and signaling. In the present study, the following lines of ev-idence support the major finding of this study, i.e., that CB2R,unlike CB1R, are not affected by perturbation of lipid rafts: 1)binding of [3H]CP55.940 to CB2R and kinetic constants of satu-ration curves of this binding were not affected by MCD, used in aconcentration range that enhances CB1R in glioma cells (34) andin breast cancer cells (69); 2) AEA-induced stimulation of thebinding of GTP�S to DAUDI cells was not enhanced by MCD atany dose, neither was 3) CB2R-dependent signaling through ACand MAPK; 4) the antiapoptotic effect of CB2R activation, exertedthrough the MAPK pathway, was not further enhanced by MCD;and 5) lipid rafts perturbation by membrane cholesterol enrichmentwas ineffective on CB2R. Remarkably, the effect of MCD onmembrane properties of DAUDI cells (Table III) was superimpos-able on that observed in C6 cells (34), ruling out that MCD treat-ment was ineffective on leukemic cell membranes. In addition,transfection of the human immune U937 cell line with CB1R- orCB2R-expressing plasmids ruled out that the gross cellular envi-ronment could be responsible per se for the different effect of MCDon CB1 and CB2 receptors (Table I). Furthermore, the effect ofMCD on the binding of CB1R, but not CB2R, in primary cells(Fig. 2) suggests that type-1 but not type-2 cannabinoid receptorsare localized within lipid rafts also in normal cells.

The molecular basis of the different sensitivity of CB1 and CB2receptors to raft integrity might be complex, and need a thoroughanalysis of the lipid environment of the receptors, along with thecharacterization of the three dimensional structures of the two re-ceptor subtypes in the context of membrane bilayers. The presentstudy suggests that CB2R, unlike CB1R, does not interact withlipid rafts, a conclusion further supported by preliminary fraction-ation studies showing that CB1R in C6 cells, but not CB2R inDAUDI cells, colocalize with the raft marker caveolin-1 (M. Bari,M. Ranalli, A. Finazzi-Agro, and M. Maccarrone, manuscript inpreparation). In this context, it seems noteworthy that in the mem-brane outer leaflet AEA takes an extended conformation that en-ables it to interact with a hydrophobic groove formed by helices 3and 6 of CB1R, where its terminal carbon is positioned close to akey cysteine residue (Cys47) in helix 6 (70). This interaction isessential for receptor activation (70), suggesting that differences in

folding between the two CB receptor subtypes might lead to dif-ferent activity and regulation. In the same line, a recent study usingcombined high resolution NMR and computer modeling hasshown that CB1 and CB2 receptors have indeed conformationalproperties and salt bridge differences in the so-called juxtamem-brane segment (or helix 8), which is critical for their activity andregulation and, more notably, is under the influence of the sur-rounding chemical environment (71). Therefore, it is tempting tospeculate that lipid rafts might regulate CB1 receptor by interact-ing with specific regions of its three-dimensional structure, likehelices 3 and 6 (70) or helix 8 (71). The lack of these interactionscould make CB2R insensitive to lipid rafts perturbation.

This study has also extended for the first time the role of lipidrafts perturbation to the metabolism of 2-AG, in both immune andneuronal cells. This seems of major interest, because DAGL is theonly enzyme that synthesizes 2-AG (18), whereas MAGL is themain enzyme that hydrolyzes it (19), with a minor contribution ofFAAH (16). Critical activities of 2-AG independent of those ofAEA are emerging both in the CNS (72) and in the periphery (73).Thus, a better understanding of DAGL and MAGL regulation, andof their role in maintaining the endocannabinoid tone in vivo, canbe of utmost importance, as it has been the case for NAPE-PLD(12) and FAAH (16) with respect to AEA. In this line, selectiveinhibitors of MAGL have been developed recently, and have iden-tified this enzyme as a novel target for drug design (74). In thepresent study, we show that neither DAGL, a membrane-boundenzyme, nor MAGL, a cytosolic enzyme, were affected by pertur-bation of lipid rafts integrity in immune or neuronal cells. How-ever, cholesterol depletion or enrichment, respectively reduced orenhanced the uptake of 2-AG by DAUDI cells or C6 cells, in muchthe same way as they modulated AEA transport by AMT. Thisobservation, along with the similar Km values of AEA or 2-AGuptake, favors the hypothesis that 2-AG might be transported bythe same AMT that takes up AEA (13, 20, 21). The inhibition ofAMT by 2-AG, and that of 2-AG uptake by AEA, seem tostrengthen this hypothesis. In this context, it should be recalled thatAMT or other endocannabinoid transporter(s) have not been iden-tified yet, and there is controversy about their existence (75).While it is clear that AEA uptake has the features of a facilitatedtransport, and there may be indeed a site through which it diffuses(76), the molecular identity of, and the gene encoding for, an AEAmembrane transporter still remain elusive (14, 15, 77). At any rate,the data demonstrate that lipid rafts integrity modulates endocan-nabinoid transport across cell membranes. In addition, the obser-vation that raft disruption reduced Vmax, without affecting Km ofAEA or 2-AG transport by DAUDI cells, seems to favor the “en-docytic hypothesis” of McFarland et al. (36), who proposed thatthe AEA transporter may be internalized upon MCD treatment,thus reducing the number (and hence the Vmax) of transportermolecules on the cell surface. However, until the existence andidentity of the putative AMT is known and specific Abs have been

Table V. Effect of MCD or cholesterol treatment on 2-AG metabolism in C6 cellsa

Parameter Control �2.5 mM MCD �Cholesterol

DAGL activity (pmol/min per mg protein)a 65 � 8 (100%) 75 � 7 (115%) 52 � 7 (80%)[3H]2-AG uptake (pmol/min per mg protein)b 40 � 5 (100%) 25 � 4 (62%)* 75 � 7 (187%)*MAGL activity (pmol/min per mg protein)c 212 � 20 (100%) 233 � 22 (110%) 176 � 18 (83%)

a Values in parentheses represent percentage of the control, set to 100.b Substrate was 10 �M sn-1-stearoyl-2-[14C]arachidonoyl-glycerol.c Substrate was 400 nM [3H]2-AG.d Substrate was 10 �M 2-oleoyl-[3H]glycerol.* Denotes p � 0.01 compared with untreated control ( p � 0.05 in all other cases).

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generated, it will not be possible to quantify the level of AMT inplasma membranes of cells.

We also extended the study to a crucial biological effect of CBRactivation, i.e., the protection against AEA-induced apoptosis (38).There is converging evidence that activation of CB1R or CB2Rprotects neurons, astrocytes and several peripheral cells againstapoptosis (reviewed in Refs. 78 and 79), and that CBR-dependentactivation of MAPK is a means to execute this anti-apoptotic ac-tivity (63, 80). In keeping with this view, we have shown that theprotective effect of MCD against apoptosis induced in glioma C6cells by AEA via TRPV1 receptors could be fully attributed toactivation of CB1R, that in turn activated the MAPK pathway (34).Like C6 glioma cells, DAUDI cells undergo apoptosis induced byAEA via TRPV1, and CB2R protect cells against this activity ofAEA (38). Therefore, our present findings that MCD does notfurther protect DAUDI cells against AEA-induced apoptosis, as itdoes not enhance CB2R binding and signaling, support the conceptthat disruption of raft integrity can contribute to control the choicebetween survival or death only in cells expressing CB1R. Thisseems of interest also in the light of the ability of endocannabi-noids to modulate growth of different cell populations (78–80),and might have implications for cancer therapy (22, 68). At anyrate, the unprecedented observation that CB1 and CB2 receptorsubtypes are differentially modulated by lipid rafts perturbation isa major finding of this investigation, which seems to open a newperspective for the understanding of the biological function andregulation of cannabinoid receptors, and for the design of receptorsubtype-specific drugs.

AcknowledgmentsWe thank Dr. Valeria Gasperi for her valuable help with the biochemicalassays and Gianna Rossi (University of L’Aquila, L’Aquila, Italy) for iso-lation and treatment of mouse Sertoli cells.

DisclosuresThe authors have no financial conflict of interest.

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