Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation
2008; Elsevier BV; Volume: 49; Issue: 6 Linguagem: Inglês
10.1194/jlr.m700486-jlr200
ISSN1539-7262
AutoresFilomena Fezza, Sergio Oddi, Monia Di Tommaso, Chiara Simone, Cinzia Rapino, Nicoletta Pasquariello, Enrico Dainese, Alessandro Finazzi‐Agrò, Mauro Maccarrone,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoAnandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid. Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid. Anandamide (N-arachidonoylethanolamine; AEA) is an endogenous agonist of type-1 (CB1R) and type-2 (CB2R) cannabinoid receptors (1.Di Marzo V. Petrocellis L.De Fezza F. Ligresti A. Bisogno T. Anandamide receptors.Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 377-391Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 2.Howlett A.C. Barth F. Bonner T.I. Cabral G. Casellas P. Devane W.A. Felder C.C. Herkenham M. Mackie K. Martin B.R. et al.International Union of Pharmacology. XXVII. Classification of cannabinoid receptors.Pharmacol. Rev. 2002; 54: 161-202Crossref PubMed Scopus (2302) Google Scholar, 3.Howlett A.C. Breivogel C.S. Childers S.R. Deadwyler S.A. Hampson R.E. Porrino L.J. Cannabinoid physiology and pharmacology: 30 years of progress.Neuropharmacology. 2004; 47: 345-358Crossref PubMed Scopus (519) Google Scholar) CB1R is localized mainly in the central nervous system (4.Egertova M. Cravatt B.F. Elphick M.R. Comparative analysis of fatty acid amide hydrolase and CB1 cannabinoid receptor expression in the mouse brain: evidence of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid signaling.Neuroscience. 2003; 119: 481-496Crossref PubMed Scopus (284) Google Scholar) but is also expressed in peripheral districts like immune cells (5.Maccarrone M. Petrocellis L.De Bari M. Fezza F. Salvati S. Marzo V.Di Finazzi-Agrò. A. Lipopolysaccharide downregulates fatty acid amide hydrolase expression and increases anandamide levels in human peripheral lymphocytes.Arch. Biochem. Biophys. 2001; 393: 321-328Crossref PubMed Scopus (141) Google Scholar, 6.Nong L. Newton C. Friedman H. Klein T.W. CB1 and CB2 receptor mRNA expression in human peripheral blood mononuclear cells (PBMC) from various donor types.Adv. Exp. Med. Biol. 2001; 493: 229-233Crossref PubMed Google Scholar, 7.Klein T.W. Newton C. Larsen K. Lu L. Perkins I. Nong L. Friedman H. The cannabinoid system and immune modulation.J. Leukoc. Biol. 2003; 74: 486-496Crossref PubMed Scopus (425) Google Scholar); conversely, CB2R is expressed predominantly in the periphery but is also present in the brain (8.Nunez E. Benito C. Pazos M.R. Barbachano A. Fajardo O. Gonzalez S. Tolon R.M. Romero J. Cannabinoid CB2 receptors are expressed by perivascular microglial cells in the human brain: an immunohistochemical study.Synapse. 2004; 53: 208-213Crossref PubMed Scopus (266) Google Scholar, 9.Van Sickle M.D. Duncan M. Kingsley P.J. Mouihate A. Urbani P. Mackie K. Stella N. Makriyannis A. Piomelli D. Davison J.S. et al.Identification and functional characterization of brainstem cannabinoid CB2 receptors.Science. 2005; 310: 329-332Crossref PubMed Scopus (1254) Google Scholar). Therefore, the activation of CB1 or CB2 receptors by AEA has many central (10.Fride E. Endocannabinoids in the central nervous system—an overview.Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 221-233Abstract Full Text PDF PubMed Scopus (158) Google Scholar) and peripheral (11.Parolaro D. Massi P. Rubino T. Monti E. Endocannabinoids in the immune system and cancer.Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 319-332Abstract Full Text PDF PubMed Scopus (116) Google Scholar) effects, which are terminated by cellular uptake (12.Battista N. Gasperi V. Fezza F. Maccarrone M. The anandamide membrane transporter and the therapeutic implications of its inhibition.Therapy. 2005; 2: 141-150Crossref Google Scholar, 13.Glaser S.T. Kaczocha M. Deutsch D.G. Anandamide transport: a critical review.Life Sci. 2005; 77: 1584-1604Crossref PubMed Scopus (117) Google Scholar, 14.Hillard C.J. Jarrahian A. Accumulation of anandamide: evidence for cellular diversity.Neuropharmacology. 2005; 48: 1072-1078Crossref PubMed Scopus (34) Google Scholar), followed by degradation to ethanolamine and arachidonic acid (AA) by the fatty acid amide hydrolase (FAAH) (15.McKinney M.K. Cravatt B.F. Structure and function of fatty acid amide hydrolase.Annu. Rev. Biochem. 2005; 74: 411-432Crossref PubMed Scopus (557) Google Scholar). AEA is biosynthesized mainly by a specific N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD), which releases "on demand" AEA from membrane NAPEs (16.Okamoto Y. Morishita J. Tsuboi K. Tonai T. Ueda N. Molecular characterization of a phospholipase D generating anandamide and its congeners.J. Biol. Chem. 2004; 279: 5298-5305Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 17.Fezza F. Gasperi V. Mazzei C. Maccarrone M. Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) activity.Anal. Biochem. 2005; 339: 113-120Crossref PubMed Scopus (45) Google Scholar). In addition, other biosynthetic pathways of AEA have been discovered recently (18.Liu J. Wang L. Harvey-White J. Osei-Hyiaman D. Razdan R. Gong Q. Chan A.C. Zhou Z. Huang B.X. Kim H.Y. et al.A biosynthetic pathway for anandamide.Proc. Natl. Acad. Sci. USA. 2006; 103: 13345-13350Crossref PubMed Scopus (360) Google Scholar, 19.Leung D. Saghatelian A. Simon G.M. Cravatt B.F. Inactivation of N-acyl phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids.Biochemistry. 2006; 45: 4720-4726Crossref PubMed Scopus (294) Google Scholar, 20.Simon G.M. Cravatt B.F. Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha/beta-hydrolase 4 in this pathway.J. Biol. Chem. 2006; 281: 26465-26472Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Together with AEA and other congeners, like 2-arachidonoylglycerol, N-arachidonoyldopamine, noladin ether, and virodhamine, the proteins that bind, transport, synthesize, and hydrolyze these lipids form the "endocannabinoid system" (21.Piomelli D. Beltramo M. Glasnapp S. Lin S.Y. Goutopoulos A. Xie X.Q. Makriyannis A. Structural determinants for recognition and translocation by the anandamide transporter.Proc. Natl. Acad. Sci. USA. 1999; 96: 5802-5807Crossref PubMed Scopus (239) Google Scholar, 22.De Petrocellis L. Cascio M.G. Marzo V.Di The endocannabinoid system: a general view and latest additions.Br. J. Pharmacol. 2004; 141: 765-774Crossref PubMed Scopus (413) Google Scholar). Also, the ability of AEA to bind to and activate type-1 vanilloid receptors [now called transient receptor potential channel vanilloid receptor subunit 1 (TRPV1)] has attracted growing interest (23.Van der Stelt M. Marzo V.Di Endovanilloids. Putative endogenous ligands of transient receptor potential vanilloid 1 channels.Eur. J. Biochem. 2004; 271: 1827-1834Crossref PubMed Scopus (353) Google Scholar). For instance, the activation of CB1R or TRPV1 by AEA can exert opposite biological effects, like protection against or induction of apoptosis in neuronal and peripheral cells (24.Sarker K.P. Maruyama I. Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts.Cell. Mol. Life Sci. 2003; 60: 1200-1208Crossref PubMed Scopus (95) Google Scholar, 25.Bari M. Battista N. Fezza F. Gasperi V. Maccarrone M. New insights into endocannabinoid degradation and its therapeutic potential.Mini Rev. Med. Chem. 2006; 6: 109-120Crossref PubMed Scopus (102) Google Scholar), respectively. A still unresolved, although critical, issue in endocannabinoid research is the mechanism by which AEA crosses the plasma membrane and is transported inside the cells (12.Battista N. Gasperi V. Fezza F. Maccarrone M. The anandamide membrane transporter and the therapeutic implications of its inhibition.Therapy. 2005; 2: 141-150Crossref Google Scholar, 13.Glaser S.T. Kaczocha M. Deutsch D.G. Anandamide transport: a critical review.Life Sci. 2005; 77: 1584-1604Crossref PubMed Scopus (117) Google Scholar, 14.Hillard C.J. Jarrahian A. Accumulation of anandamide: evidence for cellular diversity.Neuropharmacology. 2005; 48: 1072-1078Crossref PubMed Scopus (34) Google Scholar, 26.McFarland M.J. Barker E.L. Anandamide transport.Pharmacol. Ther. 2005; 104: 117-135Crossref Scopus (100) Google Scholar). Because the intracellular accumulation of AEA is known to be temperature-dependent, saturable, substrate-specific, and subjected to specific inhibition by AA derivatives, the existence of a selective AEA membrane transporter has been postulated (27.Beltramo M. Stella N. Calignano A. Lin S.Y. Makriyannis A. Piomelli D. Functional role of high-affinity anandamide transport, as revealed by selective inhibition.Science. 1997; 277: 1094-1097Crossref PubMed Scopus (722) Google Scholar, 28.Hillard C.J. Edgemond W.S. Jarrahian A. Campbell W.B. Accumulation of N-arachidonoylethanolamine (anandamide) into cerebellar granule cells occurs via facilitated diffusion.J. Neurochem. 1997; 69: 631-638Crossref PubMed Scopus (315) Google Scholar). However, the molecular identity of the purported transporter remains unknown, and at present molecular probes to test its expression at the protein or mRNA level are not available. In addition, the kinetic features of AEA uptake do not rule out other mechanisms of transmembrane transport, being compatible, for example, with a simple diffusion process driven by FAAH-catalyzed hydrolysis of AEA. In this line, whether and to what extent FAAH activity may control the uptake of AEA is still unclear, although pharmacological (29.Ortar G. Ligresti A. Petrocellis L.De Morera E. Marzo V.Di Novel selective and metabolically stable inhibitors of anandamide cellular uptake.Biochem. Pharmacol. 2003; 65: 1473-1481Crossref PubMed Scopus (146) Google Scholar, 30.Fegley D. Kathuria S. Mercier R. Li C. Goutopoulos A. Makriyannis A. Piomelli D. Anandamide transport is independent of fatty-acid amide hydrolase activity and is blocked by the hydrolysis-resistant inhibitor AM1172.Proc. Natl. Acad. Sci. USA. 2004; 101: 8756-8761Crossref PubMed Scopus (202) Google Scholar), biochemical (31.Ligresti A. Morera E. Van der Stelt M. Monory K. Lutz B. Ortar G. Marzo V.Di Further evidence for the existence of a specific process for the membrane transport of anandamide.Biochem. J. 2004; 380: 265-272Crossref PubMed Scopus (126) Google Scholar, 32.Oddi S. Bari M. Battista N. Barsacchi D. Cozzani I. Maccarrone M. Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes.Cell. Mol. Life Sci. 2005; 62: 386-395Crossref PubMed Scopus (62) Google Scholar), and immunofluorescence microscopy (32.Oddi S. Bari M. Battista N. Barsacchi D. Cozzani I. Maccarrone M. Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes.Cell. Mol. Life Sci. 2005; 62: 386-395Crossref PubMed Scopus (62) Google Scholar) studies strongly suggest that an authentic AEA membrane transporter may exist and it may be distinct from FAAH. To shed some light on the degradation pathway of AEA, which is critical for the metabolic control of its biological activity (33.Fowler C.J. Pharmacological properties and therapeutic possibilities for drugs acting upon endocannabinoid receptors.Curr. Drug Target. CNS Neurol. Disord. 2005; 4: 685-696Crossref PubMed Scopus (18) Google Scholar, 34.Ligresti A. Cascio M.G. Marzo V.Di Endocannabinoid metabolic pathways and enzymes.Curr. Drug Target. CNS Neurol. Disord. 2005; 4: 615-623Crossref PubMed Scopus (74) Google Scholar, 35.Ortega-Gutierrez S. Therapeutic perspectives of inhibitors of endocannabinoid degradation.Curr. Drug Target. CNS Neurol. Disord. 2005; 4: 697-707Crossref PubMed Scopus (20) Google Scholar), it would be very important to visualize by means of microscopy techniques the accumulation and intracellular distribution of AEA. However, analogs of AEA or other molecular tools designed to this end are not yet available. The aim of the present investigation was to characterize a biotinylated analog of AEA (b-AEA or MM22) designed to visualize the intracellular accumulation of this endocannabinoid through immunomicroscopy techniques. We chose to modify the polar head of AEA because this structural change does not influence the kinetics of AEA uptake, as reported (21.Piomelli D. Beltramo M. Glasnapp S. Lin S.Y. Goutopoulos A. Xie X.Q. Makriyannis A. Structural determinants for recognition and translocation by the anandamide transporter.Proc. Natl. Acad. Sci. USA. 1999; 96: 5802-5807Crossref PubMed Scopus (239) Google Scholar). In the present study, we used the human keratinocytes (HaCaT cells), because they have a full and functional endocannabinoid system (36.Maccarrone M. Rienzo M.Di Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò. A. The endocannabinoid system in human keratinocytes.J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and are suitable for immunomicroscopy studies (32.Oddi S. Bari M. Battista N. Barsacchi D. Cozzani I. Maccarrone M. Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes.Cell. Mol. Life Sci. 2005; 62: 386-395Crossref PubMed Scopus (62) Google Scholar). By means of biochemical assays and morphological analysis, here we show that biotinylation of the polar head of AEA does not affect its accumulation by the cells but prevents its interaction with FAAH, NAPE-PLD, CB1R, CB2R, and TRPV1. Chemicals were of the purest analytical grade. [3H]AEA (205 Ci/mmol), [3H]AA (98.60 Ci/mmol), [3H]CP55.940 (126 Ci/mmol), and [3H]resinferatoxin ([3H]RTX; 43 Ci/mmol) were from Perkin-Elmer Life Sciences, Inc. (Boston, MA). [3H]N-Arachidonoylphosphatidylethanolamine ([3H]NArPE; 200 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO). AEA, AA, and 12-O-tetradecanoylphorbol 13-acetate were from Sigma Chemical Co. (St. Louis, MO). Cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB597) and (S)-1′-(4-hydroxybenzyl)-oleoylethanolamide (OMDM-1) were from Cayman Chemical (Ann Arbor, MI), and capsazepine was from Calbiochem (La Jolla, CA). EZ-Link Biotin-PEO-Amine was from Pierce (Rockford, IL). N-Piperidino-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-3-pyrazole carboxamide (SR141716) and 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-carboxa-mide (SR144528) were kind gifts from Sanofi-Aventis Recherche (Montpellier, France). Unlabeled NArPE was synthesized from AA and phosphatidylethanolamine as reported (17.Fezza F. Gasperi V. Mazzei C. Maccarrone M. Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) activity.Anal. Biochem. 2005; 339: 113-120Crossref PubMed Scopus (45) Google Scholar). Anti-CB1R and anti-CB2R rabbit polyclonal antibodies were purchased from Cayman Chemical. Mouse anti-biotin antibody, anti-mouse conjugated to Alexa Fluor 488, or anti-rabbit conjugated to horseradish peroxidase secondary antibodies and the Prolong antifade kit were purchased from Molecular Probes (Eugene, OR). We prepared b-AEA (Fig. 1A) using the EZ-Link Biotin-PEO-Amine. Briefly, the AA was activated using an amide-coupling reagent and then treated with the biotin tag in basic medium (37.Maccarrone, M., F. Fezza, A. Finazzi-Agrò, and S. Oddi. 2006. Design and synthesis of biotinylated probes for N-acyl-ethanolamines. PCT/EP2006/061988. www.freepatentsonline.com/WO2007128344.html.Google Scholar). The synthesis of the radiolabeled [3H]b-AEA was carried out under the same experimental conditions using a mix of AA and [3H]AA (specific activity, 10 mCi/mmol). The activity of AEA uptake was studied in intact HaCaT cells, as described (36.Maccarrone M. Rienzo M.Di Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò. A. The endocannabinoid system in human keratinocytes.J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Cells were incubated for 10 min at 37°C with [3H]AEA or [3H]b-AEA as substrate and washed three times in 1 ml of PBS containing 1% BSA; then, they were resuspended in 0.5 ml (0.5 M) of NaOH and measured in a scintillation counter. To further discern non-carrier-mediated from carrier-mediated transport of [3H]AEA or [3H]b-AEA across cell membranes, control experiments were carried out at 4°C. The effect of different compounds on [3H]AEA or [3H]b-AEA uptake was determined by adding each substance directly to the incubation medium at the indicated concentrations (36.Maccarrone M. Rienzo M.Di Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò. A. The endocannabinoid system in human keratinocytes.J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). FAAH activity was assayed in HaCaT cell extracts (100 μg/test) by measuring the release of [3H]AA from [3H]AEA or [3H]b-AEA through reverse-phase (RP)-HPLC (38.Maccarrone M. Bari M. Finazzi-Agrò. A. A sensitive and specific radiochromatographic assay of fatty acid amide hydrolase activity.Anal. Biochem. 1999; 267: 314-318Crossref PubMed Scopus (30) Google Scholar). Also, the effect of cold b-AEA on the hydrolysis of [3H]AEA was ascertained by adding the biotin derivative directly to the assay buffer. NAPE-PLD activity was assayed in HaCaT cell extracts (100 μg/test) by measuring the release of [3H]AEA from [3H]NArPE (100 μM) through RP-HPLC (17.Fezza F. Gasperi V. Mazzei C. Maccarrone M. Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) activity.Anal. Biochem. 2005; 339: 113-120Crossref PubMed Scopus (45) Google Scholar). The effect of b-AEA on NAPE-PLD activity was ascertained by adding the biotin derivative directly to the assay buffer. Both AEA uptake and FAAH activity data were elaborated through nonlinear regression analysis using the Prism 4 program (GraphPAD Software for Science, San Diego, CA) to calculate apparent Km and Vmax of [3H]AEA or [3H]b-AEA. Concentrations of b-AEA able to reduce by half (IC50) the uptake, hydrolysis, and synthesis of AEA through uptake, FAAH activity, and NAPE-PLD activity, respectively, were calculated from dose-response curves drawn with b-AEA concentrations up to 10 μM. For cannabinoid receptor studies, HaCaT cells, mouse brain, or mouse spleen were homogenized in 2 mM Tris-EDTA, 320 mM sucrose, 5 mM MgCl2, and 100 μM PMSF buffer (pH 7.4) using a Potter homogenizer and were centrifuged three times at 1,000 g (10 min), discharging the pellet. The supernatant was centrifuged at 18,000 g (30 min), and the pellet was resuspended in assay buffer (50 mM Tris-HCl, 2 mM Tris-EDTA, 3 mM MgCl2, and 100 μM PMSF, pH 7.4) to a final protein concentration of 1 mg/ml. These membrane fractions were used in rapid filtration assays with [3H]AEA, [3H]b-AEA, or [3H]CP55.940 (36.Maccarrone M. Rienzo M.Di Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò. A. The endocannabinoid system in human keratinocytes.J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). For the assay of vanilloid receptors, which are not expressed by HaCaT cells (36.Maccarrone M. Rienzo M.Di Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò. A. The endocannabinoid system in human keratinocytes.J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), membrane fractions isolated from rat neuroblastoma C6 cells were used in rapid filtration assays with [3H]RTX as reported (39.Bari M. Paradisi A. Pasquariello N. Maccarrone M. Cholesterol-dependent modulation of type 1 cannabinoid receptors in nerve cells.J. Neurosci. Res. 2005; 81: 275-283Crossref PubMed Scopus (55) Google Scholar). The effects of different compounds on CBR or TRPV1 binding were tested as reported (36.Maccarrone M. Rienzo M.Di Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò. A. The endocannabinoid system in human keratinocytes.J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 39.Bari M. Paradisi A. Pasquariello N. Maccarrone M. Cholesterol-dependent modulation of type 1 cannabinoid receptors in nerve cells.J. Neurosci. Res. 2005; 81: 275-283Crossref PubMed Scopus (55) Google Scholar). Binding data from HaCaT cells and mouse brain membranes were elaborated through nonlinear regression analysis, using the Prism 4 program, to calculate apparent dissociation constant (Kd) and maximum binding (Bmax) of [3H]AEA, [3H]b-AEA, or [3H]CP55.940. IC50 values of b-AEA toward the binding of [3H]CP55.940 to CBRs and of [3H]RTX to TRPV1 were calculated from dose-response curves. Western blotting was performed according to standard procedures (36.Maccarrone M. Rienzo M.Di Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò. A. The endocannabinoid system in human keratinocytes.J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The following antibodies were used to immunodetect CB1 and CB2 receptors: anti-CB1R and anti-CB2R rabbit polyclonal antibodies. HaCaT cells were grown on collagen-coated glass coverslips and were incubated either with 5 μM biotin tag (as a negative control) or with 5 μM b-AEA for 10 min at 37°C. The effects of 5 μM OMDM-1, a selective inhibitor of AEA transport (29.Ortar G. Ligresti A. Petrocellis L.De Morera E. Marzo V.Di Novel selective and metabolically stable inhibitors of anandamide cellular uptake.Biochem. Pharmacol. 2003; 65: 1473-1481Crossref PubMed Scopus (146) Google Scholar), of 0.1 μM URB597, a selective inhibitor of the AEA hydrolase FAAH (40.Kathuria S. Gaetani S. Fegley D. Valino F. Duranti A. Tontini A. Mor M. Tarzia G. Rana G.La Calignano A. et al.Modulation of anxiety through blockade of anandamide hydrolysis.Nat. Med. 2003; 1: 76-81Crossref Scopus (1229) Google Scholar), and of 0.5 μM SR141716 or 0.5 μM SR144528, CB1 or CB2 receptor-selective antagonists, respectively (41.Pertwee R.G. Ross R.A. Cannabinoid receptors and their ligands.Prostaglandins Leukot. Essent. Fatty Acids. 2002; 66: 101-121Abstract Full Text PDF PubMed Scopus (455) Google Scholar), on the uptake of b-AEA were determined by adding each substance directly to the incubation medium at 5 min before b-AEA addition. Cells were washed, fixed with 4% paraformaldehyde for 30 min at room temperature, and then permeabilized with 0.1% Triton X-100 in PBS for 2 min at 4°C. After a blocking step in 5% BSA in PBS for 30 min at room temperature, cells were incubated for 1 h at room temperature with anti-biotin primary antibody diluted 1:100 in blocking solution. Anti-mouse secondary antibodies conjugated to Alexa Fluor 488 were diluted 1:200 in blocking solution and incubated with the specimens for 30 min at room temperature. After washing, the coverslips were mounted using an antifade reagent and visualized with a Nikon Eclipse E800 fluorescence microscope equipped with a filter pack for green fluorescence detection (excitation wavelength = 465–495, emission wavelength = 515–555) (Nikon Instruments, Tokyo, Japan). For image analysis, five fields from at least three independent experiments were examined for each treatment. Quantification of the mean fluorescence intensity in selected regions was carried out using ImageJ software http://rsb.info.nih.gov/ij/). Data reported in this paper are means ± SD of at least three independent experiments, each performed in duplicate. Statistical analysis was performed by the nonparametric Mann-Whitney U-test, elaborating experimental data by means of the InStat 3 program (GraphPAD Software for Science). Previous studies have indicated that the kinetic parameters of AEA uptake are sensitive to modification of the arachidonate moiety, whereas changes in the ethanolamide region are well tolerated (42.Muthian S. Nithipatikom K. Campbell W.B. Hillard C.J. Synthesis and characterization of a fluorescent substrate for the N-arachidonoylethanolamine (anandamide) transmembrane carrier.J. Pharmacol. Exp. Ther. 2000; 293: 289-295PubMed Google Scholar, 43.Di Marzo V. Ligresti A. Morera E. Nalli M. Ortar G. The anandamide membrane transporter. Structure-activity relationships of anandamide and oleoylethanolamine analogs with phenyl rings in the polar head group region.Bioorg. Med. Chem. 2004; 12: 5161-5169Crossref PubMed Scopus (21) Google Scholar). Therefore, we designed b-AEA, in which the biotin tag was attached to the polar head of AEA through a spacer arm (Fig. 1A). The synthetic route of b-AEA allowed us to produce both b-AEA and its tritium-labeled analog ([3H]b-AEA) with a yield of ∼50%. The identity and purity of b-AEA was checked by HPLC-ESI-MS and by 1H NMR. HPLC-ESI-MS was performed with a Waters apparatus (Milford, MA), and 1H NMR was recorded on a Bruker AM series spectrometer (Rheinstetten, Germany) at 300 K and 300 MHz. Biotin-AEA showed an MS spectrum (Fig. 1B) with m/z = 661.8 [M+H]+ and a 1H NMR (CD3CN) spectrum (Fig. 1C) with δ: 0.88 (3H, t, J = 5.1 Hz), 1.24–1.47 (6H, m), 1.49–1.74 (8H, m), 2.06–2.31 (8H, m, partially under water residual peak), 2.66 (1H, d, J = 12.9 Hz), 2.77–2.96 (7H, m), 3.14–3.23 (1H, m), 3.26–3.36 (4H, m), 3.46–3.55 (4H, m), 3.58 (4H, s), 4.18–4.29 (1H, m), 4.40–4.46 (1H, m), 5.26–5.48 (8H, m), 6.51–6.56 (bs, 2H). Interestingly, the addition of the biotin tag did not yield any major change in lipophilicity, expressed as logarithm of the partition coefficient in n-octanol/water (LogP in Fig. 2), calculated through the HyperChem™ 6.03 Molecular Modeling System (Hypercube, Inc., Gainesville, FL). Also, the analysis of low-energy conformations of AEA and b-AEA showed similar electrostatic potentials on the acyl chain moiety (Fig. 2). These conformations were obtained using molecular mechanics geometry optimization with the AMBER94 force field, followed by single-point calculations (HyperChem™ 6.03), as reported (44.Dainese E. Oddi S. Bari M. Maccarrone M. Modulation of the endocannabinoid system by lipid rafts.Curr. Med. Chem. 2007; 14: 2702-2715Crossref PubMed Scopus (50) Google Scholar). Intact HaCaT cells were able to accumulate [3H]b-AEA in a concentration-dependent manner, typical of a saturable process (Fig. 3A). Accumulation of [3H]b-AEA was similar to that of [3H]AEA (Fig. 3A) and showed apparent Km and Vmax values of 421 ± 88 nM and 116 ± 10 pmol/min/mg protein, respectively (Table 1). These kinetic constants are typical of AEA transport in HaCaT cells (36.Maccarrone M. Rienzo M.Di Battista N. Gasperi V. Guerrieri P. Rossi A. Finazzi-Agrò. A. The endocannabinoid system in human keratinocytes.J. Biol. Chem. 2003; 278: 33896-33903Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) as well as in other cell types (12.Battista N. Gasperi V. Fezza F. Maccarrone M. The anandamide membrane transporter and the therapeutic implications of its inhibition.Therapy. 2005; 2: 141-150Crossref Google Scholar, 13.Glaser S.T. Kaczocha M. Deutsch D.G. Anandamide transport: a critical review.Life Sci. 2005; 77: 1584-1604Crossref PubMed Scopus (117) Google Scholar, 14.Hillard C.J. Jarrahian A. Accumulation of anandamide: evidence for cellular diversity.Neuropharmacology. 2005; 48: 1072-1078Crossref PubMed Scopus (34) Google Scholar). In addition, 5 μM OMDM-1, a selective AEA uptake inhibitor (29.Ortar G. Ligresti A. Petrocellis L.De Morera E. Marzo V.Di Novel selective and metabolically stable inhibitors of anandamide cellular uptake.Biochem. Pharmacol. 2003; 65: 1473-1481Crossref PubMed Scopus (146) Google Scholar), minimized the uptake of [3H]b-AEA (Fig. 3A) in much the same way as it inhibited that of [3H]AEA in the same cells (data not
Referência(s)