Pitfalls and solutions in assaying anandamide transport in cells
2010; Elsevier BV; Volume: 51; Issue: 8 Linguagem: Inglês
10.1194/jlr.d004176
ISSN1539-7262
AutoresSergio Oddi, Filomena Fezza, Giuseppina Catanzaro, Chiara Simone, Mariangela Pucci, Daniele Piomelli, Alessandro Finazzi‐Agrò, Mauro Maccarrone,
Tópico(s)Neurotransmitter Receptor Influence on Behavior
ResumoNonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fluorescence microscopy. Nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fluorescence microscopy. ERRATAJournal of Lipid ResearchVol. 53Issue 6PreviewAn affiliation for Dr. Daniele Piomelli, Italian Institute of Technology, was inadvertently omitted on the following papers Dr. Piomelli co-authored: Full-Text PDF Open Access The endogenous cannabinoid anandamide [N-arachidonoylethanolamine (AEA)] carries out several biological functions, both in the central nervous system and in the periphery, by binding to type-1 and type-2 cannabinoid receptors (1.Devane W.A. Hanus L. Breuer A. Pertwee R.G. Stevenson L.A. Griffin G. Gibson D. Mandelbaum A. Etinger A. Mechoulam R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor.Science. 1992; 258: 1946-1949Crossref PubMed Scopus (4798) Google Scholar), and to transient receptor potential vanilloid 1 (TRPV1) ion channels (2.Van der Stelt M. Di Marzo V. 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Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase.Proc. Natl. Acad. Sci. USA. 2001; 98: 9371-9376Crossref PubMed Scopus (1138) Google Scholar). The process whereby AEA is transported across the plasma membrane and within the cell is considered a hot topic, because it is a potential target for treatment of several pathologies associated with endocannabinoid system dysfunctions (13.Piomelli D. The molecular logic of endocannabinoid signalling.Nat. Rev. Neurosci. 2003; 4: 873-884Crossref PubMed Scopus (1659) Google Scholar, 14.Felder C.C. Dickason-Chesterfield A.K. Moore S.A. Cannabinoids biology: the search for new therapeutic targets.Mol. Interv. 2006; 6: 149-161Crossref PubMed Scopus (44) Google Scholar, 15.McFarland M.J. Barker E.L. Anandamide transport.Pharmacol. Ther. 2004; 104: 117-135Crossref PubMed Scopus (100) Google Scholar, 16.Dainese E. Oddi S. Bari M. Maccarrone M. 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The endocannabinoid pathway in Huntington's disease: a comparison with other neurodegenerative diseases.Prog. Neurobiol. 2007; 81: 349-379Crossref PubMed Scopus (96) Google Scholar). Despite considerable experimental efforts made to clarify the mechanism of AEA transport, there is not yet general consensus on the identity of the elements responsible for this process [see (22.Yates M.L. Barker E.L. Organized trafficking of anandamide and related lipids.Vitam. Horm. 2009; 81: 25-53Crossref PubMed Scopus (28) Google Scholar) for a very recent review]. In general, five nonmutually exclusive models have been proposed: i) passive diffusion (23.Fasia L. Karava V. Siafaka-Kapadai A. Uptake and metabolism of [3H]anandamide by rabbit platelets. Lack of transporter?.Eur. J. Biochem. 2003; 270: 3498-3506Crossref PubMed Scopus (31) Google Scholar, 24.Ortega-Gutierrez S. Hawkins E.G. Viso A. Lopez-Rodriguez M.L. Cravatt B.F. Comparison of anandamide transport in FAAH wild-type and knockout neurons: evidence for contributions by both FAAH and the CB1 receptor to anandamide uptake.Biochemistry. 2004; 43: 8184-8190Crossref PubMed Scopus (83) Google Scholar); ii) facilitated transport (25.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 (320) Google Scholar, 26.Moore S.A. Nomikos G.G. Dickason-Chesterfield A.K. Schober D.A. Schaus J.M. Ying B.P. Xu Y.C. Phebus L. Simmons R.M. Li D. et al.Identification of a high-affinity binding site involved in the transport of endocannabinoids.Proc. Natl. Acad. Sci. USA. 2005; 102: 17852-17857Crossref PubMed Scopus (155) Google Scholar, 27.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 (206) Google Scholar); iii) intracellular trafficking and sequestration (24.Ortega-Gutierrez S. Hawkins E.G. Viso A. Lopez-Rodriguez M.L. Cravatt B.F. Comparison of anandamide transport in FAAH wild-type and knockout neurons: evidence for contributions by both FAAH and the CB1 receptor to anandamide uptake.Biochemistry. 2004; 43: 8184-8190Crossref PubMed Scopus (83) Google Scholar, 27.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 (206) Google Scholar, 28.Oddi S. Fezza F. Pasquariello N. De Simone C. Rapino C. Dainese E. Finazzi-Agrò A. Maccarrone M. Evidence for the intracellular accumulation of anandamide in adiposomes.Cell. Mol. Life Sci. 2008; 65: 840-850Crossref PubMed Scopus (78) Google Scholar); iv) endocytosis (29.McFarland M.J. Porter A.C. Rakhshan F.R. Rawat D.S. Gibbs R.A. Barker E.L. A role for caveolae/lipid rafts in the uptake and recycling of the endogenous cannabinoid anandamide.J. Biol. Chem. 2004; 279: 41991-41997Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 30.Bari M. Battista N. Fezza F. Finazzi-Agrò A. Maccarrone M. Lipid rafts control signaling of type-1 cannabinoid receptors in neuronal cells. Implications for anandamide-induced apoptosis.J. Biol. Chem. 2005; 280: 12212-12220Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar); and v) FAAH-mediated uptake (31.Deutsch D.G. Glaser S.T. Howell J.M. Kunz J.S. Puffenbarger R.A. Hillard C.J. Abumrad N. The cellular uptake of anandamide is coupled to its breakdown by fatty-acid amide hydrolase.J. Biol. Chem. 2001; 276: 6967-6973Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 32.Glaser S.T. Abumrad N.A. Fatade F. Kaczocha M. Studholme K.M. Deutsch D.G. Evidence against the presence of an anandamide transporter.Proc. Natl. Acad. Sci. USA. 2003; 100: 4269-4274Crossref PubMed Scopus (248) Google Scholar, 33.Kaczocha M. Hermann A. Glaser S.T. Bojesen I.N. Deutsch D.G. Anandamide uptake is consistent with rate-limited diffusion and is regulated by the degree of its hydrolysis by fatty acid amide hydrolase.J. Biol. Chem. 2006; 281: 9066-9075Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). These mechanisms might also operate simultaneously to different extents, depending on cell type and assay conditions used. It is widely accepted that conflicting literature data on AEA transport are largely due to the methodological intricacy of measuring such a lipophilic molecule (34.Fowler C.J. Tiger G. Ligresti A. Lopez-Rodriguez M.L. Di Marzo V. Selective inhibition of anandamide cellular uptake versus enzymatic hydrolysis–a difficult issue to handle.Eur. J. Pharmacol. 2004; 492: 1-11Crossref PubMed Scopus (88) Google Scholar). In particular, the main problem might be a nonspecific adsorption of AEA onto plastic plates, resulting in a very high background noise (34.Fowler C.J. Tiger G. Ligresti A. Lopez-Rodriguez M.L. Di Marzo V. Selective inhibition of anandamide cellular uptake versus enzymatic hydrolysis–a difficult issue to handle.Eur. J. Pharmacol. 2004; 492: 1-11Crossref PubMed Scopus (88) Google Scholar). Furthermore, the adsorption of AEA to cell-free plates can be even more pronounced than that to cell-containing plates. Besides binding to the plastic in a way that can be prevented by binding inhibitors, AEA can be subsequently released in a time- and temperature-dependent manner. These features could be mistaken as biological processes, thereby representing an important source of conflicting results in both uptake and release studies (24.Ortega-Gutierrez S. Hawkins E.G. Viso A. Lopez-Rodriguez M.L. Cravatt B.F. Comparison of anandamide transport in FAAH wild-type and knockout neurons: evidence for contributions by both FAAH and the CB1 receptor to anandamide uptake.Biochemistry. 2004; 43: 8184-8190Crossref PubMed Scopus (83) Google Scholar, 35.Karlsson M. Pahlsson C. Fowler C.J. Reversible, temperature-dependent, and AM404-inhibitable adsorption of anandamide to cell culture wells as a confounding factor in release experiments.Eur. J. Pharm. Sci. 2004; 22: 181-189Crossref PubMed Scopus (33) Google Scholar). Such concern has been addressed in a few papers that propose the use of fatty acid-free BSA in the uptake buffer to stabilize AEA solubility and to minimize its retention by plastic wells (24.Ortega-Gutierrez S. Hawkins E.G. Viso A. Lopez-Rodriguez M.L. Cravatt B.F. Comparison of anandamide transport in FAAH wild-type and knockout neurons: evidence for contributions by both FAAH and the CB1 receptor to anandamide uptake.Biochemistry. 2004; 43: 8184-8190Crossref PubMed Scopus (83) Google Scholar, 32.Glaser S.T. Abumrad N.A. Fatade F. Kaczocha M. Studholme K.M. Deutsch D.G. Evidence against the presence of an anandamide transporter.Proc. Natl. Acad. Sci. USA. 2003; 100: 4269-4274Crossref PubMed Scopus (248) Google Scholar, 34.Fowler C.J. Tiger G. Ligresti A. Lopez-Rodriguez M.L. Di Marzo V. Selective inhibition of anandamide cellular uptake versus enzymatic hydrolysis–a difficult issue to handle.Eur. J. Pharmacol. 2004; 492: 1-11Crossref PubMed Scopus (88) Google Scholar, 35.Karlsson M. Pahlsson C. Fowler C.J. Reversible, temperature-dependent, and AM404-inhibitable adsorption of anandamide to cell culture wells as a confounding factor in release experiments.Eur. J. Pharm. Sci. 2004; 22: 181-189Crossref PubMed Scopus (33) Google Scholar). However, the use of BSA in the uptake assay has not been widely accepted, because BSA has been reported to interfere with, or even abolish, AEA uptake by cells, possibly due to the high-affinity of AEA binding to BSA (25.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 (320) Google Scholar, 36.Di Marzo V. Fontana A. Cadas H. Schinelli S. Cimino G. Schwartz J.C. Piomelli D. Formation and inactivation of endogenous cannabinoid anandamide in central neurons.Nature. 1994; 372: 686-691Crossref PubMed Scopus (1378) Google Scholar, 37.Ligresti A. Morera E. Van Der Stelt M. Monory K. Lutz B. Ortar G. Di Marzo V. Further evidence for the existence of a specific process for the membrane transport of anandamide.Biochem. J. 2004; 380: 265-272Crossref PubMed Scopus (128) Google Scholar). In the present study, we sought to minimize the errors associated with the nonspecific binding of AEA to plasticware by developing a methodological alternative to traditional protocols. To this aim, we carried out the experiments on glass supports (coverslips) instead of plastic, exploiting the limited tendency of hydrophobic molecules to bind the hydrophilic surface of borosilicate glass (38.Palmgren J.J. Monkkonen J. Korjamo T. Hassinen A. Auriola S. Drug adsorption to plastic containers and retention of drugs in cultured cells under in vitro conditions.Eur. J. Pharm. Biopharm. 2006; 64: 369-378Crossref PubMed Scopus (109) Google Scholar). Indeed, we found that AEA does not appreciably adsorb onto the glass surface of the coverslips, thus minimizing the background noise often associated with assays performed on plastic supports. This new approach was successfully applied also to the measurement of AEA export from cells. Additionally, we demonstrated that a biotinylated derivative of AEA (b-AEA) (28.Oddi S. Fezza F. Pasquariello N. De Simone C. Rapino C. Dainese E. Finazzi-Agrò A. Maccarrone M. Evidence for the intracellular accumulation of anandamide in adiposomes.Cell. Mol. Life Sci. 2008; 65: 840-850Crossref PubMed Scopus (78) Google Scholar, 39.Fezza F. Oddi S. Di Tommaso M. De Simone C. Rapino C. Pasquariello N. Dainese E. Finazzi-Agrò A. Maccarrone M. Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation.J. Lipid Res. 2008; 49: 1216-1223Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) can be used to visualize the in-and-out traffic of this endocannabinoid by immunofluorescence techniques, providing a nonradioactive, rapid, and easy-to-use alternative for the study of anandamide metabolism. All chemicals were of the purest analytical grade and were from Sigma Chemical Co. (St. Louis, MO). Arachidonoyl-5,6,8,9,11,12,14,15-[3H]ethanolamine ([3H]AEA, 205 Ci/mmol) was from Perkin-Elmer Life Sciences, Inc. (Boston, MA). Nonradioactive AEA was obtained from Sigma Chemical Co. Cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB597) was from Cayman Chemical (Ann Arbor, MI). OMDM-1 was purchased from Alexis (San Diego, CA). b-AEA was synthesized and characterized as described (39.Fezza F. Oddi S. Di Tommaso M. De Simone C. Rapino C. Pasquariello N. Dainese E. Finazzi-Agrò A. Maccarrone M. Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation.J. Lipid Res. 2008; 49: 1216-1223Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Alexa Fluor 488-conjugated streptavidin and Prolong antifade kit were purchased from Molecular Probes (Eugene, OR). Round borosilicate glass coverslips (No.1; 12 mm diameter; cod. 0111520) were purchased from Marienfeld GmbH and Co. (Lauda-Königshofen, Germany). Culture media, sera, and supplements were from Euroclone (Milan, Italy). Human keratinocytes (HaCaT cells) and human neuroblastoma SH-SY5Y cells were cultured as already described (40.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). Briefly, cells were grown in a mixture of DMEM and F12 media (1:1, v/v) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 100 units/ml penicillin at 37°C in a 5% CO2 humidified atmosphere. For experiments carried out with [3H]AEA, cells were plated onto 24-well plates (cod. 3526; Corning Incorporated, NY) containing or not coverslips, at an initial density of 2.0 × 105 cells/well, and were incubated with supplemented medium for 18 h. For assays performed with b-AEA, 1 day before the experiment, cells were plated at 50% confluence on glass coverslips, placed into 24-well plates, and grown as described above. To increase cell attachment to the glass surface, the coverslips were coated for 2 h at room temperature with human placenta type IV collagen solution (0.1 mg/ml in 0.1 M acetic acid) and then were rinsed with sterile water (tissue culture grade) before adding cells and medium. At the beginning of each assay, the wells were washed once with 0.5 ml warm PBS. The effect of OMDM-1 on the import/export of [3H]AEA or b-AEA was determined by adding this substance (50 μM) directly to the incubation medium 10 min before the experiments (28.Oddi S. Fezza F. Pasquariello N. De Simone C. Rapino C. Dainese E. Finazzi-Agrò A. Maccarrone M. Evidence for the intracellular accumulation of anandamide in adiposomes.Cell. Mol. Life Sci. 2008; 65: 840-850Crossref PubMed Scopus (78) Google Scholar). The assay was based on the method described by Fowler et al. (34.Fowler C.J. Tiger G. Ligresti A. Lopez-Rodriguez M.L. Di Marzo V. Selective inhibition of anandamide cellular uptake versus enzymatic hydrolysis–a difficult issue to handle.Eur. J. Pharmacol. 2004; 492: 1-11Crossref PubMed Scopus (88) Google Scholar), adapted as described below. For dose-dependent accumulation experiments, the culture wells, containing or not round coverslips, were incubated both at 37°C and 4°C for 10 min with different concentrations of [3H]AEA (200–3,000 nM) with the specific activity diluted to 50 mCi/mmol with unlabeled AEA in serum-free medium (500 μl/well). For time-dependent accumulation experiments, cells were incubated at either 37°C or 4°C for different periods of time (0–30 min range) with 400 nM AEA in serum-free medium (500 μl/well). Incubations were stopped by placing culture plates on ice and rapidly aspirating the media. The wells were then washed three times with 1 ml of ice-cold PBS containing 1% (w/v) BSA. To recover cell-associated AEA, NaOH extraction was used as described elsewhere (34.Fowler C.J. Tiger G. Ligresti A. Lopez-Rodriguez M.L. Di Marzo V. Selective inhibition of anandamide cellular uptake versus enzymatic hydrolysis–a difficult issue to handle.Eur. J. Pharmacol. 2004; 492: 1-11Crossref PubMed Scopus (88) Google Scholar, 27.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 (206) Google Scholar). In particular, cells grown on plastic were directly treated in the wells with 500 μl NaOH (0.5 M) for 15 min at 75°C, whereas cells grown on glass were first removed from the wells (using a curled needle and a pair of flat tweezers to remove the coverslips), and then they were placed in a scintillation vial and were treated with NaOH as described above. The radioactivity in these extracts was measured by liquid scintillation counting. For experiments carried out in the absence of cells, the wells, containing or not coverslips, were incubated either at 37°C or at 4°C in supplemented medium for 18 h and then AEA "uptake" was measured as described for the experiment with cells. In a preliminary set of experiments, we found that the small amounts of methanol (final concentration ≤ 0.5%) used as a vehicle for the preparation of AEA solutions did not significantly alter either cell viability or AEA transport, in keeping with previous reports (28.Oddi S. Fezza F. Pasquariello N. De Simone C. Rapino C. Dainese E. Finazzi-Agrò A. Maccarrone M. Evidence for the intracellular accumulation of anandamide in adiposomes.Cell. Mol. Life Sci. 2008; 65: 840-850Crossref PubMed Scopus (78) Google Scholar). To inhibit FAAH activity, 100 nM URB597 in serum-free medium was added to wells, incubated for 10 min at 37°C, then [3H]AEA was added (500 μl, 400 nM with the specific activity diluted to 50 mCi/mmol with unlabeled AEA in serum-free medium) and was further incubated for 15 min at 37°C. After this incubation step, the plates were washed three times with warm PBS, and the coverslips were transferred into new multi-wells with 500 μl PBS containing 0.15% BSA and were incubated at either 37°C or 4°C. Next, the coverslips were withdrawn at different time points (0–60 min range), were placed into scintillation vials, and radioactivity was counted as described above. For time-dependent accumulation, cells were incubated at either 37°C or 4°C for different periods of time (0–60 min range) with 10 μM b-AEA in serum-free medium (0.25 ml/well). Because b-AEA is not hydrolyzed by FAAH (28.Oddi S. Fezza F. Pasquariello N. De Simone C. Rapino C. Dainese E. Finazzi-Agrò A. Maccarrone M. Evidence for the intracellular accumulation of anandamide in adiposomes.Cell. Mol. Life Sci. 2008; 65: 840-850Crossref PubMed Scopus (78) Google Scholar), the addition of URB597 in the culture medium was omitted. For concentration-dependent accumulation experiments, cells were incubated at either 37°C or 4°C with b-AEA in the 0–20 μM range. After incubation, the plates were placed on ice and the wells were rinsed three times with ice-cold PBS. The coverslips were then transferred into new multi-wells containing 0.3 ml of fixative solution [PBS containing 3% (w/v) paraformaldehyde and 4% (w/v) sucrose] for 20 min at room temperature. Cells were extensively washed with PBS, and the excess of fixative was removed by incubating cells for 5 min at room temperature with PBS + 0.2 M glycine. After fixation, b-AEA was revealed by incubating the coverslips with the streptavidin Alexa Fluor 488-conjugated, diluted 1:100 in PBS + 0.05% saponin for 30 min at room temperature. After three washes with PBS + 0.001% saponin, coverslips were almost air-dried, mounted with 4 μl of ProLong Gold antifade, and analyzed by a Leica TCS SP confocal microscope equipped with a 40× oil objective (Leica Microsystems, Wetzlar, Germany). Pictures were taken using the program LAS AF (Leica Microsystems) and then were processed with Adobe Photoshop CS2 (Mountain View, CA) for adjustments of brightness and contrast. 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 the ImageJ software (http://rsb.info.nih.gov/ij/). A preloading step was carried out by incubating cells with 10 μM b-AEA in serum-free medium for 10 min at 37°C. Because b-AEA is not hydrolyzed by FAAH (28.Oddi S. Fezza F. Pasquariello N. De Simone C. Rapino C. Dainese E. Finazzi-Agrò A. Maccarrone M. Evidence for the intracellular accumulation of anandamide in adiposomes.Cell. Mol. Life Sci. 2008; 65: 840-850Crossref PubMed Scopus (78) Google Scholar), the addition of URB597 in the culture medium was omitted. After incubation, the plates were washed three times with warm PBS. The coverslips were then transferred into new culture wells containing 500 μl serum-free medium with 0.15% BSA and were incubated at either 37°C or 4°C. The coverslips were withdrawn at different time points (0–60 min range), washed once with 0.5 ml ice-cold PBS, transferred into new wells containing 0.3 ml of fixative solution, and labeled with streptavidin-488 for fluorescence detection as described above. Data reported in this paper are the means ± SD or ± SEM (as indicated) of at least three independent experiments, each performed in triplicate. Statistical analysis was performed by using unpaired Student's t-test, elaborating experimental data by means of Prism 4 program (GraphPAD Software, San Diego, CA). As an initial experiment aimed at evaluating the different adsorption of [3H]AEA on plastic or glass, plastic plates as such were incubated for 10 min either at 37°C or at 4°C with increasing concentrations of [3H]AEA in buffer in the presence or absence of coverslips. As shown in Fig. 1, strong nonspecific AEA adsorption on plastic was observed that was both concentration- and temperature-dependent. Moreover, the temperature-sensitive component of this binding, obtained by subtracting the uptake at 37°C from that at 4°C, was better fitted to a rectangular hyperbola (dotted line in Fig. 1; R2= 0.945) than to a straight line (R2= 0.842), yielding apparent Km and Vmax values of 0.84 ± 0.25 μM and 0.34 ± 0.01 pmol AEA/min/cm2, respectively. These data indicate that the binding of AEA to polystyrene is indeed a high-capacity, low-affinity process that mimics a protein-mediated binding (41.Thors L. Fowler C.J. Is there a temperature-dependent uptake of anandamide into cells?.Br. J. Pharmacol. 2006; 149: 73-81Crossref PubMed Scopus (22) Google Scholar). In marked contrast, neither a concentration- nor a temperature-dependent retention of tritium-labeled AEA was found on the coverslips, demonstrating that AEA does not bind appreciably onto glass surfaces (inset to Fig. 1). In particular, by recovering the total amount of AEA adsorbed on the two types of support, we could estimate that plastic binds AEA ∼80-fold more avidly than glass. We also checked that precoating the coverslips with collagen did not affect the binding of AEA to these glass supports (see supplementary data). Incidentally, similar results were also obtained for 2-arachidonoylglycerol (2-AG), another major endocannabinoid. In
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