The Cellular Uptake of Anandamide Is Coupled to Its Breakdown by Fatty-acid Amide Hydrolase
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m003161200
ISSN1083-351X
AutoresDale G. Deutsch, Sherrye T. Glaser, Judy M. Howell, Jeffrey S. Kunz, Robyn A. Puffenbarger, Cecilia J. Hillard, Nada A. Abumrad,
Tópico(s)Pancreatic function and diabetes
ResumoAnandamide is an endogenous compound that acts as an agonist at cannabinoid receptors. It is inactivated via intracellular degradation after its uptake into cells by a carrier-mediated process that depends upon a concentration gradient. The fate of anandamide in those cells containing an amidase called fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and ethanolamine. The active site nucleophilic serine of FAAH is inactivated by a variety of inhibitors including methylarachidonylfluorophosphonate (MAFP) and palmitylsulfonyl fluoride. In the current report, the net uptake of anandamide in cultured neuroblastoma (N18) and glioma (C6) cells, which contain FAAH, was decreased by nearly 50% after 6 min of incubation in the presence of MAFP. Uptake in laryngeal carcinoma (Hep2) cells, which lack FAAH, is not inhibited by MAFP. Free anandamide was found in all MAFP-treated cells and in control Hep2 cells, whereas phospholipid was the main product in N18 and C6 control cells when analyzed by TLC. The intracellular concentration of anandamide in N18, C6, and Hep2 cells was up to 18-fold greater than the extracellular concentration of 100 nm, which strongly suggests that it is sequestered within the cell by binding to membranes or proteins. The accumulation of anandamide and/or its breakdown products was found to vary among the different cell types, and this correlated approximately with the amount of FAAH activity, suggesting that the breakdown of anandamide is in part a driving force for uptake. This was shown most clearly in Hep2 cells transfected with FAAH. The uptake in these cells was 2-fold greater than in vector-transfected or untransfected Hep2 cells. Therefore, it appears that FAAH inhibitors reduce anandamide uptake by cells by shifting the anandamide concentration gradient in a direction that favors equilibrium. Because inhibition of FAAH increases the levels of extracellular anandamide, it may be a useful target for the design of therapeutic agents. Anandamide is an endogenous compound that acts as an agonist at cannabinoid receptors. It is inactivated via intracellular degradation after its uptake into cells by a carrier-mediated process that depends upon a concentration gradient. The fate of anandamide in those cells containing an amidase called fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and ethanolamine. The active site nucleophilic serine of FAAH is inactivated by a variety of inhibitors including methylarachidonylfluorophosphonate (MAFP) and palmitylsulfonyl fluoride. In the current report, the net uptake of anandamide in cultured neuroblastoma (N18) and glioma (C6) cells, which contain FAAH, was decreased by nearly 50% after 6 min of incubation in the presence of MAFP. Uptake in laryngeal carcinoma (Hep2) cells, which lack FAAH, is not inhibited by MAFP. Free anandamide was found in all MAFP-treated cells and in control Hep2 cells, whereas phospholipid was the main product in N18 and C6 control cells when analyzed by TLC. The intracellular concentration of anandamide in N18, C6, and Hep2 cells was up to 18-fold greater than the extracellular concentration of 100 nm, which strongly suggests that it is sequestered within the cell by binding to membranes or proteins. The accumulation of anandamide and/or its breakdown products was found to vary among the different cell types, and this correlated approximately with the amount of FAAH activity, suggesting that the breakdown of anandamide is in part a driving force for uptake. This was shown most clearly in Hep2 cells transfected with FAAH. The uptake in these cells was 2-fold greater than in vector-transfected or untransfected Hep2 cells. Therefore, it appears that FAAH inhibitors reduce anandamide uptake by cells by shifting the anandamide concentration gradient in a direction that favors equilibrium. Because inhibition of FAAH increases the levels of extracellular anandamide, it may be a useful target for the design of therapeutic agents. fatty-acid amide hydrolase methylarachidonylfluorophosphonate palmitylsulfonyl fluoride phenylmethylsulfonyl fluoride N18TG2 neuroblastoma cells C6 glioma cells human laryngeal carcinoma cells Endocannabinoids, such as anandamide (arachidonyl ethanolamide) and 2-arachidonyl glycerol, are endogenous ligands that bind to the cannabinoid receptors (1Devane W.A. Hanus L. Breuer A. Pertwee R.G. Stevenson L.A. Griffin G. Gibson D. Mandelbaum A. Etinger A. Mechoulam R. 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Δ9-Tetrahydrocannabinol, the active component of marijuana, appears to mimic many of the physiological and pharmacological effects of the endogenous cannabinoids, in some cases to an extreme degree (e.g. a marijuana "high"). Anandamide is transported into the neuroblastoma, glioma, brain neuron, brain astrocyte, cerebellar granule cells, leukocyte, macrophage, leukemia, and lymphoma cells in culture (5Deutsch D.G. Chin S.A. Biochem. Pharmacol. 1993; 46: 791-796Crossref PubMed Scopus (653) Google Scholar, 6Koutek B. Prestwich G.D. Howlett A.C. Chin S.A. Salehani D. Akhavan N. Deutsch D.G. J. Biol. Chem. 1994; 269: 22937-22940Abstract Full Text PDF PubMed Google Scholar, 7Di Marzo V. Fontana A. Cadas H. Schinelli S. Cimino G. Schwartz J.C. Piomelli D. Nature. 1994; 372: 686-691Crossref PubMed Scopus (1352) Google Scholar, 8Hillard C.J. Edgemond W.S. Jarrahian A. Campbell W.B. J. Neurochem. 1997; 69: 631-638Crossref PubMed Scopus (316) Google Scholar, 9Di Marzo V. Bisogno T. Melck D. 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After being transported into the cell, anandamide is subsequently broken down into arachidonic acid and ethanolamine by an endoplasmic reticular integral membrane-bound enzyme called fatty-acid amide hydrolase (FAAH),1anandamide amidase, or anandamide amidohydrolase (for review see Ref.17Di Marzo V. Deutsch D.G. Neurobiol. Cannabinoid Transmission. 1998; 5: 386-404Google Scholar). Interestingly, the catalytic site contains at least two important serines and a lysine (not the histidine, serine, and aspartate triad found in many hydrolytic serine active site enzymes) with Ser-241 acting as the nucleophile involved in the bond breaking of substrates (18Omeir R.L. Arreaza G. Deutsch D.G. Biochem. Biophys. Res. Commun. 1999; 264: 316-320Crossref PubMed Scopus (39) Google Scholar, 19Patricelli M.P. Lovato M.A. Cravatt B.F. Biochemistry. 1999; 38: 9804-9812Crossref PubMed Scopus (142) Google Scholar, 20Goparaju S.K. Kurahashi Y. Suzuki H. Ueda N. Yamamoto S. Biochim. Biophys. Acta. 1999; 1441: 77-84Crossref PubMed Scopus (50) Google Scholar). It has been shown that FAAH is inhibited by a variety of compounds, such as methylarachidonylfluorophosphonate (MAFP) and palmitylsulfonyl fluoride (PSF) (21Deutsch D.G. Omeir R. Arreaza G. Salehani D. Prestwich G.D. Huang Z. Howlett A. Biochem. Pharmacol. 1997; 53: 255-260Crossref PubMed Scopus (219) Google Scholar, 22Deutsch D.G. Lin S. Hill W.A. Morse K.L. Salehani D. Arreaza G. Omeir R.L. Makriyannis A. Biochem. Biophys. Res. Commun. 1997; 231: 217-221Crossref PubMed Scopus (134) Google Scholar, 23De Petrocellis L. Melck D. Ueda N. Maurelli S. Kurahashi Y. Yamamoto S. Marino G. Di Marzo V. Biochem. Biophys. Res. Commun. 1997; 231: 82-88Crossref PubMed Scopus (119) Google Scholar). Employing these inhibitors, we show that the net movement of anandamide into the cells is coupled to the activity of intracellular FAAH. We propose that the inhibition of anandamide breakdown results in its intracellular build-up and the attainment of equilibrium between free intracellular and extracellular anandamide, and this disfavors further net uptake. N18TG2 neuroblastoma, C6 glioma (kindly provided by Allyn Howlett and Joel Levine, respectively), and human laryngeal carcinoma cells (Hep2), provided by our in-house cell culture facility, were grown in 35 × 10-mm dishes in 2 ml of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Gemini Bioproducts, Calabasas, CA), 1% penicillin/streptomycin, and l-glutamine (Life Technologies, Inc.). All cells were grown at 37 °C with 5% CO2. For all experiments, the number of cells plated for Hep2 and C6 was ∼1.7 × 106, and the number of cells plated for N18 was ∼7 × 105. The exact cell numbers were determined for comparison of the uptake rates in N18 neuroblastoma, C6 glioma, and Hep2 carcinoma (see below and Fig.3). The effects of MAFP and PSF on anandamide uptake were measured using arachidonyl-[5,6,8,9,11,12,14,15-3H]ethanolamide called [3H]anandamide (172 Ci/mmol, 62.2 nCi/μl) from PerkinElmer Life Sciences as substrate. Growth medium was removed from the cells and replaced with 750 μl of supplemented Dulbecco's modified Eagle's medium containing 40 nCi[3H]anandamide (100 nm anandamide). The cells were incubated for 1–6 min at 37 °C with 5% CO2. To account for nonspecific binding to cellular membrane, parallel incubations were carried out at 4 °C. The counts from the nonspecific binding were subtracted from the total amount of anandamide taken up after each incubation so only the carrier-mediated transport uptake was represented in the data. To study whether MAFP affected the cellular uptake of anandamide, incubations were carried out after a 10-min preincubation of the cells with 1 μmMAFP (Cayman Chemical, Ann Arbor, MI) or 100 nmpamitylsulfonyl fluoride (provided by Alex Makriyannis, University of Connecticut) in the medium at 37 °C. After the incubation, the medium was immediately removed, and the cells were washed with ice-cold supplemented Dulbecco's modified Eagle's medium. The cells were scraped from the plate after the addition of 0.4 ml of 2 mmEDTA in phosphate-buffered saline. This procedure was repeated two additional times to maximize yield. The labeled compounds were then extracted from both the medium and the cells by the addition of 2 volumes of chloroform:methanol (1:1) (Fisher) mixed thoroughly and spun down in a clinical centrifuge for 5 min. Finally, 100 μl of the organic layer from both the cell and medium extractions were placed in scintillation vials with 3.0 ml of ScintiVerse II scintillation fluid (Fisher). The samples were counted in an LKB Rack beta scintillation counter. Saturation kinetics were determined in N18, C6, and Hep2 cells plated at a minimal density of 1 × 106cells. They were incubated for either 3 s (to determine nonspecific binding) or 60 s at 37 °C. For N18 and C6 cells, these experiments were performed with[lsqb]3H]anandamide (0.09–3.0 nm) and unlabeled anandamide to yield total concentrations ranging from 0.03 to 1.0 μm anandamide. For Hep2 cells,[lsqb]3H]anandamide (0.01–5.5 nm) was added to unlabeled anandamide over a concentration range of 0.25–100 μm anandamide. The same procedure was used for these experiments as for the time course of anandamide uptake with the exception that the experiment was terminated by the addition of 5 ml of ice-cold supplemented Dulbecco's modified Eagle's medium, which was then immediately removed. Experiments were conducted in duplicate or triplicate and repeated two or three separate times. Calculations were performed by subtracting the nonspecific binding at 3 s from the 60-s incubation, and the picomoles taken up were corrected for the number of cells. To determine theK m and V max, the data were analyzed employing a Lineweaver-Burk plot using the linear regression program of Sigma Plot (SPSS, Chicago, IL). The same procedure described above for the time course experiments was employed for these 5-min incubations, with the exception that the level of radioactivity was increased to 600 nCi/dish to have enough counts for thin layer chromatography analysis. After the 5-min incubation, the labeled anandamide was extracted from both the medium and the cells by the addition of 2 volumes of chloroform:methanol (1:1) mixed thoroughly and spun down in a clinical centrifuge for 5 min. 100 μl of the organic layer were removed for counting from both the cellular (1.2 ml total) and medium (750 μl) extracts. The remaining portions of the organic extracts from the cells and medium were dried down. The residue was resuspended in 40 μl of chloroform:methanol (1:1), and this was spotted on silica-based thin layer chromatography plates provided by Analtech (Newark, DE). The solvent consisted of a 6:3:1 mixture of ethyl acetate, hexane, and acetic acid (Fisher). Arachidonic acid and anandamide (PerkinElmer Life Sciences) standards were run alongside the experimental samples. The plate was then treated with EN3HANCE autoradiography spray provided by PerkinElmer Life Sciences and exposed on Kodak X-Omat AR film. After film development, the areas corresponding to the bands on the x-ray film were scraped off the plates and placed in scintillation vials with scintillation fluid to quantify the products of the TLC analysis (anandamide, phospholipids, and arachidonic acid). Each experiment was performed in triplicate. Following the time course experiments and TLC analysis, the raw results were corrected to account for the percentages of the organic layer, which was taken from each sample. The inhibition of uptake values in Table I was obtained by averaging the triplicates and assuming that the uptake of controls for each cell line was 100%.Table IThe effect of MAFP upon the cellular uptake of anandamide in C6 glioma cells, N18 neuroblastoma, and Hep2 laryngeal carcinoma cellsCell lineMAFPUptakeMean ± S.E.Student's t-test, 2-sidedp valueInhibition of uptake% of control mean%%N18−98.3−97.8100.0 ± 2.0−103.9N18+56.6+54.855.7 ± 0.50.000144+55.7C6−104.6−97.7100.0 ± 2.3−97.7C6+61.9+66.064.0 ± 1.20.000136+64.0Hep2−112.4−88.8100.0 ± 6.8−98.9Hep2+128.9+130.7124.0 ± 5.80.0553−24+112.5Quantification of label in cells after 5-min incubation with [3H]anandamide. The total number of counts was determined in the absence (−) and presence (+) of 1 μm MAFP. These values were corrected for nonspecific binding by the subtraction of counts from a 4 °C incubation. The average of the controls for each cell line was normalized to 100%. A Student's t test (two sided) was used to compare the differences between those cells in the presence (+) and absence (−) of MAFP and determine the pvalue. The percent inhibition of uptake was calculated by (dpm in control cells − dpm in MAFP-treated cells) × 100/dpm in control cells. The remainder of the sample was used for thin layer chromatography analysis as shown in Fig. 2. Open table in a new tab Quantification of label in cells after 5-min incubation with [3H]anandamide. The total number of counts was determined in the absence (−) and presence (+) of 1 μm MAFP. These values were corrected for nonspecific binding by the subtraction of counts from a 4 °C incubation. The average of the controls for each cell line was normalized to 100%. A Student's t test (two sided) was used to compare the differences between those cells in the presence (+) and absence (−) of MAFP and determine the pvalue. The percent inhibition of uptake was calculated by (dpm in control cells − dpm in MAFP-treated cells) × 100/dpm in control cells. The remainder of the sample was used for thin layer chromatography analysis as shown in Fig. 2. To statistically quantify the uptake rates in these three cell lines, the percentage of the total anandamide taken up by each cell line was compared after 5 min of incubation under identical conditions at the same time. The amount of uptake was then related to the total number of cells, thus allowing the comparison of uptake rates among different cell lines. Each experiment was performed in triplicate on 35-mm plates, which were ∼50% confluent. A fourth plate was run in parallel to determine the number of cells/plate. The enzyme assay for FAAH activity in N18, C6, and Hep2 cells was conducted as described previously (24Omeir R.L. Chin S. Hong Y. Ahern D.G. Deutsch D.G. Life Sci. 1995; 56: 1999-2005Crossref PubMed Scopus (91) Google Scholar). Cells were washed with ice cold PBS and scraped in ice-cold Tris-EDTA, pH 7.6. The cells were then disrupted by sonication. Incubations were performed in triplicate at 37 °C in a water bath with shaking. Each incubation contained 10 μl of 50 mg/ml defatted bovine serum albumin in H2O, 50 μl of the cellular extract, 30 μm anandamide (Cayman Chemical Co., Ann Arbor, MI), and 0.01 mCi of 120 mCi/mmol arachidonyl[ethanolamine-1,2-14C]ethanolamide (PerkinElmer Life Sciences). The control tubes contained everything except the cell extract. The reactions were terminated after 30 min by the addition of 2 volumes of chloroform:methanol (1:1). The radioactivity in the aqueous phase was measured by liquid scintillation counting. The number of cells/plate was determined with a hemacytometer. The specific activity was expressed as nanomoles of anandamide hydrolyzed/106 cells/h. This was more meaningful than activity based on the amount of protein because protein levels varied widely among the different cell types. Cells were seeded at 3 × 105 cells/35-mm dish on day 1. On day 2, cells were transfected with 2.5 μg of DNA using 2.5 μl of LipofectAMINE according to the manufacturer's instructions (Life Technologies, Inc.). On day 4, cells were either harvested in 150 μl of Tris-EDTA, pH 7.4, for FAAH assays or used for uptake experiments. The control Hep2 cells were either untransfected and serum-starved or were transfected with the pcDNA3 vector (Invitrogen, Carlsbad, CA), and the experimental Hep2 cells were transfected with rat pcDNA3-FAAH cDNA (30Arreaza G. Devane W.A. Omeir R.L. Sajnani G. Kunz J. Cravatt B.F. Deutsch D.G. Neurosci. Lett. 1997; 234: 59-62Crossref PubMed Scopus (71) Google Scholar). To quantify the anandamide uptake rates of untransfected, vector-transfected, and FAAH-transfected cells, the total anandamide taken up was compared after 5 min of incubation under identical conditions. The cells were used for uptake 48 h after transfection. The same procedure that was used for the time course experiments was used for these incubations, with the exception that 200 nCi/dish was used, nonspecific binding to the cellular membrane was determined by incubating parallel plates in 37 °C uptake medium for 3 s, and all incubations were stopped by adding 4 °C medium. Anandamide uptake in N18, C6, and Hep2 cells was saturable and temperature- and time-dependent. The apparentK m and V max values were 1.8 μm and 17.4 pmol/min/106 cells for N18 cells, 0.7 μm and 3.9 pmol/min/106 cells for C6 cells, and 20.9 μm and 5.9 pmol/min/106 cells for Hep2 cells, respectively. Time course experiments with N18, C6, and Hep2 cells were conducted to qualitatively determine the effects of two FAAH inhibitors, MAFP and PSF, on anandamide uptake. Fig.1 A shows that after preincubating C6 cells with MAFP for 10 min, the amount of anandamide that enters the cell is reduced at each time point from 2 to 6 min. At 6 min, cellular net uptake of labeled anandamide in the control without inhibitor is about double the net cellular uptake of labeled anandamide in the presence of MAFP. The results of the N18 cell time course experiment in Fig. 1 C demonstrate that the effect of MAFP on cellular uptake is not restricted to C6 cells. After 6 min, the control cells have taken up approximately twice as much labeled anandamide as did the MAFP-treated cells. To study the effects of PSF, the other hydrolase inhibitor used in these studies, on anandamide uptake, identical experiments were conducted in N18 and C6 cell lines. Fig. 1,B and D, shows that preincubating C6 and N18 cells with 100 nm PSF for 10 min also reduces the amount of anandamide entering the cell at each time point from 2 up to 6 min. In experiments similar to those described above for C6 and N18 cells, uptake in Hep2 cells was characterized in the presence of MAFP and PSF. In contrast to the results found with N18 and C6 cells, net uptake was not decreased in the presence of either inhibitor (Fig. 1, Eand F). The shapes of the curves resulting from the time course experiments are interesting. First, anandamide is accumulated at a greater rate during the first min than during the rest of the experiment, and the amount taken up is approximately the same with or without the inhibitor. Second, the cells with uninhibited FAAH show a linear uptake after 1 min, whereas those that are inhibited or lack FAAH appear to level slowly to a plateau. The results in Fig. 1 are qualitatively representative of at least three experiments conducted with N18, C6, and Hep2 cells. To more accurately characterize the effect of FAAH inhibition on anandamide uptake, the total cellular radioactivity resulting from anandamide incorporation was quantified in N18, C6, and Hep2 cells after an incubation of 5 min in the absence or presence of MAFP (TableI). MAFP inhibited the incorporation by ∼40% in both N18 and C6, and this effect was highly significant for both of these cell lines (p = 0.0001). However, MAFP did not inhibit anandamide incorporation in Hep2. In fact, it seemed to cause a slight stimulation, although this effect was not quite significant (p = 0.0553). Whereas 1 μmMAFP was chosen for these experiments, similar results were observed at 100 nm (data not shown). The observed inhibition in N18 and C6 cells in the presence of MAFP was not due to toxicity because cells exposed to MAFP do not exhibit decreased viability as observed after trypan blue staining (data not shown). To determine the fate of [lsqb]3H]anandamide (arachidonyl[5, 6, 8, 9, 11, 12, 14, 15-3H]ethanolamide) after being taken up by the cells, thin layer chromatography was performed on those control and experimental C6, N18, and Hep2 cells, which were quantified in Table I. After incubation of C6 and N18 with anandamide in the absence of inhibitor (−), the main radioactive product formed inside the cell was phospholipids (83% for C6, 93% for N18), which remain at the origin in this TLC development system (Fig.2). For the C6 and N18 cells, only 14 and 3% of the radioactivity in the cell was free anandamide, respectively, and only 3% of the radioactivity was detected as arachidonic acid in the C6 cells and none in the N18 cells. Because of overexposure, the TLC chromatogram for N18 cells (Fig. 2) gives the impression of quite high anandamide levels in the samples without inhibitor (−), although counting of the samples scraped from the plate actually shows only 3% free anandamide. In the presence of MAFP (+), the entire radioactivity in the C6 and N18 cells is accounted for by anandamide (Fig. 2). Anandamide accumulated in these cells because the breakdown reaction by FAAH was rendered inactive. These experiments raise the possibility that MAFP exerts its effect by inhibiting the transporter. However, it seems unlikely that MAFP exerts its effect in this way as shown by the TLC experiments with Hep2 cells (Fig. 2). The anandamide transport mechanism is operational in these cells in the absence of MAFP (−). However, unlike the situation with control C6 and N18 cells, anandamide is not broken down (>90% of the radioactivity in the cell is anandamide) after being transported into the cells because these cells lack FAAH (5Deutsch D.G. Chin S.A. Biochem. Pharmacol. 1993; 46: 791-796Crossref PubMed Scopus (653) Google Scholar). Significantly, in the presence of MAFP (+) transport is not inhibited because there is no significant difference in the amount of anandamide on the TLC, and as mentioned above (Table I), there seems to be a slight stimulation. The TLC pattern for Hep2 cells with (+) or without (−) MAFP treatment is the same as that observed in N18 and C6 cells that have been treated with MAFP. An experiment was performed to make a side by side comparison of N18, C6, and Hep2 in terms of their uptake and enzyme activity. Interestingly, the absolute rate of uptake was found to vary among the different cell types when the cell numbers were carefully quantified, and this correlated approximately with the amount of FAAH activity (Fig. 3). The N18 cells had the greatest total uptake with the C6 and Hep2 cells having ∼64 and 51% of the uptake of the N18, respectively. The FAAH activity in C6 was found to be approximately 60% of that found in N18, although there was no measurable FAAH activity in Hep2 cells as mentioned above. FAAH transfection experiments were undertaken to demonstrate that uptake is dependent upon FAAH activity in Hep2 cells. The Hep2 cells, which have no measurable FAAH activity, were transfected with a pcDNA3 vector as a control or with pcDNA3-FAAH cDNA (Fig.4). The vector-transfected Hep2 cells had no measurable FAAH activity, whereas the Hep2 cells transfected with pcDNA3-FAAH cDNA were very active (2.92 nmol ± 0.19/h/106 cells), demonstrating the expression of FAAH (p < 0.0001). Significantly, anandamide uptake in the transfected cells doubled (8.27 ± 1.0 pmol for transfectedversus 4.28 ± 0.21 pmol for untransfected cells and 4.29 ± 0.34 pmol for vector-transfected cells) (Fig. 4). This finding demonstrates that uptake is dependent upon FAAH activity. The intracellular concentration of anandamide was calculated for each of the cell types (Table II). The intracellular concentration of anandamide in N18, C6, and Hep2 cells was determined to be ∼5-, 7-, and 18-fold greater, respectively, than the extracellular concentration of 100 nm that was used in these experiments. These data strongly suggest some sequestering mechanisms for accumulating anandamide inside cells without FAAH or when FAAH is inhibited with MAFP.Table IIIntracellular concentrations of anandamide in the presence of inhibitorCell lineUptake of anandamide/cellIntracellular volumeExtracellular concentration of anandamideIntracellular concentration of anandamide-Fold intracellular accumulationpmollitersnmnmN18TG24.28 × 10−68.5100503.85C65.31 × 10−67.5100707.97Hep27.90 × 10−64.51001756.118The intracellular concentrations of anandamide were calculated in N18, C6, and Hep2 cells after a preincubation with 1 μm MAFP for 10 min followed by a 5-min incubation at 37 °C in supplemented Dulbecco's modified Eagle's medium containing 100 nmanandamide and 1 μm MAFP. Cell volumes of N18 and C6 cells were described previously (37Chan P.H. Kerlan R. Fishman R.A. J. Neurosci. Res. 1982; 8: 67-72Crossref PubMed Scopus (10) Google Scholar). The intracellular volume of Hep2 cells was kindly provided by Janikke Ludt and Kirsten Sandvig (personal communication) from calculations related to prior experiments (38Madshus I.H. Tonnessen T.I. Olsnes S. Sandvig K. J. Cell. Physiol. 1987; 131: 6-13Crossref PubMed Scopus (26) Google Scholar). Open table in a new tab The intracellular concentrations of anandamide were calculated in N18, C6, and Hep2 cells after a preincubation with 1 μm MAFP for 10 min followed by a 5-min incubation at 37 °C in supplemented Dulbecco's modified Eagle's medium containing 100 nmanandamide and 1 μm MAFP. Cell volumes of N18 and C6 cells were described previously (37Chan P.H. Kerlan R. Fishman R.A. J. Neurosci. Res. 1982; 8: 67-72Crossref PubMed Scopus (10) Google Scholar). The intracellular volume of Hep2 cells was kindly provided by Janikke Ludt and Kirsten Sandvig (personal communication) from calculations related to prior experiments (38Madshus I.H. Tonnessen T.I. Olsnes S. Sandvig K. J. Cell. Physiol. 1987; 131: 6-13Crossref PubMed Scopus (26) Google Scholar). It is known that anandamide uptake is an energy-independent, reversible process and is
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