Báo cáo khoa học: Uptake and metabolism of [3H]anandamide by rabbit platelets Lack of transporter? ppt

9 309 0
Báo cáo khoa học: Uptake and metabolism of [3H]anandamide by rabbit platelets Lack of transporter? ppt

Đang tải... (xem toàn văn)

Thông tin tài liệu

Uptake and metabolism of [ 3 H]anandamide by rabbit platelets Lack of transporter? Lambrini Fasia, Vivi Karava and Athanassia Siafaka-Kapadai Department of Chemistry (Biochemistry), University of Athens, Greece Anandamide is an endogenous ligand for cannabinoid receptor and its protein-mediated transport across cellular membranes has been demonstrated in cells derived from brain as well as in cells of the immune system. This lipid is inactivated via intracellular degradation by a fatty acid amidohydrolase (FAAH). In the present study, we report that rabbit platelets, in contrast to human platelets, do not possess a carrier-mediated mechanism for the transport of [ 3 H]anandamide into the cell, i.e. cellular uptake was not temperature dependent and its accumulation was not satu- rable. This endocannabinoid appears to enter the cell by simple diffusion. Once taken up by rabbit platelets, [ 3 H]anandamide was rapidly metabolized into compounds which were secreted into the medium. Small amounts of free arachidonic acid as well as phospholipids were amongst the metabolic products. FAAH inhibitors did not decrease anandamide uptake, whereas these compounds inhibited anandamide metabolism. In conclusion, anandamide is rapidly taken up by rabbit platelets and metabolized mainly into water-soluble metabolites. Interestingly, the present study also suggests the absence of a transporter for anand- amide in these cells. Keywords: anandamide; anandamide transporter; fatty acid amidohydrolase; rabbit platelets. Anandamide (N-arachidonoylethanolamine) is the most important member of a class of endogenous lipids called N-acylethanolamines that have been proposed as the physiological ligands for the cannabinoid (CB) receptors [1,2]. In addition to anandamide, there is another endo- cannabinoid namely 2-arachidonoylglycerol that has been isolated from rat brain and canine gut [3,4]. For anandamide signaling via CB receptors, an active uptake mechanism to transport anandamide into the cell has been reported. The properties of this transport process together with the transport mechanisms of the related endogenous compounds 2-arachidonoylglycerol and palmi- toylethanolamide have recently been reviewed [5]. Cellular uptake is followed by the rapid degradation of anandamide by an endoplasmic reticular integral membrane-bound enzyme called fatty-acid amide hydrolase (FAAH) [6,7]. Anandamide uptake has been demonstrated in neuro- blastoma and glioma cells [8], cortical neurons [9], cerebellar granule neurons [10], cerebral cortical neurons and astrocytes [11], macrophages and basophils [12], human platelets [13], human mast cells [14] and human endothelial cells [15]. In these cells, anandamide transport has the characteristics of facilitated diffusion rather than an active cotransport system or passive diffusion, as it follows saturation kinetics, is temperature- and time-dependent, shows structural specificity among N-acylethanolamines, is bidirectional and lacks the requirement of ATP or extracel- lular sodium [9–11,16]. Several specific inhibitors of anand- amide transport have been described including various structural analogs of anandamide [11,17–22]. Ligand struc- tural requirements of anandamide transporter are very different from those for the CB1 receptor. However the transporter and the FAAH do share some of them [21]. The kinetic parameters of anandamide accumulation among different cell types is varied suggesting the existence of different subtypes of anandamide carrier [16]. For example, in the cerebellar granule neurons, K m ¼ 41 ± 15 l M [10] while in the human umbelical vein endothelial cells K m ¼ 190 ± 10 n M [15]. It should be noted that Bisogno et al. demonstrated that different kinetics might depend on the experimental protocol [22]. Studies in several cell types have shown that the net movement of anandamide into the cells is coupled to the activity of intracellular FAAH [23,24]. FAAH is responsible for the catabolism of anandamide and it contributes to anandamide uptake by making and maintaining a concen- tration gradient between the extracellular space and the interior of the cell. Therefore, in the presence of various inhibitors of FAAH (e.g. phenylmethylsulfonyl fluoride), the uptake is limited by the shifting of anandamide concentration gradient in a direction that leads to equilib- rium [23,24]. FAAH is the main catabolic enzyme in the conversion of anandamide into free arachidonic acid and ethanolamine Correspondence to A. Siafaka-Kapadai, Department of Chemistry (Biochemistry), University of Athens, Panepistimioupolis, 157 71 Athens, Greece. Fax: +30 210 7274476, Tel.: +30 210 7274493, E-mail: siafaka@chem.uoa.gr Abbreviations: CB, cannabinoid receptors; FAAH, fatty acid amidohydrolase; COX, cyclooxygenase; POPOP, 1,4-di[2-(5-phenyl- oxazole)] benzene; PPO, 2,5-diphenyloxazole; ACD, acid-citrate- dextrose; Tg/Ca, Tyrodes/gelatin/Ca 2+ ; PL, phospholipids; LOX, lipoxygenase. Enzymes: fatty acid amide hydrolase (arachidonoylethanolamide amidohydrolase; EC 3.5.1.4). (Received 4 December 2002, revised 6 May 2003, accepted 19 June 2003) Eur. J. Biochem. 270, 3498–3506 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03724.x [25,26]. FAAH is also capable of metabolizing several other fatty acid amides [7] and esters such as oleamide [27] and 2-arachidonoylglycerol [28] as well as catalyzing the reverse condensation of free arachidonic acid and ethanolamine to the formation of anandamide [6,29,30]. FAAH is a mem- brane-bound protein localized mainly in the microsomal and mitochondrial fractions [6,31–33]. The K m of the enzyme for anandamide has been reported to be 2–67 l M depending on the enzyme preparations and assay conditions and the optimum pH is 8.5–10 [25,26]. The existence and secretion of a FAAH from the unicellular eucaryote Tetrahymena pyriformis, has been recently reported by our laboratory [34]. Other enzymes also directly metabolize anandamide. Various purified lipoxygenases as well as lipoxygenases derived from tissues convert anandamide to polar metabolites (hydroperoxides and hydroperoxy-deriv- atives) [35–37]. Recombinant human cyclooxygenase-2 (hCOX-2) but not hCOX-1 has the ability to directly oxidize anandamide to yield prostaglandin E 2 ethanolamide [38]. Here, we report the results of a study aimed at assessing the possibility that anandamide is taken up by rabbit platelets via a carrier-mediated transport system as it has been suggested for a number of cells including human platelets, and subsequently exploring anandamide meta- bolic fate. Materials and methods Materials [ 3 H]Anandamide (200 CiÆmmol )1 ), radiolabelled at the arachidonic moiety, was purchased from American Radio- labelled Chemicals Inc. (St Louis, MO, USA). Anandamide, arachidonic acid, phosphatidylethanolamine, phenyl- methylsulfonyl fluoride, caffeic acid, indomethacin, bovine serum albumin, BSA, 1,4-di[2-(5-phenyloxazole)] benzene (POPOP), 2,5-diphenyloxazole (PPO) and naphthalene were from Sigma Chemicals Co. VDM11 was from Tocris Cookson Ltd (UK). Other chemicals were of the highest purity available. Buffers The anticoagulant solution, acid/citrate/dextrose (ACD), contained 1.36% citric acid, 2.5% trisodium citrate and 2.0% dextrose (w/v). The resuspension buffers for the washed rabbit platelets were: (a) Tyrodes/gelatin/Ca 2+ pH 7.2 (Tg/Ca) which contained 0.8% NaCl, 0.02% KCl, 0.02% MgCl 2 , 0.1% dextrose, 0.25% gelatin and 0.02% CaCl 2 (w/v) and (b) 10 m M NaCl/P i pH 7.4 which con- tained 0.14% Na 2 HPO 4 ,0.12%NaH 2 PO 4 and 0.82% NaCl (w/v). Preparation of washed rabbit platelets Blood was drawn from the central vein of the ear of male rabbits and was collected into an ACD anticoagulant solution. Platelets were washed as described previously [39,40]. Platelets were finally resuspended in Tg/Ca pH 7.2 or a 10 m M NaCl/P i pH 7.4, at a concentration of 3 · 10 8 plateletsÆmL )1 . Measurement of [ 3 H]anandamide accumulation by rabbit platelets Platelet suspension (3 · 10 8 plateletsÆmL )1 )inTg/Cawas incubated with 1.25 n M [ 3 H]anandamide at 37 °Cfor various time intervals. The incubation was stopped by the addition of 4% v/v ice-cold formol and the suspension was placed on ice. Platelets were separated from the medium by centrifugation at 13 000 g for 2 min, washed with 0.9% v/v NaCl and extracted according to Bligh and Dyer [41]. [ 3 H]Anandamide (100 n M ) uptake was also studied in platelet suspension in 10 m M NaCl/P i . In this case, after the incubation with [ 3 H]anandamide, platelets were washed with NaCl/P i containing 1% w/v BSA and the study was repeated at various temperatures (0–4, 25 and 37 °C) as well as in the presence of VDM11 (20 l M )at37°C. For the kinetic studies, [ 3 H]anandamide at concentrations between 100 and 2000 n M was used and the incubation took place at 37 °C. The concentration of phenylmethylsulfonyl fluor- ide used was 2 m M and the preincubation time was 15 min. Metabolism of [ 3 H] anandamide by intact rabbit platelets Washed rabbit platelets were resuspended in Tg/Ca pH 7.2 or in 10 m M NaCl/P i resulting in a final concen- tration of 3 · 10 8 plateletsÆmL )1 . The platelet suspension was incubated with [ 3 H]anandamide 1.25 n M (specific activity 200 CiÆmmoL )1 )or450n M in certain experiments, at 37 °C for different time intervals. Lipids from 0.5-mL aliquots of the platelet suspension were extracted accord- ing to Bligh and Dyer [41] and were separated by TLC on heat-activated silica-gel G-plates using CHCl 3 /CH 3 OH/ NH 4 OH 80 : 20 : 2 (v/v). After visualization by exposure to iodine vapors, lipids corresponding to free arachidonic acid, phosphatidylethanolamine and anandamide were scraped off the plates and their radioactivity was meas- ured by liquid scintillation counting using a toluene base (5 g PPO and 0.3 g POPOP in 1 L toluene) as the scintillation fluid. The radioactivity of the methanol/water phase was also measured using dioxan/water base (100 g napthalene, 7 g PPO, 0.3 g POPOP, 200 mL water in 1 L dioxan) as the scintillation fluid. The liquid scintillation counter used was a Wallac 1209 Rackbeta, Pharmacia. Inthesamemanner,for[ 3 H]anandamide accumulation experiments, after incubation of platelets with radiolabeled anandamide, the cells were separated from the medium by centrifugation at 13 000 g for 2 min, washed and the lipids from platelets and media were extracted and separated as described above. The experiments were repeated after preincubation of platelet suspension with either 2 m M phenylmethylsulfonyl fluoride or 100 l M caffeic acid, or 0.5 and 75 l M indomethacin. Results [ 3 H]Anandamide uptake and metabolism [ 3 H]Anandamide was rapidly taken up by rabbit platelets as shown in Fig. 1. At 2 min, there was a high percentage of radioactivity incorporated into platelets (32.6 ± 2.7%). Ó FEBS 2003 Uptake and metabolism of anandamide by rabbit platelets (Eur. J. Biochem. 270) 3499 Surprisingly, the amount of radioactivity associated with the platelets was reduced over time in parallel with an increase in extracellular radioactivity. This uptake and metabolism was completed within 20 min and then the amount of radioactivity remained constant in both the platelets and the extracellular space. Our studies with intact rabbit platelets showed that these cells were capable of rapidly metabolizing [ 3 H]anandamide (Fig. 2). The main metabolic products in the control cells were found in the water/methanol phase of the Bligh–Dyer extraction and referred throughout this study as Ôwater-solubleÕ com- pounds, and the metabolism was completed at 20 min when a plateau was reached. The metabolism occurred rapidly, since at 5 min almost 50% of the exogenously added [ 3 H]anandamide had been catabolized. Only a small percentage of [ 3 H]anandamide metabolic products were free arachidonic acid and phospholipids ( 2% and  10% at 20 min, respectively, Fig. 2). Very similar results were obtained when the metabolism experiment was performed in the presence of 100 l M caffeic acid (a LOX inhibitor). The main metabolic products were water-soluble compounds and the metabo- lism occurred rapidly ( 50% at 5 min) and reached a plateau at 20 min (Fig. 3A). In the presence of 0.5 l M indomethacin (a COX inhibitor) a small inhibition was observed ( 10% at 5, 10 and 20 min) and reached a plateau at 40 min. The extent of the metabolism at 40 min was the same compared to the control cells (not shown). In the presence of 75 l M indomethacin, a significant inhibition of anandamide metabolism was observed. At 5 min, the inhibition was  65% and the metabolism reached a plateau at 20 min when the inhibition was approximately 50% (Fig. 3A). This inhibi- tion was almost totally attributed to water-soluble metabolites (Fig. 3B). In subsequent studies, attempts were made to further identify the metabolic products of [ 3 H]anandamide in intact platelets and in the extracellular space. For this purpose, lipids were extracted from platelets as well as from the medium and were separated by TLC. The results are presented in Fig. 4. After a 2-min incubation, the radioactivity incorporated in platelets corresponded mainly to [ 3 H]anandamide. Thereafter, the amount of [ 3 H]anandamide in platelets was reduced rapidly with time while the amount of other radiolabeled products, phospholipids, free arachidonic acid or water-soluble compounds remained constant. The radioactivity profile of the medium was completely different. At 2 min, the total amount of radioactivity found in the media was low and corresponded mainly to [ 3 H]anandamide, which had not been bound to platelets. This is consistent with the observation that this amount was unchangeable with time. On the other hand, the total amount of radio- activity in the medium increased rapidly with time and this increase was attributed totally to water-soluble compounds. In order to test the possible involvement of FAAH on [ 3 H]anandamide uptake and metabolism by rabbit platelets, the effect of phenylmethylsulfonyl fluoride on [ 3 H]ananda- mide uptake was determined (Fig. 5). Preincubation of the platelet suspension with phenylmethylsulfonyl fluoride had no effect on the rapid uptake of [ 3 H]anandamide by Fig. 1. Distribution of radioactivity in platelets and medium. Platelet suspension in Tg/Ca (3 · 10 8 plateletsÆmL )1 ) was incubated with [ 3 H]anandamide (1.25 n M )at37°C. At the time intervals indicated, 0.5 mL of platelet suspension was centrifuged, the supernatant (Y-1) was removed and the cells were washed with 0.9% NaCl (w/v). The supernatant (Y-2) was removed, lipids were extracted from platelets and their radioactivity was measured. The sum of radioactivity in Y-1 and Y-2 is the radioactivity of extracellular space. Values are the means ± SD of duplicate samples of three independent experiments. Total c.p.m., 15 000–45 000. Fig. 2. [ 3 H]Anandamide metabolism by intact rabbit platelets. Platelet suspension in Tg/Ca (3 · 10 8 plateletsÆmL )1 ) was incubated with [ 3 H]anandamide (1.25 n M )at37°C. At the time intervals indicated, 0.5 mL of platelet suspension were extracted according to Bligh and Dyer [41]. Lipids were subjected to TLC and radioactivity was meas- ured. The radioactivity of water-methanol phase was also measured. (j) Water-soluble compounds (h) phospholipids (m) free arachidonic acid (d) anandamide. Values are the means ± SD of duplicate sam- ples of three independent experiments. Total c.p.m., 15000–45 000. 3500 L. Fasia et al. (Eur. J. Biochem. 270) Ó FEBS 2003 platelets. The amount of radioactivity in both platelets and extracellular space remained constant with time. Phenyl- methylsulfonyl fluoride is a well-known, strong inhibitor of FAAH. The effect of phenylmethylsulfonyl fluoride on [ 3 H]anandamide accumulation may be due to the inhibition of the anandamide metabolism. Similar results were obtained using a more specific FAAH inhibitor, namely arachidonoyltrifluoromethyl-ketone (data not shown). After preincubation of platelets with phenylmethylsulfonyl fluoride, the distribution of radioactivity in platelets corres- ponded to nonmetabolized [ 3 H]anandamide (Fig. 6). Based on these results, we assumed at the time that the uptake of anandamide was carrier-mediated, coupled to its metabo- lism and reached a plateau in 2 min when the metabolism was inhibited by phenylmethylsulfonyl fluoride. In that case, the uptake should be also temperature- and concentration- dependent, well known characteristics of a facilitated diffusion. Effect of temperature on [ 3 H]anandamide uptake and metabolism Studies were then undertaken to determine if anandamide is transported across the cellular membrane via facilitated diffusion as has been shown for a number of cell types. Since this type of transport is temperature dependent, ananda- mide uptake at 37 °C, 25 °C and 0–4 °C was examined. In these experiments, 100 n M [ 3 H]anandamide in 10 m M NaCl/P i was used in order to have experimental conditions comparable to those previously reported in human platelets [13] as well as other cells [12,15]. The profile of [ 3 H]anand- amide uptake was the same (Fig. 7) although the percentage of radioactivity of platelet fraction was lower in NaCl/P i compared to that in Tg/Ca (Fig. 1). This difference could be attributed to the higher [ 3 H]anandamide concentration used (100 n M instead of 1.25 n M ) as well as to the presence of 1% w/v BSA in the washing buffer. BSA apparently removed Fig. 3. Effect of caffeic acid and indomethacin on [ 3 H]anandamide metabolism by intact rabbit platelets. (A) Platelet suspension in 10 m M NaCl/P i (3 · 10 8 plateletsÆmL )1 ) was incubated with [ 3 H]anandamide (450 n M )at37 °C. At the time intervals indicated, the medium (extracellular space) of 0.5 mL of the platelet suspension was removed by centrifugation. The platelets were extracted twice according to Bligh and Dyer [41]. Lipids were subjected to TLC and radioactivity corresponding to nonmetabolized anandamide was measured. (B) Distribution of the radioactivity in water- soluble compounds and in lipids extracted from platelets. Incubation time with [ 3 H]anandamide: 40 min. W: water-soluble compounds, PL: phospholipids, FFA: free arachidonic acid. Values are the means ± SD of duplicate samples of three independent experiments. Total c.p.m., 6000–25 000. Fig. 4. Distribution of the radioactivity of the platelets (A) and the extracellular space (B) into various lipids. Platelet suspension in Tg/Ca (3 · 10 8 plateletsÆmL )1 ) was incubated with [ 3 H]anandamide (1.25 n M )at37°C. At the time intervals indicated, the medium (extracellular space) of 0.5 mL of the platelet suspension was removed by centrifugation. The platelets as well as the extracellular medium were extracted twice according to Bligh- Dyer [41] method. Lipids were subjected to TLC and radioactivity was measured. (j) Water-soluble compounds (m) phospholipids ())free arachidonic acid (s) anandamide. Values are the means ± SEM of duplicate samples of one representative experiment. Ó FEBS 2003 Uptake and metabolism of anandamide by rabbit platelets (Eur. J. Biochem. 270) 3501 [ 3 H]anandamide that had not entered the platelets and could have been nonspecifically bound to the plasma membrane. In order to further test this hypothesis, platelets were incubated with [ 3 H]anandamide for 1–2 min (low metabolism) and were then separated from the medium (M 1 ) by centrifugation and washed with NaCl/P i containing BSA (M 2 ). Interestingly, the radioactivity found in M 2 (corresponded mainly to [ 3 H]anandamide) was higher than that found in M 1 which corresponded mainly to water- soluble compounds produced after [ 3 H]anandamide meta- bolism (data not shown). These results suggest that [ 3 H]anandamide, at least in part, may not be transferred into the cells but is possibly nonspecifically bound to the plasma membranes from which it was removed after treatment with BSA. As shown in Fig. 7, the profile of [ 3 H]anandamide uptake was identical at 37 °Cand25°C. At these two tempera- tures, the metabolism of [ 3 H]anandamide took place to the same extent and resulted in the same products. When the uptake of [ 3 H]anandamide at 0–4 °C was studied, the amount of radioactivity found in platelets was larger than that at 37 °C (Fig. 7). This observation can be explained by thesmallerextentof[ 3 H]anandamide metabolism by intact rabbit platelets at this low temperature. [ 3 H]Anandamide that was not metabolized remained bound to the platelets, and resulted in a smaller amount of radioactivity being released into the extracellular space at 0–4 °C compared to 37 °C.Inthecasethat[ 3 H]anandamide had been trans- ferred via facilitated diffusion, the uptake at 4 °C should have been much lower. Fig. 5. Effect of phenylmethylsulfonyl fluoride on [ 3 H]anandamide uptake by rabbit platelets. Platelet suspension in Tg/Ca (3 · 10 8 plateletsÆmL )1 ) was incubated with [ 3 H]anandamide (1.25 n M )at 37 °C in the absence or presence of 2 m M phenylmethylsulfonyl fluoride. At the time intervals indicated, the medium (extracellular space) of 0.5 mL of the platelet suspension was removed by centrifu- gation. The platelets as well as the extracellular medium were extracted twice according to Bligh and Dyer [41]. The radioactivity of platelets and extracellular space was measured. Values are the means ± SD of duplicate samples of three independent experiments. Total c.p.m., 15 000–45 000. Fig. 6. Distribution of the radioactivity into various lipids in the presence of phenylmethylsulfonyl fluoride. Platelet suspension in Tg/Ca (3 · 10 8 plateletsÆmL )1 ) preincubated with phenylmethylsulfonyl fluoride (2 m M ), was then incubated with [ 3 H]anandamide (1.25 n M )at37°C. At the time intervals indicated, the medium (extracellular space) of 0.5 mL of the platelet suspension was removed by centrifugation. The platelets were extracted twice according to Bligh and Dyer [41]. Lipids weresubjectedtoTLCandradioactivitywasmeasured.(j)Water soluble compounds (m) phospholipids (h) free arachidonic acid ()) anandamide. Values are the means ± SD of duplicate samples of three independent experiments. Total c.p.m., 15 000–45 000. Fig. 7. Effect of temperature and VDM11 on [ 3 H]anandamide uptake by rabbit platelets. Platelet suspension in NaCl/P i (3 · 10 8 plate- letsÆmL )1 )wasincubatedwith[ 3 H]anandamide (100 n M )atvarious temperatures or after preincubation with 20 l ı ` VDM11 (for 10 min). At the time intervals indicated, the medium (extracellular space) of 0.5 mL of the platelet suspension was removed by centrifugation. After centrifugation, the supernatant was decanted and the platelets were washed with NaCl/P i containing 1% w/v BSA. The platelets as well as the extracellular medium were extracted twice according to Bligh and Dyer [41]. The radioactivity of platelets and extracellular space was measured. Values are the means ± SD of duplicate samples of two to five independent experiments. Total c.p.m., 8000–45 000. 3502 L. Fasia et al. (Eur. J. Biochem. 270) Ó FEBS 2003 To explore this hypothesis further, the experiments at 37 °C were repeated in the presence of VDM11, a specific anandamide membrane transporter inhibitor. Platelets were preincubated for 10 min with 20 l M VDM11 as the 50% inhibitory concentration (IC 50 ) reported for other cells was 10–11 l M [19]. As shown in Fig. 7, although a small inhibition ( 20%) of uptake was observed at 2 min, the profile of [ 3 H]anandamide uptake was almost identical with or without VDM11 at 37 °C. Thus, these results indicate the absence of a membrane transporter in rabbit platelets. Effect of concentration on [ 3 H]anandamide uptake and metabolism Among the criteria for the characterization of a transport process across cellular membranes as carrier-mediated, is its saturation at high ligand concentrations. Therefore studies were undertaken at [ 3 H]anandamide concentrations of 100–2000 n M . Similar concentrations were used for human platelets [13]. Cellular uptake was estimated from the total radioactivity in platelets and the extracellular space after 1–2 min of incubation with [ 3 H]anandamide. The catabol- ism of [ 3 H]anandamide was low during this time (Fig. 2). The amount of radioactivity found in platelets was a linear function of [ 3 H]anandamide concentration (Fig. 8). These results indicate that anandamide crossed the platelet plasma membrane by simple diffusion and not by a carrier- mediated transport. Again, the accumulation of [ 3 H]anand- amide in platelets was higher at 0–4 °Cthanat37°C due to decreased metabolism. Interestingly, the uptake was also linear but higher both in the presence of phenylmethylsul- fonyl fluoride, and 0–4 °C apparently due to the inhibition of FAAH activity, or decreased metabolism, respectively (Fig. 8A and B). Discussion Initially, the purpose of these experiments was to investigate the existence of a transporter in rabbit platelets, as it has been suggested for human platelets [13] as well as for a number of cells [8–12,14,15]. Additionally, it has been reported by Braud et al. [42] and by our laboratory [43], that aggregation of rabbit platelets caused by anandamide is accomplished through its conversion to arachidonic acid by the action of a FAAH; this is in contrast to human platelets in which the process is independent of the arachidonate cascade. The effect on rabbit platelets was blocked by the FAAH inhibitor, phenylmethylsulfonyl fluoride. Having in mind that in almost every cell type tested, FAAH is localized in the membrane of microsomes (endoplasmic reticulum) or mitochondria [6,31–33], it was assumed at thetimethat[ 3 H]anandamide should be taken up by a facilitated diffusion process. Subsequently, [ 3 H]anandamide would be hydrolyzed to arachidonic acid, which in turn would induce platelet aggregation through a well-known process [44,45]. It should be noted that enzymes involved in arachidonic acid metabolism, such as LOX and COX are also localized inside the cell [44,46]. Surprisingly, the results presented here indicate that rabbit platelets do not possess a carrier-mediated mechanism for the transport of [ 3 H]anand- amide into the cell, i.e. the process was not temperature dependent (Fig. 7) and was not saturable (Fig. 8) in contrast to the results reported for human platelets [13] and for other cells [8–12,14,15]. The uptake of [ 3 H]anandamide was exactly the same at 37 and 25 °C (Fig. 7) but was higher at 0–4 °C, apparently due to the lower degree of metabolism at 0–4 °Ccomparedto that at 37 °Corat25°C. At these temperatures, even after only 1–2-min incubation there was some anandamide metabolism (Fig. 2). Therefore, the higher degree of uptake was probably due to the diminished anandamide meta- bolism. Furthermore, very interesting results came from experi- ments in which FAAH activity was inhibited; inhibition resulted in an increase of the uptake (Figs 5, 6 and 8). If a transporter is present, inhibition of anandamide hydrolysis decreases rather than enhances the uptake as it has been shown in previous studies [23]. On the contrary, if a membrane transporter is really absent, as we suggest here, Fig. 8. Concentration dependence of [ 3 H]anandamide uptake by rabbit platelets. Platelet suspension in NaCl/P i (3 · 10 8 plateletsÆmL )1 )wasincu- bated with various concentrations of [ 3 H]anandamide for 1 min at 0–4 °Cor37°C (A) or in the presence of 2 m M phenylmethylsulfonyl fluoride (B). At the time intervals indicated, the medium (extracellular space) of 0.5 mL of the platelet suspension was removed by centrifugation. After centrifugation, the supernatant was decanted and the platelets were washed with NaCl/P i containing 1% w/v BSA. The platelets were extracted twice according to Bligh and Dyer. The radioactivity of platelets was measured. Values are the means ± SD of duplicate samples of three independent experiments. Ó FEBS 2003 Uptake and metabolism of anandamide by rabbit platelets (Eur. J. Biochem. 270) 3503 the inhibition of FAAH should increase the extent of the apparent uptake. Hence, these results demonstrate the absence of a membrane transporter in rabbit platelets. Time dependence experiments (Fig. 1) revealed that radioactivity was rapidly associated with platelets and then gradually decreased within the cell and increased in the extracellular space, suggesting the existence of a metabolic process. Preincubation of platelets with phenylmethylsulfo- nyl fluoride resulted in the rapid uptake of anandamide ( 90% of the total [ 3 H]anandamide at 2 min) which remained almost unchanged up to 40 min (Fig. 5). In the presence of phenylmethylsulfonyl fluoride, anandamide was neither metabolized nor released into the medium (Fig. 6). The phenomenon seems to be rather a passive diffusion and/or a nonspecific binding to the membrane of platelets according to the results presented in Fig. 8. The concentration dependence curve was linear up to 2000 n M anandamide, while in human platelets, the uptake was saturable (K m ¼ 200 ± 20 n M at 37 °C). In the presence of phenylmethylsulfonyl fluoride, the concentration dependence curve was also linear but higher (e.g. at 2000 n M [ 3 H]anandamide the uptake in the presence of phenylmethylsulfonyl fluoride was threefold higher than in control cells). On the contrary, for human platelets it has been reported that 100 l M phenylmethylsulfonyl fluoride reduced anandamide uptake by  40% of the untreated control [13] as it should be expected if a membrane transporter is present [23]. Additionally, the uptake was also linear but higher at 0–4 °C compared to 37 °C (Fig. 8A) also suggesting the absence of a carrier-mediated process, since this kind of transport could not take place at low temperature. Although, the possibility of the existence of a hidden carrier-mediated transport along with the passive diffusion could not be totally excluded, since the uptake was almost the same at 1–2 min in the presence of phenylmethylsulfo- nyl fluoride (Figs 5 and 8) and there is a small inhibition at 2 min by VDM11 (Fig. 7), our data do not provide any other evidence to support this hypothesis. The coexistence of passive diffusion and a facilitated transport has been reported for palmitoylethanolamide in Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leukaemia cells, but the uptake was temperature sensitive in these cells [47]. Rabbit platelets could play a role in rapidly removing anandamide from the extracellular space and in metaboli- zing it as it has been suggested for other cells [16], and/or anandamide could be a precursor for the strong agonist arachidonic acid and its metabolic products. The [ 3 H]anandamide metabolism in intact cells was investigated in order to determine the assumed main metabolic products, e.g. arachidonic acid and phospho- lipids (PL) [13,23] in both cells and extracellular space, using a TLC separation of total lipids extracted by the Bligh–Dyer method. Our results showed that most of radioactivity found in platelets after 2 min, was non- metabolized [ 3 H]anandamide, which was subsequently metabolized mainly to methanol/water-soluble products that increased dramatically and reached a plateau after 10–20 min. Interestingly, no initial increase in free fatty acid was detected ( 2% after 2 min, and reached a plateau ( 5%) after 20 min) (Fig. 4). Results obtained in the presence of caffeic acid (a LOX inhibitor) suggested that LOX was not involved in the metabolism since no inhibition was observed. When indo- methacin (a COX inhibitor) was used at 0.5 l M ,a concentration that inhibited platelet aggregation induced by 14 l M anandamide [43], only a small inhibition was observed. On the contrary, a significant inhibition ( 50%) of [ 3 H]anandamide metabolism by 75 l M indomethacin was observed, suggesting the involvement of COX. This inhibi- tion could be also attributed to the inhibition of FAAH as it has been reported that indomethacin is a competitive inhibitor of rat brain FAAH enzyme (K i ¼ 120 l M )[48]. On the other hand, it is well known that platelets possess two major enzymatic routes for arachidonic acid metabo- lism, the cyclooxygenase (COX) and the lipoxygenase (LOX) pathways. In the COX pathway, the main product is thromboxane A 2 and other prostaglandins while in the LOX pathway the main product is 12-monohydroxyeicosa- tetraenoic acid. Another minor pathway for arachidonic acid metabolism in platelets has also been reported: a nonenzymatic free-radical catalyzed peroxidation to iso- prostanes such as 8-Epi-PGF 2a [49,50]. The products of arachidonic acid peroxidation are water soluble, as reported in the literature in experiments with rabbit platelets labeled with [ 3 H]arachidonic acid [51]. The identification of meth- anol/water-soluble products of [ 3 H]anandamide metabolism was not addressed in the present study but it could be speculated that these were oxidation products of ananda- mide and/or arachidonic acid produced by the action of FAAH activity. Additionally, data from studies performed in our laboratory with rabbit platelet homogenate showed the presence of FAAH activity which is localized mainly in the plasma membrane-rich fraction of rabbit platelets and is much higher than that in human platelets (L. Fasia and A. Siafaka, unpublished data). Moreover, the localization of endogenous FAAH in the plasma membrane has been also reported for the rat liver [7]. In conclusion, the above results revealed a major difference between human and rabbit platelets, since [ 3 H]anandamide is not taken up by a carrier-mediated process in rabbit platelets in contrast to human platelets. Anandamide is taken up by rabbit platelets through passive diffusion, and subsequently rapidly metabolized apparentlybytheactionofaFAAH,incontrasttorat platelets where no FAAH expression was found [52]. Rabbit platelets could act as modulators to control anandamide concentration and keep it at physiological levels. Alternatively, anandamide could be a precursor for arachidonic acid and its metabolic products. Further studies are required to conclusively prove this suggestion and clarify the possibility of the involvement of other enzyme(s) (besides FAAH) in the metabolism of anand- amide for the production of water-soluble metabolites. These metabolites could be products of arachidonic acid produced by the action of FAAH, but the direct action of other enzyme(s) on anandamide could not be excluded. Acknowledgements This work was supported in part by University of Athens Special Account for Research Grants (70/4/3351). The authors would like to thank L. McManus (UTHSCSA, USA) for reading the manuscript. 3504 L. Fasia et al. (Eur. J. Biochem. 270) Ó FEBS 2003 References 1. Devane,W.A.,Hanus,L.,Breuer,A.,Pertwee,R.G.,Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. & Mechoulam, R. (1992) Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, 1946–1949. 2. Hanus, L., Gopher, A., Almog, S. & Mechoulam, R. (1993) Two new unsaturated fatty acid ethanolamides in brain that bind to the cannabinoid receptor. J. Med. Chem. 36, 3032–3034. 3. Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M., Kaminski, N.E., Schatz, A.R., Gopher, A., Almog, S., Martin, B.R.,Compton,D.R.,Pertwee,R.G.,Griffin,G.,Bayewitch,M., Barg, J. & Vogel, Z. (1995) Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem. Pharmacol. 50, 83–90. 4. Sugiura, T., Kondo, S., Sukagawa, A., Nakane, S., Shinoda, A., Itoh, K., Yamashita, A. & Waku, K. (1995) 2-Arachidonoylgly- cerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem. Biophys. Res. Commun. 215, 89–97. 5. Fowler, C.J. & Jacobsson, S.O.P. (2002) Cellular transport of anandamide, 2-arachidonoylglycerol and palmitoylethanolamide- targets for drug development? Prostaglandins Leukot. Essent. Fatty Acids 66, 193–200. 6. Ueda, N., Kurahashi, Y., Yamamoto, S. & Tokunaga, T. (1995) Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide. J. Biol. Chem. 270, 23823–23827. 7. Cravatt, B.F., Giang, D.K., Mayfield, S.P., Boger, D.L., Lerner, R.A. & Gilula, N.B. (1996) Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides. Nature 384, 83–87. 8. Deutsch, D. & Chin, S. (1993) Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist. Biochem. Pharmacol. 46, 791–796. 9. DiMarzo,V.,Fontana,A.,Cadas,H.,Schinelli,S.,Cimino,G., Schwartz, J.C. & Piomelli, D. (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372, 686–691. 10. Hillard, C.J., Edgemond, W.S., Jarrahian, A. & Campbell, W.B. (1997) Accumulation of N-arachidonoylethanolamine (ananda- mide) into cerebellar granule cells occurs via facilitated diffusion. J. Neurochem. 69, 631–638. 11. Beltramo, M., Stella, N., Calignano, A., Lin, S.Y., Makriyannis, A. & Piomelli, D. (1997) Functional role of high-affinity ana- ndamide transport, as revealed by selective inhibition. Science 277, 1094–1097. 12. Bisogno, T., Maurelli, S., Melck, D., De Petrocellis, L. & Di Marzo, V. (1997) Biosynthesis, uptake and degradation of ana- ndamide and palmitoylethanolamide in leukocytes. J. Biol. Chem. 272, 3315–3323. 13. Maccarrone, M., Bari, M., Menichelli, A., Del Principe, D. & Finazzi Agro ` , A. (1999) Anandamide activates human platelets through a pathway independent of the arachidonate cascade. FEBS Lett. 447, 277–282. 14. Maccarrone, M., Fiorucci, L., Erba, F., Bari, M., Finazzi Agro ` ,A. & Ascoli, F. (2000) Human mast cells take up and hydrolyze anandamide under the control of 5-lipoxygenase and do not express cannabinoid receptors. FEBS Lett. 468, 176–180. 15. Maccarrone, M., Bari, M., Lorenzon, T., Bisogno, T., Di Marzo, V. & Finazzi Agro ` , A. (2000) Anandamide uptake by human endothelial cells and its regulation by nitric oxide. J. Biol. Chem. 275, 13484–13492. 16. Hillard, C.J. & Jarrahian, A. (2000) The movement of N-arachi- donoylethanolamine (anandamide) across cellular membranes. Chem. Phys. Lipids 108, 123–134. 17. Piomelli, D., Beltramo, M., Glasnapp, S., Lin, S.Y., Goutopoulos, A., Xie, X.Q. & Makriyannis, A. (1999) Structural determinants for recognition and translocation by the anandamide transporter. Proc.Nat.Acad.Sci.USA96, 5802–5807. 18. Di Marzo, V., Bisogno, T., Melck, D., Ross, R., Brockie, H., Stevenson, L., Pertwee, R. & De Petrocellis, L. (1998) Interactions between synthetic vanilloids and the endogenous cannabinoid system. FEBS Lett. 436, 449–454. 19. De Petrocellis, L., Bisogno, T., Davis, J.B., Pertwee, R.G. & Di Marzo, V. (2000) Overlap between the ligand recognition prop- erties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsai- cin-like activity. FEBS Lett. 483, 52–56. 20. Lopez-Rodriguez, M.L., Viso, A., Ortega-Gutierrez, S., Lastres- Becker, I., Gonzalez, S., Fernandez-Ruiz, J. & Ramos, J.A. (2001) Design, synthesis and biological evaluation of novel arachidonic acid derivatives as highly potent and selective endocannabinoid transporter inhibitors. J. Med. Chem. 44, 4505–4508. 21. Jarrahian, A., Manna, S., Edgemond, W.S., Campbell, W.B. & Hillard, C.J. (2000) Structure-activity relationships among N-arachidonylethanolamine (Anandamide) head group analogues for the anandamide transporter. J. Neurochem. 74, 2597–2606. 22. Bisogno, T., Maccarrone, M., De Petrocellis, L., Jarrahian, A., Finazzi-Agro, A., Hillard, C. & Di Marzo, V. (2001) The uptake by cells of 2-arachidonoylglycerol, an endogenous agonist of cannabinoid receptors. Eur. J. Biochem. 268, 1982–1989. 23. Deutsch, D.G., Glaser, S.T., Howell, J.M., Kunz, J.S., Puffen- barger, R.A., Hillard, C.J. & Abumrad, A. (2001) The cellular uptake of anandamide is coupled to its breakdown by fatty-acid amide hydrolase. J. Biol. Chem. 276, 6967–6973. 24. Day,T.A.,Rakhshan,F.,Deutsch,D.G.&Barker,E.L.(2001) Role of fatty acid amide hydrolase in the transport of the endogenous cannabinoid anandamide. Mol. Pharmacol. 59, 1369–1375. 25. Ueda, N., Puffenbarger, R.A., Yamamoto, S. & Deutsch, D.G. (2000) The fatty acid amide hydrolase (FAAH). Chem. Phys. Lipids 108, 107–121. 26. Deutsch, D.G., Ueda, N. & Yamamoto, S. (2002) The fatty acid amide hydrolase (FAAH). Prostaglandins Leukot. Essent. Fatty Acids 66, 173–192. 27. Maurelli, S., Bisogno, T., De Petrocellis, L., Di Luccia, A., Mar- ino, G. & Di Marzo, V. (1995) Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma Ôanandamide amidohydrolaseÕ. FEBS Lett. 377, 82–86. 28. Goparaju, S.K., Ueda, N., Yamaguchi, H. & Yamamoto, S. (1998) Anandamide amidohydrolase reacting with 2-arachido- noylglycerol, another cannabinoid receptor ligand. FEBS Lett. 422, 69–73. 29. Devane, W.A. & Axelrod, J. (1994) Enzymatic synthesis of ana- ndamide, an endogenous ligand for the cannabinoid receptor, by brain membranes. Proc. Natl Acad. Sci. USA 91, 6698–6701. 30. Arreaza,G.,Devane,W.A.,Omeir,R.L.,Sajnani,G.,Kunz,J., Cravatt, B.F. & Deutsch, D.G. (1997) The cloned rat hydrolytic enzyme responsible for the breakdown of anandamide also cata- lyzes its formation via the condensation of arachidonic acid and ethanolamine. Neurosci. Lett. 234, 59–62. 31. Schmid, P.C., Zuzarte-Augustin, M.L. & Schmid, H.H.O. (1985) Properties of rat liver N-acylethanolamine amidohydrolase. J. Biol. Chem. 260, 14145–14149. 32. Desarnaud, F., Cadas, H. & Piomelli, D. (1995) Anandamide amidohydrolase activity in rat brain microsomes. Identification and partial characterization. J. Biol. Chem. 270, 6030–6035. 33. Hillard, C.J., Wilkison, D.M., Edgemond, W.S. & Campbell, W.B. (1995) Characterization of the kinetics and distribution of N-arachidonylethanolamine (anandamide) hydrolysis by rat brain. Biochim. Biophys. Acta 1257, 249–256. Ó FEBS 2003 Uptake and metabolism of anandamide by rabbit platelets (Eur. J. Biochem. 270) 3505 34. Karava, V., Fasia, L. & Siafaka-Kapadai, A. (2001) Anandamide amidohydrolase activity, released in the medium by Tetrahymena pyriformis. Identification and partial characterization. FEBS Lett. 508, 327–331. 35. Hampson, A.J., Hill, W.A.G., Zan-Phillips, M., Makriyannis, A., Leung, E., Eglen, R.M. & Bornheim, L.M. (1995) Anandamide hydroxylation by brain lipoxygenase: metabolite structures and potencies at the cannabinoid receptor. Biochim. Biophys. Acta 1259, 173–179. 36. Ueda, N., Yamamoto, K., Yamamoto, S., Tokunaga, T., Shi- rakawa, E., Shinkai, H., Ogawa, M., Sato, T., Kudo, I., Inoue, K., Takizawa, H., Nagano, T., Hirobe, M., Matsuki, N. & Saito, H. (1995) Lipoxygenase-catalyzed oxygenation of arachidonyletha- nolamide, a cannabinoid receptor agonist. Biochim. Biophys. Acta 1254, 127–134. 37. Edgemond, W.S., Hillard, C.J., Falck, J.R., Kearn, C.S. & Campbell, W.B. (1998) Human platelets and polymorphonuclear leukocytes synthesize oxygenated derivatives of arachidonyletha- nolamide (anandamide): their affinities for cannabinoid receptors and pathways of inactivation. Mol. Pharmacol. 54, 180–188. 38. YuM., Ives, D. & Ramesha, C.S. (1997) Synthesis of pros- taglandin E 2 ethanolamide from anandamide by cyclooxygenase- 2. J. Biol. Chem. 272, 21181–21186. 39. Pinckard, R.N., Farr, R.S. & Hanahan, D.J. (1979) Physico- chemical and functional identity of rabbit platelet-activating factor (PAF)releasedinvivoduringIgEanaphylaxiswithPAFreleased in vitro from IgE sensitized basophils. J. Immunol. 123, 1847–1857. 40. Siafaka-Kapadai, A. & Hanahan, D.J. (1993) An endogenous inhibitor of PAF-induced platelet aggregation, isolated from rat liver, has been identified as free fatty acid. Biochim. Biophys. Acta 1166, 217–221. 41. Bligh, E.G. & Dyer, W. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Pharmacol. 37, 911–917. 42. Braud, S., Bon, C., Touqui, L. & Mounier, C. (2000) Activation of rabbit blood platelets by anandamide through its cleavage into arachidonic acid. FEBS Lett. 471, 12–16. 43. Fasia, L. & Siafaka-Kapadai, A. (1997) Effects of anandamide and other N-acylethanolamines on rabbit platelet function. Proc. 11th Balkan Biochemical Biophysical Days, May 15–17, Thes- saloniki, Greece, pp. 141. 44. Lagarde, M. (1988) Metabolism of fatty acids by platelets and the functions of various metabolites in mediating platelet function. Prog. Lipid Res. 27, 135–152. 45. Kroll, M.H. & Schafer, A.I. (1989) Biochemical mechanisms of platelet activation. Blood 74, 1181–1195. 46. Yamamoto, S. (1992) Mammalian lipoxygenases: molecular structures and functions. Biochim. Biophys. Acta 1128, 117–131. 47. Jacobsson, S.O.P. & Fowler, C.J. (2001) Characterization of pal- mitoylethanolamide transport in mouse Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leukaemia cells: comparison with anandamide. Br. J.Pharmacol. 132, 1743–1754. 48. Fowler, C.J., Jonsson, K O. & Tiger, G. (2001) Fatty acid amide hydrolase: biochemistry, pharmacology, and therapeutic possibi- lities for an enzyme hydrolyzing anandamide, 2-arachidonoylgly- cerol, palmitoylethanolamide, and oleamide. Biochem. Pharmacol. 62, 517–526. 49. Morrow, J.D., Awad, J.A., Boss, H.J., Blair, I.A. & Roberts, L.J. (1992) Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids. Proc. Natl Acad. Sci. USA 89, 10721–10725. 50. Pratico, D., Lawson, J.A. & Fitzgerald, G.A. (1994) Cyclooxy- genase-dependent formation of the isoprostane 8-epi-prosta- glandin F2 alpha. Ann. N.Y. Acad. Sci. 744, 139–145. 51. Hashizume, T., Yamaguchi, H., Kawamoto, A., Tamura, A., Sato, T. & Fujii, T. (1991) Lipid peroxide makes rabbit platelet hyperaggregable to agonists through PLA 2 activation. Arch. Bio- chem. Biophys. 86, 47–52. 52. Di Marzo, V., Bisogno, T., De Petrocellis, L., Melck, D., Orlando, P., Wagner, J.A. & Kunos, G. (1999) Biosynthesis and inactivation of the endocannabinoid 2-arachidonoylglycerol in circulating and tumoral macrophages Eur. J. Biochem. 264, 258–267. 3506 L. Fasia et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . Uptake and metabolism of [ 3 H]anandamide by rabbit platelets Lack of transporter? Lambrini Fasia, Vivi Karava and Athanassia Siafaka-Kapadai Department. high percentage of radioactivity incorporated into platelets (32.6 ± 2.7%). Ó FEBS 2003 Uptake and metabolism of anandamide by rabbit platelets (Eur. J.

Ngày đăng: 08/03/2014, 08:20

Từ khóa liên quan

Tài liệu cùng người dùng

Tài liệu liên quan