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Carrier-mediated transport and enzymatic hydrolysis of the endogenous cannabinoid 2-arachidonylglycerol

Carrier-mediated transport and enzymatic hydrolysis of the endogenous cannabinoid... UC Irvine UC Irvine Previously Published Works Title Carrier-mediated transport and enzymatic hydrolysis of the endogenous cannabinoid 2- arachidonylglycerol Permalink https://escholarship.org/uc/item/92w2j8v2 Journal Neuroreport, 11(6) ISSN 0959-4965 Authors Beltramo, Massimiliano Piomelli, Daniele Publication Date 2000-04-01 DOI 10.1097/00001756-200004270-00018 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed eScholarship.org Powered by the California Digital Library University of California NEUROPHARMACOLOGY NEUROREPORT Carrier-mediated transport and enzymatic hydrolysis of the endogenous cannabinoid 2- arachidonylglycerol CA,1 Massimiliano Beltramo and Daniele Piomelli Department of Pharmacology, University of California, Irvine, CA 92697, USA Present address: Schering-Plough Research Institute, 58 via Olgettina, 20132 Milan, Italy CA Corresponding Author Received 6 January 2000; accepted 8 February 2000 Acknowledgements: We thank Mr Thien Dinh for critical reading of the manuscript. Additional support was provided by the National Institute of Drug Abuse (under grant number 12447, to D.P.). Initial experiments were supported by the Neuroscience Research Foundation, which receives major support from Novartis. The human astrocytoma cell line CCF-STTG1 accumulates [ H]2-AG, radioactivity is recovered in phospholipids, mono- 3 ‡ 3 [ H]2-AG through an Na - and energy-independent process, acylglycerols (unmetabolized [ H]2-AG), free fatty acids with a K of 0.7 0.1 ìM. Non-radioactive 2-AG, anandamide ([ H]arachidonate) and, to a minor extent, diacylglycerols and or the anandamide transport inhibitor 4-hydroxyphenyl arachi- triacylglycerols. Arachidonic acid (100 ìM) and triacsin C donamide inhibit [ H]2-AG uptake with half-maximal inhibitory (10 ìM), an acyl-CoA synthetase inhibitor, prevent incorpora- concentrations (IC ) of 5.5 1.0 ìM, 4.2 0.3 ìM and tion of [ H]arachidonic acid in phospholipids and signi®cantly 1.8 0.1 ìM, respectively. A variety of lipid transport sub- reduce [ H]2-AG transport. Thus, the driving force for 2-AG strates and inhibitors interfere with neither [ H]2-AG nor internalization may derive from the hydrolysis of 2-AG to [ H]anandamide uptake. These results suggest that 2-AG and arachidonate and the subsequent incorporation of this fatty anandamide are internalized in astrocytoma cells through a acid into phospholipids. NeuroReport 11:1231±1235 & 2000 common carrier-mediated mechanism. After incubation with Lippincott Williams & Wilkins. Key words: 2-Arachidonylglycerol; Anandamide; Endogenous cannabinoid; Human astrocytoma cells; Transport INTRODUCTION for anandamide amidohydrolase [17,18] suggest that 2-AG Two endogenous cannabinoid compounds, anandamide and anandamide may share a common inactivation route. (arachidonylethanolamide) and 2-arachidonylglycerol (2- As a test of this hypothesis, here we have investigated AG), have been identi®ed in the brain [1 ± 6]. Both com- the biological disposition of [ H]2-AG in human astrocyto- pounds activate cannabinoid receptors producing a spec- ma cells. Our results suggest that 2-AG and anandamide trum of cannabimimetic effects, which include movement may be internalized in these cells via a common carrier- inhibition, pain relief and vasodilation [7]. mediated process. By contrast, the intracellular hydrolysis Anandamide is released from neurons in an activity- of 2-AG to arachidonic acid and glycerol may be catalyzed dependent manner [2,6] and is inactivated by transport by an enzyme activity distinct from anandamide amidohy- into cells followed by intracellular hydrolysis. Anandamide drolase. transport is mediated by a high-af®nity, Na -independent carrier and is selectively inhibited by 4-hydroxyphenyl MATERIALS AND METHODS arachidonamide (AM404) [8,9]. Hydrolysis of anandamide Chemicals: Anandamide and palmitylethanolamide were to arachidonic acid and ethanolamine is catalyzed by a prepared as described [1]; 2-AG was from Deva Biotech membrane-bound amidohydrolase enzyme [10 ± 12], which (Hartboro, PA); arachidonic acid from Nu Check-Prep has been puri®ed and molecularly cloned [13,14]. 2-AG (Elysian, MN); AM404 from Tocris (Ballwin, MO); triacsin may be produced by cleavage of 1,2-diacylglycerol gener- C from Biomol (Plymouth Meeting, PA); prostaglandin E ated by phospholipase C (PLC) acting on phosphatidylino- and (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)- sitol bisphosphate [15]. The mechanisms mediating 2-AG 2H-pyran-2-one from Cayman (Ann Arbor, MI); sulphobro- inactivation are still only partially understood, but the mophthalein, digoxin, taurocholic acid, quinidine, verapa- ability of 2-AG to inhibit [ H]anandamide transport in mil and phloretin from Sigma (St. Louis, MO). All organic human astrocytoma cells [16] and to serve as a substrate solvents were from Burdick and Jackson. 0959-4965 & Lippincott Williams & Wilkins Vol 11 No 6 27 April 2000 1231 NEUROREPORT M. BELTRAMO AND D. PIOMELLI Transport assay: Human astrocytoma CCF-STTG1 cells Statistical analyses: Statistically signi®cant differences (American Type Culture Collection, Rockville, MD) were were determined by ANOVA, followed by Bonferroni's grown in 24-well plates with RPMI 1640 medium contain- multiple comparison test. ing FBS (10%) and glutamine (1 mM) [16]. Con¯uent cultures were rinsed with Tris ± Krebs' buffer at 378C, incu- RESULTS 3 3 bated for 4 min with buffer containing [ H]2-AG (for [ H]2-AG accumulation in astrocytoma cells is rapid and kinetic analyses: 28 ± 1600 nM; for inhibition assays: 30 nM; saturable: Lineweaver ± Burk analyses of this accumulation 100 mCi/mmol; New England Nuclear, custom synthe- yield a Michaelis constant (K ) of 0.7 0.1 ìM and a ÿ1 sized) either at 378Cor0±48C, rinsed with buffer contain- maximal accumulation rate (V )of28 6 pmol min max ÿ1 ing bovine serum albumin (BSA, 0.1%), and disrupted by mg protein ( nˆ 9). sonication in buffer containing Triton X-100 (1%). Radio- Non-radioactive 2-AG inhibits [ H]2-AG accumulation activity in samples was measured by liquid scintillation in a concentration-dependent manner, with a half-maximal counting. In some experiments, anandamide transport was inhibitory concentration (IC ) of 5.5 1.0 ìM( nˆ 6). At 3 3 measured using [ H]anandamide (30 nM, 220 Ci/mmol, 100 ìM, non-radioactive 2-AG reduces [ H]2-AG accumula- New England Nuclear). For inhibition assays, the cells tion in cells to 24 1% of control (control: 8381 393 were preincubated for 10 min with test compounds at d.p.m./well; 2-AG: 2017 87 d.p.m./well; nˆ 6). We con- appropriate concentrations. The same concentrations of test sidered this value to be an estimate of the non-speci®c compounds were also added to the ®nal incubations, association of [ H]2-AG with the cells, and used it in which were carried out for an additional 4 min period. subsequent experiments as a background value to calculate Concentrations required for half-maximal inhibition of potency and ef®cacy of test compounds. transport (IC ) were determined by non-linear least square Non-radioactive anandamide inhibits [ H]2-AG accumu- ®tting of the data, using the software GraphPad Prism lation with an IC of 4.2 0.3 ìM and a maximal effect of (GraphPad Software, San Diego, CA). 100% ( nˆ 6). The anandamide transport inhibitor AM404 is also very effective in interfering with [ H]2-AG uptake (IC ˆ 1.8 0.1 ìM, nˆ 6). Conversely, a variety of sub- Hydrolysis assay: Following incubations with [ H]2-AG strates and inhibitors of lipid transport systems (including or [ H]anandamide, the cells were rinsed and disrupted by prostaglandin E , sulphobromophthalein, digoxin, tauro- sonication in methanol at 0 ± 48C for 30 s. Lipids were cholic acid, quinidine, verapamil and phloretin) have no extracted with chloroform (chloroform/methanol/buffer, effect on the accumulation of [ H]2-AG (data not shown). 2:1:1; vol./vol./vol.). The lipid extracts were analyzed by On the other hand, arachidonic acid (100 ìM) substantially thin-layer chromatography (TLC) on silica gel G plates reduces the accumulation of [ H]2-AG in astrocytoma cells (Analtech). TLC analyses were carried out using either (Fig. 1b). TLC analyses revealed that astrocytoma cells ethylacetate/methanol/water/ammonium hydroxide (20: rapidly metabolize [ H]2-AG (Fig. 2). After a 4 min incuba- 1:10:1) or chloroform/methanol/ammonium hydroxide tion, cell-associated radioactivity is distributed among TLC (80:20:1) as solvent system. The lipids were visualized with fractions migrating with monoacylglycerols (unmetabo- a 10% solution of phosphomolybdic acid (Sigma, Saint lized [ H]2-AG; 1384 46 d.p.m./well), free fatty acids Louis, MO) in ethanol followed by heating at 2508C. Bands (probably [ H]arachidonic acid, 560 64 d.p.m./well), with the mobility of authentic standards were scraped off phospholipids (6928 504 d.p.m./well), diacylglycerols the TLC plates for radioactivity determination. and triacylglycerols (370 52 d.p.m./well; results are from 120 (a) 120 (b) *** *** 0 0 3 3 Fig. 1. Effects of various compounds on [ H]2-AG and [ H]anandamide accumulation by astrocytoma cells. Intracellular accumulation of 3 3 [ H]anandamide (a) and [ H]2-AG (b) was measured in cells at con¯uence in the presence of the anandamide amidohydrolase inhibitor BTNP (5 ìM), the acyl-CoA synthetase inhibitor triacsin C (10 ìM), or the hydrolysis product, arachidonic acid (100 ìM). Results represent the mean s.e.m. ( nˆ 6± 9). p , 0.001 (ANOVA followed by Bonferroni multiple comparison test). 1232 Vol 11 No 6 27 April 2000 Control BTNP AA TrC Control BTNP AA TrC Total radioactivity accumulation (% of control) Total radioactivity accumulation (% of control) 2-ARACHIDONYLGLYCEROL INACTIVATION NEUROREPORT 2-AG AA 125 125 100 100 75 75 *** 50 50 *** 25 25 0 0 DAG-TAG PL 125 400 *** ** *** *** Fig. 2. Effects of various compounds on [ H]2-AG metabolism in astrocytoma cells. Cells at con¯uence were incubated in Tris ± Krebs' buffer containing [ H]2-AG with or without one of the following compounds: AM404, an anandamide transport inhibitor (10 ìM); BTNP, an anandamide amidohydrolase inhibitor (5 ìM); arachidonic acid (AA, 100 ìM); and triacsin C, an acyl CoA synthetase inhibitor (TrC, 10 ìM). Reactions were stopped by addition of cold methanol, and the lipids were extracted and analyzed by TLC. The lipids were stained with phosphomolybdic acid, identi®ed by comparison with authentic standards and quanti®ed by liquid scintillation counting. PL, phospholipids; DAG, diacylglycerols; TAG, triacylglycerols. p , 0.01, p , 0.001 (ANOVA followed by Bonferroni multiple comparison test). 3 3 one experiment representative of three). AM404 (10 ìM), with [ H]2-AG (1222 152 d.p.m./well) and [ H]- signi®cantly reduces the incorporation of radioactivity in arachidonic acid (631 75 d.p.m./well), but drastically the TLC fractions migrating with [ H]2-AG (413 54 reduces the radioactivity in phospholipids (to 1369 287 d.p.m./well), [ H]arachidonic acid (203 26 d.p.m./well) d.p.m./well). Arachidonic acid also augments incorpora- and phospholipids (2906 141 d.p.m./well). These results tion of radioactivity in diacylglycerols and triacylglycerols are in agreement with the ability of AM404 to inhibit (to 1335 42 d.p.m./well; Fig. 2). However, this increase is [ H]2-AG accumulation in astrocytoma cells, and under- not suf®cient to compensate for the substantial loss of score the intracellular localization of [ H]2-AG hydrolysis. radioactivity in the phospholipid fraction and we did not Conversely, the anandamide amidohydrolase inhibitor, (E)- investigate further its molecular basis. These ®ndings sug- 6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyr- gest that arachidonic acid inhibits [ H]2-AG accumulation an-2-one (BTNP; 5 ìM) [19] has no effect on [ H]2-AG indirectly, possibly by hindering the esteri®cation into accumulation (Fig. 1b) or metabolism (Fig. 2). phospholipids of the [ H]arachidonate produced by the 3 3 If arachidonic acid directly interfered with [ H]2-AG hydrolysis of [ H]2-AG. transport it should produce an effect similar to that of To test this possibility, we investigated the effects of AM404 (i.e. a complete inhibition of [ H]2-AG transport). triacsin C, a selective acyl-coenzyme A synthetase inhibitor In contrast with this prediction, arachidonic acid (100 ìM) [20], on [ H]2-AG uptake and metabolism. Incubation of does not affect radioactivity in the TLC fractions migrating astrocytoma cells with triacsin C (10 ìM) signi®cantly Vol 11 No 6 27 April 2000 1233 Control AM404 BTNP AA TrC Control Control AM404 AM404 BTNP BTNP AA AA TrC TrC Control AM404 BTNP AA TrC Radioactivity (% of control) Radioactivity (% of control) Radioactivity (% of control) Radioactivity (% of control) NEUROREPORT M. BELTRAMO AND D. PIOMELLI reduces both [ H]2-AG uptake (Fig. 1b) and incorporation player in 2-AG degradation in intact cells [23]. The trans- of [ H]arachidonate in phospholipids (Fig. 2). By contrast, port inhibitor AM404 dramatically reduces the intracellular the inhibitor has no effect on the levels of radioactivity in accumulation of [ H]2-AG whereas administration of ara- 3 3 3 [ H]2-AG, [ H]arachidonic acid, diacylglycerols and tria- chidonic acid has no effect on intracellular [ H]2-AG levels. cylglycerols (Fig. 2). In addition, triacsin C does not affect These ®ndings suggest that arachidonic acid may inhibit 3 3 [ H]anandamide accumulation (Fig. 1a) or its intracellular [ H]2-AG accumulation indirectly, possibly by hindering hydrolysis (data not shown). the esteri®cation into phospholipids of the [ H]- arachidonate produced by the hydrolysis of [ H]2-AG. DISCUSSION These results imply that arachidonate esteri®cation in membrane phospholipids may be a primary driving force Human astrocytoma CCF-STTG1 cells internalize [ H]- anandamide by a transport mechanism functionally indis- for [ H]2-AG transport. tinguishable from that found in rat brain neurons and CONCLUSIONS astrocytes [8,16]. Therefore, we used these cells to test the hypothesis that [ H]2-AG may be a substrate for the Our ®ndings suggest that 2-AG and anandamide are internalized in astrocytoma cells via a common carrier- anandamide transporter. The kinetic of [ H]2-AG accumu- lation in astrocytoma cells is consistent with a single high- mediated transport system. Four key observations support this hypothesis: (1) the kinetic properties of [ H]2-AG and af®nity transport process. In the same cells and under 3 3 identical conditions, [ H]anandamide transport displays [ H]anandamide accumulation are very similar; (2) 2-AG and anandamide mutually displace each other's accumula- similar characteristics (K of 0.6 0.1 ìM and V of m max ÿ1 ÿ1 14.7 1.5 pmol min mg protein) [16]. Several lines of tion; (3) [ H]2-AG uptake is blocked by the anandamide 3 3 3 transport inhibitor, AM404; (4) [ H]2-AG and [ H]- evidence indicate that [ H]2-AG accumulation in astrocyto- ma cells is mediated by a transport system akin to that anandamide uptake are insensitive to several substrates involved in anandamide internalization. First, non-radio- and inhibitors of lipid transporters and Na - and energy- active anandamide inhibits [ H]2-AG accumulation while, independent. By contrast, the intracellular degradation of conversely, 2-AG inhibits [ H]anandamide transport (IC 2-AG may differ substantially from that of anandamide: whereas anandamide hydrolysis is blocked by BTNP, an of 18.5 0.7 ìM) [16]. Second, the anandamide transport inhibitor AM404 is also effective, and even more potent in inhibitor of amidohydrolase activity, 2-AG hydrolysis is insensitive to this drug. This indicates that 2-AG break- interfering with [ H]2-AG uptake than anandamide or 2- AG. Third, a variety of substrates and inhibitors of lipid down in intact cells may depend on other hydrolase enzymes, such as monoacylglycerol lipase [23]. The role of transport systems have no effect on the accumulation of 3 3 either [ H]2-AG or [ H]anandamide [8,16]. Finally, ananda- hydrolysis in 2-AG disposition may differ from ananda- mide in another important way. We found that treating the mide and 2-AG transport are both Na and energy independent [8,16]. cells with arachidonic acid or the acyl-CoA synthetase inhibitor, triacsin C, prevents the transport of 2-AG, but There are, however, a number of differences between anandamide and 2-AG inactivation. [ H]Anandamide not that of anandamide. A plausible interpretation of these transport is not affected by arachidonic acid (Fig. 1a) and, results is that 2-AG hydrolysis into free arachidonic acid and the subsequent incorporation of arachidonic acid into vice versa,[ H]arachidonic acid transport is not affected by anandamide (data not shown), indicating that distinct membrane phospholipids may be a primary driving force for 2-AG internalization. This difference between the bio- membrane mechanisms mediate the internalization of these two lipid compounds [8,16]. By contrast, arachidonic acid logical dispositions of anandamide and 2-AG might be exploited for the development of selective inactivation substantially reduces the accumulation of [ H]2-AG (Fig. 1b). A possible interpretation of this ®nding is that inhibitors. arachidonic acid may directly interfere with the putative carrier involved in [ H]2-AG uptake, implying the exis- REFERENCES 1. Devane WA, Hanus L, Breuer A et al. Science 258, 1946 ± 1949 (1992). tence of different transport mechanisms for [ H]2-AG and 3 2. Di Marzo V, Fontana A, Cadas H et al. Nature 372, 686 ± 691 (1994). [ H]anandamide (sensitive and insensitive, respectively, to 3. Sugiura T, Kondo S, Sukagawa A et al. Biochem Biophys Res Commun 215, arachidonic acid). An alternative possibility is that arachi- 89 ± 97 (1995). donic acid may inhibit [ H]2-AG accumulation indirectly, 4. Mechoulam R, Ben-Shabat S, Hanus L et al. Biochem Pharmacol 50,83±90 for example by interfering with the intracellular disposition (1995). of [ H]2-AG. To evaluate these alternative possibilities, we 5. Stella N, Schweitzer P and Piomelli D. Nature 388, 773ÿ778 (1997). 6. Giuffrida A, Parsons LH, Kerr TM et al. Nature Neurosci 2, 358 ± 363 investigated the fate of [ H]2-AG in astrocytoma cells. The (1999). routes of 2-AG metabolism in cells are still only partially 7. Pertwee RG. Pharmacol Ther 74, 129 ± 180 (1997). understood, but two enzymes are thought to be primarily 8. Beltramo M, Stella N, Calignano A et al. Science 277, 1094 ± 1097 (1997). involved: anandamide amidohydrolase and monoacylgly- 9. Hillard CJ, Edgemond WS, Jarrahian A and Campbell WB. J Neurochem cerol lipase [17,18,21]. Both enzymes catalyze the hydro- 69, 631 ± 638 (1997). lytic cleavage of 2-AG to arachidonate and glycerol. Free 10. Schmid PC, Zuzarte-Augustin ML and Schmid HH. J Biol Chem 260, 14145 ± 14149 (1985). arachidonate is short-lived in cells, being rapidly converted 11. Deutsch GG and Chin S. Biochem Pharmacol 46, 791 ± 796 (1993). to arachidonyl-coenzyme A (CoA) and then incorporated 12. Desarnaud F, Cadas H and Piomelli D. J Biol Chem 270, 6030 ± 6035 into phospholipids [22]. Blockade of anandamide amidohy- (1995). drolase activity completely abrogates [ H]anandamide de- 13. Ueda N, Kurahashi Y, Yamamoto S and Tokunaga T. J Biol Chem 270, gradation [8,16], but it does not affect [ H]2-AG hydrolysis, 23823 ± 23827 (1995). suggesting that monoacylglycerol lipase may be a major 14. Cravatt BF, Giang DK, May®eld SP et al. Nature 384, 83 ± 87 (1996). 1234 Vol 11 No 6 27 April 2000 2-ARACHIDONYLGLYCEROL INACTIVATION NEUROREPORT 15. Piomelli D, Beltramo M, Giuffrida A and Stella N. Neurobiol Dis 5, (1997). 462 ± 473 (1998). 20. Tomoda H, Igarashi K and Omura S. Biochim Biophys Acta 921, 595 ± 598 16. Piomelli D, Beltramo M, Glasnapp S et al. Proc Natl Acad Sci USA 96, (1987). 5802 ± 5807 (1999). 21. Goparaju SK, Ueda N, Taniguchi K and Yamamoto S. Biochem Pharmacol 17. Di Marzo V, Bisogno T, Sugiura T et al. Biochem J 331, 15 ± 19 (1998). 57, 417 ± 423 (1999). 18. Goparaju SK, Ueda N, Yamaguchi H and Yamamoto S. FEBS Lett 422, 22. Waku K. Biochim Biophys Acta 1124, 101 ± 111 (1992). 69 ± 73 (1998). 23. Karlsson M, Contreras JA, Hellman U et al. J Biol Chem 272, 27218 ± 27223 19. Beltramo M, di Tomaso E and Piomelli D. FEBS Lett 403, 263 ± 267 (1997). Vol 11 No 6 27 April 2000 1235 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Neuroreport Unpaywall

Carrier-mediated transport and enzymatic hydrolysis of the endogenous cannabinoid 2-arachidonylglycerol

Beltramo, Massimiliano; Piomelli, Daniele
Neuroreport – Apr 1, 2000
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UC Irvine UC Irvine Previously Published Works Title Carrier-mediated transport and enzymatic hydrolysis of the endogenous cannabinoid 2- arachidonylglycerol Permalink https://escholarship.org/uc/item/92w2j8v2 Journal Neuroreport, 11(6) ISSN 0959-4965 Authors Beltramo, Massimiliano Piomelli, Daniele Publication Date 2000-04-01 DOI 10.1097/00001756-200004270-00018 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed eScholarship.org Powered by the California Digital Library University of California NEUROPHARMACOLOGY NEUROREPORT Carrier-mediated transport and enzymatic hydrolysis of the endogenous cannabinoid 2- arachidonylglycerol CA,1 Massimiliano Beltramo and Daniele Piomelli Department of Pharmacology, University of California, Irvine, CA 92697, USA Present address: Schering-Plough Research Institute, 58 via Olgettina, 20132 Milan, Italy CA Corresponding Author Received 6 January 2000; accepted 8 February 2000 Acknowledgements: We thank Mr Thien Dinh for critical reading of the manuscript. Additional support was provided by the National Institute of Drug Abuse (under grant number 12447, to D.P.). Initial experiments were supported by the Neuroscience Research Foundation, which receives major support from Novartis. The human astrocytoma cell line CCF-STTG1 accumulates [ H]2-AG, radioactivity is recovered in phospholipids, mono- 3 ‡ 3 [ H]2-AG through an Na - and energy-independent process, acylglycerols (unmetabolized [ H]2-AG), free fatty acids with a K of 0.7 0.1 ìM. Non-radioactive 2-AG, anandamide ([ H]arachidonate) and, to a minor extent, diacylglycerols and or the anandamide transport inhibitor 4-hydroxyphenyl arachi- triacylglycerols. Arachidonic acid (100 ìM) and triacsin C donamide inhibit [ H]2-AG uptake with half-maximal inhibitory (10 ìM), an acyl-CoA synthetase inhibitor, prevent incorpora- concentrations (IC ) of 5.5 1.0 ìM, 4.2 0.3 ìM and tion of [ H]arachidonic acid in phospholipids and signi®cantly 1.8 0.1 ìM, respectively. A variety of lipid transport sub- reduce [ H]2-AG transport. Thus, the driving force for 2-AG strates and inhibitors interfere with neither [ H]2-AG nor internalization may derive from the hydrolysis of 2-AG to [ H]anandamide uptake. These results suggest that 2-AG and arachidonate and the subsequent incorporation of this fatty anandamide are internalized in astrocytoma cells through a acid into phospholipids. NeuroReport 11:1231±1235 & 2000 common carrier-mediated mechanism. After incubation with Lippincott Williams & Wilkins. Key words: 2-Arachidonylglycerol; Anandamide; Endogenous cannabinoid; Human astrocytoma cells; Transport INTRODUCTION for anandamide amidohydrolase [17,18] suggest that 2-AG Two endogenous cannabinoid compounds, anandamide and anandamide may share a common inactivation route. (arachidonylethanolamide) and 2-arachidonylglycerol (2- As a test of this hypothesis, here we have investigated AG), have been identi®ed in the brain [1 ± 6]. Both com- the biological disposition of [ H]2-AG in human astrocyto- pounds activate cannabinoid receptors producing a spec- ma cells. Our results suggest that 2-AG and anandamide trum of cannabimimetic effects, which include movement may be internalized in these cells via a common carrier- inhibition, pain relief and vasodilation [7]. mediated process. By contrast, the intracellular hydrolysis Anandamide is released from neurons in an activity- of 2-AG to arachidonic acid and glycerol may be catalyzed dependent manner [2,6] and is inactivated by transport by an enzyme activity distinct from anandamide amidohy- into cells followed by intracellular hydrolysis. Anandamide drolase. transport is mediated by a high-af®nity, Na -independent carrier and is selectively inhibited by 4-hydroxyphenyl MATERIALS AND METHODS arachidonamide (AM404) [8,9]. Hydrolysis of anandamide Chemicals: Anandamide and palmitylethanolamide were to arachidonic acid and ethanolamine is catalyzed by a prepared as described [1]; 2-AG was from Deva Biotech membrane-bound amidohydrolase enzyme [10 ± 12], which (Hartboro, PA); arachidonic acid from Nu Check-Prep has been puri®ed and molecularly cloned [13,14]. 2-AG (Elysian, MN); AM404 from Tocris (Ballwin, MO); triacsin may be produced by cleavage of 1,2-diacylglycerol gener- C from Biomol (Plymouth Meeting, PA); prostaglandin E ated by phospholipase C (PLC) acting on phosphatidylino- and (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)- sitol bisphosphate [15]. The mechanisms mediating 2-AG 2H-pyran-2-one from Cayman (Ann Arbor, MI); sulphobro- inactivation are still only partially understood, but the mophthalein, digoxin, taurocholic acid, quinidine, verapa- ability of 2-AG to inhibit [ H]anandamide transport in mil and phloretin from Sigma (St. Louis, MO). All organic human astrocytoma cells [16] and to serve as a substrate solvents were from Burdick and Jackson. 0959-4965 & Lippincott Williams & Wilkins Vol 11 No 6 27 April 2000 1231 NEUROREPORT M. BELTRAMO AND D. PIOMELLI Transport assay: Human astrocytoma CCF-STTG1 cells Statistical analyses: Statistically signi®cant differences (American Type Culture Collection, Rockville, MD) were were determined by ANOVA, followed by Bonferroni's grown in 24-well plates with RPMI 1640 medium contain- multiple comparison test. ing FBS (10%) and glutamine (1 mM) [16]. Con¯uent cultures were rinsed with Tris ± Krebs' buffer at 378C, incu- RESULTS 3 3 bated for 4 min with buffer containing [ H]2-AG (for [ H]2-AG accumulation in astrocytoma cells is rapid and kinetic analyses: 28 ± 1600 nM; for inhibition assays: 30 nM; saturable: Lineweaver ± Burk analyses of this accumulation 100 mCi/mmol; New England Nuclear, custom synthe- yield a Michaelis constant (K ) of 0.7 0.1 ìM and a ÿ1 sized) either at 378Cor0±48C, rinsed with buffer contain- maximal accumulation rate (V )of28 6 pmol min max ÿ1 ing bovine serum albumin (BSA, 0.1%), and disrupted by mg protein ( nˆ 9). sonication in buffer containing Triton X-100 (1%). Radio- Non-radioactive 2-AG inhibits [ H]2-AG accumulation activity in samples was measured by liquid scintillation in a concentration-dependent manner, with a half-maximal counting. In some experiments, anandamide transport was inhibitory concentration (IC ) of 5.5 1.0 ìM( nˆ 6). At 3 3 measured using [ H]anandamide (30 nM, 220 Ci/mmol, 100 ìM, non-radioactive 2-AG reduces [ H]2-AG accumula- New England Nuclear). For inhibition assays, the cells tion in cells to 24 1% of control (control: 8381 393 were preincubated for 10 min with test compounds at d.p.m./well; 2-AG: 2017 87 d.p.m./well; nˆ 6). We con- appropriate concentrations. The same concentrations of test sidered this value to be an estimate of the non-speci®c compounds were also added to the ®nal incubations, association of [ H]2-AG with the cells, and used it in which were carried out for an additional 4 min period. subsequent experiments as a background value to calculate Concentrations required for half-maximal inhibition of potency and ef®cacy of test compounds. transport (IC ) were determined by non-linear least square Non-radioactive anandamide inhibits [ H]2-AG accumu- ®tting of the data, using the software GraphPad Prism lation with an IC of 4.2 0.3 ìM and a maximal effect of (GraphPad Software, San Diego, CA). 100% ( nˆ 6). The anandamide transport inhibitor AM404 is also very effective in interfering with [ H]2-AG uptake (IC ˆ 1.8 0.1 ìM, nˆ 6). Conversely, a variety of sub- Hydrolysis assay: Following incubations with [ H]2-AG strates and inhibitors of lipid transport systems (including or [ H]anandamide, the cells were rinsed and disrupted by prostaglandin E , sulphobromophthalein, digoxin, tauro- sonication in methanol at 0 ± 48C for 30 s. Lipids were cholic acid, quinidine, verapamil and phloretin) have no extracted with chloroform (chloroform/methanol/buffer, effect on the accumulation of [ H]2-AG (data not shown). 2:1:1; vol./vol./vol.). The lipid extracts were analyzed by On the other hand, arachidonic acid (100 ìM) substantially thin-layer chromatography (TLC) on silica gel G plates reduces the accumulation of [ H]2-AG in astrocytoma cells (Analtech). TLC analyses were carried out using either (Fig. 1b). TLC analyses revealed that astrocytoma cells ethylacetate/methanol/water/ammonium hydroxide (20: rapidly metabolize [ H]2-AG (Fig. 2). After a 4 min incuba- 1:10:1) or chloroform/methanol/ammonium hydroxide tion, cell-associated radioactivity is distributed among TLC (80:20:1) as solvent system. The lipids were visualized with fractions migrating with monoacylglycerols (unmetabo- a 10% solution of phosphomolybdic acid (Sigma, Saint lized [ H]2-AG; 1384 46 d.p.m./well), free fatty acids Louis, MO) in ethanol followed by heating at 2508C. Bands (probably [ H]arachidonic acid, 560 64 d.p.m./well), with the mobility of authentic standards were scraped off phospholipids (6928 504 d.p.m./well), diacylglycerols the TLC plates for radioactivity determination. and triacylglycerols (370 52 d.p.m./well; results are from 120 (a) 120 (b) *** *** 0 0 3 3 Fig. 1. Effects of various compounds on [ H]2-AG and [ H]anandamide accumulation by astrocytoma cells. Intracellular accumulation of 3 3 [ H]anandamide (a) and [ H]2-AG (b) was measured in cells at con¯uence in the presence of the anandamide amidohydrolase inhibitor BTNP (5 ìM), the acyl-CoA synthetase inhibitor triacsin C (10 ìM), or the hydrolysis product, arachidonic acid (100 ìM). Results represent the mean s.e.m. ( nˆ 6± 9). p , 0.001 (ANOVA followed by Bonferroni multiple comparison test). 1232 Vol 11 No 6 27 April 2000 Control BTNP AA TrC Control BTNP AA TrC Total radioactivity accumulation (% of control) Total radioactivity accumulation (% of control) 2-ARACHIDONYLGLYCEROL INACTIVATION NEUROREPORT 2-AG AA 125 125 100 100 75 75 *** 50 50 *** 25 25 0 0 DAG-TAG PL 125 400 *** ** *** *** Fig. 2. Effects of various compounds on [ H]2-AG metabolism in astrocytoma cells. Cells at con¯uence were incubated in Tris ± Krebs' buffer containing [ H]2-AG with or without one of the following compounds: AM404, an anandamide transport inhibitor (10 ìM); BTNP, an anandamide amidohydrolase inhibitor (5 ìM); arachidonic acid (AA, 100 ìM); and triacsin C, an acyl CoA synthetase inhibitor (TrC, 10 ìM). Reactions were stopped by addition of cold methanol, and the lipids were extracted and analyzed by TLC. The lipids were stained with phosphomolybdic acid, identi®ed by comparison with authentic standards and quanti®ed by liquid scintillation counting. PL, phospholipids; DAG, diacylglycerols; TAG, triacylglycerols. p , 0.01, p , 0.001 (ANOVA followed by Bonferroni multiple comparison test). 3 3 one experiment representative of three). AM404 (10 ìM), with [ H]2-AG (1222 152 d.p.m./well) and [ H]- signi®cantly reduces the incorporation of radioactivity in arachidonic acid (631 75 d.p.m./well), but drastically the TLC fractions migrating with [ H]2-AG (413 54 reduces the radioactivity in phospholipids (to 1369 287 d.p.m./well), [ H]arachidonic acid (203 26 d.p.m./well) d.p.m./well). Arachidonic acid also augments incorpora- and phospholipids (2906 141 d.p.m./well). These results tion of radioactivity in diacylglycerols and triacylglycerols are in agreement with the ability of AM404 to inhibit (to 1335 42 d.p.m./well; Fig. 2). However, this increase is [ H]2-AG accumulation in astrocytoma cells, and under- not suf®cient to compensate for the substantial loss of score the intracellular localization of [ H]2-AG hydrolysis. radioactivity in the phospholipid fraction and we did not Conversely, the anandamide amidohydrolase inhibitor, (E)- investigate further its molecular basis. These ®ndings sug- 6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyr- gest that arachidonic acid inhibits [ H]2-AG accumulation an-2-one (BTNP; 5 ìM) [19] has no effect on [ H]2-AG indirectly, possibly by hindering the esteri®cation into accumulation (Fig. 1b) or metabolism (Fig. 2). phospholipids of the [ H]arachidonate produced by the 3 3 If arachidonic acid directly interfered with [ H]2-AG hydrolysis of [ H]2-AG. transport it should produce an effect similar to that of To test this possibility, we investigated the effects of AM404 (i.e. a complete inhibition of [ H]2-AG transport). triacsin C, a selective acyl-coenzyme A synthetase inhibitor In contrast with this prediction, arachidonic acid (100 ìM) [20], on [ H]2-AG uptake and metabolism. Incubation of does not affect radioactivity in the TLC fractions migrating astrocytoma cells with triacsin C (10 ìM) signi®cantly Vol 11 No 6 27 April 2000 1233 Control AM404 BTNP AA TrC Control Control AM404 AM404 BTNP BTNP AA AA TrC TrC Control AM404 BTNP AA TrC Radioactivity (% of control) Radioactivity (% of control) Radioactivity (% of control) Radioactivity (% of control) NEUROREPORT M. BELTRAMO AND D. PIOMELLI reduces both [ H]2-AG uptake (Fig. 1b) and incorporation player in 2-AG degradation in intact cells [23]. The trans- of [ H]arachidonate in phospholipids (Fig. 2). By contrast, port inhibitor AM404 dramatically reduces the intracellular the inhibitor has no effect on the levels of radioactivity in accumulation of [ H]2-AG whereas administration of ara- 3 3 3 [ H]2-AG, [ H]arachidonic acid, diacylglycerols and tria- chidonic acid has no effect on intracellular [ H]2-AG levels. cylglycerols (Fig. 2). In addition, triacsin C does not affect These ®ndings suggest that arachidonic acid may inhibit 3 3 [ H]anandamide accumulation (Fig. 1a) or its intracellular [ H]2-AG accumulation indirectly, possibly by hindering hydrolysis (data not shown). the esteri®cation into phospholipids of the [ H]- arachidonate produced by the hydrolysis of [ H]2-AG. DISCUSSION These results imply that arachidonate esteri®cation in membrane phospholipids may be a primary driving force Human astrocytoma CCF-STTG1 cells internalize [ H]- anandamide by a transport mechanism functionally indis- for [ H]2-AG transport. tinguishable from that found in rat brain neurons and CONCLUSIONS astrocytes [8,16]. Therefore, we used these cells to test the hypothesis that [ H]2-AG may be a substrate for the Our ®ndings suggest that 2-AG and anandamide are internalized in astrocytoma cells via a common carrier- anandamide transporter. The kinetic of [ H]2-AG accumu- lation in astrocytoma cells is consistent with a single high- mediated transport system. Four key observations support this hypothesis: (1) the kinetic properties of [ H]2-AG and af®nity transport process. In the same cells and under 3 3 identical conditions, [ H]anandamide transport displays [ H]anandamide accumulation are very similar; (2) 2-AG and anandamide mutually displace each other's accumula- similar characteristics (K of 0.6 0.1 ìM and V of m max ÿ1 ÿ1 14.7 1.5 pmol min mg protein) [16]. Several lines of tion; (3) [ H]2-AG uptake is blocked by the anandamide 3 3 3 transport inhibitor, AM404; (4) [ H]2-AG and [ H]- evidence indicate that [ H]2-AG accumulation in astrocyto- ma cells is mediated by a transport system akin to that anandamide uptake are insensitive to several substrates involved in anandamide internalization. First, non-radio- and inhibitors of lipid transporters and Na - and energy- active anandamide inhibits [ H]2-AG accumulation while, independent. By contrast, the intracellular degradation of conversely, 2-AG inhibits [ H]anandamide transport (IC 2-AG may differ substantially from that of anandamide: whereas anandamide hydrolysis is blocked by BTNP, an of 18.5 0.7 ìM) [16]. Second, the anandamide transport inhibitor AM404 is also effective, and even more potent in inhibitor of amidohydrolase activity, 2-AG hydrolysis is insensitive to this drug. This indicates that 2-AG break- interfering with [ H]2-AG uptake than anandamide or 2- AG. Third, a variety of substrates and inhibitors of lipid down in intact cells may depend on other hydrolase enzymes, such as monoacylglycerol lipase [23]. The role of transport systems have no effect on the accumulation of 3 3 either [ H]2-AG or [ H]anandamide [8,16]. Finally, ananda- hydrolysis in 2-AG disposition may differ from ananda- mide in another important way. We found that treating the mide and 2-AG transport are both Na and energy independent [8,16]. cells with arachidonic acid or the acyl-CoA synthetase inhibitor, triacsin C, prevents the transport of 2-AG, but There are, however, a number of differences between anandamide and 2-AG inactivation. [ H]Anandamide not that of anandamide. A plausible interpretation of these transport is not affected by arachidonic acid (Fig. 1a) and, results is that 2-AG hydrolysis into free arachidonic acid and the subsequent incorporation of arachidonic acid into vice versa,[ H]arachidonic acid transport is not affected by anandamide (data not shown), indicating that distinct membrane phospholipids may be a primary driving force for 2-AG internalization. This difference between the bio- membrane mechanisms mediate the internalization of these two lipid compounds [8,16]. By contrast, arachidonic acid logical dispositions of anandamide and 2-AG might be exploited for the development of selective inactivation substantially reduces the accumulation of [ H]2-AG (Fig. 1b). A possible interpretation of this ®nding is that inhibitors. arachidonic acid may directly interfere with the putative carrier involved in [ H]2-AG uptake, implying the exis- REFERENCES 1. Devane WA, Hanus L, Breuer A et al. Science 258, 1946 ± 1949 (1992). tence of different transport mechanisms for [ H]2-AG and 3 2. Di Marzo V, Fontana A, Cadas H et al. Nature 372, 686 ± 691 (1994). [ H]anandamide (sensitive and insensitive, respectively, to 3. Sugiura T, Kondo S, Sukagawa A et al. Biochem Biophys Res Commun 215, arachidonic acid). An alternative possibility is that arachi- 89 ± 97 (1995). donic acid may inhibit [ H]2-AG accumulation indirectly, 4. Mechoulam R, Ben-Shabat S, Hanus L et al. 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