From - Wed Mar 25 00:26:44 1998

Subject: HT: very technical article on anandamide R

Source: Science, August 22, 1997 v277 n5329 p1094(4). Title: Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Author: M. Beltramo and D. Piomelli Author's Abstract: COPYRIGHT 1997 American Association for the Advancement of Science

Anandamide, an endogenous ligand for central cannabinoid receptors, is released from neurons on depolarization and rapidly inactivated. Anandamide inactivation is not completely understood, but it may occur by transport into cells or by enzymatic hydrolysis. The compound N-(4-hydroxyphenyl)arachidonylamide (AM404) was shown to inhibit high-affinity anandamide accumulation in rat neurons and astrocytes in vitro, an indication that this accumulation resulted from carrier-mediated transport. Although AM404 did not activate cannabinoid receptors or inhibit anandamide hydrolysis, it enhanced receptor-mediated anandamide responses in vitro and in vivo. The data indicate that carrier-mediated transport may be essential for termination of the biological effects of anandamide, and may represent a potential drug target. Subjects: Ligands (Biochemistry) - Research Cannabinoids - Research Electronic Collection: A20171331 RN: A20171331

Anandamide (arachidonylethanolamide) is an endogenous lipid that activates brain cannabinoid receptors and mimics the pharmacological effects of [Delta][sup.9]-tetrahydrocannabinol, the active principle of hashish and marijuana[1]. In humans, such effects include euphoria, calmness, dream states, and drowsiness[2]. Depolarized neurons release anandamide[3] through a mechanism that may require the calcium-dependent cleavage of a phospholipid precursor in neuronal membranes[4]. Like other modulatory substances, extracellular anandamide is thought to be rapidly inactivated, but the exact nature of this inactivating process is still unclear. A possible pathway is hydrolysis to arachidonic acid and ethanolamine, catalyzed by a membrane-bound fatty acid amide hydrolase (FAAH) highly expressed in rat brain and liver[5]. Nevertheless, the low FAAH activity found in brain plasma membranes indicates that this enzyme may be intracellular[5], a possibility that is further supported by sequence analysis of rat FAAH[6]. Although anandamide could gain access to FAAH by passive diffusion, the transfer rate is expected to be low because of the molecular size of this lipid mediator[7]. In that other lipids including polyunsaturated fatty acids and prostaglandin [E.sub.2] ([PGE.sub.2]) enter cells by carrier-mediated transport[8, 9], it is possible that anandamide uses a similar mechanism. Indeed, the existence of a rapid, saturable process of anandamide accumulation into neural cells has been reported[3]. This accumulation may result from the activity of a transmembrane carrier, which may thus participate in termination of the biological actions of anandamide. Accordingly, we developed drug inhibitors of anandamide transport and investigated their pharmacological properties in cultures of rat cortical neurons or astrocytes.

The accumulation of exogenous [[sup.3H]anandamide by neurons or astrocytes fulfills several criteria of a carrier-mediated transport (Fig. 1)[10]. It is a rapid process that reaches 50% of its maximum within about 4 min (Fig. 1A). Furthermore, [[sup.3H]anandamide accumulation is temperature-dependent (Fig. 1A) and saturable (Fig. 1, B and C). Kinetic analyses revealed that accumulation in neurons can be represented by two components of differing affinities (lower affinity: Michaelis constant, [K.sub.m], = 1.2 [mu]M, maximum accumulation rate, [V.sub.max], = 90.9 pmol/min per milligram of protein; higher affinity: [K.sub.m] = 0.032 [mu]M, [V.sub.max] = 5.9 pmol/min per milligram of protein) (Fig. 1B). The higher affinity component may reflect a binding site, however, as it is displaced by the cannabinoid receptor antagonist, SR-141716-A (100 nM)[11]. In astrocytes, [[sup.3H]]anandamide accumulation is represented by a single high-affinity component ([K.sub.m] = 0.32 [mu]M, [V.sub.max] = 171 pmol/min per milligram of protein) (Fig. 1C). Such apparent [K.sub.m] values are similar to those of known neurotransmitter uptake systems[12] and are suggestive therefore of high-affinity carrier-mediated transport.

To characterize further this putative anandamide transport, we used cortical astrocytes in culture. As expected from a selective process, the temperature-sensitive component of [[sup.3H]]anandamide accumulation was prevented by nonradioactive anandamide, but not by palmitoylethanolamide, arachidonate, prostanoids, or leukotrienes (Fig. 2A). Replacement of extracellular [Na.sup.+] with N-dimethylglucosamine or choline had no effect (as percentage of control: N-dimethylglucosamine, 124 [+ or -] 12%; choline, 98 [+ or -] 14%; mean [+ or -] SEM, n = 6), suggesting that [[sup.3H]]anandamide accumulation is mediated by a [Na.sup.+]-independent mechanism, which has been observed with other lipids[8, 9]. Moreover, inhibition of FAAH activity by treating the cells with (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (25 [mu]M) or linoleyl trifluoromethyl ketone (15 [mu]M)[13, 14] had no effect (Fig. 2, B and C). This indicates that anandamide hydrolysis did not provide the driving force for anandamide transport into astrocytes within the time frame of our experiments. Finally, the cannabinoid receptor agonist WIN-55212-2 (1 [mu]M) and antagonist SR-141716-A (10 [mu]M) also had no effect, suggesting that receptor internalization was not involved (Fig. 2A).

A primary criterion for defining carrier-mediated transport is pharmacological inhibition. To identify inhibitors of anandamide transport, we first examined compounds that prevent the cellular uptake of other lipids, such as fatty acids (phloretin, 50 [mu]M), phospholipids (verapamil, 100 [mu]M; quinidine, 50 [mu]M), or [PGE.sub.2] (bromcresol green, 0.1 to 100 [mu]M)[15]. Among the compounds tested, only bromcresol green interfered with anandamide transport, albeit with limited potency and partial efficacy (Fig. 3, A and B). Bromcresol green inhibited [[sup.3H]]anandamide accumulation with an [IC.sub.50] (concentration needed to produce half-maximal inhibition) of 4 [mu]M in neurons and 12 [mu]M in astrocytes and acted noncompetitively[16]. Moreover, bromcresol green had no significant effect on the binding of [[sup.3H]]WIN-55212-2 to rat cerebellar membranes (inhibition constant, [K.sub.i], = 22 [mu]M), FAAH activity in rat brain microsomes ([IC.sub.50] [is greater than] 50 [mu]M), and uptake of [[sup.3H]]arachidonate or [[sup.3H]]ethanolamine in astrocytes (121 [+ or -] 13% and 103 [+ or -] 12%, respectively, at 50 [mu]M bromcresol green, n = 3)[17]. The sensitivity to bromcresol green, which blocks [PGE.sub.2] transport, raised the question of whether anandamide accumulation occurred by means of a [PGE.sub.2] carrier. That this is not the case was shown by the lack of [[sup.3]]H[PGE.sub.2] accumulation in neurons or astrocytes[18] and by the inability of [PGE.sub.2] to interfere with [[sup.3H]]anandamide accumulation (Fig. 2A). Previous results indicating that expression of [PGE.sub.2] transporter mRNA in brain tissue is not detectable further support this conclusion[9].

To search for more potent anandamide transport inhibitors, we synthesized and tested a series of structural analogs of anandamide[19]. From this screening, we selected the compound N-(4-hydroxyphenyl)arachidonylamide (AM404), which was both efficacious and relatively potent (Fig. 3, C and D; [IC.sub.50] was 1 [mu]M in neurons and 5 [mu]M in astrocytes). As we anticipated from its chemical structure, AM404 acted as a competitive inhibitor[20], suggesting that it may serve as a transport substrate or pseudosubstrate. In contrast, at the concentrations tested AM404 had no effect on FAAH activity ([IC.sub.50] [is greater than] 30 [mu]M) or on uptake of [[sup.3H]]arachidonate or [[sup.3H]]ethanolamine (102 [+ or -] 4% and 96 [+ or -] 14%, respectively, at 20 [mu]M AM404, n = 6). Furthermore, a positional isomer of AM404, N-(3-hydroxyphenyl)-arachidonylamide (AM403), was significantly less effective than AM404 in inhibiting transport (Fig. 3, C and D). These data provide pharmacological evidence for the existence of a specific anandamide transporter and suggest (i) that neurons and astrocytes may act synergistically in the brain to dispose of extracellular anandamide and (ii) that the transport systems in these two cell types may differ kinetically and pharmacologically (Fig. 1, B and C, and Fig. 3, C and D).

The identification of inhibitors allowed us to examine whether transmembrane transport participates in terminating anandamide responses mediated by cannabinoid receptor activation. Cannabinoid receptors of the CB1 subtype are expressed in neurons[21] where they are negatively coupled to adenylyl cyclase activity[22]. Accordingly, in cultures of rat cortical neurons the cannabinoid receptor agonist WIN-55212-2 inhibited forskolin-stimulated adenosine 3',5'-monophosphate (cAMP) accumulation (control: 39 [+ or -] 4 pmol per milligram of protein; 3 [mu]M forskolin: 568 [+ or -] 4 pmol per milligram of protein; forskolin plus 1 [mu]M WIN-55212-2: 220 [+ or -] 24 pmol per milligram of protein), and this inhibition was prevented by the antagonist SR-141716-A (1 [mu]M) (555 [+ or -] 39 pmol/mg of protein, n = 9)[23]. Anandamide produced a similar effect, but with a potency ([IC.sub.50], 1 [mu]M) that was 1/20 of that expected from its binding constant for CB1 cannabinoid receptors ([K.sub.i] [is nearly equal to] 50 nM)[1] (Fig. 4A). The transport inhibitor AM404 bound to CB1 receptors with low affinity ([K.sub.i] = 1.8 [mu]M)[19] and did not reduce cAMP concentrations when applied at 10 [mu]M (Fig. 4B). Nevertheless, the drug enhanced the effects of anandamide, increasing the potency (by a factor of[10] and decreasing the threshold (by a factor of 1/100), an effect that was prevented by SR-141716-A (Fig. 4A). Thus, a concentration of anandamide that was below threshold when applied alone (0.3 [mu]M) produced an almost maximal effect when applied with AM404 (Fig. 4B). Bromcresol green and AM403, which were less effective than AM404 in inhibiting anandamide transport (Fig. 3), were also less effective in enhancing the anandamide response (Fig. 4B). Furthermore, the decreases in cAMP concentrations produced by WIN-55212-2 (which stimulates CB1 receptors but is not subject to physiological clearance) or glutamate [which stimulates metabotropic receptors negatively coupled to adenylyl cyclase[24] and is cleared by a selective transporter[25]] are not affected by any of the anandamide transport inhibitors tested[26].

These results suggest that pharmacological blockade of carrier-mediated transport protects anandamide from physiological inactivation, enhancing the potency of anandamide to nearly that expected from its affinity for CB1 cannabinoid receptors in vitro. To find out whether this potentiation occurs in vivo, we tested the effects of AM404 on the antinociceptive activity of anandamide in mice. Intravenous anandamide (20 mg per kilogram of body weight) elicited a modest but significant analgesia, as measured by the hot plate test[27] (P [is less than] 0.05, Student's t test); this analgesia disappeared 60 min after injection and was prevented by SR-141716-A (Fig. 4C)[28]. Administration of AM404 (10 mg/kg, intravenously) had no antinociceptive effect within 60 min of injection but significantly enhanced and prolonged anandamide-induced analgesia (Fig. 4C) (P [is less than] 0.01, Student's t test).

Our findings indicate that a high-affinity transport system present in neurons and astrocytes has a role in anandamide inactivation by removing this lipid mediator from the extracellular space and delivering it to intracellular metabolizing enzymes such as FAAH[5, 6]. Therefore, the identification of selective inhibitors of anandamide transport should be instrumental in understanding the physiological roles of the endogenous cannabinoid system and may lead to the development of therapeutic agents.

REFERENCES AND NOTES

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[10.] Cultures of cortical neurons [N. Stella, L. Pellerin, P. Magistretti, J. Neurosci. 15, 3307 (1995)] or astrocytes (13) were prepared from rat embryos and were used after 4 to 6 days and 21 to 25 days in vitro, respectively. Accumulation of [[sup.3H]]anandamide (221 Ci/mmol, New England Nuclear, Wilmington, DE) was measured by incubating the cells (six-well plates) for various times in Krebs buffer [136 mM NaCl, 5 mM KCI, 1.2 mM Mg[Cl.sub.2], 2.5 mM Ca[Cl.sub.2], 10 mM glucose, and 20 mM Trizma base; (pH 7.4), at 37[degrees]C containing [[sup.3H]]anandamide (0.45 nM, brought to 100 nM with nonradioactive anandamide). Incubations were stopped by aspirating the media, and cells were rinsed with Krebs buffer containing bovine serum albumin (BSA, 0.1% w/v) and subjected to extraction with methanol and chloroform. Radioactivity in the extracts was measured directly or after fractionation of cell lipids by thin-layer chromatography[13]. For kinetic analyses, the neurons were incubated for 4 min at 37[degrees]C in the presence of 10 to 500 nM anandamide containing 0.05 to 2.5 nM [[sup.3H]]anandamide. We subtracted nonspecific accumulation (measured at 0[degrees] to 4[degrees]C before determining kinetic constants by Lineweaver-Burk analysis.

[11.] M. Beltramo and D. Piomelli, unpublished data.

[12.] E. L. Barker and R. D. Blakely, in Psychopharmacology: The Fourth Generation of Progress, F. E. Bloom and D. J. Kupfer, Eds. (Raven, New York, 1995), pp. 321-334. In a previous study with mixed cultures of rat cortical neurons and astrocytes, a [K.sub.m] of 30 [mu]M for [[sup.H]]anandamide accumulation was obtained (3). Such a high value likely resulted from the low specific radioactivity (0.3 mCi/mmol) of the [[sup.3H]]anandamide used.

[13.] M. Beltramo, E. di Tomaso, D. Piomelli, FEBS Lett. 403, 263 (1997).

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[16.] In astrocytes, [V.sub.max] values for [[sup.3H]]anandamide accumulation were 200 pmol/min per milligram of protein without bromcresol green, and 111 pmol/min per milligram of protein with bromcresol green (10 [mu]M). Apparent [K.sub.m] values were 0.24 and 0.25 [mu]M, respectively (n = 6).

[17.] Displacement of [[sup.3H]]WIN-55212-2 binding (40 to 60 Ci/mmol; New England Nuclear) to rat cerebellar membranes (0.1 mg/ml) was determined as described [J. E. Kuster et a/., J. Pharmacol. Exp. Ther. 264, 1352 (1993)]. Nonspecific binding was measured in the presence of 1 [mum]M nonradioactive WIN-55212-2. FAAH activity was measured in rat brain particulate fractions as described (13). The uptake of [[sup.3H]]arachidonate (Amersham, 200 Ci/mmol; 5 nM brought to 100 nM) and [[sup.3H]]ethanolamine (Amersham, 50 Ci/mmol; 20 nM brought to 100 nM) was determined on cortical astrocytes for 4 min as described (10). The control uptake for [[sup.3H]]arachidonate was 16729 [+ or -] 817 dpm per well and for [[sup.3H]]ethanolamine it was 644 [+ or -] 100 dpm per well (n = 6).

[18.] Neurons or astrocytes were incubated for 4 min at 37[degrees]C in Krebs buffer containing [[sup.3H]][PGE.sub.2] (0.67 nM brought to 100 nM with nonradioactive [PGE.sub.2]; 171 Ci/mmol, New England Nuclear). After rinsing with Krebs buffer containing BSA, we subjected the cells to lipid extraction and counted radioactivity in the extracts. On average, neurons contained 245 [+ or -] 65 dpm per well and astrocytes 302 [+ or -] 20 dpm per well; nonspecific accumulation in astrocytes at 0[degrees] to 4[degrees]C was 355 [+ or -] 28 dpm per well (n = 6).

[19.] A. Khanolkar et al., J. Med. Chem. 39, 4515 (1996).

[20.] In astrocytes, apparent [K.sub.m] values for [[sup.3H]]anandamide accumulation were 0.11 [mu]M without AM404 and 0.27 [mu]M with AM404 (10 [mu]M).[V.sub.max] values were 29 pmol/min per milligram of protein without AM404 and 26 pmol/min per milligram of protein with AM404, respectively (n = 6).

[21.] M. Herkenham et al., Proc. Natl. Acad. Sci. U.S.A. 87, 1932 (1990); L. A. Matsuda, T. I. Bonner, S. J. Lolait, J. Comp. Neurol. 327, 535 (1993).

[22.] A. C. Howlett, J. M. Qualy, L. L. Khachatran, Mol. Pharmacol. 29, 307 (1986); M. Jung et al., J. Neurochem. 68 402 (1997).

[23.] Cortical neurons were prepared in 12-well plates and used after 4 to 6 days in vitro. Incubations were carried out in the presence of forskolin (3 [mu]M) and isobutyl methyl xanthine (1 mM). The cAMP concentrations were measured by radioimmunoassay with a commercial kit (Amersham, Arlington, IL) and following manufacturer's instructions.

[24.] L. Prezeau et al., Proc. Natl. Acad. Sci. U.S.A. 89, 8040 (1992).

[25.] R. Maki, M. B. Robinson, M. A. Dichter, J. Neurosci. 14, 6754 (1994).

[26.] The amounts of cAMP in the presence of a concentration of WIN-55212-2 below threshold (1 nM, determined in preliminary experiments) were 96.7 [+ or -] 2.5% of forskolin alone and were not significantly affected by 10 [mu]M AM404 (89.8 [+ or -] 2.6%,), 10 [mu]M AM403 (92.4 [+ or -] 2.3%), or 10 [mu]M bromcresol green (92.9 [+ or -] 2.3%) (n = 3). In the presence of a concentration of glutamate below threshold (3 [mu]M) (24), cAMP concentrations were 91.6 [+ or -] 2% of forskolin alone and were not significantly affected by AM404 (84.4 [+ or -] 4.9%), AM403 (89.5 [+ or -] 2.4%), or bromcresol green (84.4 [+ or -] 3%) (n = 3).

[27.] E. Fride and R. Mechoulam, Eur. J. Pharmacol. 231, 313 (1993); P. B. Smith et al., J. Pharmacol. Exp. Ther. 270, 219 (1994).

[28.] The hot plate test (55.5[degrees]C was carried out on male Swiss mice (25 to 30 g, Nossan, Italy) following standard procedures [F. Porreca, H. L. Mosberg, R. Hurst, V. J. Hruby, T. F. Burks, J. Pharmacol. Exp. Ther. 230, 341 (1994)]. Anandamide and AM404 were dissolved in 0.9% NaCl solution containing 20% dimethyl sulfoxide and injected intravenously at 20 mg/kg and 10 mg/kg, respectively. To determine whether cannabinoid receptors participate in the effect of anandamide, we administered anandamide (20 mg/kg intravenously) or anandamide plus SR141716-A (2 mg/kg, subcutaneously) to two groups of six mice each. In mice that received anandamide alone, latency to jump increased from 21.7 [+ or -] 1.5 s to 30.7 [+ or -] 0.8 s (P [is less than] 0.05, ANOVA) 20 min after injection. In contrast, in mice that received anandamide plus SR141716-A, the latency to jump was not affected (19.6 [+ or -] 3.1 s).

[29.] We thank E. di Tomaso and H. Cadas for help and E. Barker, L. Parsons, and P. Schweitzer for critical reading of the manuscript. Supported by the Neuroscience Research Foundation, which receives major support from Novartis. -- End --