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Reversal of Dopamine D2 Receptor Responses by an Anandamide Transport Inhibitor

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Reversal of Dopamine D2 Receptor Responses by an Anandamide Transport Inhibitor The Journal of Neuroscience, May 1, 2000, 20(9):3401–3407 Reversal of Dopamine D2 Receptor Responses by an Anandamide Transport Inhibitor Massimiliano Beltramo,1 Fernando Rodrı´guez de Fonseca,2 Miguel Navarro,2 Antonio Calignano,4 Miquel Angel Gorriti,2 Georgios Grammatikopoulos,5 Adolfo G. Sadile,5 Andrea Giuffrida,3 and Daniele Piomelli1 1The Neurosciences Institute, San Diego, CA 92121, 2Department of Psychobiology, Complutense University, Madrid, Spain 28233, 3Department of Pharmacology, University of Naples, Italy, 80131, 4Laboratory of Neurophysiology, Behavior and Neural Networks, II University of Naples, Italy, 80138, and 5Department of Pharmacology, University of California, Irvine, California 92697-4625 We characterized the pharmacological properties of the anan- produced by systemic administration of a low dose of apomor- damide transport inhibitor N-(4-hydroxyphenyl)-arachidonamide phine, an effect that was dose-dependent and blocked by (AM404) in rats and investigated the effects of this drug on SR141716A. Furthermore, AM404 reduced the stimulation of behavioral responses associated with activation of dopamine motor behaviors elicited by the selective D2 family receptor 3 D2 family receptors. Rat brain slices accumulated [ H]anand- agonist quinpirole. Finally, AM404 reduced hyperactivity in ju- amide via a high-affinity transport mechanism that was blocked venile spontaneously hypertensive rats, a putative model of by AM404. When administered alone in vivo, AM404 caused a attention deficit hyperactivity disorder. The results support a mild and slow-developing hypokinesia that was significant primary role of the endocannabinoid system in the regulation of 60 min after intracerebroventricular injection of the drug and psychomotor activity and point to anandamide transport as a was reversed by the CB1 cannabinoid receptor antagonist potential target for neuropsychiatric medicines. SR141716A. AM404 produced no significant catalepsy or an- Key words: AM404; anandamide transport; cannabinoid re- algesia, two typical effects of direct-acting cannabinoid ago- ceptors; dopamine receptors; spontaneously hypertensive rats; nists. However, AM404 prevented the stereotypic yawning Wistar-Kyoto rats Cannabinoid receptors, the target of the marijuana constituent to locally administered CB1 receptor agonists (San˜udo-Pen˜a et ⌬ 9-tetrahydrocannabinol (Pertwee, 1997), are densely expressed al., 1996, 1998; San˜udo-Pen˜a and Walker, 1998). Finally, chronic in basal ganglia and cortex, regions of the CNS that are critical for treatment with D2 family antagonists results in upregulated ex- the control of cognition, motivation, and movement (Herkenham pression of CB1 receptor mRNA in striatum (Mailleux and et al., 1990; Matsuda et al., 1993; Tsou et al., 1998). This distri- Vanderhaeghen, 1993). Together, these findings suggest that one bution provides multiple opportunities for functional interactions of the functions of anandamide in the CNS may be to modulate between endogenous cannabinoid substances, such as anandam- dopamine D2 receptor-induced facilitation of psychomotor activ- ide (Devane et al., 1992; Di Marzo et al., 1994), and ascending ity. In agreement with this possibility, anandamide and other CB1 dopamine pathways. That these interactions may occur in vivo is agonists inhibit movement, produce catalepsy, and attenuate indicated by several observations. First, in the striatum of freely d-amphetamine-induced hyperactivity and stereotypy (Pryor et moving rats, anandamide release is greatly increased after acti- al., 1978; Gorriti et al., 1999), whereas disruption of the CB1 vation of dopamine D2 family receptors with the selective agonist receptor gene profoundly affects movement control (Ledent et quinpirole (Giuffrida et al., 1999). Second, pretreatment with the al., 1999; Zimmer et al., 1999). CB1 cannabinoid antagonist SR141716A enhances the stimula- When anandamide is administered as a drug, its effects are tion of motor behavior elicited by systemic administration of curtailed by a two-step mechanism consisting of transport into quinpirole (Giuffrida et al., 1999), although it has little effect per cells, mediated by a high-affinity carrier system (Beltramo et al., se on basal motor activity (Rinaldi-Carmona et al., 1994; Comp- 1997b; Hillard et al., 1997; Piomelli et al., 1999), followed by ton et al., 1996; Navarro et al., 1997). Third, injection of D2 family intracellular hydrolysis, catalyzed by a relatively nonselective agonists into basal ganglia nuclei opposes the behavioral response amidohydrolase enzyme (Deutsch and Chin, 1993; De´sarnaud et al., 1995; Cravatt et al., 1996). Consequently, the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) Received Dec. 23, 1999; accepted Feb. 11, 2000. This work was supported by National Institute on Drug Abuse Grants DA12413 prolongs and enhances several responses to exogenous anandam- and DA12447 (to D.P.), DGICYT, Comunidad de Madrid and Plan Nacional sobre ide, including analgesia (Beltramo et al., 1997b) and vasodilata- Drogas (to M.N. and F.R.F.), and Telethon Italy Grant E513 (to A.G.S.). F.R.F. and tion (Calignano et al., 1997a). We hypothesized that blockade of M.N. are Jaime del Amo Research Fellows at the Complutense University. We thank A. Bilbao, U. Gironi Carnevale, J. Mun˜oz, and M. Pignatelli for their expert anandamide transport, by causing this lipid to accumulate at its assistance. We also thank Dr. L. H. Parsons for critical reading of this manuscript. sites of release, may help uncover a participation of anandamide Correspondence should be addressed to Daniele Piomelli, Department of Phar- macology, 360 MSR II, University of California, Irvine, CA 92697-4625. E-mail: in the control of dopamine neurotransmission and might offer a [email protected]. pharmacological strategy to correct pathological conditions char- Copyright © 2000 Society for Neuroscience 0270-6474/00/203401-07$15.00/0 acterized by dopaminergic dysfunction. To test this hypothesis, 3402 J. Neurosci., May 1, 2000, 20(9):3401–3407 Beltramo et al. • Endocannabinoid Modulation of Dopamine Effects we investigated the pharmacological properties of AM404 in the (Calignano et al., 1997a), and inhibition of intestinal motility in mice rat CNS and examined the effects of this drug on behavioral (Calignano et al., 1997b). These results indicate that AM404 does not act as an agonist at CB1 receptors either in vitro or in vivo. Moreover, responses elicited by the activation of D2 family receptors. AM404 did not prevent the inhibition of forskolin-induced cAMP accu- mulation produced in cortical neurons by the application of WIN- MATERIALS AND METHODS 55212–2, indicating that the drug does not act as a partial agonist on antagonist at CB1 receptors (Beltramo et al., 1997a). CB2 receptors do Characterization of anandamide transport. Coronal slices (0.45-mm-thick) not appear to be expressed in the CNS (Ledent et al., 1999; Zimmer et from adult rat brain were prepared with a vibratome and split along the al., 1999); thus, the interaction of AM404 with these receptors was not midline with a razor blade. Each half was collected separately and investigated in the present experiments. allowed to equilibrate for 2.5 hr at 37°C in Tris-Krebs’ buffer (in mM: Surgery. Implantation of stainless steel guide cannulas and intracere- NaCl 136, KCl 5, MgCl2 1.2, CaCl2 2.5, glucose 10, and Trizma base 20, broventricular injections were performed in lateral ventricles of male pH 7.4) aerated with 5% CO in O . The slices were incubated under 2 2 Wistar rats (Ͼ8 weeks old, 300–350 gm) as described previously (Rodrı´- agitation for 10 min in Tris-Krebs’ buffer containing test compounds, guez de Fonseca et al., 1996). AM404 (dissolved in 5 ␮l of DMSO; Tocris followed bya5minincubation in the presence of [ 3H]anandamide (30 5 Cookson, Ballwin, MO) or DMSO was injected via an 8 mm 30 gauge nM, 1.8 ϫ 10 dpm/ml, 221 Ci/mmol; NEN, Wilmington, DE) [the injector connected to a calibrated polyethylene-10 tubing. Doses were not Michaelis constant (K ) for anandamide transport in rat brain astrocytes M corrected for recovery after passage through the polyethylene tubing (see is 0.32 ␮M (Beltramo et al., 1997b)] and appropriate concentrations of above); thus, they represent an overestimate of the actual amount deliv- vehicle or test compounds. In all experiments, SR141716A [0.1 ␮M; ered to the tissue. Cannula placements were evaluated by injection of a provided by Research Biochemicals (Natick, MA) as part of the Chem- blue dye, and only those rats with proper intracerebroventricular place- ical Synthesis Program of the National Institute of Mental Health (Grant ments were included in the data analysis. NO1MH30003)] was added to the incubations to prevent binding of Effects of AM404 on apomorphine-induced yawning. Apomorphine- [ 3H]anandamide to CB1 receptors. At the end of the incubation period, induced yawning was measured in transparent plastic boxes (35 ϫ 30 ϫ the slices were rinsed with Tris-Krebs’ buffer containing fatty acid-free 17 cm) following established procedures (Yamada and Furukawa, 1980; bovine serum albumin (0.1%) and homogenized in Tris-Krebs’ buffer/ ␮ ␮ methanol (1:1, v/v), and radioactivity was measured by liquid scintillation Dourish et al., 1989). AM404 (2 g/rat) or vehicle (DMSO, 5 l/rat) were administered 5 min before subcutaneous injection of apomorphine counting. ␮ Selectivity of AM404. [ 3H]AM404 (arachidonyl-5,6,8,11,12,14,15- 3H; (80 g/kg) or vehicle (aqueous 0.9% NaCl containing 40% DMSO, 0.2 200–240 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO) ml/kg). Yawning was measured for a 30 min period after apomorphine was radioactively pure (Ͼ99%) by HPLC. [ 3H]AM404 [10 ␮g containing injection. Intraperitoneal injections of AM404 (10 and 20 mg/kg), anan- 2.8 ϫ 10 6 dpm in 5 ␮l of dimethylsulphoxide (DMSO)] was administered damide (0.1, 1, and 10 mg/kg), or vehicle (0.2 ml of aqueous 0.9% NaCl by intracerebroventricular injection in cannulated rats (Taconic Farms, containing 10% DMSO) were done 30 min before apomorphine admin- Germantown, NY) through a calibrated polyethylene-10 tubing. Average istration. DMSO alone had no effect on yawning (data not shown).
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