Reversal of dopamine D(2) receptor responses by an anandamide transport inhibitor

Abstract

We characterized the pharmacological properties of the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) in rats and investigated the effects of this drug on behavioral responses associated with activation of dopamine D(2) family receptors. Rat brain slices accumulated [(3)H]anandamide via a high-affinity transport mechanism that was blocked by AM404. When administered alone in vivo, AM404 caused a mild and slow-developing hypokinesia that was significant 60 min after intracerebroventricular injection of the drug and was reversed by the CB1 cannabinoid receptor antagonist SR141716A. AM404 produced no significant catalepsy or analgesia, two typical effects of direct-acting cannabinoid agonists. However, AM404 prevented the stereotypic yawning produced by systemic administration of a low dose of apomorphine, an effect that was dose-dependent and blocked by SR141716A. Furthermore, AM404 reduced the stimulation of motor behaviors elicited by the selective D(2) family receptor agonist quinpirole. Finally, AM404 reduced hyperactivity in juvenile spontaneously hypertensive rats, a putative model of attention deficit hyperactivity disorder. The results support a primary role of the endocannabinoid system in the regulation of psychomotor activity and point to anandamide transport as a potential target for neuropsychiatric medicines.

Fig. 1.

Selectivity of [³H]anandamide transport in rat brain slices. Accumulation was measured in coronal half-slices after a 5 min incubation with [³H]anandamide at 37°C in the absence (control) or presence of nonradioactive anandamide (AEA; 100 μm), palmitylethanolamide (PEA; 100 μm), arachidonic acid (AA; 100 μm), prostaglandin E₂ (PGE₂; 100 μm), digoxin (100 μm), AM404 (10 μm), or (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BTNP; 5 μm). Nonspecific association of [³H]anandamide to the slices was measured at 0–4°C. Results are expressed as mean ± SEM; number of independent determinations are indicated within the bars. **p < 0.01 by ANOVA, followed by Dunnett's test.

Fig. 2.

Effects of the anandamide transport inhibitor AM404 on motor activity and pain threshold in rats. A1, Time course of the effects of vehicle (open bars), AM404 (filled bars), and AM404 plus SR141716A (hatched bars) on time spent in immobility.A2, Cumulative time spent in immobility. AM404 (10 μg/rat) or vehicle (DMSO, 5 μl) were administered by intracerebroventricular injection. The CB1 antagonist SR141716A (1 mg/kg) or vehicle (aqueous 0.9% NaCl containing 10% DMSO, 0.2 ml/kg) was administered intraperitoneally 60 min before intracerebroventricular injection of AM404. Values shown represent the mean ± SEM; number of experiments is indicated within the bars. *p < 0.05 compared with vehicle or AM404 plus SR141716A; one-way ANOVA, followed by Student–Newman–Keuls test for pairwise comparisons. B1–B5, Lack of effect of AM404 on various motor behaviors measured 60 min after drug injection.B6, Lack of effect of AM404 on latency to jump measured in the hot plate test 60 min after drug injection.

Fig. 3.

Effects of AM404 on apomorphine-induced yawning.A, The effect of AM404 (2 μg/rat) or vehicle (DMSO, 5 μl), injected intracerebroventricularly 5 min before apomorphine, was prevented by intravenous administration of SR141716A (SR; 0.2 mg/kg, 15 min before apomorphine).B, Effects of systemic injections of vehicle (subcutaneously, aqueous 0.9% NaCl containing 10% DMSO), AM404 (10–20 mg/kg, s.c.), SR141716A (0.2 mg/kg, i.v.), and AM404 plus SR141716A on apomorphine-induced yawning. AM404 or vehicle were injected 30 min before and SR141716A 45 min before apomorphine.C, Effects of systemic injections of vehicle (subcutaneously, aqueous 0.9% NaCl containing 10% DMSO), anandamide (AEA; 0.1–10 mg/kg), and anandamide plus SR141716A on apomorphine-induced yawning. Values shown represent the mean ± SEM; number of experiments is indicated within the bars. **p < 0.01 compared with apomorphine.

Fig. 4.

Effects of AM404 on the behavioral responses elicited by the dopamine D₂ family agonist quinpirole. Time course of the effects of vehicle (open bars), AM404 (filled bars), quinpirole (striped bars), and AM404 plus quinpirole (hatched bars) on locomotor activity (A) and time spent in immobility (B). Quinpirole (0.25 mg/kg) or vehicle (aqueous 0.9% NaCl, 0.2 ml/kg) were administered intraperitoneally 30 min after intracerebroventricular injection of AM404 (10 μg/rat) or vehicle (DMSO, 5 μl). Values shown represent the mean ± SEM; number of experiments is indicated within the bars. *p < 0.05 compared with vehicle.

Fig. 5.

Effects of AM404 on horizontal and vertical activity in SHR and control WKY rats. Time-dependent effects of vehicle (DMSO, 1 ml/kg; open bars) or AM404 (1 mg/kg, s.c.;filled bars) on duration of rearing episodes (A) and horizontal activity (assessed as frequency of corner crossings; P < 0.001 compared with vehicle) (B) in SHR and WKY rats. Values shown represent the mean ± SEM of n = 5 per group. *p < 0.05 compared with vehicle.