The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB 1 receptors, is still a matter of debate. In the present study, we report that CB 1 and adenosine A 2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB 1 receptor signaling was found to be completely dependent on A 2A receptor activation. Accordingly, blockade of A 2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB 1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A 2A and CB 1 receptors.

Adenosine, by acting on adenosine A 1 and A 2A receptors, exerts opposite modulatory roles on striatal extracellular levels of glutamate and dopamine, with activation of A 1 inhibiting and activation of A 2A receptors stimulating glutamate and dopamine release. Adenosine-mediated modulation of striatal dopaminergic neurotransmission could be secondary to changes in glutamate neurotransmission, in view of evidence for a preferential colocalization of A 1 and A 2A receptors in glutamatergic nerve terminals. By using in vivo microdialysis techniques, local perfusion of NMDA (3, 10 lM), the selective A 2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5¢-N-ethylcarboxamidoadenosine (CGS 21680; 3, 10 lM), the selective A 1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 300, 1000 lM), or the non-selective A 1 -A 2A receptor antagonist in vitro caffeine (300, 1000 lM) elicited significant increases in extracellular levels of dopamine in the shell of the nucleus accumbens (NAc). Significant glutamate release was also observed with local perfusion of CGS 21680, CPT and caffeine, but not NMDA. Co-perfusion with the competitive NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV; 100 lM) counteracted dopamine release induced by NMDA, CGS 21680, CPT and caffeine. Co-perfusion with the selective A 2A receptor antagonist MSX-3 (1 lM) counteracted dopamine and glutamate release induced by CGS 21680, CPT and caffeine and did not modify dopamine release induced by NMDA. These results indicate that modulation of dopamine release in the shell of the NAc by A 1 and A 2A receptors is mostly secondary to their opposite modulatory role on glutamatergic neurotransmission and depends on stimulation of NMDA receptors. Furthermore, these results underscore the role of A 1 vs. A 2A receptor antagonism in the central effects of caffeine.

The cyclic peptide zyklophin {[N-benzylTyr 1 ,cyclo(D-Asp 5 ,Dap 8 )-dynorphin A-(1-11)NH 2, Patkar KA, et al. (2005) J Med Chem 48: 4500 -4503} is a selective peptide kappa opioid receptor (KOR) antagonist that shows activity following systemic administration. Systemic (1-3 mg/kg s.c.) as well as central (0.3-3 nmol intracerebroventricular, i.c.v.) administration of this peptide dose-dependently antagonizes the antinociception induced by the selective KOR agonist U50,488 in C57BL/6J mice tested in the 55°C warm water tail withdrawal assay. Zyklophin administration had no effect on morphine-or SNC-80-mediated antinociception, suggesting that zyklophin selectively antagonizes KOR in vivo. Additionally, the antagonism of antinociception induced by centrally (i.c.v.) administered U50,488 following peripheral administration of zyklophin strongly suggests that the peptide crosses the blood-brain barrier to antagonize KOR in the CNS. Most importantly, the antagonist activity of zyklophin (3 mg/kg s.c.) lasts less than 12 h, which contrasts sharply with the exceptionally long duration of antagonism reported for the established small-molecule selective KOR antagonists such as norbinaltorphimine (nor-BNI) that last weeks after a single administration. Systemically administered zyklophin (3 mg/kg s.c.) also prevented stress-induced reinstatement of cocaine-seeking behavior in a conditioned place preference assay. In conclusion, the peptide zyklophin is a KOR-selective antagonist that exhibits the desired shorter duration of action, and represents a significant advance in the development of KOR-selective antagonists.cocaine abuse ͉ dynorphin A analog ͉ kappa opioid receptor antagonist ͉ opioid peptide ͉ stress I nterest in kappa opioid receptor (KOR) ligands has focused on agonists that have potential therapeutic applications, such as anti-inflammatory activity (1) and the acute suppression of the rewarding effects of cocaine (2) [see (3) for a review], in addition to their analgesic properties. Until recently, KOR-selective antagonists have only been used as pharmacological tools. However, recent reports describing antidepressant-like effects (4-6), anxiolytic-like effects (7,8), efficacy against opiate dependence (9), and the ability to prevent stress-induced reinstatement of cocaine-seeking behavior (5, 10) indicate that KOR antagonists could be promising therapeutic agents [see (3) for a review].Several selective non-peptide antagonists, in particular norbinaltorphimine (nor-BNI) (11), 5Ј-guanidinonaltrindole (GNTI) (12), and the phenylpiperidine JDTic (13), have been studied extensively in animal models (4-8, 14-16). All of these compounds, however, exhibit exceptionally long activity in vivo, lasting weeks to more than a month after a single dose [see (14) for a detailed review], an effect that could limit their therapeutic use. While the mechanism producing this prolonged activity is poorly understood, to date there have been no reports of small molecule-selective KOR antagonists with activity lasting less than 1...

BACKGROUND AND PURPOSECyclic peptides are resistant to proteolytic cleavage, therefore potentially exhibiting activity after systemic administration. We hypothesized that the macrocyclic k opioid receptor ( CONCLUSIONS AND IMPLICATIONSThe macrocyclic tetrapeptide [D-Trp]CJ-15,208 is a short-duration KOR antagonist with weak KOR agonist activity that is active after oral administration and demonstrates blood-brain barrier permeability. These data validate the use of systemically active peptides such as [D-Trp]CJ-15,208 as potentially useful therapeutics. Abbreviations