We present here the pharmacological characterization of cannabinoid-binding sites in zebrafish brain homogenates using radiolabeled binding techniques. The nonselective agonist [3H]-CP55940 binds with high affinity (KD = 0.50 ± 0.06 nM and a Bmax = 1047 ± 36.01 fmol/mg protein), displaying one binding site. The slightly CB2 selective agonist [3H]-WIN55212-2 also binds with high affinity to zebrafish brain membranes displaying two different binding sites with affinities KD1 = 0.35 ± 0.09 nM and KD2 = 105.81 ± 66.36 nM. Competition binding assays using [3H]-WIN55212-2 and several unlabeled ligands were performed. WIN55212-2 significantly displaced the tritiated ligand binding showing the two binding sites observed with its tritiated homologous, while the slightly selective CB1 cannabinoid ligand HU-210, the nonselective cannabinoid ligand CP55940 and the endogenous cannabinoid ligand anandamide presented one binding site. Also, the functionality of these cannabinoid sites was analyzed using the known [35S]GTPγS assay. All the agonist used presented an agonist profile and the rank order for potency was HU-210 > WIN55212-2 > CP55940 > anandamide. Our results provide evidence that, although some of the typical cannabinoid ligands for mammalian receptors do not fully recognize the cannabinoid-binding sites in zebrafish brain, the activity of the endogenous zebrafish cannabinoid system might not significantly differ from that displayed by the cannabinoid system described in other species. Hence the study of zebrafish cannabinoid activity may contribute to an understanding of the endogenous cannabinoid system in higher vertebrates.
Bibliographical noteFunding Information:
This study was supported by grants from Ministerio de Educación y Ciencia (SAF2004-05144) and Junta de Castilla y León (SA080A05).
- Cannabinoid ligands
- Cannabinoid receptors
- Competition binding
- G protein activation
- Saturation binding