Behavioral, pharmacological, and molecular characterization of an amphibian cannabinoid receptor

Investigation of cannabinoid pharmacology in a vertebrate with a phylogenet ic history distinct from that of mammals may allow better understanding of the physiological significance of cannabinoid neurochemistry. Taricha granu losa, the roughskin newt, was used here to characterize an amphibian cannab inoid receptor. Behavioral experiments demonstrated that the cannabinoid ag onist levonantradol inhibits both newt spontaneous locomotor activity and c ourtship clasping behavior, Inhibition of clasping was dose-dependent and p otent (IC50 = 1.2 mu g per animal). Radioligand binding studies using [H-3] CP-55940 allowed identification of a specific binding site (K-D = 6.5 nM, B -max = 1,853 fmol/mg of protein) in brain membranes. Rank order of affinity of several ligands was consistent with that reported for mammalian species (K-D, nM): CP-55940 (3.8) > levonantradol (13.0) > WIN55212-2 (25.7) much greater than anandamide (1,665) approximate to anandamide + 100 mu M phenyl methylsulfonyl fluoride (2,398). The cDNA encoding the newt CB1 cannabinoid receptor was cloned, and the corresponding mRNA of 5.9 kb was found to be highly expressed in brain. A nonclonal Chinese hamster ovary cell line stab ly expressing the newt CB1 cannabinoid receptor was prepared that allowed d emonstration of cannabinoid-mediated inhibition of adenylate cyclase (EC 4. 6.1.1) activity. This inhibition was dose-dependent and occurred at concent rations consistent with affinities determined through radioligand binding e xperiments. The behavioral, pharmacological, and molecular cloning results demonstrate that a CB1 cannabinoid receptor is expressed in the CNS of the roughskin newt. This amphibian CB1 is very similar in density, ligand bindi ng affinity, ligand binding specificity, and amino acid sequence to mammali an CB1. The high degree of evolutionary conservation of cannabinoid signali ng systems implies an important physiological role in vertebrate brain func tion.