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Behavioral, pharmacological, and molecular characterization of an amphibian cannabinoid receptor
by
Soderstrom K, Leid M, Moore FL, Murray TF
ABSTRACT
Investigation of cannabinoid pharmacology in a vertebrate
with a phylogenetic history distinct from that of mammals may allow better
understanding of the physiological significance of cannabinoid neurochemistry.
Taricha granulosa, the roughskin newt, was used here to characterize an
amphibian cannabinoid receptor. Behavioral experiments demonstrated that
the cannabinoid agonist levonantradol inhibits both newt spontaneous locomotor
activity and courtship clasping behavior. Inhibition of clasping was dose-dependent
and potent (IC(50) = 1.2 microgram per animal). Radioligand binding studies
using [(3)H]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) >> anandamide (1,665) approximately anandamide 100 microM phenylmethylsulfonyl
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 stably expressing
the newt CB1 cannabinoid receptor was prepared that allowed demonstration
of cannabinoid-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity.
This inhibition was dose-dependent and occurred at concentrations consistent
with affinities determined through radioligand binding experiments. 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 binding affinity,
ligand binding specificity, and amino acid sequence to mammalian CB1. The
high degree of evolutionary conservation of cannabinoid signaling systems
implies an important physiological role in vertebrate brain function.
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