Pharmacological Properties of Cannabinoid Receptors in the Avian Brain: Similarity of Rat and Chicken Cannabinoid1 Receptor Recognition Sites and Expression of Cannabinoid2 Receptor-Like Immunoreactivity in the Embryonic Chick Brain

Abstract

The pharmacological properties of brain cannabinoid receptors were investigated in brains of 35 day-old chickens, since little is known about the avian cannabinoid system. The cannabinoid1 receptor-selective antagonist ligand [3H]SR 141716A bound to chicken brain membranes with K(D) and Bmax values of 0.92+/-0.28 nM and 790+/-58 fmol/mg protein, respectively. The binding was inhibited by CP 55,940 with a pI50 value of 7.63+/-0.14 and by a series of compounds with the order of potency CP 55,940>R(+)WIN 55,212-2>R-1 methanandamide approximately DAK. S(-)WIN 55,212-3 and AM404 were without inhibitory effect at 1 microM. Similar results were found for rat brain membranes. For both rat and chicken brain membranes, addition of the non-hydrolysable GTP analogues Gpp[NH]p and GTPgammaS shifted the CP 55,940 inhibition curve to the right, consistent with an intact coupling to G-proteins in the preparations. Fatty acid amidohydrolase in chicken brain membranes was less sensitive to inhibition by phenylmethylsulphonyl fluoride and arachidonoyl serotonin than its rodent equivalent. However, when fatty acid amidohydrolase activity in the preparations was reduced by use of a lower assay membrane concentration, anandamide was found to inhibit the binding of [3H]SR 141716A to chicken membranes with a pI50 value of 6.39+/-0.16. Using a novel antibody raised to amino acids 346-359 from the C-terminal tail of the human cannabinoid2 receptor, it was found that embryonic chick brain tissue (and embryonic chick neurones in primary culture) expressed a approximately 53 kDa immunoreactive band. This immunoreactivity, which was prevented by preincubation of the antibody with the immunising peptide, was also seen in cells expressing the recombinant human cannabinoid, receptor, but was not seen in adult chicken brain homogenates or in rat cerebellar homogenates. However, a "classical" cannabinoid2-receptor component of [3H]WIN 55212-2 binding (i.e. a fraction inhibited by low concentrations of the cannabinoid2-receptor-selective antagonist SR 144528) was not found.

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