Establishment of recombinant cannabinoid receptor assays and characterization of several natural and synthetic ligands

Abstract

Cannabinoid receptors (CBR) are important drug targets for the treatment of various inflammatory, metabolic and neurol. diseases. Therefore, sensitive test systems for the assessment of ligands are needed. In this study, a steady-state GTPase assay for human CBR subtypes 1 and 2 was developed to characterize the pharmacol. property of ligands at a very proximal point of the signal transduction cascade. Establishing these in vitro test sytems, we studied cell or tissue membranes heterogenously or endogenously expressing CBR, such as CBR-infected Human Embryonic Kidney (HEK) 293 cells, rat cerebellum and spleen cells. The lack of effects in the GTPase assay and in [35S]GTPgamma S binding expts. in these expression system, directed us to use Spodoptera frugiperda (Sf9) cells. Co-expressing CBR, different Galpha -subunits, Gbeta gamma heterodimer, and RGS (Regulator of G-protein signaling)-proteins in Sf9 cell membranes greatly improved the sensitivity of the assay, with highest GTPase activation in the CBR + Galpha i2 + Gbeta 1gamma 2 + RGS4 system. We examd. exogenous and endogenous std. ligands as well as secondary metabolites as Delta 9-tetrahydrocannabinol (Delta 9-THC), dodeca-2E,4E-dienoic acid isobutylamide, an alkylamide from Echinacea purpurea, and an E. purpurea hexane ext. according their agonistic and antagonistic properties. The suitability of the assay for screening procedures was also proven by detecting the activity of Delta 9-THC in a matrix of other less active compds. (Delta 9-THC-free Cannabis sativa ext.). In conclusion, we have developed highly sensitive test systems for the anal. of CBR ligands.