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

Cannabinoid receptors (CBR) are important drug targets for the treatment of various inflammatory, metabolic and neurological 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 pharmacological 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 [(35)S]GTPgammaS binding experiments in these expression system, directed us to use Spodoptera frugiperda (Sf9) cells. Co-expressing CBR, different Galpha-subunits, Gbetagamma 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(1)gamma(2) + RGS4 system. We examined exogenous and endogenous standard 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 extract 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 compounds (Delta(9)-THC-free Cannabis sativa extract). In conclusion, we have developed highly sensitive test systems for the analysis of CBR ligands.