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 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.

Characterization of cannabinoid receptor ligands in tissues natively expressing cannabinoid CB2 receptors

“…Thus, the percentage stimulation of [ 35 S]GTPγS binding with the high efficacy CB 2 receptor agonist, CP 55,940 is only 21, 29 and 35% in human, mouse and rat spleens, respectively. This is in line with a recent report demonstrating a low signal detected with CB 2 receptor ligands in rat spleen cells using the [ 35 S]GTPγS binding assay (Geiger et al ., ). However, motivated by the imperative for assessment of CB 2 ligand pharmacology in native tissues, we progressed with compound characterization.…”

Magnolia Extract, Magnolol, and Metabolites: Activation of Cannabinoid CB2 Receptors and Blockade of the Related GPR55

“…12 CB 1 activation mediates analgesia, stimulation of appetite, and euphoria, among other effects. 13 CB 2 receptor activation results in analgesic and antiinflammatory effects. 9 A related GPCR is the orphan GPR55, which has been described to be activated by lysophosphatidylinositol (LPI).…”

Impact of fusion to Gαi2 and co-expression with RGS proteins on pharmacological properties of human cannabinoid receptors CB1R and CB2R

“…The GTPase assay was performed as described previously [17] . Assay tubes contained membranes (G α i2 co‐transfected membranes, 10 µg of protein/tube; G α i2 fusion protein membranes, 5 µg of protein/tube), 1.0 m m MgCl 2 , 0.1 m m EDTA, 0.1 m m ATP, 100 n m GTP, 0.1 m m adenylyl imidodiphosphate, 5 m m creatine phosphate, 40 µg of creatine kinase and 0.2% (w/v) bovine serum albumin in 50 m m Tris/HCl, pH 7.4 to prevent binding of protein or ligand to the polystyrol tubes, CB 1 R and CB 2 R ligands at various concentrations and 20 µl of [ γ ‐ 32 P]GTP (0.1 µCi/tube).…”

“…Data shown in Tables 2 and 3 were analysed statistically by one‐way ANOVA, followed by Dunnett's multiple comparison test [17] …”