Org 27569

Binding assays are performed with the CB1 receptor agonist [³H]CP 55,940 (0.7 nM) and the CB1 receptor antagonist [³H]SR 141716A (1.2 nM), 1 mg/ml BSA and 50 mM Tris buffer containing 0.1 mM EDTA and 0.5 mM MgCl₂, pH 7.4, in a total assay volume of 500 μl. Binding is initiated by the addition of mouse brain membranes (30 μg). Assays are carried out at 37°C for 60 min before termination by addition of ice-cold wash buffer (50 mM Tris buffer and 1 mg/ml BSA) and vacuum filtration using a 24-well sampling manifold and Whatman GF/B glass-fibre filters that have been soaked in wash buffer at 4°C for 24 h. Each reaction tube is washed five times with a 4-ml aliquot of buffer. The filters are oven-dried for 60 min and then placed in 5 ml of scintillation fluid, and radioactivity is quantitated by liquid scintillation spectrometry. Specific binding is defined as the difference between the binding that occurred in the presence and absence of 1 μM concentrations of the corresponding unlabelled ligand and is 70 to 80% of the total binding.

HEK293

~10 μM

5 to 15 min

Cells expressing CB1 receptors are exposed to ORG27569 (10 μM) for 5 to 15 min. For toxin treatment to abrogate Gi coupling effects, PTX is added to the medium at 5 ng/ml. Following an 18-h incubation in the presence of toxin, cells are washed twice with PBS and treated with compounds. Cells are washed with ice-cold PBS, and cell lysates are obtained by harvesting the cells with ice-cold lysis buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 7.5 containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin as protease inhibitors).