The cannabinoid agonists are ACEA (CB1), WIN55 (CB1, CB2) and CB65 (CB2) and the assay is performed on wild type male winstar rat whole spinal cord membrane homogenates. The compounds were ordered from tocris, stored at -20 in 1mM stock solutions. The ligands are in a final concentration of 10 mikroM(CB65, WIN55, ACEA) or 1 mikroM (ACEA) and each time in both concentrations they reduce [35S]GTPgammaS total specific binding, while they should do the opposite, since they are agonists.

The compounds from the same batch worked fine before and worked fine now in other type of samples (for instance in whole brain membrane homogenates). So most probably the problem is the sample, however I do not really understand why they behave particularly as inverse agonists.

Also here are some details of our experimental setup:

The assay buffer contains 50 mM Tris HCl, 1 mM EGTA, 3 mM MgCl2, 100 mM NaCl, containing ~0.05 nM [35S]GTPgammaS and 30 mikroM GDP. The incubation was done in a final volume of 1 ml at 30 oC for 60 min. The non-specific binding was determined by 10 mikroM unlabeled GTPgammaS. The bound [35S]GTPgammaS was seperated from the unbound by vacuum filtration on a fiberglass filter (whatmann GF/B).

Thank you in advance