cAMP assay

Functional studies were performed using CisBio (Codolet, France) HTRF dynamic-cAMP assay kit in 384-well format with 600–800 cells per well. Cells were pre-incubated with test compound incubated for 15 min and then agonist (typically dopamine) added, and the incubation continued for a further minute after which cAMP levels were determined in the presence of IBMX (0.1 mM final). Results were always compared to wild type control and data was analyzed in GraphPad PRISM5.0 using 4-paramater logistic function. Initial data where effects were seen were reproduced in a second batch of transiently transfected cells.

For initial screening assays, the ability of the test compound to potentiate cAMP levels in Lmtk-cells expressing human dopamine D1 receptor was determined in the presence and absence of a concentration of dopamine, which gave a 20% response compared to a maximal effect (10 mM dopamine). Cells (20,000 per well) were incubated for 60 min at room temperature. Results were analysed using the 4-paramater logistic equation and expressed as pEC50 and Erel (the maximal effect relative to the maximal response to dopamine over background expressed as a %).