Rotigotine hydrochloride

The concentration of cAMP was determined in cell lysates using cAMP ELISA chemiluminescence kit (STA-500; Cell Biolabs Inc., San Diego, CA, USA). Three days after transfection, the HEK293 cells were stimulated with 1 µM rotigotine hydrochloride (Sigma Aldrich, Poznań, Poland), a D₂R agonist, for 10 min. Prior to stimulation, the cells transfected with Gαi were incubated in a medium containing 1 µM forskolin (Sigma Aldrich, Poznań, Poland) for 5 min. These prestimulation and stimulation procedures were conducted in MEM supplemented with 0.5% FBS. After stimulation, the cells were harvested and their cAMP concentration was determined according to the manufacturer’s instructions. In each case, four independent experiments were performed in duplicates. Nontransfected cells were used as controls, and the concentrations of cAMP in transfected cells were normalized in comparison with the values determined in controls in each experiment.