Fig. 4.
Pharmacological characterization of SKF83959 on D1R + D2LR–mediated calcium mobilization. HEK293T cells were transfected with D1R + D2LR as described, plated 24 hours later in 384-well plates, and assayed for calcium accumulation the following day. (A) Cells were stimulated with one of the following conditions as indicated: DA, SKF83959, the D2R-selective agonist quinpirole, or both SKF83959 and quinpirole combined. (B) Cells were incubated with SKF83959 or the D1R-selective antagonist SCH23390, then stimulated with an ∼EC80 of DA (1 μM). Data are expressed as a percentage of control maximum DA-stimulated response and are representative of two or three independent experiments performed with the same assay conditions on different days. Error bars indicate S.E.M. from multiple wells within the representative experiment. (C) HEK293 cells stably transfected with D1R (Codex Biosolutions, Inc., Gaithersburg, MD) were grown and membranes harvested as described in Materials and Methods. Membranes were incubated with various concentrations of SKF83959 and 0.5 nM [3H]SCH23390 as indicated. Graph is representative of two independent experiments done on different days. Data are expressed as specific binding in units of fmol/mg. Ki value was calculated using the Cheng-Prushoff equation and a radioligand Kd value of 0.5 nM as determined via saturation binding isotherms (unpublished data). Average Ki for SKF83959 on D1R was 2.6 nM ± 0.7.