FIGURE 8.

DAR function after ouabain inhibition of NKAα1. HEK293T cells were co-transfected with 15 μg of DAR (D_2L (A) or D₁ (B)) and 7.5 μg of either empty pcDNA vector or NKAα1. Cells were washed 24 h later and re-plated into 24-well plates at a density of 200,000 cells per well; assays were conducted 48 h post-transfection. Five min prior to agonist treatment, cells were placed into media with (test) or without (control) ouabain. Cells were then stimulated with forskolin and dopamine (A) or dopamine alone (B) for 10 min in the presence or absence of ouabain (OUA), and cAMP accumulation was measured as described under “Experimental Procedures.” Data were normalized for individual experiments as a percentage of the E_max value for DAR alone and are expressed as mean ± S.E. of five independent experiments, each conducted in triplicate; all statistical analyses were performed using one-way ANOVA with Bonferroni's post test. For D_2L alone (A), raw values for E_max (maximal dopamine-induced inhibition) ranged from 6.6 to 10.1 pmol of cAMP/200,000 cells. For D₁ alone (B), raw values for E_max ranged from 37.9 to 68.4 pmol of cAMP/200,000 cells. Complete E_max and EC₅₀ data are summarized in Table 3.