Fig. 5.

Effect of cocaine on D₁R-mediated cAMP production. (A) RT-PCR was performed using total RNA from CHO cells (lanes 1–4) and primers for Chinese hamster σ₁R (lane 2), DAT (lane 3), or GAPDH (lane 4). RNA from cells without primers (lane 1) was included as negative control. (B) CHO cell membranes were analyzed by SDS/PAGE and immunoblotted with the anti-σ₁R antibody (top blot) or anti-D₁R antibody (bottom blot, lanes 1 and 2: cells transfected or not transfected with D₁R cDNA, respectively). (C) CHO cells transfected with D₁R cDNA (1.5 μg, filled bars) or cotransfected with D₁ receptor cDNA and 125 pmol σ₁R siRNA (open bars) were treated with increasing concentrations of D₁R agonist SKF 81297 for 10 min in the absence or presence of 150 μM cocaine or with cocaine alone. Results are mean ± SEM of three to six independent experiments performed in triplicate. Bifactorial ANOVA of results of samples without or with siRNA transfection showed significant effect of SKF (P < 0.0001 and P < 0.001, respectively), but only in samples without siRNA transfection was there a highly significant effect of cocaine (***P < 0.0001, compared with samples with the same concentration of SKF 81297 and without RNAi transfection and in the absence of cocaine; Bonferroni post hoc tests).