Figure 5.

Dopamine decreases intracellular cAMP levels and inhibits VPF/VEGF- and NE-stimulated Src-activation. (A) DA at 10 and 50 μM caused a significant decrease of intracellular cAMP levels in MOEC and SKOV3ip1 cells. Values are means ± SE of three independent experiments; *p<0.001 for MOEC; *p<0.05 for SKOV3 ip1 cells. (B) In MOEC, pre-incubation with DA 10 μM for 10 min inhibited the VPF/VEGF stimulated phosphorylation of Src^Y416. In SKOV3 ip1 cells, DA reverted the stimulatory effect of NE on Src^Y419 phosphorylation by combined treatment of NE+DA for 5 and 15 min. Phospho- Src densitometric data were normalized to total Src; these are representative of two independent experiments. (C) Dopamine induces association of DR2 to Gαi-protein. MOEC were exposed to 10 μM DA, for 10 and 30 min. MOEC lysates were then immunoprecipitated with DR2 antibody and analyzed by Western blotting. Gαi-protein was immunodetected using a specific anti-Gαi antibody (Abcam). DA caused a 1.6 fold higher interaction between DR2 and Gαi-protein after 10 min of exposure, compared to untreated cells. The densitometry data were normalized to a loading control (β-actin from original lysate) and are representative of two independent experiments.