FIGURE 6.

β-Arrestin-dependent activation of Cox1-PGES3 dependent PGE₂ synthesis by SII. A, SII stimulation increases the association of endogenous PGES3 with FLAG-β-arrestin 1. HEK-AT1AR cells transiently expressing FLAG epitope-tagged β-arrestin 1 were stimulated for 5 min with SII or AngII, after which FLAG immunoprecipitates (IP) were probed for the presence of coimmunoprecipitating PGES3. A representative immunoblot (IB) is shown above a bar graph depicting the amount of PGES3 protein normalized to the amount of FLAG-β-arrestin 1 in the immunoprecipitate. *, t test p < 0.05, greater than NS (n = 3). B, reconstitution of SII-induced PGE₂ synthesis in HEK-AT_1AR cells. Cells were transfected with plasmids encoding Cox 1, PGES3, or both and then stimulated with SII for 30 min before enzyme-linked immunoassay of PGE₂ released into the culture medium. Data shown are the mean ± S.E. of the rate of PGE₂ release determined under each condition. *, p < 0.05, greater than NS (n = 7). C, effect of silencing β-arrestin 1/2 expression on SII-induced PGE₂ synthesis. Down-regulation of β-arrestin 1/2 expression using siRNA was performed in HEK-AT_1AR transiently coexpressing Cox1 and PGES3, after which the effects of SII and AngII on PGE₂ synthesis was determined. The upper panel immunoblot and lower panel bar graph show the effect of siRNA treatment on endogenous β-arrestin 1/2 expression. *, t test p < 0.05, less than control siRNA-transfected cells (n = 4). The upper panel bar graph shows the effect of siRNA treatment on SII and AngII stimulated PGE₂ release into the culture medium. *, t test p < 0.05, greater than NS; †, p < 0.05, less than control siRNA (n = 4). D, effect of SII and AngII on PGE₂ synthesis by primary rat aortic vascular smooth muscle cells (VSMC). Some wells were preincubated for 30 min with the Cox 1/2 inhibitor indomethacin (10 μm) prior to stimulation. *, p < 0.05, greater than NS; †, p < 0.05, less than without indomethacin (n = 3).