We have previously reported that dopamine D1‐like receptors (D1R and D5R) negatively regulate NADPH oxidase isoform expression and activity in renal proximal tubule (RPT) cells from rodents and humans. We tested the hypothesis that D1‐like dopamine receptors (D1R and D5R) differentially regulate the anti‐oxidant enzyme paraoxonase2 (PON2) in HEK‐293 cells heterologously expressing human D1R and D5R. Fenoldopam (1µM, 15 min), an agonist for both D1R and D5R, increased PON2 co‐immunoprecipitation with D1R (160±4% vs. control, 101±1%) and D5R (132±0.1% vs. control, 102±1%) in both cell lines. Short‐term (15, 30, and 60min) stimulation of D1R and D5R with fenoldopam (1µM) did not alter total PON2 protein. However, in D1R cells, at 15min, fenoldopam increased PON2 protein in both lipid rafts (LRs) (144±3% vs. control, 100±3%) and non‐LRs (163±25% vs. control, 100±21%) (P<0.05, n=6/group, ANOVA). In contrast, in D5R cells, fenoldopam increased PON2 protein only in non‐LRs (132±5% vs. control, 100±1.6%, n=3). Long‐term (2, 4, 8, and 24h) fenoldopam (1µM) stimulation increased PON2 protein in a time dependent manner (146% ±7/8h, and 162% ±10/24h vs. control, 100±9%) (P<0.05, n=4, ANOVA) in D5R but not D1R cells. Fenoldopam also increased PON2 mRNA (1.25‐fold) (n=3, P<0.05, t‐test) at 24h in D5R but not D1R cells. Silencing PON2 gene with PON2‐siRNA (10nM/48h) in D5R cells decreased PON2 protein (‐40±4% vs. mock‐siRNA, 100±17%, n=3) and mRNA (‐17±4% vs. mock‐siRNA, 100±2%, P<0.05, n=3, t‐test) and increased basal ROS production by 1.36‐fold, relative to mock‐siRNA. PON2 protein was decreased in D5R‐/‐ mice (‐44±13%) relative to D5R+/+ mice (100±8%, n=4‐6/group). We conclude that short‐term stimulation of either D1R or D5R leads to PON2 protein recruitment to non‐LRs and also to LRs in the case of D1R. However, long‐term stimulation of PON2 and negative ROS regulation are mainly due to D5R.
Grant Funding Source: HL023081, HL074940, DK039308
