P22PHOX KNOCKDOWN ATTENUATES NOREPINEPHRINE TRANSPORTER REDUCTION IN RESPONSE TO ENDOTHELIN‐1 IN PC12 CELLS

Abstract

In DOCA-salt hypertension, superoxide anion (O2-·) levels are increased in sympathetic ganglion neurons as compared to normotensive controls; this increase is mediated in part by endothelin-1 (ET-1) via the activation of NADPH oxidase. Increased O2-· reduces norepinephrine uptake via norepinephrine transporter (NET). We tested the hypothesis that ET-1 reduces NET expression in PC12 cells via increases in O2-·, and this effect can be abolished by knockdown of p22phox, an indispensable component of the O2-·-generating NADPH oxidase. Undifferentiated PC12 cells were transfected with short hairpin RNA (shRNA) targeting p22phox. Cells with stable expression of p22phox shRNA (shRNA-p22) had reduced p22phox mRNA (71% knockdown) and protein (55% knockdown). Compared to wild type PC12 cells (WT), O2-· production induced by ET-1 was diminished in shRNA-p22. To assess ET-1 effects mediated by NADPH oxidase on NET, both shRNA-p22 and WT were exposed to ET-1 (100nM) for 30min to 24hrs. ET-1 reduced NET mRNA and protein levels in WT cells; the mRNA levels decreased over time and reached their minimum at 2 hrs (3.41 fold decrease, n=3), while protein levels remained reduced for 24 hrs (68% decrease, n=3). In shRNA-p22 cells, neither NET mRNA nor protein levels were changed by ET-1 treatment. These results indicate that the effects of ET-1 on O2-· production and NET expression in PC12 cells are p22phox -dependent.