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P22PHOX KNOCKDOWN ATTENUATES NOREPINEPHRINE TRANSPORTER REDUCTION IN RESPONSE TO ENDOTHELIN‐1 IN PC12 CELLS
Author(s) -
Cao Xian,
Sordillo Lorraine M,
Kreulen David L
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a817
Subject(s) - p22phox , small hairpin rna , gene knockdown , nadph oxidase , messenger rna , norepinephrine transporter , chemistry , endothelin 1 , transfection , microbiology and biotechnology , medicine , endocrinology , biology , norepinephrine , receptor , apoptosis , biochemistry , oxidative stress , gene , dopamine
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.