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Regulation of Dimethylarginine Dimethylaminohydrolase (DDAH) in Rat Preglomerular Vascular Smooth Muscle Cells (PGVSMCs) by Reactive Oxygen Species (ROS)
Author(s) -
Luo Zaiming,
Gill Primohinder S.,
Welch William,
Jose Pedro A.,
Wilcox Christopher S
Publication year - 2008
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.22.1_supplement.758.24
Subject(s) - reactive oxygen species , chemistry , superoxide , transfection , microbiology and biotechnology , endogeny , blot , vascular smooth muscle , messenger rna , medicine , biochemistry , endocrinology , gene , biology , enzyme , smooth muscle
DDAH degrades asymmetric dimethylarginine (ADMA) which is an endogenous NOS inhibitor and risk factor for cardiovascular disorders. We tested the hypothesis that ROS regulate the transcription, translation or activity of DDAH. We created a stable rat PGVSMC line overexprissing p22 phox (S‐p22 phox ) using a p22 phox ‐pcDNA4/HisMax construct (a generous gift from Kathy Griendling). Quantitative real time PCR and Western blotting confirmed a marked increase in p22 phox mRNA and protein of 4.7±0.6 and 2.3±0.5 fold, (p<0.01), compared to wild type cells (WT). S‐p22 phox cells had elevated superoxide and hydrogen peroxid generation (2.3±0.09 fold and 1.8±0.1 fold p<0.01). The mRNAs for DDAH ‐1 and ‐2 were increased by 2.8 ± 0.2 and 1.8 ± 0.1 fold in S‐p22 phox cells p< 0.01). DDAH‐2 promoter activity, determined by transient transfection and luciferase assay, was 4.1 fold increased in S‐p22 phox cells (p < 0.01). Interestingly, both DDAH‐1 and ‐2 proteins were significantly lower in S‐p22 phox cells (0.9±0.2 and 0.9±0.1 fold; p<0.01), yet β‐actin was not different. DDAH activity measured by a colorimetric assay (Kidney Int. 72:886–9, 2007) was reduced by 62±2% in S‐p22 phox (p<0.01). The results reveal for the first time an important effect but complex of p22 phox – dependent ROS generation on DDAH. Thus, ROS upregulate the expression of DDAH‐1 and ‐2 genes via activating a promoter, at least for DDAH‐2, yet downregulate both DDAH‐1 and ‐2 proteins and their enzyme activity. This is a specific effect since ROS do not change the expression of cell structure proteins. These findings in PGVSMCs provide a novel regulatory pathway whereby ROS may modulate renal hemodynamics, and thereby BP and salt homeostasis.