Nitric oxide synthase (NOS2) mutation in Dahl/Rapp rats decreases enzyme stability.
The pathogenesis of salt-sensitive hypertension remains poorly defined, but a role for nitric oxide (NO) has been suggested. The Dahl/Rapp salt-sensitive rat possesses a defect in NO synthesis that is overcome by supplementation with L-arginine, which increases NO and cGMP production and prevents salt-sensitive hypertension. An S714P mutation of inducible NO synthase (NOS2) was subsequently identified. The current report examined the functional significance of an S714P mutation in NOS2. COS-7 cells were transiently transfected with cDNA of wild-type NOS2 and S714P and S714A mutants of NOS2, and enzyme function was determined. Whereas steady-state mRNA levels did not differ, immunoblot analysis demonstrated decreased levels of NOS2 protein. Metabolic labeling experiments confirmed a reduced half-life of the S714P mutation. Nitrite production, which was dependent on the concentration of L-arginine in the medium, was diminished in cells transfected with the S714P mutant, compared with the wild type and the S714A mutant. These data provide a biochemical explanation of the physiological abnormalities of NOS2 in the Dahl/Rapp salt-sensitive rat and suggest that a posttranslational mechanism involving the proteasome may be responsible for the diminished NO production observed in response to increased dietary salt intake in these animals.
Ying
W Z
WZ
Nephrology Research and Training Center, Comprehensive Cancer Center, Division of Nephrology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294-0007, USA.
Xia
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H
Sanders
P W
PW
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000378:metabolism; D015152:Blotting, Northern; D019556:COS Cells / Q000187:drug effects; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000235:genetics*; D006973:Hypertension / Q000201:enzymology* / Q000235:genetics; D015151:Immunoblotting; D007783:Lactones / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020303:Rats, Inbred Dahl; D013329:Structure-Activity Relationship; D014162:Transfection
D007783:Lactones; D009097:Multienzyme Complexes; D009573:Nitrites; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C098869:clasto-lactacystin beta-lactone; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex