Endothelial cell dysfunction in a model of oxidative stress.
BACKGROUND
We have investigated the role of L-arginine in hyperhomocysteinemia (HHCY). L-arginine is the substrate required for NO production by endothelial NOS (eNOS). When L-arginine is limited, NOS acts principally upon O2 to form superoxide (O2.-). Because HHCY causes formation of reactive oxygen species and reduced endothelial-dependent vasodilation, we hypothesized that HHCY decreases NO formation by limiting the cellular supply of L-arginine.


METHODS
Studies with cultured bovine aortic endothelial cells (ECs) determined effects of HCY on transport of [3H] L-arginine. Effects on L-arginine transporter protein CAT-1 and eNOS protein were assessed by immunoblotting. Peroxynitrite formation was evaluated by an immunoassay for nitrotyrosine levels. eNOS activity in forming NO was determined by assay for 3H-L-arginine to 3H-citrulline conversion.


RESULTS
HCY had a depressive effect on arginine transport in ECs. HCY treatment for a 24 hr period decreased arginine uptake by 27%. HCY treatment for 24 hr significantly reduced cellular levels of the CAT-1 arginine transporter protein ( approximately 30%) and increased nitrotyrosine formation, whereas levels of eNOS protein and basal NOS activity were not altered. Nevertheless, total NO production as indicated by citrulline conversion was significantly decreased. Treatment with the antioxidant N-acetylcysteine reversed the HCY effect on arginine transport, suggesting that transporter oxidation may contribute to the endothelial dysfunction.


CONCLUSIONS
The association of HCY-induced decreases in NO formation with decreases in function and expression of the arginine transporter in the absence of alterations in eNOS expression or activity suggests a primary role for arginine transport alterations in HHCY. The action of HCY to reduce arginine uptake may accentuate endothelial dysfunction due to generation of O2.- and peroxynitrite formation, which may cause further oxidative injury.
Jin
L
L
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912, USA.
Abou-Mohamed
G
G
Caldwell
R B
RB
Caldwell
R W
RW
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000503:physiopathology*; D006710:Homocysteine / Q000502:physiology*; D008954:Models, Biological; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D018384:Oxidative Stress
D006710:Homocysteine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III