Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase.
BACKGROUND
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). Plasma levels of ADMA are elevated in individuals with hypercholesterolemia or atherosclerosis. We postulated that reduced degradation of ADMA may play a role in the accumulation of ADMA in these individuals. Accordingly, we studied the effects of oxidized LDL (oxLDL) or tumor necrosis factor-alpha (TNF-alpha) on the accumulation of ADMA by transformed human umbilical vein endothelial cells (ECV304) and on the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which degrades ADMA.


RESULTS
ECV304 were incubated with or without native LDL (100 micrograms/mL), oxLDL (100 micrograms/mL), or TNF-alpha (250 U/mL) for 48 hours. The concentration of ADMA in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. We assayed DDAH activity by determining L-citrulline formation from ADMA. The addition of oxLDL or TNF-alpha to ECV304 significantly increased the level of ADMA in the conditioned medium. The effect of oxLDL or TNF-alpha was not due to a change in DDAH expression but rather to the reduction of DDAH activity. To determine whether dysregulation of DDAH also occurred in vivo, New Zealand White rabbits were fed normal chow or a high-cholesterol diet. Hypercholesterolemia significantly reduced aortic, renal, and hepatic DDAH activity.


CONCLUSIONS
These results suggest that the endothelial vasodilator dysfunction observed in hypercholesterolemia may be due to reduced degradation of ADMA, the endogenous inhibitor of NOS.
Ito
A
A
Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA, USA.
Tsao
P S
PS
Adimoolam
S
S
Kimoto
M
M
Ogawa
T
T
Cooke
J P
JP
D000581:Amidohydrolases; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000503:physiopathology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006867:Hydrolases / Q000032:analysis / Q000378:metabolism*; D006937:Hypercholesterolemia / Q000201:enzymology / Q000503:physiopathology; D015151:Immunoblotting; D008074:Lipoproteins / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000494:pharmacology; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D011817:Rabbits; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
D000596:Amino Acids; D008074:Lipoproteins; D008077:Lipoproteins, LDL; D014409:Tumor Necrosis Factor-alpha; C099252:oxidized low density lipoprotein; D009569:Nitric Oxide; D006867:Hydrolases; D000581:Amidohydrolases; C059999:dimethylargininase