Effect of homocysteine on bovine aortic endothelial cell function

Endothelial cell dysfunction is generally defined as impaired endothelial-dependent vasodilation (e.g. diminished NO production). Reactive oxygen species (ROS) such as superoxide anion (O₂·−), nitric oxide (NO), H₂O₂ and peroxynitrite (ONOO− ) can be produced in vessels by certain pathological conditions to initiate or accentuate endothelial cell dysfunction. Hyperhomocysteinemia is an independent risk for cardiovascular diseases. About 5–7% of the general population has hyperhomocysteinemia. Hyperhomocysteinemia produces endothelial injury and dysfunction. Hyperhomocysteinemia also leads to increased ROS levels through auto-oxidation of homocysteine (HCY) and diminished anti-oxidative activities. Transport of L-arginine into endothelial cells appears to be critical for its availability to endothelial NOS (eNOS) for production of NO. When L-arginine availability to NOS is restricted, NOS acts principally upon O ₂ to form O₂·−. We hypothesize that hyperhomocysteinemia reduces cellular uptake of arginine, augments O ₂·− production and causes endothelial dysfunction.; Aortic strips pretreated with HCY showed a substantial reduction in vasorelaxant response to acetylcholine as evidenced by a rightward shift (7 fold) and diminished maximal relaxation (80%) of the dose-response curve. These effects were substantially prevented by treatment with supplemental arginine. This indicates that HCY impairs endothelial-dependent relaxation by a mechanism involving arginine availability.; Treatment with HCY caused a biphasic effect on arginine transport. Acute treatment with HCY for 15 min and 2 hr did not alter arginine uptake in growth medium. After 6 hr incubation, uptake of arginine was increased in bovine aortic endothelial cells (BAECs) while arginine transport was reduced by 27% after 24 hr treatment in growth medium. However, in Hepes buffer HCY suppressed uptake of arginine after 15 min and 2 hr treatment. This suggests that arginine and proteins in growth medium had a protective effect.; Treatment with HCY also significantly reduced cellular levels of the CAT-1 arginine transporter protein (∼30%) after 24 hr treatment but not after 6 hr treatment, whereas levels of eNOS protein and activity were not altered. Moreover, HCY treatment caused membrane hyperpolarization after 6 hr and 24 hr treatment indicating that the drive for arginine uptake was maintained. Treatment with the antioxidants N-acetylcysteine and ascorbic acid reversed the HCY effect on arginine uptake, suggesting that oxidation of the transporter may contribute to the endothelial dysfunction. This concept was supported by observation of a significant increase in peroxynitrite formation (∼30%) after 24 hr treatment but not 6 hr treatment.; In summary, HCY-induced endothelial dysfunction appears to be due to enhanced oxidative stress, including formation of superoxide anion and peroxynitrite. Superoxide formation can occur from eNOS when the availability of arginine is limited. The action of HCY to reduce arginine uptake by reducing CAT-1 levels and possibly oxidizing the transporter may increase ROS formation and endothelial dysfunction.