Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production

It is commonly believed that the activity of NO synthase (NOS) solely controls NO production from its substrates, L-Arg and O₂. The Michaelis- Menten constant (K(m)) of NOS for L-Arg is in the micromolar range; cellular levels of L-Arg are much higher. However, evidence strongly suggests that cellular supply of L-Arg may become limiting and lead to reduced NO and increased superoxide anion (O₂/^-·) formation, promoting cardiovascular dysfunction. Uptake of L-Arg into cells occurs primarily (~85%) through the actions of a Na⁺-independent, carrier-mediated transporter (system y⁺). We have examined the effects of NOS agonists (substance P, bradykinin, and ACh) and NO donors (S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport of L-Arg into bovine aortic endothelial cells (BAEC). Our results demonstrate that NOS agonists increase y⁺ transporter activity. A rapidly acting NO donor initially increases L-Arg uptake; however, after longer exposure, L-Arg uptake is suppressed. Exposure of BAEC without L-Arg to substance P and a Ca²⁺ ionophore (A-23187) increased O₂/^-· formation, which was blocked with concurrent presence of L-Arg or the NOS antagonist N(ω)-nitro-L-arginine methyl ester. We conclude that factors including NO itself control y⁺ transport function and the production of NO and O₂/^-·.