Substrate inhibition of nitric oxide synthase in pulmonary artery endothelial cells in culture

The effects of arginine on nitric oxide synthase (NOS) activity and NO production were studied in pulmonary artery endothelial cells (PAEC). Incubation of PAEC with 0-100 μM arginine increased NO production, detected as nitrite in the culture medium, in a dose- dependent manner. In contrast, incubation with concentrations of arginine in excess of 100 μM resulted in a reversible dose-dependent inhibition of NO production, even though intracellular arginine content increased in these cells. The NOS enzyme kinetics were studied in a total membrane preparation and in purified NOS protein and revealed that the K(m) of arginine as a substrate for NOS is 3-5 μM, the V(max) occurred at 100 μM arginine, and substrate inhibition occurred at >100 μM arginine. Oxyhemoglobin, carboxy-PTIO, catalase, SOD, citrulline, hydroxyarginine, and D-arginine did not change NOS kinetics. Those results indicate that substrate inhibition of eNOS exists in porcine PAEC in vitro.