Quantitative Measurement for Endothelial Constitutive Nitric Oxide Synthase in Cultured Human Endothelial Cells

Constitutively expressed endothelial nitric oxide synthase (ecNOS) produces nitric oxide (NO) froml-arginine and is important for the maintenance of cardiovascular homeostasis. We report the development of a capture ELISA which is specific for ecNOS. The assay detection limit is 0.5 ng/ml ecNOS protein, allowing the measurement of ecNOS from as few as 6000 human endothelial cells cultured in 96-well microtiter plates. This ELISA has been used to measure a downregulation of ecNOS with 24-h TNFα treatment, consistent with results obtained by Western blot analysis. Quantitation of ecNOS in human endothelial cells showed a higher expression of ecNOS in human aortic endothelial cells (18.3 ± 1.35 ng ecNOS per 106cells,n= 3 donors) than in human umbilical vein endothelial cells (10.4 ± 0.48 ng ecNOS per 106cells,n= 3 donors). These studies demonstrate that this convenient, quantitative assay is currently the most sensitive method for investigating ecNOS protein regulation.