Jun 1, 1997

Quantitative measurement for endothelial constitutive nitric oxide synthase in cultured human endothelial cells

Nitric Oxide : Biology and Chemistry
S AberleK Kauser

Abstract

Constitutively expressed endothelial nitric oxide synthase (ecNOS) produces nitric oxide (NO) from L-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 TNFalpha 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 10(6) cells, n = 3 donors) than in human umbilical vein endothelial cells (10.4+/-0.48 ng ecNOS per 10(6) cells, n = 3 donors). These studies demonstrate that this convenient, quantitative assay is currently the most sensitive method for investigating ecNOS protein regulation.

Mentioned in this Paper

Tumor Necrosis Factor-alpha
Chinese Hamster Ovary Cell
Nitric Oxide Synthase Type III
Western Blot
Endothelium, Vascular
Human Umbilical Vein Endothelial Cells
NOS3 protein, human
Down-Regulation
Aorta
Arginine

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