Quantitative measurement for endothelial constitutive nitric oxide synthase in cultured human endothelial cells.
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.
Department of Cardiovascular Research, Berlex Biosciences, Richmond, California 94804-0099, USA.
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Aberle
T A
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Young
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Medberry
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Parkinson
G M
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Rubanyi
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Kauser
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D012680:Sensitivity and Specificity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D014409:Tumor Necrosis Factor-alpha; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III