Description
Introduction: Nitric oxide (NO) is produced by a group of enzymes called nitric oxide synthases (NOS). These enzymes catalyze the production of NO and L-citrulline from L-arginine, O2, and NADPH derived electrons. Mammalian systems contain three well-characterized isoforms of the enzyme: neuronal NOS (nNOS, also called NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3). The cell types that express eNOS include vascular endothelial cells, cardiomyocytes and others. In blood vessels, NO produced by the eNOS in endothelial cells functions as a vasodilator thereby regulating blood flow and pressure. Mutant eNOS knockout mice have blood pressure that is 30% higher than wild-type littermates. Within cardiomyocytes, eNOS affects Ca2+ currents and contractility. Expression of eNOS is usually reported to be constitutive, though modest degrees of regulation occur in response to factors such as shear stress, exercise, chronic hypoxia, and heart failure. The unique N-terminal sequence of eNOS is about 70 residues long and functions to localize the enzyme to membranes. Upon myristoylation at one site and palmitoylation at two other sites within this segment, the enzyme is exclusively membrane-bound. Palmitoylation is a reversible process that is influenced by some agonists and is essential for membrane localization. Within the membrane, eNOS is targeted to the caveolae, small invaginations characterized by the presence of proteins called caveolins. These regions serve as sites for the sequestration of signaling molecules such as receptors, G proteins and protein kinases. The oxygenase domain of eNOS contains a motif that binds to caveolin-1, and calmodulin is believed to competitively displace caveolin resulting in eNOS activation. Bound calmodulin is required for activity of eNOS, and this binding occurs in response to transient increases in intracellular Ca2+. Thus, eNOS occurs at sites of signal transduction and produces short pulses of NO in response to agonists that elicit Ca2+ transients. Physiological concentrations of eNOS-derived NO are in the picomolar range. Within the cardiovascular system, eNOS generally has protective effects. Studies with nNOS and eNOS knockout mice clearly indicate that eNOS plays a protective role in cerebral is chemia by preserving cerebral blood flow. During inflammation and atherosclerosis, low concentrations of NO prevent apoptotic death of endothelial cells and preserve the integrity of the endothelial cell monolayer. NO also acts as an inhibitor of platelet aggregation, adhesion molecule expression, and vascular smooth muscle cell proliferation.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to eNOS. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for eNOS and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain eNOS, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of eNOS in the samples is then determined by comparing the O.D. of the samples to the standard curve