Cigarette smoke constituents cause endothelial nitric oxide synthase dysfunction and uncoupling due to depletion of tetrahydrobiopterin with degradation of GTP cyclohydrolase.

Cigarette smoking (CS) is a well-established risk factor for cardiovascular disease (CVD). Endothelial dysfunction (ED) with loss of nitric oxide (NO) production is a central mechanism leading to the advent of CVD. Despite many prior studies of this major health problem, the exact mechanism by which CS induces ED is not well understood. This study examines the mechanism by which CS induces ED with altered endothelial NO synthase (eNOS) function in aortic endothelial cells (AECs). Exposure of AECs to cigarette smoke extract (CSE) resulted in a marked decrease in NO production with concomitant increase in superoxide (O2.-) generation and accumulation of 4-hydroxy-2-nonenal protein adducts. CSE exposure led to depletion of the essential eNOS cofactor tetrahydrobiopterin (BH4) as well as total biopterin levels and decreased the expression level of guanosine triphosphate cyclohydrolase (GTPCH), the rate limiting enzyme in BH4 biosynthesis. Moreover, exposure of AECs to CSE increased the level of ubiquitinated proteins and increased 26 S proteasomal activity in a concentration-dependent manner. Pre-treatment with MG132, a 26 S proteasome inhibitor, partially prevented CSE-induced loss of BH4, total biopterin, GTPCH, and increased NO production following CSE exposure, indicating a role of the ubiquitin-proteasome system in CSE-induced eNOS dysfunction. In conclusion, CSE-induced eNOS dysfunction and uncoupling occurs due to BH4 depletion with BH4de novo synthesis limited by diminished GTPCH expression.

Effect of temperature, pH and heme ligands on the reduction of Cygb(Fe(3+)) by ascorbate.

Cytoglobin (Cygb) plays a role in regulating vasodilation in response to changes in local oxygen concentration by altering the rate of nitric oxide (NO) metabolism. Because the reduction of Cygb(Fe(3+)) by a reductant is the control step for Cygb-mediated NO metabolism, we examined the effects of temperature, pH, and heme ligands on the Cygb(Fe(3+)) reduction by ascorbate (Asc) under anaerobic conditions. The standard enthalpy of Cygb(Fe(3+)) reduction by Asc was determined to be 42.4 ± 3.1 kJ/mol. The rate of Cygb(Fe(3+)) reduction increased ~6% per °C when temperature varied from 35°C to 40°C. The yield and the rate of Cygb(Fe(3+)) reduction significantly increases with pH (2-3 times per pH unit), paralleling the formation of the Asc ion (A(2-)) and the increased stability of reduced state of heme iron at high pH values. Heme ligand cyanide (CN(-)) decreased the yield and the rate of Cygb(Fe(3+)) reduction, but ligands CO and NO allowed the process of Cygb(Fe(3+)) reduction to continue to completion. Critical information is provided for modeling and prediction of the process of Cygb-mediated NO metabolism in vessels in a range of temperature and pH values.

Differences in oxygen-dependent nitric oxide metabolism by cytoglobin and myoglobin account for their differing functional roles.

The endogenous vasodilator nitric oxide (NO) is metabolized in tissues in an oxygen-dependent manner. In skeletal and cardiac muscle, high concentrations of myoglobin (Mb) function as a potent NO scavenger. However, the Mb concentration is very low in vascular smooth muscle, where low concentrations of cytoglobin (Cygb) may play a major role in metabolizing NO. Questions remain regarding how low concentrations of Cygb and Mb differ in terms of NO metabolism, and the basis for their different cellular roles and functions. In this study, electrode techniques were used to perform comparative measurements of the kinetics of NO consumption by Mb and Cygb. UV/Vis spectroscopic methods and computer simulations were performed to study the reaction of Mb and Cygb with ascorbate (Asc) and the underlying mechanism. It was observed that the initial rate of Cygb(3+) reduction by Asc was 415-fold greater than that of Mb(3+). In the low [O2] range (0-50 µM), the Cygb-mediated NO consumption rate is ~ 500 times more sensitive to changes in O2 concentration than that of Mb. The reduction of Cygb(3+) by Asc follows a reversible kinetic model, but that of Mb(3+) is irreversible. A reaction mechanism for Cygb(3+) reduction by Asc is proposed, and the reaction equilibrium constants are determined. Our results suggest that the rapid reduction of Cygb by cellular reductants enables Cygb to efficiently regulate NO metabolism in the vascular wall in an oxygen-dependent manner, but the slow rate of Mb reduction does not show this oxygen dependence.

Characterization of the function of cytoglobin as an oxygen-dependent regulator of nitric oxide concentration.

The endogenous vasodilator nitric oxide (NO) is metabolized in tissues in an O(2)-dependent manner. This regulates NO levels in the vascular wall; however, the underlying molecular basis of this O(2)-dependent NO consumption remains unclear. While cytoglobin (Cygb) was discovered a decade ago, its physiological function remains uncertain. Cygb is expressed in the vascular wall and can consume NO in an O(2)-dependent manner. Therefore, we characterize the process of the O(2)-dependent consumption of NO by Cygb in the presence of the cellular reductants and reducing systems ascorbate (Asc) and cytochrome P(450) reductase (CPR), measure rate constants of Cygb reduction by Asc and CPR, and propose a reaction mechanism and derive a related kinetic model for this O(2)-dependent NO consumption involving Cygb(Fe(3+)) as the main intermediate reduced back to ferrous Cygb by cellular reductants. This kinetic model expresses the relationship between the rate of O(2)-dependent consumption of NO by Cygb and rate constants of the molecular reactions involved. The predicted rate of O(2)-dependent consumption of NO by Cygb is consistent with experimental results supporting the validity of the kinetic model. Simulations based on this kinetic model suggest that the high efficiency of Cygb in regulating the NO consumption rate is due to the rapid reduction of Cygb by cellular reductants, which greatly increases the rate of consumption of NO at higher O(2) concentrations, and binding of NO to Cygb, which reduces the rate of consumption of NO at lower O(2) concentrations. Thus, the coexistence of Cygb with efficient reductants in tissues allows Cygb to function as an O(2)-dependent regulator of NO decay.