Antioxidant/Oxidant Status and Cardiac Function in Bradykinin B1- and B2-Receptor Null Mice

Kinin-vasoactive peptides activate two G-protein-coupled receptors (R), B1R (inducible) and B2R (constitutive). Their complex role in cardiovascular diseases could be related to differential actions on oxidative stress. This study investigated impacts of B1R or B2R gene deletion in mice on the cardiac function and plasma antioxidant and oxidant status. Echocardiography-Doppler was performed in B1R (B1R-/-) and B2R (B2R-/-) deficient and wild type (WT) adult male mice. No functional alteration was observed in B2R-/- hearts. B1R-/- mice had significantly lowered fractional shortening and increased isovolumetric contraction time. The diastolic E and A waves velocity ratio was similar in all mice groups. Thus B1R-/- mice provide a model of moderate systolic dysfunction, whereas B2R-/- mice displayed a normal cardiac phenotype. Plasma antioxidant capacity (ORAC) was significantly decreased in both B1R-/- and B2R-/- mice whereas the vitamin C levels were decreased in B2R-/- mice only. Plasma ascorbyl free radical was significantly higher in B1R-/- compared to WT and B2R-/- mice. Therefore, the oxidative stress index, ascorbyl free radical to vitamin C ratio, was increased in both B1R-/- and B2R-/- mice. Hence, B1R and B2R deficiency are associated with increased oxidative stress, but there is a differential imbalance between free radical production and antioxidant defense. The interrelationship between the differential B1R and B2R roles in oxidative stress and cardiovascular diseases remain to be investigated.

Spectral characterization and chemical modification of FMN-containing ascorbyl free-radical reductase from Pleurotus ostreatus

Ascorbyl free-radical reductase was purified 1143-fold with an overall yield of 9.9% from the cytosolic fraction of Pleurotus ostreatus. The native enzyme had a molecular mass of 127 kDa and SDS/PAGE revealed that the enzyme consists of two subunits, each with a molecular mass of 62 kDa. The enzyme utilized only NADH as an electron donor. The enzyme was highly specific for ascorbyl free radical as an electron acceptor and capable of catalysing the reduction of ferricyanide and 2,6-dichloroindophenol as artificial electron acceptors. The apparent Km values of the enzyme towards NADH and ascorbyl free radical were 35±0.22 and 2.1±0.03 μM, respectively. The catalytic mechanism of this enzyme is of Ping Pong type. The enzyme contained FMN as a prosthetic group and showed the characteristic absorption spectrum ascribed to the charge-transfer interaction of thiolate anion with FMN. The enzyme contained eight cysteine residues per monomer and was inactivated more rapidly by mercurials than by thiol-alkylating reagents. Kinetic analysis of the inactivation process revealed that the enzyme had 1 mol of thiol group/mol of subunit in the active site with a pKa of 6.9. The modification of the thiol group of the enzyme caused the loss of charge-transfer absorbance centred at 540 nm and blocked the electron-transfer process from NADH to FMN. The modification of lysine, arginine and histidine residues led to the loss of its activity. Unlike the active enzyme, the fluorescence quenching of NADH was not observed in the lysine-modified enzyme, which implies that lysine residues can participate in the interaction with NADH.