Elevated NADPH-oxidase activity in neutrophils from bile-duct-ligated rats: Changes in the kinetic parameters and in the oxidase cytosolic factor p47

Abstract

Stimulated superoxide generation was 2-fold higher in neutrophils from 20 rats with common bile-duct ligation (CBDL) compared to that of 20 sham-operated control rats. In order to study the mechanism of the higher NADPH oxidase activity in CBDL rats, the kinetic parameters of NADPH oxidase were analyzed. The V max of the NADPH oxidase in CBDL rat neutrophils was significantly higher than that of control rat neutrophils (10.2 and 5.3 nmol/min, respectively). The membrane and cytosol fractions of the oxidase were studied in a cell-free system. Neutrophil cytosol from CBDL rats added to neutrophil membranes from either CBDL or control rats produced 22.4 ± 1.6 and 21.0 ± 1.4 nmol/10 6 cells per 10 min, respectively. When neutrophil cytosol from control rats was mixed with neutrophil membranes from control or CBDL rats the generation of superoxide was 10.6 ± 1.4 and 10.0 ± 1.5 nmol/10 6 cells per 10 min, respectively. These results suggest that the cytosol components of the oxidase regulate its activity. By immunoblot analysis it was shown that the amount of the cytosolic factor p47 in neutrophils of CBDL rats is higher than that present in an equal number of neutrophils from control rats.