Reconstitution Of NADPH Oxidase Research Articles

Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils

In neutrophils (PMNs), superoxide anion (O2−), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b558. In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural or synthetic compounds on the enzyme activity or assembly. The cell-free assay was validated with diphenyleneiodonium chloride and Gp91ds-tat, two inhibitors largely described for human NADPH oxidase. The anti-oxidant effects of curcumin and resveratrol at final concentration ranging from 10−4 to 10−6 M were studied on whole cells by chemiluminescence (CL) and by cell-free assay, in which the molecule was added before or after the enzyme assembly. The CL assay demonstrated that curcumin efficiently inhibited the O2− production and easily entered into PMNs or interacted with their membrane. Cell-free assay showed that curcumin acted on the reconstitution of NADPH oxidase even at 10−5M, while resveratrol appeared to be an O2− scavenger rather than an inhibitor of NADPH oxidase activity, since it acted from outside the cell in CL and after the complex assembly in cell-free assay. By acting directly on NADPH oxidase, curcumin should be a good candidate for the treatment of acute or inflammatory diseases involving an excessive ROS production.

A Critical Role of Protein Kinase Cδ Activation Loop Phosphorylation in Formyl-Methionyl-Leucyl-Phenylalanine-Induced Phosphorylation of p47phox and Rapid Activation of Nicotinamide Adenine Dinucleotide Phosphate Oxidase

Generation of superoxide by professional phagocytes is an important mechanism of host defense against bacterial infection. Several protein kinase C (PKC) isoforms have been found to phosphorylate p47(phox), resulting in its membrane translocation and activation of the NADPH oxidase. However, the mechanism by which specific PKC isoforms regulate NADPH oxidase activation remains to be elucidated. In this study, we report that PKCdelta phosphorylation in its activation loop is rapidly induced by fMLF and is essential for its ability to catalyze p47(phox) phosphorylation. Using transfected COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS-phox cells), we found that a functionally active PKCdelta is required for p47(phox) phosphorylation and reconstitution of NADPH oxidase. PKCbetaII cannot replace PKCdelta for this function. Characterization of PKCdelta/PKCbetaII chimeras has led to the identification of the catalytic domain of PKCdelta as a target of regulation by fMLF, which induces a biphasic (30 and 180 s) phosphorylation of Thr(505) in the activation loop of mouse PKCdelta. Mutation of Thr(505) to alanine abolishes the ability of PKCdelta to catalyze p47(phox) phosphorylation in vitro and to reconstitute NADPH oxidase in the transfected COS-phox cells. A correlation between fMLF-induced activation loop phosphorylation and superoxide production is also established in the differentiated PLB-985 human myelomonoblastic cells. We conclude that agonist-induced PKCdelta phosphorylation is a novel mechanism for NADPH oxidase activation. The ability to induce PKCdelta phosphorylation may distinguish a full agonist from a partial agonist for superoxide production.

Elevated superoxide generation in mononuclear phagocytes by treatment with 1 alpha hydroxyvitamin D3

The effect of 1 alpha-hydroxyvitamin D3 (1 alpha OHD3) treatment on the superoxide production of phagocytic cells in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) was studied. A 3-day treatment of CAPD patients with 3 micrograms per day of 1 alpha OHD3 (high-dose treatment) significantly increased NADPH oxidase and killing activities in peritoneal macrophages and peripheral blood monocytes (P < 0.001) as compared with low-dose (0.25 microgram/day of 1 alpha OHD3) treatment or nontreatment. The high oxidase activity observed in macrophages and monocytes after the treatment with 1 alpha OHD3, correlated significantly to the increase in the amount of the cytosolic factor p47 of the NADPH oxidase as detected by western blotting analysis. Superoxide production by the peripheral blood neutrophils of these patients only slightly increased with 1 alpha OHD3 treatment, and the amount of p47 was not affected by 1 alpha OHD3 administration. In order to evaluate the significance of the oxidase cytosolic factor in dictating oxidase activity, a reconstitution of NADPH oxidase was conducted by mixing macrophage cytosols and membranes in a cell-free system. The addition of macrophage cytosol from patients on high-dose treatment to macrophage membranes from patients in all of the categories of treatment resulted in significantly higher (P < 0.001) superoxide production as opposed to the macrophage cytosol from nontreated patients. These results suggest that 1,25(OH)2D3 causes an increase in NADPH oxidase activity in the peritoneal macrophages and monocytes of CAPD patients by inducing synthesis and elevating the amount of the cytosolic factor p47.(ABSTRACT TRUNCATED AT 250 WORDS)