%0 Journal Article
%T The Importance of Myeloperoxidase in Apocynin-Mediated NADPH Oxidase Inhibition
%A Ana Carolina de Almeida
%A Maria Marluce dos Santos Vilela
%A Antonio Condino-Neto
%A Valdecir F. Ximenes
%J ISRN Inflammation
%D 2012
%R 10.5402/2012/260453
%X Apocynin is widely used as an inhibitor of the NADPH oxidase. Since myeloperoxidase (MPO) has been considered as essential for the mechanism of action of apocynin, here we used cells with different levels of MPO and compared their sensitivity to apocynin. HL-60 cells were differentiated with DMSO or IFN¦Ã/TNF¦Á and compared with peripheral mononuclear (PBMC) and polymorphonuclear cells (PMN). The relative MPO activity was PBMC = HL60 DMSO < HL60 IFN¦Ã < PMN. Apocynin inhibited the intracellular reactive oxygen species production by PMN (80%) and IFN¦Ã/TNF¦Á-differentiated HL-60 cells (45%) but showed a minor effect in PBMC and DMSO differentiated HL-60 cells (20%). The addition of azide decreased the efficiency of apocynin in PMN and the addition of peroxidase increased the inhibition in PBMC. We also determined the gene expression of the components gp91phox, p47phox, p22phox and p67phox in the resting cells. Apocynin did not change gp91phox, p47phox or p22phox gene expression in nonstimulated PBMC, HL60 DMSO, HL60 IFN¦Ã/TNF¦Á, and PMN and has a subtle increase in p67phox in HL60 IFN¦Ã/TNF¦Á. The results from this work suggest that a rational search for better inhibitors of NADPH oxidase in leukocytes should include a correlation with their affinity as substrates for MPO. 1. Introduction NADPH oxidases (NOXs) comprise a family of multicomponent enzymatic systems that catalyze the reduction of molecular oxygen to superoxide anion radical [1¨C3]. The best characterized and studied member of the NOX family is NOX2, which is expressed in phagocytic cells and has its primordial microbicidal function in the innate immune system as the primary source of reactive oxygen species (ROS). NOX2 is comprised by cytosolic factors p47phox, p67phox, and p40phox and the membrane-linked p22phox and gp91phox factors, which are the catalytic subunit. NOX2 also has a regulatory GTPase, Rac2 (in neutrophils), or Rac1 (in monocytes) [4, 5]. NOXs, including NOX2, are also expressed in endothelial cells and have direct influence in the oxidative imbalance, which is considered a pivotal event in the endothelial dysfunction and, consequently, in the initiation and/or progression of chronic inflammatory and vascular diseases [5]. Hence, the inhibition or modulation of NOXs activities may have direct benefit in the treatment of these pathologies [5]. Apocynin (4-hydroxy-3-methoxyacetophenone) is widely used as an inhibitor of NOXs and of the concomitant ROS production in experimental models involving phagocytic [6¨C8] and nonphagocytic cells [9¨C13]. The effects of apocynin include the
%U http://www.hindawi.com/journals/isrn.inflammation/2012/260453/