Apocynin, Reduced Nicotinamide-Adenine Dinucleotide Phosphate Oxidase, and Vascular Cells

Apocynin (4-hydroxy-3-methoxyacetophenone), isolated from the traditional medicinal plant Picrorhiza kurroa, is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase.1 Activated neutrophil NADPH oxidase, a multisubunit complex composed of membrane-associated gp91phox (Nox2) and p22phox and cytosolic subunits, p47phox, p67phox, and p40phox, produces superoxide anion, a precursor of microbicidal reactive oxygen species (ROS), thereby playing a crucial role in host defense. Apocynin inhibits the release of superoxide anion by NADPH oxidase by blocking migration of p47phox to the membrane, critically involved in initiating assembly of the functional NADPH oxidase complex.1 The underlying chemistry of apocynin’s actions has been elucidated in phagocytic cells.2 Apocynin needs to be activated (oxidized) for it to inhibit the oxidase. In the presence of H2O2 and myeloperoxidase (MPO), abundant in neutrophils, apocynin is oxidized and has as products dimer and trimer derivatives resulting in diapocynin formation, the metabolically active compound of apocynin. The reactivity of apocynin radical with thiol compounds is possibly the mechanism involved in the inhibitory effect of apocynin on the NADPH oxidase complex.2,3 In the early 1990s, with the discovery that cardiovascular cells possess functionally active NADPH oxidase critically involved in ROS generation and oxidative stress in the vascular wall, many investigators extended neutrophil findings and used apocynin as a pharmacological agent to specifically inhibit the vascular cell oxidase. Convincing data from numerous in vitro studies demonstrated that, in endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts, apocynin blocks NADPH oxidase activity and superoxide anion generation.4,5 In vivo studies, in which mice and rats were treated with apocynin, confirmed these cellular findings.6,7 Such experiments facilitated progress in the field of ROS and vascular biology and elucidated the importance of NADPH oxidase in oxidative stress and cardiovascular, renal, and cerebrovascular disease. The above studies were all based on the presumption that apocynin regulation in nonphagocytic cells is similar to that in phagocytic cells. However, this may not be true, as highlighted in the article by Heumueller et al8 in the current issue, where it is shown that apocynin is not an inhibitor of vascular NADPH oxidase but an important antioxidant. These provocative conclusions were based on the premise that vascular cells (and other nonphagocytic cells) do not possess MPO, and, therefore, activated apocynin is not formed. In their comprehensive experiments, using HEK 293 cells overexpressing Nox subunits, Nox1, Nox2, or Nox 4, and in endothelial and vascular smooth muscle cells, in which MPO is absent, the investigators demonstrate that apocynin does not inhibit NADPH oxidase activation or the generation of superoxide. Exposure to MPO induced apocynin dimer formation and, as predicted, inhibition of the oxidase. Despite no effect on NADPH oxidase, apocynin did reduce ROS bioavailability, probably through an antioxidant mechanism. A number of important questions arise from the study by Heumueller et al, as described below.8 First, is apocynin activity MPO specific? Peroxidasedeficient cells appear to be insensitive to apocynin. However, peroxidases other than MPO might influence apocynin activity. Vejražka et al9 showed that horseradish peroxidase, like MPO, induces apocynin dimer formation, with consequent NADPH oxidase inhibitory effects. Hence it is possible that vascular cells possess peroxidases, other than MPO, which may activate apocynin. This is supported by findings in endothelial cells, where apocynin dimers were identified and found to dose-dependently inhibit NADPH oxidase activity, ROS formation, and cell proliferation.4 Second, can apocynin inhibit vascular cell NADPH oxidase even if these cells do not possess MPO themselves? In vivo studies demonstrated that neutrophils secrete MPO, which can be taken up by endothelial cells, in which apocynin can then be metabolized to active dimers to inhibit vascular cell NADPH oxidase (Figure). Cytokeratin 1 has been shown recently to be the protein responsible for MPO internalization in endothelial cells.10 Such a process may explain the NADPH oxidase inhibitory effects observed in many animal studies and does not necessarily refute the use of apocynin as a specific inhibitor in this context. However, such a situation is different in cultured cells, which are not exposed to The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada. Correspondence to Rhian M. Touyz, Kidney Research Centre, University of Ottawa/Ottawa Health Research Institute, 451 Smyth Rd, Ottawa, Ontario, KIH 8M5 Canada. E-mail [email protected] (Hypertension. 2008;51:1-3.) © 2007 American Heart Association, Inc.