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Apocynin inhibits NADPH oxidase in phagocytes but stimulates ROS production in non-phagocytic cells

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Abstract

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH oxidase. Since it acts as a potent inhibitor in studies with neutrophils and macrophages, no inhibitory effect can often be found in non-phagocyte cells. In our experiments, apocynin even stimulated reactive oxygen species (ROS) production by vascular fibroblasts. Even when added to macrophages, apocynin initially caused an increase in ROS production. The inhibition of ROS formation followed, suggesting that in the presence of leukocyte myeloperoxidase and hydrogen peroxide, apocynin is converted to another compound. Apocynin pre-activated with H2O2 and horseradish peroxidase (HRP) inhibited ROS production immediately. In non-phagocytes, apocynin stimulated ROS production and no inhibition was observed even after 60 min. Apocynin treated with H2O2 and HRP, however, decreased ROS production in the same manner as in macrophages. The stimulatory effect on ROS production can be abolished by tiron and superoxide dismutase (SOD), suggesting that superoxide was the produced species. The effect of apocynin was inhibited by diphenylene iodinium (DPI), a non-scavenging NADPH oxidase inhibitor. It can be summarized that apocynin stimulates cell superoxide production. In the presence of peroxidase and hydrogen peroxide, however, it is converted into another compound that acts as an inhibitor of superoxide production. It strongly suggests that under conditions in vivo, apocynin can have opposite effects on phagocytes and non-phagocyte cells. It acts as an inhibitor of phagocyte NADPH oxidase but also as a ROS production stimulator in non-phagocyte cells.

Introduction

Apocynin (4-hydroxy-3-methoxyacetophenone, acetovanillone, CAS 498-02-2) is a methoxy-substituted catechol originally extracted from the roots of Picrorrhiza kurroa [1], a small perennial herb that grows in the Himalayas. Like other methoxy-substituted catechols [2], [3], it can decrease the production of superoxide (O2.−) by activated neutrophils and macrophages while the ability of phagocytosis remains unaffected. The formation of superoxide and consequently of other reactive oxygen species is the key to microcidal and cytotoxic function of phagocytes; however, it also contributes to the collateral damage of tissues in inflammation. In traditional medicine, various extracts of P. kurroa are used for treating diseases associated with chronic inflammation. Nowadays, this compound is investigated as a possible anti-inflammatory drug that could be useful, e.g. in the treatment of arthritis [1]. The positive effects of apocynin in treating atherosclerosis [4] or preventing ischemia-reperfusion lung injury [5] were proved in experiments with animals.

Apocynin inhibits the formation of superoxide by phagocyte NADPH oxidase. The likely mechanism of this effect is the inhibition of enzyme assembly, probably by blocking sulfhydryl groups [2]. Apocynin itself does not act as a ROS scavenger [6].

Apocynin is used as an experimental tool to inhibit phagocyte NADPH oxidase. We intended to use this compound, like other authors [7], [8], to inhibit NADPH oxidase in non-phagocytes. The addition of apocynin, however, did not lead to a decrease in ROS formation but surprisingly to a significant increase. Some authors suggested earlier that apocynin does not act directly as an inhibitor but that it must be pre-activated by hydrogen peroxide and a peroxidase [2]. In this way it is converted into a symmetrical dimer by means of the formation of a 5,5′carbon–carbon bound [9]. In the locus of inflammation, hydrogen peroxide as well as myeloperoxidase are secreted by phagocytes; in non-phagocyte cells, however, they are practically absent. Therefore we tested the hypothesis that apocynin has different effects on immune stimulated phagocytes and on non-phagocyte cells. In the first case it acts as an inhibitor of NADPH oxidase and its presence leads to a decrease in ROS formation; in the latter case, apocynin on the contrary increases ROS production.

Section snippets

Materials and methods

All reagents were obtained from regular commercial sources and were of at least an analytical grade. Apocynin, cell culture media, Hank's balanced salt solution (HBSS), zymosan A from Saccharomyces cerevisiae, Histopaque, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), catalase, superoxide dismutase, diphenylene iodinium, hydrogen peroxide and tiron (4,5-dihydroxy-benzenedisulfonic acid) were purchased from Sigma-Aldrich Corp., USA. Horseradish peroxidase (HRP) was purchased from ICN

Determination of ROS production

2′,7′-Dichlorofluorescein diacetate was employed to measure ROS formation in intact cells. This fluorescence probe passes through the cell membrane to cytoplasm where it is cleaved by cell esterases to impermeable 2′,7′-dichlorofluorescin (DCFH). DCFH can be easily oxidized by ROS to fluorescent DCF [11], [12].

Fibroblasts or leukocytes were re-suspended in MEM without phenol red supplemented with 10 μmol l−1 DCFH-DA. After incubation for 30 min at 37 °C, the cells were washed in fresh medium

Effects of apocynin on ROS production by vascular fibroblasts

Apocynin increased the fluorescence of DCFH-DA treated fibroblasts (Fig. 1). The increase of fluorescence may be due to the oxidation of DCFH trapped in cells by ROS. However, such observation does not correspond to the fact that apocynin was assumed to reduce ROS formation by cells as it acts as an inhibitor of NADPH oxidase.

We therefore tested whether the increase in fluorescence is not caused by a direct interaction of apocynin with DCFH or whether a fluorescing metabolite of apocynin is not

Discussion

Apocynin is a widely used inhibitor of phagocyte NADPH oxidase. NADPH oxidase is also present in non-phagocyte cells where it plays an important role in ROS production for cell signaling. As phagocyte and non-phagocyte NADPH oxidases share most subunits and properties, we tried to use apocynin as an NADPH oxidase inhibitor in experiments with vascular cells. Surprisingly, apocynin had no inhibitory effect on ROS production but even enhanced ROS formation.

The reactive oxygen species formed after

Acknowledgements

This study was supported by a grant from the Charles University in Prague, No. 16/00/C/1.LF and the Grant Agency of the Czech Republic N303/02/1282.

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