To specify the site of action of the synthetic coumarin derivatives 7-hydroxy-3-(4′-hydroxyphenyl) coumarin (HHC) and 7-hydroxy-3-(4′-hydroxyphenyl) dihydrocoumarin (HHDC), we evaluated their effects on extra- and intracellular reactive oxygen species (ROS) formation in phorbol-myristate-13-acetate (PMA) stimulated human neutrophils. We studied also the effects of HHC and HHDC on possible molecular mechanisms which participate in the activation of NADPH oxidase, that is, on PKC activity, on phosphorylation of some PKC isoforms (α, βII, and δ), and on phosphorylation of the NADPH oxidase subunit p40phox. Without affecting cytotoxicity, both coumarines tested were effective inhibitors/scavengers of ROS produced by neutrophils on extracellular level. HHC markedly diminished oxidant production and also, intracellularly, decreased PKC activity and partly phosphorylation of PKCα, βII. On the other hand, we did not observe any effect of coumarin derivatives on phosphorylation of PKCδ and on phosphorylation of the NADPH oxidase subunit p40phox, which were suggested to be involved in the PMA-dependent intracellular activation process. In agreement with our previous findings, we assume that the different molecular structures of HHC and HHDC with their different physicochemical and free radical scavenging characteristics are responsible for their diverse effects on the parameters tested. 1. Introduction Neutrophils are key cells of the first line of defense, but they are also considered potent inflammatory cells causing tissue damage. Thus the ability of compounds which prevent extensive and potentially dangerous activation of neutrophils has been proposed as an important injury-limiting way. Coumarins belong to the group of plant-derived polyphenolic compounds possessing broad biochemical and pharmacological effects, like anti-HIV, anti-inflammatory, antioxidant, antibacterial, anticoagulant, and anticancer activities [1–4]. Over the last years, natural as well as synthetic coumarins were extensively studied and many of them are considered attractive candidates in therapeutic development. Production of reactive oxygen species (ROS) in neutrophils and other phagocytic cells is linked to the activation of NADPH oxidase, a multiprotein enzyme complex, which plays an essential role in innate immunity. Yet excessive ROS generation by phagocytes is involved in tissue injury associated with a number of chronic inflammatory diseases [5–7]. In resting cells, NADPH oxidase is inactive and its components are distributed between the cytosol and membranes. When cells are
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