Naturally appearing N-feruloylserotonin isomers suppress oxidative burst of human neutrophils at the protein kinase C level Rado Nosá¾ 1 , Tomáš Pereèko 1 , Viera Janèinová 1 , Katarína Drábiková 1 , Juraj Harmatha 2 , Klara Sviteková 3 Abstract: N-feruloylserotonin (N-f-5HT) isomers, isolated from seeds of Leuzea carthamoides (Wild) DC, inhibited dose-dependent oxidative burst in human whole blood and isolated neutrophils in vitro, which were measured by luminol- and/or isoluminol-enhanced chemi- luminescence in the following rank order of stimuli: PMA > OpZ > calcium ionophore A23187. In isolated neutrophils that were stimulated with PMA, N-f-5HT isomers were effective against extracellular and intracellular reactive oxygen species. Liberation of ATP, analysis of apoptosis, and recombinant caspase-3 activity revealed that N-f-5HTisomers, used in concentrations up to 100 μM, did not alter the viability and integrity of isolated neutrophils. Western blot analysis documented that in concentrations of 10 and 100 μM, N-f-5HT isomers significantly decreased PMA-induced phosphorylation of PKC a/b II. The results suggest that N-f-5HT isomers are an effective, naturally occurring substance with a potent pharmacological effect on the oxidative burst of human neutro- phils. It should be further investigated for its pharmacological activity against oxidative stress in ischemia-reperfusion, inflamma- tion and other pathological conditions. Keywords: neutrophils, N-feruloylserotonin, extracellular and intracellular chemiluminescence, protein kinase C phosphorylation Abbreviations: A23187 – calcium ionophore, CL – chemilu- minescence, HRP – horseradish peroxidase, N-f-5HT – N- feruloylserotonin isomers, PMA – phorbol-myristate-acetate, RLU – relative light units, ROS – reactive oxygen species Introduction Neutrophils, leukocytes and macrophages represent professional phagocytic cells of the innate immune system that are important in host defense and inflam- matory responses. When appropriately stimulated by a variety of agents, they undergo dramatic physiologi- cal and biochemical changes. This results in phagocy- tosis, chemotaxis and degranulation, with the activa- tion of reactive oxygen species (ROS) production in a metabolic pathway, which is known as the respira- tory burst [32], where they consume oxygen and pro- duce superoxide anions [35]. The respiratory burst can be stimulated either by specific receptor-operated stimuli, such as the chemoattractant peptide n-formyl- methionyl-leucyl-phenylalanine (fMLP) and opson- ized zymosan (OpZ), or it can be stimulated by recep- tor-bypassing stimuli like calcium ionophore A23187