MELATONIN REGULATES THE RESPIRATORY BURST OF HUMAN NEUTROPHILS AND THEIR DEPOLARIZATION

The effect of different doses of melatonin on the respiratory burst as well as on the membrane potential changes of human neutrophils stimul ated with phorbol 12-myristate 13-acetate (PMA) was investigated. The intracellular production of reactive oxygen species (ROS) in stimulate d neutrophils was quantified in individual cells by flow cytometry, me asuring the oxidation of nonfluorescent dihydrorhodamine 123 to the gr een fluorescent rhodamine 123. The transmembrane potential change was measured using the fluorescent probe oxonol. Preincubating the cells w ith micromolar concentrations of the indole resulted in an increase of the response to PMA. In two of six subjects investigated, the respira tory burst was also increased by a 10 nM concentration of the indole, but when the melatonin concentration was increased to 2 mM the respira tory burst was inhibited. The change in the transmembrane potential of neutrophils paralleled the respiratory burst. Indeed, the treatment o f the cells with doses of melatonin up to 0.5 mM increased the depolar ization occurring subsequent to PMA stimulation, whereas 2 mM melatoni n concentration decreased the extent of depolarization. To investigate whether melatonin could directly affect the transmembrane potential c hanges of neutrophils, the extent of depolarization, induced by increa sing the extracellular potassium concentration, was measured in cells preincubated with 2 mM melatonin. This treatment resulted in a decreas e of the extent of depolarization, which suggests that melatonin can d irectly alter membrane ion conductance in human neutrophils.