Inhibition of superoxide generation and myeloperoxidase release by carvedilol after receptor and nonreceptor stimulation of human neutrophils

OBJECTIVES: To compare three stimuli which activate human neutrophils with different signal transduction mechanisms, in order to better localize the effect of the beta-adrenoceptor antagonist carvedilol (CARV) on superoxide generation (O-2(center dot-)) and myeloperoxidase release (MPO). The effect of CARV [0.1-100 mu mol/l] on O-2(center dot-) generation and MPO release from isolated human neutrophils was studied after specific receptor activator N-formyl-methionyl-leucyl-phenylalanine (fMLP) and nonreceptor phorbol-12-myristate-13-acetate (PMA) and calcium ionophor (A23187) stimuli. METHODS: O-2(center dot-) generation was measured as superoxide dismutase inhibitable reduction of cytochrome c and MPO release as the oxidation of o-dianisidine in the presence of hydrogen peroxide in a spectrophotometer Hewlet Packard 8452 A at respective 550 and 463 nm. RESULTS: CARV had no effect on O-2(center dot-) generation and MPO release in nonstimulated cells. In the concentration 10 and 100 mu mol/l, it significantly decreased fMLP and PMA stimulated O-2(center dot-) generation and MPO release. Incubation of neutrophils with CARV [100 mu mol/l] caused significant inhibition of O-2(center dot-) generation and MPO release induced by A23187. Wortmannin, a specific inhibitor of 1-phosphatidylinositol-3-kinase, inhibited significantly only fMLP stimulated O-2(center dot-) generation. CARV [100 mu mol/l] with wortmannin [50 nmol/l] further decreased O-2(center dot-) generation after the same stimulus. CONCLUSION: CARV decreased O-2(center dot-) generation and MPO release from isolated human neutrophils both by membrane-operating stimulus - fMLP and membrane bypassing activators - PMA and A 23187. This fact, together with effect the of wortmannin, indicates that the inhibition may be attributed to the non-specific action of CARV and its interference with phospholipase D signaling pathway, which plays only a minor role in proteinkinase C stimulated O-2(center dot-) generation.