Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca 2+ and protein kinase C activity

Abstract

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O 2 − ) generation with a slow onset and long lasting profile. The maximum response (16.4 ± 2.3 nmol O 2 − /10 min per 10 6 cells) was observed at 3–10 μM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O 2 − generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O 2 − generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O 2 − generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O 2 − generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 μM cyclocommunin. The membrane-associated PKC-θ and PKC-β were increased following the stimulation of neutrophils with 30 and 100 μM cyclocommunin, respectively. Cyclocommunin reduced the [ 3H]phorbol 12,13-dibutyrate ([ 3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (≥3 μM) significantly evoked a slow and long lasting [Ca 2+] i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca 2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP 2 and IP 3) levels were observed in neutrophils stimulated with 30 μM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca 2+] i elevation in rat neutrophils.