Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity.
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 (O2*-) generation with a slow onset and long lasting profile. The maximum response (16.4+/-2.3 nmol O2*-/10 min per 10(6) cells) was observed at 3-10 microM 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 O2*- generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O2*-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 O2*- 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 O2*- generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 microM cyclocommunin. The membrane-associated PKC-theta and PKC-beta were increased following the stimulation of neutrophils with 30 and 100 microM cyclocommunin, respectively. Cyclocommunin reduced the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (> or =3 microM) significantly evoked a slow and long lasting [Ca2+]i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP2 and IP3) levels were observed in neutrophils stimulated with 30 microM 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 [Ca2+]i elevation in rat neutrophils.
Wang
J P
JP
Department of Medical Research, Taichung Veterans General Hospital, Taiwan.
Tsao
L T
LT
Raung
S L
SL
Lin
P L
PL
Lin
C N
CN
D000818:Animals; D000440:Butanols / Q000494:pharmacology; D002118:Calcium / Q000097:blood*; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology; D004533:Egtazic Acid / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000097:blood; D007700:Kinetics; D019255:NADPH Oxidases / Q000097:blood; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000097:blood*; D051381:Rats; D016897:Respiratory Burst / Q000187:drug effects* / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D013481:Superoxides / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000440:Butanols; D005419:Flavonoids; D007527:Isoenzymes; D010566:Virulence Factors, Bordetella; D013481:Superoxides; D004533:Egtazic Acid; D019255:NADPH Oxidases; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium