THE SIGNAL-TRANSDUCTION MECHANISM INVOLVED IN KAZINOL B-STIMULATED SUPEROXIDE ANION GENERATION IN RAT NEUTROPHILS

1 In this study, the underlying mechanism of stimulation of respirator y burst by kazinol B, a natural isoprenylated flavan, in rat neutrophi ls in vitro was investigated. 2 Kazinol B concentration-dependently st imulated the superoxide anion (O-2(.-)) generation, with a lag but tra nsient activation profile, in neutrophils but not In a cell-free syste m. The maximum response (13.2+/-1.4 nmol O-2(.-) 10 min(-1) per 10(6) cells) was observed at 10 mu M kazinol B. 3 Pretreatment of neutrophil s with phorbol 12-myristate 13-acetate (PMA) or formylmethionyl-leucyl phenylalanine (fMLP) significantly enhanced the O-2(.-) generation fol lowing the subsequent stimulation of cells with kazinol B. 4 Cells pre treated with EGTA or a protein kinase inhibitor taurosporine effective ly attenuated the kazinol B-induced O-2(.-) generation. However, 3 p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and a phos phoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect on the kazinol B-induced response. 5 Kazinol B significantly stimulated [Ca2 +](i) elevation in neutrophils, with a lag and slow rate of rise activ ation profile, and this response was attenuated by a phospholipase C ( PLC) inhibitor U73122. Kazinol B also stimulated the inositol bis- and trisphosphate (IP2 and IP3) formation with a 1 min lag time.6 The mem brane-associated PKC-alpha and PKC-theta but not PKC-iota were increas ed following the stimulation of neutrophils with kazinol B. It was mor e rapid and sensitive in the activation of PKC-theta than PKC-alpha by kazinol B. Kazinol B partially inhibited the [H-3]phorbol 12,13-dibut yrate ([H-3]PDB) binding to the neutrophil cytosolic PKC. 7 Neither th e cellular mass of phosphatidic acid (PA) and phosphatidylethanol (PEt ), in the presence of ethanol, nor the protein tyrosine phosphorylatio n were stimulated by kazinol B. In addition, the kazinol B-induced O-2 (.-) generation remained relatively unchanged in cells pretreated with ethanol or a tyrosine kinase inhibitor genistein. 8 Collectively, the se results indicate that the stimulation of the respiratory burst by k azinol B is probably mediated by the synergism of PKC activation and [ Ca2+](i) elevation in rat neutrophils.