Daphnoretin-induced respiratory burst in rat neutrophils is, probably, mainly through protein kinase C activation

Abstract

Daphnoretin, a dicoumarin isolated from Wikstroemia indica C.A. Mey. ( Thymelaceae), induced superoxide anion (O 2 −) formation in rat neutrophils in a concentration-dependent manner. Addition of staurosporine reduced daphnoretin-induced respiratory burst. Removal of extracellular free Ca 2+ by EGTA did not affect the respiratory burst of neutrophils in response to daphnoretin. Prior exposure of neutrophils to phorbol 12-myristate 13-acetate (PMA) or daphnoretin reduced the O 2 − formation caused by a subsequent challenge with PMA and daphnoretin, but potentiated the response caused by a subsequent addition of formyl-Met-Leu-Phe (fMLP). Like PMA, daphnoretin did not increase the [Ca 2+] i during cell activation. In neutrophil suspension, daphnoretin increased the membrane associated protein kinase C activity. In the presence of Ca 2+ and phosphatidylserine, daphnoretin also activated protein kinase C isolated from cytosolic fraction of resting neutrophils. Staurosporine inhibited the direct activation of protein kinase C caused by daphnoretin as well as by PMA. Daphnoretin reduced the [ 3H]Phorbol-12,13-dibutyrate ([ 3H]PDB) binding to the neutrophil cytosolic protein kinase C in a concentration-dependent manner with an IC 50 value of 1.77±0.37 μM. These results indicate that daphnoretin, like PMA, may direct activation of protein kinase C which in turn activated NADPH oxidase and elicited respiratory burst.