Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

BACKGROUND: The role of protein kinase C (PKC) in regulating the activity of phospholipase C (PLC) in neutrophils activated with the chemoattractant, platelet-activating factor (PAF, 20 and 200 nM), was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 μM) and staurosporine (400 nM). METHODS: Alterations in cytosolic Ca(2+), Ca(2+ )influx, inositol triphosphate (IP(3)), and leukotriene B(4 )production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively. RESULTS: Activation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca(2+ )followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP(3 )production with associated enhanced Ca(2+ )release from storage vesicles, prolongation of the peak cytosolic Ca(2+ )transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB(4). The alterations in Ca(2+ )fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca(2+)-resequestering endomembrane ATPase. Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP(3 )production and down-regulation of Ca(2+ )mediated pro-inflammatory responses of PAF-activated neutrophils. CONCLUSION: Although generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils.