Abstract
BackgroundThe 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).MethodsAlterations in cytosolic Ca2+, Ca2+ influx, inositol triphosphate (IP3), and leukotriene B4 production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively.ResultsActivation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca2+ followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP3 production with associated enhanced Ca2+ release from storage vesicles, prolongation of the peak cytosolic Ca2+ transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB4. The alterations in Ca2+ fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca2+-resequestering endomembrane ATPase.Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP3 production and down-regulation of Ca2+ mediated pro-inflammatory responses of PAF-activated neutrophils.ConclusionAlthough 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.
Highlights
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)
PLC activity is modulated by depletion of enzyme substrate [4], and decay of receptor-mediated signaling [4], it has been proposed that in some cell types, namely vascular endothelial cells [9] and platelets [10], protein kinase C (PKC) negatively regulates PLC
Effects of staurosporine and GF10903X on the fura-2 responses of PAF- or FMLP-activated neutrophils These results are shown in Figures 1 and 2
Summary
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). Chemoattractants, including the bioactive phospholipid, platelet-activating factor (PAF), interact with G-protein coupled receptors on the plasma membrane of human neutrophils to activate phospholipase C (PLC), which is followed by rapid and transient increases in cytosolic calcium concentrations [1,2]. PLC activity is modulated by depletion of enzyme substrate [4], and decay of receptor-mediated signaling [4], it has been proposed that in some cell types, namely vascular endothelial cells [9] and platelets [10], protein kinase C (PKC) negatively regulates PLC. The existence and physiologic consequences of cross-talk between PKC and PLC in activated human neutrophils has, received little attention despite the potential of this mechanism to expedite restoration of Ca2+ homeostasis and attenuate the Ca2+-dependent pro-inflammatory activities of these cells
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