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

Abstract

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 muM) and staurosporine (400 nM).

Methods: Alterations 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.

Results: Activation 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.

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.

Figure 1

Fura-2 fluorescence responses of PAF (20 nM)-activated neutrophils (A), pretreated with staurosporine 400 nM (B), GF10903X 0.5 μM (C) and 1 μM (D), in the presence (_ _ _) or absence (____) of rolipram (2 μM), as well as those of FMLP (1 μM)-activated cells (E), with (_ _ _) and without (____) GF10903X (1 μM). These are traces from a single representative experiment with a total of 3 8 in each series. Addition of the chemoattractant is denoted by the arrow (↓).

Figure 2

Fura-2 fluorescence responses of PAF (200 nM)-activated neutrophils (A), pre-treated with staurosporine 400 nM (B), GF10903X 0.5 μM (C), and 1 μM (D) in the presence (_ _ _), or absence (____) of EGTA or U73122 (2 μM) (.....) added 10 - 15 sec after PAF. These are traces from a single representative experiment with a total of 4 - 12 in each series. The arrows denote addition of PAF (↓) or U73122 (↑).

Figure 3

Effects of GF10903X 0.5 μM (.....) or 1 μM (_ _ _) on the Mn²⁺ quenching of fura-2 fluorescence assay in PAF 20 nM (A)- or 200 nM (B)-activated neutrophils. PAF was added as indicated (↓). These are traces from a single representative experiment with a total of 5 - 8 in each series.