Effect of silybin on phorbol myristate actetate-induced protein kinase C translocation, NADPH oxidase activity and apoptosis in human neutrophils

Stimulation of the respiratory burst of human neutrophils by fMet-Leu-Phe (in the absence of cytochalasin B) is largely unaffected when the activities of protein kinase C and phospholipase D are inhibited. This has been confirmed using three separate assays to measure the respiratory burst. However, whilst these enzymes are not required for the initiation or maximal rate of oxidant generation, they are required to sustain oxidase activity. In contrast, in the presence of cytochalasin B, fMet-Leu-Phe stimulated oxidase activity is much more dependent on phospholipase D activity. It is proposed that (in the absence of cytochalasin B) activation of the NADPH oxidase utilises cytochrome b molecules that are already present on the plasma membrane and activation occurs independently of phospholipase D and protein kinase C. Once these complexes are inactivated, then new cytochrome b molecules must be recruited from sub-cellular stores. This translocation and/or activation of these molecules is phospholipase D dependent. Some support for this model comes from the finding that the translocation of CD11b (which co-localises with cytochrome b) onto the cell surface is phospholipase D dependent.

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Protein kinase C-dependent and -independent activation of the NADPH oxidase of human neutrophils.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)89465-4 ◽

1991 ◽

Vol 266 (12) ◽

pp. 7432-7439

Author(s):

F Watson ◽

J Robinson ◽

S W Edwards

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Nadph Oxidase ◽

Human Neutrophils ◽

Kinase C

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Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C

Biochemical Journal ◽

10.1042/bj2920781 ◽

1993 ◽

Vol 292 (3) ◽

pp. 781-785 ◽

Cited By ~ 48

Author(s):

G C Kessels ◽

K H Krause ◽

A J Verhoeven

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Nadph Oxidase ◽

Phospholipase D ◽

Respiratory Burst ◽

Feedback Inhibition ◽

Human Neutrophils ◽

Kinase C ◽

Pkc Activation

Stimulation of human neutrophils by the receptor agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) results in a respiratory burst, catalysed by an NADPH oxidase. Concomitantly, phospholipase D (PLD) is activated. To investigate the role of protein kinase C (PKC) in these neutrophil responses, we have compared the effects of staurosporine and a structural analogue of staurosporine (cgp41251), that reflects a higher selectivity towards PKC [Meyer, Regenass, Fabbro, Alteri, Rösel, Müller, Caravatti and Matter (1989) Int. J. Cancer 43, 851-856]. Both staurosporine and cgp41251 dose-dependently inhibited the production of superoxide induced by phorbol 12-myristate 13-acetate (PMA). Both compounds also caused inhibition of the fMLP-induced respiratory burst, but with a lower efficacy during the initiation phase of this response. This latter observation cannot be taken as evidence against PKC involvement in the activation of the respiratory burst, because pretreatment of neutrophils with ionomycin before PMA stimulation also results in a lower efficacy of inhibition. Activation of PLD by fMLP was enhanced in the presence of staurosporine, but not in the presence of cgp41251. Enhancement of PLD activation was also observed in the presence of H-89, an inhibitor of cyclic-AMP-dependent protein kinase (PKA). Both staurosporine and H-89 reversed the dibutyryl-cyclic-AMP-induced inhibition of PLD activation, whereas cgp41251 was without effect. These results indicate that the potentiating effect of staurosporine on PLD activation induced by fMLP does not reflect a feedback inhibition by PKC activation, but instead a feedback inhibition by PKC activation. Taken together, our results indicate that in human neutrophils: (i) PKC activity is not essential for fMLP-induced activation of PLD; (ii) PKC activity does play an essential role in the activation of the respiratory burst by fMLP, other than mediating or modulating PLD activation; (iii) there exists a negative-feedback mechanism on fMLP-induced PLD activation by concomitant activation of PKA.

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Protein kinase C-β contributes to NADPH oxidase activation in neutrophils

Biochemical Journal ◽

10.1042/bj3470285 ◽

2000 ◽

Vol 347 (1) ◽

pp. 285-289 ◽

Cited By ~ 85

Author(s):

Lodewijk V. DEKKER ◽

Michael LEITGES ◽

Gabriel ALTSCHULER ◽

Nishil MISTRY ◽

Aileen MCDERMOTT ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Nadph Oxidase ◽

Specific Inhibitor ◽

Human Neutrophils ◽

Cell Fractionation ◽

Coated Particles ◽

Kinase C ◽

Pkc Β ◽

Nadph Oxidase Activation

We have analysed the involvement of the β isotype of the protein kinase C (PKC) family in the activation of NADPH oxidase in primary neutrophils. Using immunofluorescence and cell fractionation, PKC-β is shown to be recruited to the plasma membrane upon stimulation with phorbol ester and to the phagosomal membrane upon phagocytosis of IgG-coated particles (Fcγ-receptor stimulus). The time course of recruitment is similar to that of NADPH oxidase activation by these stimuli. The PKC-β specific inhibitor 379196 inhibits the response to PMA as well as to IgG-coated bacteria. Partial inhibition occurs between 10 and 100 nM of inhibitor, the concentration at which PKC-β, but not other PKC isotypes, is targeted. Neutrophils isolated from a mouse that lacks PKC-β also showed an inhibition of NADPH oxidase activation by PMA and IgG-coated particles. The level of inhibition is comparable to that achieved with 379196 in human neutrophils. Thus the PKC-β isotype mediates activation of NADPH oxidase by PMA and by stimulation of Fcγ receptors in neutrophils.

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D1 Dopamine Receptor Down‐regulates NADPH Oxidase Activity via a Protein Kinase C and Protein kinase A Cross‐talk in Human Kidney Cells

The FASEB Journal ◽

10.1096/fasebj.22.1_supplement.969.31 ◽

2008 ◽

Vol 22 (S1) ◽

Author(s):

Weixing Han ◽

Francis B Arnaldo ◽

Pedro A Jose ◽

Peiying Yu

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Kinase A ◽

Oxidase Activity ◽

Human Kidney ◽

Kidney Cells ◽

Nadph Oxidase Activity ◽

Kinase C ◽

D1 Dopamine Receptor ◽

Kinase A

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Early signalling events implicated in leukotriene B4-induced activation of the NADPH oxidase in eosinophils: role of Ca2+, protein kinase C and phospholipases C and D

Biochemical Journal ◽

10.1042/bj3100795 ◽

1995 ◽

Vol 310 (3) ◽

pp. 795-806 ◽

Cited By ~ 32

Author(s):

R S Perkins ◽

M A Lindsay ◽

P J Barnes ◽

M A Giembycz

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Nadph Oxidase ◽

Respiratory Burst ◽

Leukotriene B4 ◽

Human Neutrophils ◽

H2o2 Production ◽

Intact Cells ◽

Kinase C ◽

H2o2 Generation

The early signalling events that may ultimately contribute to the assembly and subsequent activation of the NADPH oxidase in guinea-pig peritoneal eosinophils were investigated in response to leukotriene B4 (LTB4). LTB4 promoted a rapid, transient and receptor-mediated increase in the rate of H2O2 generation that was potentiated by R 59 022, a diradylglycerol (DRG) kinase inhibitor, implicating protein kinase C (PKC) in the genesis of this response. This conclusion was supported by the finding that the PKC inhibitor, Ro 31-8220, attenuated (by about 30%) the peak rate of LTB4-induced H2O2 generation under conditions where the same response evoked by 4 beta-phorbol 12,13-dibutyrate (PDBu) was inhibited by more than 90%. Paradoxically, Ro 31-8220 doubled the amount of H2O2 produced by LTB4 which may relate to the ability of PKC to inhibit cell signalling through phospholipase C (PLC). Indeed, Ro 31-8220 significantly enhanced LTB4-induced Ins(1,4,5)P3 accumulation and the duration of the Ca2+ transient in eosinophils. Experiments designed to assess the relative importance of DRG-mobilizing phospholipases in LTB4-induced oxidase activation indicated that phospholipase D (PLD) did not play a major role. Thus, although H2O2 generation was abolished by butan-1-ol, this was apparently unrelated to the inhibition of PLD, as LTB4 failed to stimulate the formation of Ptd[3H]BuOH in [3H]butan-1-ol-treated eosinophils. Rather, the inhibition was probably due to the ability of butan-1-ol to increase the eosinophil cyclic AMP content. In contrast, Ca(2+)- and PLC-driven mechanisms were implicated in H2O2 generation, as LTB4 elevated the Ins(1,4,5)P3 content and intracellular free Ca2+ concentration in intact cells, and cochelation of extracellular and intracellular Ca2+ significantly attenuated LTB4-induced H2O2 generation. Pretreatment of eosinophils with wortmannin did not affect LTB4-induced H2O2 production at concentrations at which it abolished the respiratory burst evoked by formylmethionyl-leucylphenylalanine in human neutrophils. Collectively, these data suggest that LTB4 activates the NADPH oxidase in eosinophils by PLD- and PtdIns 3-kinase-independent mechanisms that involve Ca2+, PLC and PKC. Furthermore, the activation of additional pathways that do not require Ca2+ is also suggested by the finding that LTB4 evoked a significant respiratory burst in Ca(2+)-depleted cells.

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