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.
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.
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam.
G C
GC
Kessels
K H
KH
Krause
A J
AJ
Verhoeven
D000470:Alkaloids / Q000494:pharmacology*; D003994:Bucladesine / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007546:Isoquinolines / Q000494:pharmacology*; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000097:blood*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood*; D019311:Staurosporine / Q000031:analogs & derivatives; D013449:Sulfonamides; D013481:Superoxides / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D007546:Isoquinolines; D013449:Sulfonamides; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003994:Bucladesine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011493:Protein Kinase C; D010739:Phospholipase D; D019311:Staurosporine; C059539:midostaurin; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D013755:Tetradecanoylphorbol Acetate