Studies with protein kinase C inhibitors presently available cannot elucidate the role of protein kinase C in the activation of NADPH oxidase

Abstract

The effects of various protein kinase C (PKC) inhibitors on NADPH oxidase (NO) activation by the phorbol ester PMA and by the chemotactic peptide FMLP were studied. H-7 reduced the effects of both stimuli in human neutrophils (HN) and HL-60 cells by 13-63%. Polymyxin B did not inhibit NO activation by PMA and FMLP in HN and reduced the effects of both stimuli in HL-60 cells by 27-55%. Retinal and retinoic acid enhanced the effects of PMA and FMLP in HL-60 cells and of FMLP in HN up to 4.5-fold. In contrast, retinoic acid inhibited the effect of PMA in HN, In the presence of cytochalasin B, retinal inhibited the effect of FMLP in HN, whereas retinoic acid inhibited NO activation by FMLP in both cell types. The dual PKC/calmodulin inhibitors trifluoperazine and W-7 abolished NO activation by PMA and FMLP in HN and HL-60 cells. Thus, the effects of PKC inhibitors on NO activation exhibit (I) cell type specificity, (2) stimulus dependency and (3) no correlation with in vitro inhibition of PKC. Our results suggest that studies with PKC inhibitors presently available cannot clarify the role of PKC in NO activation. ~1988Ac~d .... Press, Inc.Human neutrophils possess an NADPH oxidase which catalyzes 0 2" formation and can be activated by cell-permeable diacylglycerol and PMA via PKC [I,2] and by the chemotactic peptide, FMLP [3]. The mechanism by which FMLP activates 0 2 -formation is not known. There is a debate concerning the involvement of PKC in cellular activation by FMLP [3]. In recent studies, certain PKC

Nucleotide-, Chemotactic Peptide- and Phorbol Ester-Induced Exocytosis in HL-60 Leukemic Cells

“…To mduce differentiation, HL-60 cells were seeded at IO 6 cells/ml and were cultured for 48 h with 0.2 mM Bt2cAMP (27,28). Pappenheim-stained smears showed that Bt2cAMP-differentiated HL-60 cells consisted of more than 98 % myelocytes or cells beyond this stage.…”

“…O2" formation was monitored by continuous measurement of ferricytochrome C reduction inhibitable by superoxide dismutase using a Uvikon 810 dual-beam spectrophotometer (Kontron, Eching, FRG) (28,32). Reaction mixtures (0.5 ml) contained 2.5 x IO 6 Bt2cAMP-differentiated HL-60 cells, 100 uM ferricytochrome C and the buffer used for the determination of P-glucuronidase release.…”

“…Reaction mixtures were incubated for 5 min at 37 0 C in the absence or presence of CB (5 ug/ml) prior to the addition of stimuli. Vmax of O2 formation was calculated (28,32).…”

Role of protein kinase c in interleukin 1, anti‐T3, and mitogenic lectin‐induced interleukin 2 secretion

“…The putative PKC inhibitor, 1-(5-isoquinolinylsulfony1)-2-methylpiperazine (H7) has been re-ported to inhibit both IL2 production (Mukaida et al, 1987) and T-cell proliferation (Clark et al, 1987;Nel et al, 1987), which is an IL2-dependent event. However, recent reports suggest that H7 may not be an effective PKC inhibitor in intact cells (Watson et al, 1988;Seifert et al, 1988). Nevertheless, activation of PKC by perturbation of the T-cell receptor, combined with the augmentation of IL2 secretion by phorbol esters and inhibition of IL2 secretion by H7 support the hypothesis that activation of PKC may be required for IL2 secretion.…”

Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins

“…Reaction mixtures were preincubated for 3 min at 37°C. 0; generation was initiated by the addition of stimuli [40]. All assays were performed in the absence of cytochalasin B.…”