Abstract
Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.
Highlights
Pretreatment (“priming”)of neutrophils witha non- formylmethionylleucylphenylalanine(fMLP)’and phorbol activating concentration (2 nM)of phorbol myristate myristate acetate (PMA, in concentrations above 5 nM) (2, acetate (PMA) augments superoxide(OF)production in 3). response to the chemoattractant formylmethionylleu- The mechanism by whichthe oxidase is activated is controcylphenylalanine
Effect of Sphinganine on PMA-primed Diglyceride Generation-We used sphinganine to investigate the possibility that PMA priming ofDG generation was mediated by protein kinase C.Fig. 5 shows an early time course for fMLP-stimulated DG generation in unprimed and primed cells: In separate experiments, sphinganine alone did not affect fMLP-stimulated DG generation during the first few minutes, as we reported previously [14].Treatment of cells with sphinganine before priming eliminated most of the enhanced DG generation to levels seen in unprimed cellsT. hus, thepriming effect of PMA on fMLP-activated DG production can be blocked by sphinganine
We havepreviously observed that sphinganine, a potent in vitro inhibitor of protein kinase C [12]b, locks activation of 0; generation [13] andof secretion of lactoferrin from specific granules [44], and we report that theinhibitor is effective in PMA-primed cells
Summary
Pretreatment (“priming”)of neutrophils witha non- formylmethionylleucylphenylalanine(fMLP)’and phorbol activating concentration (2 nM)of phorbol myristate myristate acetate (PMA, in concentrations above 5 nM) (2, acetate (PMA) augments superoxide(OF)production in 3). response to the chemoattractant formylmethionylleu- The mechanism by whichthe oxidase is activated is controcylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependentenzyme), on activation of primed neutrophils In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supportingcoammon inhibited site. An obligatory role for protein kinase C has been contested, since some compounds which actas protein kinase C inhibitorsunder in vitro conditions (isoquinolinesulfonamides, polymyxin B, and retinal) fail in whole cells to antagonize neutrophil activation by fMLP [22,23,24] Another line of evidence which seems to contradict a role for DG/protein kinase C is that certain pretreatments appear to inhibit agonist-induced phosphoinositide turnover (as measured by inositol phosphate release) while simultaneously augmenting 0; generation [25,26,27]. The abbreviations used are: fMLP, N-formylmethionylleucylincrease in non-mitochondrial oxygen consumption, with gen- phenylalanine; PMA, pborbol 12-myristate13-acetate; DG, sn-1,2-
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