Neutrophils stimulated with formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) in the presence of butanol and ethanol formed phosphatidyl alcohols through a phospholipase D mechanism. The alcohols inhibited phosphatidic acid and diradylglycerol (DRG) formation, but did not block inositol 1, 4, 5-trisphosphate release. fMet-Leu-Phe-stimulated superoxide production was inhibited by alcohol concentrations which blocked DRG formation, whereas opsonized-zymosan-stimulated superoxide production was only partially decreased. These results suggest that phospholipase D activation is functionally linked to superoxide production in the human neutrophil.

The tyrosine kinase inhibitors ST271, ST638 and erbstatin inhibited phospholipase D (PLD) activity in human neutrophils stimulated by fMet-Leu-Phe, platelet-activating factor and leukotriene B4. These compounds did not inhibit phorbol ester-stimulated PLD, indicating that they do not inhibit PLD per se, but probably act at a site between the receptor and the phospholipase. In contrast, the protein kinase C inhibitor Ro-31-8220 inhibited phorbol 12,13-dibutyrate- but not fMet-Leu-Phe-stimulated PLD activity, arguing against the involvement of protein kinase C in the receptor-mediated activation of PLD. ST271 did not inhibit Ins(1,4,5)P3 generation, but did inhibit protein tyrosine phosphorylation stimulated by fMet-Leu-Phe. The phosphotyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation and stimulated PLD. These results suggest that tyrosine kinase activity is involved in receptor coupling to PLD but not to PtdIns(4,5)P2-specific phospholipase C in the human neutrophil.

1The fungal metabolite, wortmannin, has recently been shown to inhibit fMet-Leu-Phe-stimulated superoxide production and phospholipase D (PLD) activation in the human neutrophil. 2 We have found that a close structural analogue of wortmannin, demethoxyviridin, has a similar inhibitory profile but in addition blocks phosphatidylinositol 4,5-bisphosphate-specific phospholipase C and hence inositol 1,4,5-trisphosphate (1P3) formation. 3 Inhibition of fMet-Leu-Phe-stimulated PLD by demethoxyviridin was characteristically noncompetitive (IC50 = 31 + 10nM).4 Inhibition of fMet-Leu-Phe-stimulated 1P3 formation required concentrations almost 10 times higher (IC50 = 250 + 130 nM).5 Surprisingly, demethoxyviridin only inhibited fMet-Leu-Phe-induced intracellular calcium mobilization at concentrations 100 times greater than those needed to block IP3 formation. 6 Demethoxyviridin also inhibited PLD activation induced by sodium fluoride or phorbol myristate acetate (PMA) but the concentrations required were 100 times those needed to block fMet-Leu-Phestimulated PLD. 7 These observations support the contention that PLD plays an important role in signal transduction in the human neutrophil and indicate that wortmannin and demethoxyviridin inhibit PLD activation at a common step in the signalling pathway. 8 Furthermore, these results suggest that demethoxyviridin may block the interaction between the chemotactic peptide receptor and a GTP-binding protein that is intimately involved in PLD activation.

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