Related Experiment Videos
Demethoxyviridin and wortmannin block phospholipase C and D activation in the human neutrophil
R W Bonser1, N T Thompson, R W Randall
1Biochemical Sciences Department, Wellcome Research Laboratories, Beckenham, Kent.
Abstract:
1. The 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 (IP3) formation. 3. Inhibition of fMet-Leu-Phe-stimulated PLD by demethoxyviridin was characteristically non-competitive (IC50 = 31 +/- 10 nM). 4. Inhibition of fMet-Leu-Phe-stimulation IP3 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-Phe-stimulated 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.
Insights
Demethoxyviridin inhibits phospholipase D (PLD) and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, impacting neutrophil signaling. This suggests demethoxyviridin may disrupt chemotactic peptide receptor and GTP-binding protein interactions.
Area of Science:
- Immunology
- Cellular Signaling
- Biochemistry
Background:
- Wortmannin, a fungal metabolite, inhibits neutrophil superoxide production and phospholipase D (PLD) activation.
- Human neutrophils utilize signaling pathways involving PLD and phosphatidylinositol 4,5-bisphosphate-specific phospholipase C.
Purpose of the Study:
- To investigate the inhibitory effects of demethoxyviridin, a wortmannin analogue, on neutrophil signaling pathways.
- To elucidate the role of PLD in neutrophil signal transduction.
Main Methods:
- Investigated the inhibition of fMet-Leu-Phe-stimulated superoxide production and PLD activation.
- Assessed the impact of demethoxyviridin on phosphatidylinositol 4,5-bisphosphate-specific phospholipase C and inositol 1,4,5-trisphosphate (IP3) formation.
- Examined the effect on intracellular calcium mobilization and PLD activation induced by sodium fluoride or phorbol myristate acetate (PMA).
Main Results:
- Demethoxyviridin inhibited PLD activation non-competitively (IC50 = 31 nM) and IP3 formation (IC50 = 250 nM).
- Intracellular calcium mobilization was inhibited at much higher concentrations.
- Demethoxyviridin also inhibited PLD activation induced by sodium fluoride or PMA, but required higher concentrations.
Conclusions:
- PLD plays a significant role in human neutrophil signal transduction.
- Wortmannin and demethoxyviridin likely inhibit PLD activation at a common step.
- Demethoxyviridin may impede the interaction between chemotactic peptide receptors and GTP-binding proteins involved in PLD activation.