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Wortmannin inhibits mitogen-activated protein kinase activation induced by platelet-activating factor in guinea pig
I M Ferby1, I Waga, C Sakanaka
1Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Abstract:
Stimulation of guinea pig neutrophils with platelet-activating factor (PAF) caused a rapid and transient activation of mitogen-activated protein kinase (MAPK). Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, partially (approximately 50%) inhibited PAF-induced MAPK activation. Half-maximal inhibition was observed with 200-300 nM wortmannin, while it did not inhibit phorbol ester-induced MAPK activation. Neutrophils preloaded with 1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM) failed to raise cytosolic Ca2+ concentrations toward PAF, while they still responded to PAF with a 40-50% activation of MAPK. However, when cells were treated with BAPTA/AM and wortmannin in combination, the MAPK activation was completely inhibited. These results suggest that PAF activates MAPK through two distinct pathways in guinea pig neutrophils, one Ca(2+)-dependent, and the other Ca(2+)-independent but wortmannin-sensitive.
Insights
Platelet-activating factor (PAF) activates mitogen-activated protein kinase (MAPK) in neutrophils via two pathways. One pathway depends on calcium (Ca2+), while the other is independent but sensitive to wortmannin.
Area of Science:
- Immunology
- Cell Signaling
Background:
- Platelet-activating factor (PAF) is a potent lipid mediator involved in inflammation and immune responses.
- Mitogen-activated protein kinases (MAPKs) are crucial signaling molecules regulating various cellular processes, including immune cell activation.
Purpose of the Study:
- To investigate the signaling pathways by which PAF activates MAPK in guinea pig neutrophils.
- To elucidate the roles of calcium (Ca2+) and phosphatidylinositol 3-kinase (PI3K) in PAF-induced MAPK activation.
Main Methods:
- Neutrophil stimulation with PAF.
- Inhibition of MAPK activation using wortmannin (a PI3K inhibitor) and BAPTA/AM (a Ca2+ chelator).
- Assessment of MAPK activation levels via biochemical assays.
Main Results:
- PAF rapidly activated MAPK in guinea pig neutrophils.
- Wortmannin partially inhibited PAF-induced MAPK activation, indicating a PI3K-dependent component.
- Ca2+ chelation with BAPTA/AM partially inhibited MAPK activation, suggesting a Ca2+-dependent component.
- Combined treatment with BAPTA/AM and wortmannin completely abolished MAPK activation, revealing two distinct signaling pathways.
Conclusions:
- PAF activates MAPK in neutrophils through two parallel signaling pathways.
- One pathway is Ca2+-dependent, while the other is Ca2+-independent and sensitive to wortmannin, implicating PI3K in this latter pathway.