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Regulation of platelet-activating factor synthesis by acetyl-coenzyme A
C Tetta1, G Camussi, F Bussolino
1Laboratorio di Immunopatologia, Università di Torino, Italy.
Abstract:
Human neutrophils (PMN) stimulated by tumor necrosis factor (TNF) synthesize and release platelet-activating factor (PAF) transiently. In the present investigation, we have examined the mechanism responsible for the down-regulation of PAF synthesis. The response of PMN is proportional to the occupancy of high-affinity TNF receptors on the PMN plasma membrane, as shown by binding assays with [125I]TNF. These receptors are down-regulated within 10 min of the addition of TNF; the receptors reappear after 60 min, but the PMN do not resume PAF synthesis. Further PAF synthesis is obtained by adding acetyl-coenzyme A (CoA) to the culture medium. However, this compound does not diffuse into PMN, as shown by incubating these cells with labeled acetyl-CoA. This finding suggests that PAF synthesis is regulated by the amount of acetyl-CoA available to the lyso-PAF: acetyltransferase on the cell plasma membrane. This acetyl-CoA is accessible to chemicals in the culture medium, since it is hydrolysed by hydroxylamine. The inhibition of PAF synthesis by hydroxylamine is reversed by adding acetyl-CoA. PAF synthesis in TNF-treated cells appears to be regulated either by the amount of acetyl-CoA available or by the ability to transfer acetyl-CoA from the cellular pool to the lyso-PAF: acetyltransferase.
Insights
Tumor necrosis factor (TNF) triggers platelet-activating factor (PAF) synthesis in neutrophils. This process is regulated by acetyl-CoA availability to the lyso-PAF: acetyltransferase, not receptor levels.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Neutrophils (PMN) synthesize and release platelet-activating factor (PAF) upon stimulation with tumor necrosis factor (TNF).
- The transient nature of PAF synthesis suggests a regulatory mechanism controlling its production.
Purpose of the Study:
- To investigate the mechanism behind the down-regulation of PAF synthesis in TNF-stimulated human neutrophils.
- To determine the role of TNF receptor occupancy and acetyl-CoA availability in regulating PAF production.
Main Methods:
- Binding assays using [125I]TNF to measure TNF receptor occupancy on PMN plasma membrane.
- Incubation of PMN with labeled acetyl-CoA to assess its diffusion into cells.
- Assessing PAF synthesis after adding acetyl-CoA to the culture medium.
- Investigating the effect of hydroxylamine on PAF synthesis and its reversal by acetyl-CoA.
Main Results:
- PMN response to TNF is proportional to high-affinity TNF receptor occupancy.
- TNF receptors are down-regulated within 10 minutes, with partial reappearance after 60 minutes, but PAF synthesis does not resume.
- Adding acetyl-CoA to the medium enhances PAF synthesis, but it does not diffuse into PMN.
- Acetyl-CoA is accessible externally and hydrolyzed by hydroxylamine, with inhibition reversed by acetyl-CoA addition.
Conclusions:
- PAF synthesis regulation is linked to the availability of acetyl-CoA to the plasma membrane-bound lyso-PAF: acetyltransferase.
- The cellular pool of acetyl-CoA or its transfer to the enzyme may be the rate-limiting step in TNF-induced PAF synthesis.
- Down-regulation of PAF synthesis is primarily mediated by factors affecting acetyl-CoA availability rather than TNF receptor dynamics alone.