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Cholera toxin and pertussis toxin stimulate prostaglandin E2 synthesis in a murine macrophage cell line
R M Burch1, C Jelsema, J Axelrod
1Section on Pharmacology, National Institute of Mental Health, Bethesda, MD 20892.
Abstract:
When RAW264.7 murine macrophages were incubated with cholera toxin or pertussis toxin, prostaglandin E2 (PGE2) synthesis was enhanced markedly. Cholera toxin and pertussis toxin added together synergistically stimulated PGE2 synthesis. Cholera toxin and pertussis toxin also stimulated cyclic AMP (cAMP) accumulation. However, PGE2 synthesis was independent of increases in cAMP, as neither forskolin nor isoproterenol, which increased cAMP accumulation, nor dibutyryl-cAMP had any effect on PGE2 synthesis. In intact cells, cholera toxin and pertussis toxin stimulated phospholipase A2 to enhance metabolism of phosphatidylinositol to lysophosphatidylinositol and glycerophosphoinositol, with time courses similar to their stimulation of PGE2 synthesis. Cholera toxin catalyzed ADP-ribosylation of proteins of Mr 45,000 and 49,000 in intact cells, whereas an additional substrate of Mr 41,000 was observed in vitro. Preincubation of intact cells with pertussis toxin blocked subsequent in vitro labeling of the Mr 41,000 protein by cholera toxin, suggesting that the same protein was ADP-ribosylated by both toxins. Western blot analysis using specific antisera against Gi, Go and Gs revealed that the Mr 41,000 substrate was bound by the anti-Gi and anti-Go but not anti-Gs. The present data suggest that guanine nucleotide binding regulatory proteins are involved in the regulation of arachidonic acid metabolism to PGE2 in RAW264.7 cells. Furthermore, the possibility is raised that phospholipase A2 is regulated by both stimulatory and inhibitory guanine nucleotide binding proteins.
Insights
Cholera and pertussis toxins enhance prostaglandin E2 (PGE2) synthesis in macrophages, independent of cyclic AMP (cAMP). These toxins activate phospholipase A2 via guanine nucleotide binding proteins, suggesting a novel regulatory pathway for arachidonic acid metabolism.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Prostaglandin E2 (PGE2) plays a crucial role in inflammatory responses.
- Macrophage activation is a key event in immunity and inflammation.
- Toxins like cholera toxin and pertussis toxin are known modulators of cellular signaling pathways.
Purpose of the Study:
- To investigate the effect of cholera toxin and pertussis toxin on PGE2 synthesis in RAW264.7 murine macrophages.
- To elucidate the signaling mechanisms underlying toxin-induced PGE2 production.
- To determine the role of cyclic AMP (cAMP) and guanine nucleotide binding proteins (G proteins) in this process.
Main Methods:
- Incubation of RAW264.7 macrophages with cholera toxin and/or pertussis toxin.
- Measurement of PGE2 synthesis and cAMP accumulation.
- Assessment of phospholipase A2 activity and its metabolites.
- Analysis of protein ADP-ribosylation and identification of substrate proteins using Western blot with specific antisera against G proteins (Gi, Go, Gs).
Main Results:
- Both cholera toxin and pertussis toxin markedly enhanced PGE2 synthesis, with synergistic effects when applied together.
- PGE2 synthesis was found to be independent of cAMP accumulation.
- Toxins stimulated phospholipase A2 activity, leading to increased metabolism of phosphatidylinositol.
- Cholera toxin ADP-ribosylated proteins of Mr 41,000, 45,000, and 49,000. Pertussis toxin pre-incubation blocked ADP-ribosylation of the Mr 41,000 protein by cholera toxin.
- The Mr 41,000 protein was identified as a substrate for both Gi and Go proteins, but not Gs.
Conclusions:
- Guanine nucleotide binding regulatory proteins (Gi and Go) are involved in the regulation of arachidonic acid metabolism to PGE2 in RAW264.7 cells.
- The findings suggest that phospholipase A2 may be regulated by both stimulatory and inhibitory G proteins.
- This study reveals a novel signaling pathway for PGE2 production in macrophages mediated by bacterial toxins and G proteins.