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Altered responses to modulators of guanine nucleotide binding protein activity in endotoxin tolerance
K A Coffee1, P V Halushka, W C Wise
1Department of Physiology, Medical University of South Carolina, Charleston 29425.
Abstract:
The effects of cholera toxin or pertussis toxin and nonhydrolyzable GTP analogs on Salmonella enteritidis endotoxin stimulation of iTxB2 and i6-keto-PGF1 alpha synthesis in control and endotoxin tolerant rat peritoneal macrophages were determined. Pretreatment with pertussis toxin alone had no effect on basal macrophage iTxB2 or i6-keto-PGF1 alpha production, but pertussis toxin (0.1, 1.0 and 10 ng/ml) significantly inhibited endotoxin-stimulated iTxB2 and i6-keto-PGF1 alpha synthesis. Pretreatment with cholera toxin, which did not affect basal iTxB2 or i6-keto-PGF1 alpha synthesis, significantly enhanced endotoxin-induced synthesis of iTxB2 and i6-keto-PGF1 alpha. The effects of pertussis and cholera toxin with or without endotoxin were significantly (P less than 0.05) less in macrophages from endotoxin tolerant rats compared to control macrophages. GTP[gamma-S] (100 microM) significantly increased iTxB2 synthesis and significantly augmented endotoxin-stimulated iTxB2 synthesis in control macrophages (P less than 0.05). However, in macrophages from endotoxin tolerant rats the effect of GTP[gamma-S] on iTxB2 synthesis was significantly less (P less than 0.05) compared to control macrophages. Collectively, these data suggest that: (1) guanine nucleotide binding regulatory proteins mediate endotoxin-stimulated arachidonic acid metabolism in rat peritoneal macrophages; and (2) endotoxin tolerance induces alterations in guanine nucleotide binding protein activity.
Insights
Guanine nucleotide-binding proteins regulate endotoxin-stimulated arachidonic acid metabolism in rat macrophages. Endotoxin tolerance alters these protein activities, impacting inflammatory responses.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Endotoxin (lipopolysaccharide) from Salmonella enteritidis triggers inflammatory responses in macrophages.
- Arachidonic acid metabolites, such as thromboxane B2 (iTxB2) and 6-keto-prostaglandin F1 alpha (i6-keto-PGF1 alpha), are key mediators of inflammation.
- Guanine nucleotide-binding regulatory proteins (G proteins) are involved in cellular signaling pathways.
Purpose of the Study:
- To investigate the role of G proteins in mediating endotoxin-stimulated arachidonic acid metabolism in rat peritoneal macrophages.
- To determine the effects of cholera toxin and pertussis toxin on endotoxin-induced synthesis of iTxB2 and i6-keto-PGF1 alpha.
- To examine how endotoxin tolerance influences G protein activity and subsequent inflammatory mediator production.
Main Methods:
- Primary rat peritoneal macrophages were pretreated with pertussis toxin or cholera toxin.
- Macrophages were then stimulated with Salmonella enteritidis endotoxin.
- Synthesis of iTxB2 and i6-keto-PGF1 alpha was measured using established assays.
- The effects of nonhydrolyzable GTP analogs (GTP[gamma-S]) were assessed in both control and endotoxin-tolerant macrophages.
Main Results:
- Pertussis toxin inhibited endotoxin-stimulated iTxB2 and i6-keto-PGF1 alpha synthesis.
- Cholera toxin enhanced endotoxin-induced synthesis of these mediators.
- Effects of both toxins were significantly reduced in macrophages from endotoxin-tolerant rats.
- GTP[gamma-S] increased and augmented endotoxin-stimulated iTxB2 synthesis in control macrophages, but this effect was diminished in tolerant macrophages.
Conclusions:
- G protein signaling pathways are crucial for mediating endotoxin-stimulated arachidonic acid metabolism in macrophages.
- Endotoxin tolerance is associated with alterations in G protein activity, leading to modulated inflammatory mediator production.