Endotoxin-induced arachidonic acid metabolism requires de novo protein synthesis and protein kinase C activation.
The mechanisms whereby bacterial endotoxins stimulate arachidonic acid metabolism in macrophages are uncertain. Both protein kinase C activation and de novo protein synthesis occur in macrophages in response to endotoxin. In this study we evaluated the time course and role of protein kinase C and de novo protein synthesis in endotoxin stimulated arachidonic acid metabolism in resident rat peritoneal macrophages. Thromboxane (TX) B2 was measured as the representative arachidonic acid metabolite synthesized in response to Salmonella enteritidis endotoxin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate (PMA). The effect of inhibition of protein kinase C by 1-(5-isoquinolinsulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine on endotoxin- and A23187-induced TXB2 synthesis was examined. The potential roles of transcriptional and translational events in endotoxin- and A23187-stimulated TXB2 synthesis were determined by utilizing the transcriptional inhibitors camptothecin (10 microM) or actinomycin D (0.08 microM), and the translational inhibitor cycloheximide (0.1 microM). Whereas, A23187 stimulated maximal TXB2 synthesis within 15 min, endotoxin showed a more prolonged time course with a 12-fold increase in TXB2 synthesis above basal levels after 3 h (P less than 0.05). PMA induced an approx. 8-fold increase above basal TXB2 levels that was blocked by inhibition of transcription with actinomycin D. H-7 (10 microM to 50 microM) inhibited endotoxin- and A23187-stimulated eicosanoid synthesis. Staurosporine (0.2 microM) produced a selective 66% inhibition of endotoxin, but not A23187-stimulated TXB2 synthesis. Endotoxin-induced TXB2 production was significantly (P less than 0.05) inhibited by staurosporine, camptothecin, actinomycin D or cycloheximide at intervals from 30 min prior to, through 60 min after endotoxin stimulation. These studies suggest a role for protein kinase C activation and de novo protein synthesis in endotoxin signal transduction events leading to increased macrophage arachidonic acid metabolism. These intracellular events are essential in sustaining the prolonged inflammatory response to endotoxin.
Geisel
J
J
Department of Physiology, Medical University of South Carolina, Charleston.
Cook
J A
JA
Coffee
K A
KA
Wise
W C
WC
Halushka
P V
PV
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D001427:Bacterial Toxins / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002166:Camptothecin / Q000494:pharmacology; D002470:Cell Survival; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004731:Endotoxins / Q000494:pharmacology*; D004768:Enterotoxins / Q000494:pharmacology; D004789:Enzyme Activation; D007546:Isoquinolines / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism; D008297:Male; D010879:Piperazines / Q000494:pharmacology; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013929:Thromboxane B2 / Q000096:biosynthesis
D000470:Alkaloids; D001095:Arachidonic Acids; D001427:Bacterial Toxins; D004731:Endotoxins; D004768:Enterotoxins; D007546:Isoquinolines; D010879:Piperazines; C027952:salmonella toxin; D003609:Dactinomycin; D016718:Arachidonic Acid; D000001:Calcimycin; D013929:Thromboxane B2; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D003513:Cycloheximide; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002166:Camptothecin