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Mouse peritoneal macrophages release leukotriene C in response to a phagocytic stimulus
Abstract:
Mouse peritoneal macrophages that had ingested zymosan particles released a polar metabolite of arachidonic acid possessing slow-reacting substance activity in the guinea pig ileum assay. The metabolite was purified by solvent extraction, Sephadex G-25 column chromatography. The purified metabolite absorbed light at 280 nm and contained a free amino group. When macrophages were preincubated overnight with [3H]arachidonic acid, [35H]cysteine, or [14C]glutamic acid, each radiolabel was incorporated into the compound. Direct amino acid analysis revealed glycine, glutamic acid, and cysteine at molar ratios of 0.97:1.00:0.82. The above data were consistent with the structure of leukotriene C, an adduct of arachidonic acid and glutaathione. Quantification of the leukotriene C based on incorporation of [3H]arachidonic acid or amino acid analysis indicated that 6 X 10(7) macrophages (3.6 mg of cell protein) released 7.5 nmol after a maximal phagocytic stimulus. The purified leukotriene C had a slow reacting substance activity of 11,500 units/nmol (1 unit has the activity of 5 ng of histamine in a guinea pig ileum contraction assay).
Insights
Mouse macrophages release leukotriene C (a metabolite of arachidonic acid) after ingesting zymosan. This compound exhibits significant slow-reacting substance activity, identified through biochemical analysis and bioassays.
Area of Science:
- Biochemistry
- Immunology
- Pharmacology
Background:
- Macrophages play a key role in inflammatory responses.
- Arachidonic acid metabolites are important signaling molecules in inflammation.
Purpose of the Study:
- To identify and characterize the polar metabolite of arachidonic acid released by macrophages.
- To determine the biological activity of the isolated metabolite.
Main Methods:
- Phagocytosis of zymosan particles by mouse peritoneal macrophages.
- Purification of the released metabolite using solvent extraction and Sephadex G-25 chromatography.
- Radiolabeling studies with [3H]arachidonic acid, [35H]cysteine, and [14C]glutamic acid.
- Amino acid analysis and UV spectroscopy.
- Guinea pig ileum assay for slow-reacting substance activity.
Main Results:
- A polar metabolite of arachidonic acid was isolated and purified.
- The metabolite incorporated radiolabels from arachidonic acid, cysteine, and glutamic acid.
- Amino acid analysis confirmed the presence of glycine, glutamic acid, and cysteine.
- The data strongly suggested the metabolite was leukotriene C.
- Quantification revealed 7.5 nmol of leukotriene C released per 6 X 10(7) macrophages.
- Purified leukotriene C demonstrated potent slow-reacting substance activity (11,500 units/nmol).
Conclusions:
- Mouse peritoneal macrophages release leukotriene C upon stimulation with zymosan.
- Leukotriene C is a significant mediator of slow-reacting substance activity.
- This finding contributes to understanding inflammatory pathways involving arachidonic acid metabolism.