Related Experiment Videos
Biochemical and functional responses stimulated by platelet-activating factor in murine peritoneal macrophages
V Prpic1, R J Uhing, J E Weiel
1Department of Pathology, Duke University, Durham, North Carolina 27710.
Abstract:
Platelet-activating factor (PAF) is a potent stimulant of leukocytes, including macrophages. To analyze the mechanisms of its effects upon macrophages, we determined whether macrophages bear specific surface receptors for PAF. By competitive radioactive binding assays, we determined two classes of specific receptors to be present on purified membranes derived from murine peritoneal macrophages (one having a Kd of approximately 1 X 10(-10) M and one a Kd of approximately 2 X 10(-9) M). When the macrophages were incubated with PAF, rapid formation of several inositol phosphates including inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate were observed. PAF also elevated intracellular levels of calcium to 290 +/- 27% of basal levels which were 82.7 +/- 12 nM. Increases in calcium were observed first in submembranous areas of the macrophages. PAF also led to increases of 1,2-diacylglycerol of approximately 200 pmol/10(7) cells. A characteristic pattern of enhanced protein phosphorylation, similar to that initiated by both phorbol 12,13-myristate and lipopolysaccharide, was observed and involved enhanced phosphorylation of proteins of 28, 33, 67, and 103 kD. The half-maximal dose of PAF for initiating all the above effects was approximately 5 X 10(-9) M. PAF also initiated significant chemotaxis of the cells; the half-maximal dose for this effect was approximately 1 X 10(-11) M. Taken together, these observations suggest that murine mononuclear phagocytes bear specific membrane receptors for PAF and that addition of PAF leads to generation of break-down products of polyphosphoinositides, subsequent changes in intracellular calcium and protein phosphorylation, and chemotaxis.
Insights
Murine macrophages possess specific receptors for Platelet-Activating Factor (PAF). PAF binding triggers intracellular signaling pathways, including calcium mobilization and protein phosphorylation, leading to macrophage chemotaxis.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Platelet-Activating Factor (PAF) is a known stimulant of leukocytes, particularly macrophages.
- Understanding the precise mechanisms of PAF's action on macrophages is crucial for immunology research.
Purpose of the Study:
- To investigate the presence of specific surface receptors for PAF on macrophages.
- To elucidate the intracellular signaling pathways activated by PAF in macrophages.
Main Methods:
- Competitive radioactive binding assays were used to identify and characterize PAF receptors on murine peritoneal macrophage membranes.
- Intracellular levels of inositol phosphates, calcium, and 1,2-diacylglycerol were measured following PAF incubation.
- Protein phosphorylation patterns and macrophage chemotaxis were analyzed in response to PAF.
Main Results:
- Two classes of specific PAF receptors with distinct binding affinities (Kd ~1x10^-10 M and Kd ~2x10^-9 M) were identified on macrophage membranes.
- PAF induced rapid formation of inositol phosphates, elevated intracellular calcium, and increased 1,2-diacylglycerol levels.
- PAF triggered enhanced phosphorylation of specific proteins (28, 33, 67, 103 kD) and significant chemotaxis, with a half-maximal effective dose of approximately 1x10^-11 M for chemotaxis.
Conclusions:
- Murine mononuclear phagocytes express specific membrane receptors for PAF.
- PAF binding initiates a cascade of intracellular events, including polyphosphoinositide breakdown, calcium signaling, and protein phosphorylation, ultimately driving macrophage chemotaxis.