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Identification of platelet-activating factor receptors in P388D1 murine macrophages
1Veterans Administration Medical Center, Cancer Research Institute, San Francisco, CA 94121.
Abstract:
Platelet-activating factor (PAF) binding and metabolism by eight murine and human cell lines was analyzed. Only the murine P388D1 macrophage line had specific, high affinity PAF binding sites. PAF binding reached saturation within 10 min at room temperature and was irreversible. Minimal PAF metabolism was observed at the time binding saturation was achieved. Scatchard analysis of PAF binding revealed a single class of PAF receptors (7872 +/- 1310/cell) which had a dissociation constant of 0.08 +/- 0.01 nM (mean +/- SEM, eta = 6). The dissociation constant was confirmed independently by quantifying the kinetics of initial specific PAF binding. PAF binding was stereospecific, required an sn-2 acetyl substituent, and was inhibited by structurally diverse PAF antagonists including kadsurenone, BN 52021, triazolam, and CV3988. The fact that the receptors are functionally active was shown by the observation that 1 to 100 pM PAF increased free intracellular calcium in P388D1 cells in a dose-related manner. These studies demonstrate that P388D1 macrophages have functional PAF receptors whose affinity and structural specificities are similar to PAF receptors in other cells. The availability of a stable cell line that binds but does not metabolize PAF will greatly facilitate studies of the PAF receptor.
Insights
Murine P388D1 macrophages possess specific, high-affinity Platelet-Activating Factor (PAF) receptors. These functional receptors bind PAF without significant metabolism, aiding future PAF receptor research.
Area of Science:
- Immunology
- Cell Biology
- Pharmacology
Background:
- Platelet-Activating Factor (PAF) is a potent lipid mediator involved in inflammation and immune responses.
- Understanding PAF receptor interactions is crucial for developing targeted therapies.
Purpose of the Study:
- To characterize Platelet-Activating Factor (PAF) binding and metabolism in various cell lines.
- To identify and validate a cell line with functional PAF receptors for further study.
Main Methods:
- Analysis of PAF binding and metabolism across eight murine and human cell lines.
- Saturation binding assays, Scatchard analysis, and kinetic studies to determine receptor characteristics.
- Functional assays measuring intracellular calcium changes in response to PAF stimulation.
Main Results:
- Murine P388D1 macrophage line exhibited specific, high-affinity PAF binding sites.
- PAF binding was rapid, irreversible, stereospecific, and inhibited by known PAF antagonists.
- P388D1 cells showed a dose-dependent increase in intracellular calcium upon PAF stimulation, indicating functional receptors.
- Minimal PAF metabolism was observed in P388D1 cells at saturation binding.
Conclusions:
- P388D1 macrophages possess functional PAF receptors with high affinity and specific structural requirements.
- These receptors share similarities with PAF receptors found in other cell types.
- The P388D1 cell line serves as a valuable tool for studying PAF receptor biology due to its binding capacity and low metabolism.