Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils: Relationship to calcium mobilization,aggregation and superoxide radical generation |
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Authors: | Charles N. Serhan M.Johan Broekman Helen M. Korchak James E. Smolen Aaron J. Marcus Gerald Weissmann |
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Affiliation: | 1. Division of Rheumatology, Department of Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016 U.S.A.;2. Division of Hematology/Oncology, Department of Medicine, New York Veterans Administration Medical Center and Cornell University Medical College, New York, NY 10010 U.S.A. |
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Abstract: | Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to ‘stage’ events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10?7 M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 μg/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up 45Ca2+ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O?2 (10–15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O?2 generation and aggregation (30–35 s). |
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Keywords: | Superoxide radical Phosphatidate Phosphatidylinositol (Human neutrophil) EGTA PI phosphatidylinositol |
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