Abstract
Polyphosphate (polyP) is a pro-inflammatory agent and a potent modulator of the human blood-clotting system. The presence of polyP of 60 phosphate units was identified in rat basophilic leukemia (RBL-2H3) mast cells using specific enzymatic assays, urea-polyacrylamide gel electrophoresis of cell extracts, and staining of cells with 4,6-diamidino-2-phenylindole (DAPI), and the polyP-binding domain of Escherichia coli exopolyphosphatase. PolyP co-localizes with serotonin- but not with histamine-containing granules. PolyP levels greatly decreased in mast cells stimulated to degranulate by IgE. Mast cell granules were isolated and found to be acidic and decrease their polyP content upon alkalinization. In agreement with these results, when RBL-2H3 mast cells were loaded with the fluorescent calcium indicator fura-2 acetoxymethyl ester to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)), they were shown to possess a significant amount of Ca(2+) stored in an acidic compartment different from lysosomes. PolyP derived from RBL-2H3 mast cells stimulated bradykinin formation, and it was also detected in human basophils. All of these characteristics of mast cell granules, together with their known elemental composition, and high density, are similar to those of acidocalcisomes. The results suggest that mast cells polyP could be an important mediator of their pro-inflammatory and pro-coagulant activities.
Highlights
The presence of polyP of 60 phosphate units was identified in rat basophilic leukemia (RBL-2H3) mast cells using specific enzymatic assays, urea-polyacrylamide gel electrophoresis of cell extracts, and staining of cells with 4,6-diamidino-2-phenylindole (DAPI), and the polyP-binding domain of Escherichia coli exopolyphosphatase
These results suggest that RBL-2H3 cells possess two independent acidic calcium stores, the lysosome, which is sensitive to GPN, and the secretory granules, which respond to Kϩ ionophores and alkalinizing agents
Isolated mast cell granules were acidic, enriched in polyP compared with whole lysates, and lost their polyP after their alkalinization
Summary
PolyP derived from RBL-2H3 mast cells stimulated bradykinin formation, and it was detected in human basophils All of these characteristics of mast cell granules, together with their known elemental composition, and high density, are similar to those of acidocalcisomes. Basophils are a population of circulating white blood cells that are functionally similar to mast cells Both cell types share many common features, such as high affinity IgE receptor expression, metachromatic staining, and histamine release, among others [18]. Mast cells secrete their cytoplasmic granules to the media, releasing a wide selection of cell modulators such as heparin, histamine, serotonin, neutral proteases, growth factors, and proinflammatory cytokines [21]. Mast cell polyP greatly decreased when cells were stimulated to degranulate by IgE and was able to stimulate bradykinin formation, suggesting that mast cell polyP is an important mediator of mast cell pro-inflammatory and pro-coagulant activities
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