sq-23377 and arginyl-glycyl-aspartyl-serine

In pathological situations, different modes of cell death are observed, and information on the role and uptake of nonapoptotic corpses is scarce. Here, we modeled two distinct forms of death in human Jurkat T cells treated with staurosporine: classical apoptosis under normal culture conditions and programmed death with necrotic morphology under ATP-depleting conditions (necPCD). When offered to phagocytes, both types of cell corpses (but not heat-killed unscheduled necrotic cells) reduced the release of the proinflammatory cytokine TNF from the macrophages. The necPCD cells were efficiently engulfed by macrophages and microglia, and from mixtures of necPCD and apoptotic cells macrophages preferentially engulfed the necrotic cells. Using a newly developed assay, we demonstrated that phosphatidylserine is translocated to the surface of such necrotic cells. We demonstrate that this can occur independently of calcium signals, and that surface phosphatidylserine is essential for the uptake of necrotic cells by both human macrophages and murine microglia.

Topics: Animals; Annexin A5; Antibodies, Monoclonal; Apoptosis; Calcium; CD36 Antigens; Cell Line; Cell Membrane; Cells, Cultured; Escherichia coli; Formaldehyde; Humans; Inflammation; Ionomycin; Jumonji Domain-Containing Histone Demethylases; Jurkat Cells; Lipopolysaccharide Receptors; Liposomes; Macrophages; Membrane Lipids; Mice; Microglia; Microscopy, Confocal; Microscopy, Fluorescence; Necrosis; Oligomycins; Oligopeptides; Phagocytosis; Phosphatidylserines; Polymers; Receptors, Cell Surface; Staurosporine; Tumor Necrosis Factor-alpha

The relationship between platelet aggregation, calpain activation, PKC activities and the secretory response have been examined in PMA-and ionomycin-stimulated platelets. Co-addition of PMA and ionomycin resulted in a maximal synergistic secretion of [14C]5-hydroxytryptamine ([14C]5-HT) from platelet dense granules. However, prior addition of PMA for 5 or 10 min resulted in a reduction of this secretory response. Inclusion of either RGDS (to inhibit platelet aggregation) or E64-d (to inhibit calpain activity) resulted in full restoration of the secretory response. In experiments to determine the activity status of PKC, PMA was found to induce a loss in cytosolic and total PKC activity without an increase in membrane-associated activities during this time period. Inhibition of either platelet aggregation or calpain activity resulted in preservation of total and cytosolic activities with a measurable increase in membrane translocated activity. PMA-induced phosphorylation of a number of PKC substrates was measured in 32P-labelled platelets. PMA induced potent phosphorylation of the 45 and 20 kDa species and also proteins of the molecular masses 66, 80, 97 and 119 kDa. Phosphorylation was maximal at either 1 or 2 min after which dephosphorylation occurred. Inclusion of either RGDS or E64-d resulted in a reduction of the dephosphorylation rates, and sustained phosphorylation of the 66, 80, 97 and 119 kDa proteins. These studies suggest that the activity status of PKC is an important factor in the level of secretion obtained and that platelet aggregation is involved in calpain-initiated down-regulation of PKC.

Topics: Blood Platelets; Calpain; Down-Regulation; Humans; Ionomycin; Male; Oligopeptides; Phosphorylation; Platelet Aggregation; Platelet Aggregation Inhibitors; Protein Kinase C; Serotonin; Tetradecanoylphorbol Acetate