COMPLEMENT FRAGMENT C3A STIMULATES CA2-TOXIN-SENSITIVE G-PROTEIN( INFLUX IN NEUTROPHILS VIA A PERTUSSIS)

The signal pathways of neutrophils following stimulation with the comp lement fragment C3a (C3a) were studied in neutrophils and compared to the pathways activated by complement fragment C5a (C5a) . Analysis of polyphosphoinositol lipid turnover showed that C5a, but not C3a, activ ated phosphatidylinositol-bisphosphate-3-kinase (PtdInsP2 3-kinase) in dicating that different signal pathways are activated by the two anaph ylatoxins. To examine whether C3a stimulated Ca2+ transients, cytosoli c free Ca2+ levels were analyzed in Fluo-3-labelled neutrophils by flo w cytometry. C3a stimulated a fast and concentration-dependent increas e of cytosolic free Ca2+. Comparison of the C3a response with that of C5a revealed a more pronounced C5a-triggered Ca2+ rise. Addition of EG TA to the extracellular buffer prior to stimulation did not significan tly alter the initial Ca2+ rise at low C5a concentrations, but reduced the time course of the Ca2+ transients at high concentrations. In mar ked contrast, EGTA completely blocked the Ca2+ response stimulated by C3a in neutrophils labeled with either Indo-1/AM or Fluo-3. Preincubat ion of neutrophils with pertussis toxin inhibited both C3a- and C5a-st imulated Ca2+ transients, indicating the involvement of guanine-nucleo tide-binding proteins (G proteins) in these processes. In order to exa mine whether the C3a receptor is coupled to G proteins, binding of gua nosine 5'-O-(3-[S-35]thiotriphosphate) ([S-35]GTP[S]) to purified neut rophil plasma membranes was studied. Both C3a and C5a stimulated high- affinity binding of [S-35]GTP[S] up to 1.5-fold and 3-fold, respective ly. These data suggest that the two anaphylatoxins activate pertussis- toxin-sensitive G proteins, which then trigger different signal transd uction pathways. C3a specifically stimulated Ca2+ influx from the extr acellular medium, whereas C5a additionally activated the PtdInsP2 3-ki nase and stimulated Ca2+ mobilization from intracellular stores.