Complement fragment C3a stimulates Ca2+ influx in neutrophils via a pertussis-toxin-sensitive G protein.
The signal pathways of neutrophils following stimulation with the complement 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, activated phosphatidylinositol-bisphosphate-3-kinase (PtdInsP2 3-kinase) indicating that different signal pathways are activated by the two anaphylatoxins. To examine whether C3a stimulated Ca2+ transients, cytosolic free Ca2+ levels were analyzed in Fluo-3-labelled neutrophils by flow cytometry. C3a stimulated a fast and concentration-dependent increase of cytosolic free Ca2+. Comparison of the C3a response with that of C5a revealed a more pronounced C5a-triggered Ca2+ rise. Addition of EGTA to the extracellular buffer prior to stimulation did not significantly alter the initial Ca2+ rise at low C5a concentrations, but reduced the time course of the Ca2+ transients at high concentrations. In marked contrast, EGTA completely blocked the Ca2+ response stimulated by C3a in neutrophils labeled with either Indo-1/AM or Fluo-3. Preincubation of neutrophils with pertussis toxin inhibited both C3a- and C5a-stimulated Ca2+ transients, indicating the involvement of guanine-nucleotide-binding proteins (G proteins) in these processes. In order to examine whether the C3a receptor is coupled to G proteins, binding of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[S]) to purified neutrophil plasma membranes was studied. Both C3a and C5a stimulated high-affinity binding of [35S]GTP[S] up to 1.5-fold and 3-fold, respectively. These data suggest that the two anaphylatoxins activate pertussis-toxin-sensitive G proteins, which then trigger different signal transduction pathways. C3a specifically stimulated Ca2+ influx from the extracellular medium, whereas C5a additionally activated the PtdInsP2 3-kinase and stimulated Ca2+ mobilization from intracellular stores.
Norgauer
J
J
Hautklinik Freiburg, Germany.
Dobos
G
G
Kownatzki
E
E
Dahinden
C
C
Burger
R
R
Kupper
R
R
Gierschik
P
P
D002118:Calcium / Q000097:blood*; D002462:Cell Membrane / Q000378:metabolism; D015926:Complement C3a / Q000494:pharmacology*; D015936:Complement C5a / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005434:Flow Cytometry; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000378:metabolism*; D037342:Pertussis Toxin; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D015926:Complement C3a; D015936:Complement C5a; D037342:Pertussis Toxin; D017853:Phosphotransferases (Alcohol Group Acceptor); C068961:phosphatidylinositol 4,5-biphosphate kinase; D019204:GTP-Binding Proteins; D002118:Calcium