Complement component C9-dependent cytosolic free Ca2+ rise and recovery in neutrophils.
The cytosolic free Ca2+ concentration in rat neutrophils was determined by ratiometric fluorometry and imaging of Fura-2. Transient elevations of cytosolic free Ca2+ concentration were provoked by addition of C9 to neutrophils pre-coated with C5b-8. The rate of rise of the cytosolic free Ca2+ concentration was dependent upon the concentration of C9. These changes in cytosolic free Ca2+ concentration occurred in the absence of cell lysis, since there was no release of Fura-2, and were the result of increased permeability to extracellular Ca2+. More than 96% of the rise in cytosolic free Ca2+ generation by C9 was dependent upon the presence of extracellular Ca2+, but did not occur via channels which were inhibited by the Ca2+ channel blocker SKF96365. The decrease in the permeability of the membrane to Ca2+ after C9 was not triggered by the rise in cytosolic free Ca2+. After attack by C9, individual neutrophils remained responsive to f-met-leu-phe, or further attack by C9, both producing Ca2+ transients. The recovery of the Ca2+ signal was consistent with the complement membrane attack complex generating a series of permeability thresholds in the plasma membrane. These data have implications for the understanding of the mechanisms underlying the inappropriate responsiveness of neutrophils at inflammatory sites.
Laffafian
I
I
University Department of Surgery, University of Wales College of Medicine, Cardiff, UK.
Davies
E V
EV
Campbell
A K
AK
Hallett
M B
MB
D000599:Amino Acids, Diamino / Q000502:physiology*; D000600:Amino Acids, Dicarboxylic / Q000502:physiology*; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D015938:Complement Membrane Attack Complex / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D051381:Rats
D000599:Amino Acids, Diamino; D000600:Amino Acids, Dicarboxylic; D015938:Complement Membrane Attack Complex; D007093:Imidazoles; D009240:N-Formylmethionine Leucyl-Phenylalanine; C025074:alpha,alpha'-diaminononanedicarboxylic acid; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium