Ca influx shutdown in neutrophils induced by Fas (CD95) cross-linking.
In neutrophils, as in most other cell types, Ca(2+) signalling is important for a number of cellular activities. Although inositol(1,4,5)trisphosphate-mediated release of Ca(2+) from intracellular stores is a necessary prelude, it is the Ca(2+) influx that is responsible for many of the neutrophil responses. We report here that although elevations of cytosolic Ca(2+) do not accompany Fas-mediated apoptosis in neutrophils, the Ca(2+) influx component of the response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) becomes selectively inactived as the neutrophils progress towards accelerated apoptosis induced by Fas (CD95) cross-linking. After 4 hr incubation at 37 degrees, untreated neutrophils display an exaggerated Ca(2+) influx phase in response to FMLP. This was absent in neutrophils that had been Fas-activated at the same time. No Ca(2+) influx component was demonstrable by the removal of extracellular Ca(2+) or by Ca(2+) channel blockade with Ni(2+) and no Mn(2+) influx was detectable. The defect could not be attributed to a decrease in receptor sensitivity, receptor coupling or receptor number because the release of stored Ca(2+) remained constant during incubation and was unaffected by Fas activation. Ca(2+) influx became uncoupled from store release before detectable gross morphological changes or phosphatidyl serine externalization and was also insensitive to caspase 3 and 8 inhibitors. These results suggest a mechanism other than caspase-mediated proteolytic damage to components important for Ca(2+) influx.
Ayub
Khurram
K
Neutrophil Signalling Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Laffafian
Iraj
I
Dewitt
Sharon
S
Hallett
Maurice B
MB
D017209:Apoptosis; D002118:Calcium / Q000032:analysis; D020013:Calcium Signaling / Q000502:physiology*; D053148:Caspase 3; D053181:Caspase 8; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D003429:Cross Reactions; D003600:Cytosol / Q000737:chemistry; D003911:Dextrans; D016257:Fura-2; D006801:Humans; D008856:Microscopy, Fluorescence; D009504:Neutrophils / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D019014:fas Receptor / Q000378:metabolism*
D061945:Caspase Inhibitors; D003911:Dextrans; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; D019014:fas Receptor; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D002118:Calcium; D016257:Fura-2