The kinetics of secretion from permeabilized human neutrophils: release of elastase and correlations with other granule constituents and right angle light scatter.
We have previously reported that human neutrophils can be permeabilized with the cholesterol-complexing agent digitonin. These permeabilized cells can be induced to secrete lysosomal constituents when exposed to micromolar levels of free Ca2+, a process that is enhanced by certain guanine nucleotides. We examined the kinetics in this system by employing both direct and indirect measures of secretion. A continuous, fluorescent assay of elastase permits real-time monitoring of secretion from azurophil granules. The kinetics of elastase release proved to be rapid, beginning within 3-10 sec and reaching a maximum at 1-2 min. Changes in the Ca2+ concentration did not affect the "lag period" for release. A comparison of the Ca2+ dose-response curves for release of the various granule constituents indicated that elastase was being secreted along with other contents of the azurophil granules. Changes in right angle light scatter (RLS), which have been shown to correlate closely with secretion, also commenced rapidly after the addition of Ca2+; when measured simultaneously, both the Ca2+ dose-response characteristics for changes in RLS and elastase release were very similar. Changes in RLS could be halted within 5 sec by excess EGTA and restarted promptly by repletion with secretory concentrations of Ca2+. In addition, neomycin, a phospholipase C inhibitor, profoundly diminished degranulation as monitored by RLS and end-point techniques. A continuous assay employing 9-aminoacridine self-quenching as a measure of secretion proved far less satisfactory, but, nonetheless, produced similar kinetics and dose-response characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)
Smolen
J E
JE
Stoehr
S J
SJ
Traynor
A E
AE
Sklar
L A
LA
D000585:Aminacrine; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008027:Light; D009355:Neomycin / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D010196:Pancreatic Elastase / Q000378:metabolism*; D010539:Permeability; D012542:Scattering, Radiation
D000585:Aminacrine; D010196:Pancreatic Elastase; D009355:Neomycin; D002118:Calcium