Origin of intracellular calcium and quantitation of mobilizable calcium in neutrophils stimulated with chemotactic peptide.
The origin and amount of mobilized Ca2+ in chemotactic peptide-stimulated guinea pig neutrophils were examined using biochemical techniques. The total amount of releasable Ca2+ by 20 microM A23187 from the unstimulated intact cells was 0.91 nmol/4 X 10(6) cells, as assessed by change in absorbance of the antipyrylazo III-Ca2+ complex. Two types of internal vesicular Ca2+ pool, mitochondrial and non-mitochondrial pool were identified in the saponin-permeabilized cells. The total amount of releasable Ca2+ was comparable to that accumulated by the non-mitochondrial pool at (1-2) X 10(-7) M of a free Ca2+ concentration. The mitochondrial uncoupler, capable of releasing Ca2+ from the mitochondrial pool, neither modified the basal cytosolic free Ca2+ in quin 2-loaded cells nor caused a Ca2+ efflux from the intact cells. These results suggest that the releasable Ca2+ may be located in the non-mitochondrial pool of unstimulated intact cells, and the mitochondrial pool contains little releasable Ca2+. The addition of fMet-Leu-Phe increased the cytosolic free Ca2+ by two processes: Ca2+ mobilization from internal stores and Ca2+ influx through the surface membrane. The Ca2+ mobilized and effluxed from the intact cells by stimulation with the maximal doses of fMet-Leu-Phe was estimated to be 0.27 nmol/4 X 10(6) cells. Almost equal amounts were released by the maximal doses of inositol 1,4,5-trisphosphate from the non-mitochondrial pool of saponin-treated cells that had accumulated Ca2+ at a free Ca2+ concentration of 1.4 X 10(-7) M. The mechanism related to the Ca2+ influx by fMet-Leu-Phe stimulation was also examined. The addition of nifedipine or phosphatidic acid did not affect the change in the cytosolic free Ca2+ induced by fMet-Leu-Phe, thereby suggesting that the receptor-mediated Ca2+ channel may be involved in the Ca2+ influx.
D000818:Animals; D001386:Azides / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000097:blood*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D006168:Guinea Pigs; D008928:Mitochondria / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D012430:Ruthenium Red / Q000494:pharmacology; D019810:Sodium Azide
D001386:Azides; D009840:Oligomycins; D012430:Ruthenium Red; D000001:Calcimycin; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019810:Sodium Azide; D009543:Nifedipine; D002118:Calcium