Origin of intracellular calcium and quantitation of mobilizable calcium in neutrophils stimulated with chemotactic peptide

Abstract

The origin and amount of mobilized Ca 2+ in chemotactic peptide-stimulated guinea pig neutrophils were examined using biochemical techniques. The total amount of releasable Ca 2+ by 20 μM A23187 from the unstimulated intact cells was 0.91 nmol 4·10 6 cells , as assessed by change in absorbance of the antipyrylazo III-Ca 2+ complex. Two types of internal vesicular Ca 2+ pool, mitochondrial and non-mitochondrial pool were identified in the saponin-permeabilized cells. The total amount of releasable Ca 2+ was comparable to that accumulated by the non-mitochondrial pool at (1−2)·10 −7 M of a free Ca 2+ concentration. The mitochondrial uncoupler, capable of releasing Ca 2+ from the mitochondrial pool, neither modified the basal cytosolic free Ca 2+ in quin 2-loaded cells nor caused a Ca 2+ efflux from the intact cells. These results suggest that the releasable Ca 2+ may be located in the non-mitochondrial pool of unstimulated intact cells, and the mitochondrial pool contains little releasable Ca 2+. The addition of fMet-Leu-Phe increased the cytosolic free Ca 2+ by two processes: Ca 2+ mobilization from internal stores and Ca 2+ influx through the surface membrane. The Ca 2+ mobilized and effluxed from the intact cells by stimulation with the maximal doses of fMet-Leu-Phe was estimated to be 0.27 nmol 4·10 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 Ca 2+ at a free Ca 2+ concentration of 1.4·10 −7 M. The mechanism related to the Ca 2+ 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 Ca 2+ induced by fMet-Leu-Phe, thereby suggesting that the receptor-mediated Ca 2+ channel may be involved in the Ca 2+ influx.