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 μM A23187 from the unstimulated intact cells was 0.91 nmol 4·10⁶ 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)·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 4·10⁶ 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 1.4·10^-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.