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. 1986 Jun 1;102(6):2197–2204. doi: 10.1083/jcb.102.6.2197

Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils

PMCID: PMC2114244  PMID: 3011810

Abstract

Cytosolic free calcium concentration, [Ca2+]i, and exocytosis of azurophil granules (beta-glucuronidase), specific granules (vitamin B12- binding protein), and secretory vesicles (gelatinase) were measured concomitantly in intact human neutrophils under steady state [Ca2+]i. The cells were loaded with the fluorescent calcium indicator quin2 in the presence or absence of extracellular Ca2+, and steady state [Ca2+]i levels ranging from 20 to greater than 2,000 nM were obtained by adding the Ca2+ ionophore ionomycin at various concentrations of extracellular calcium. The extent of exocytosis from the three granule populations was found to be a function of [Ca2+]i. The minimal [Ca2+]i that caused significant release (threshold [Ca2+]i) was approximately 200-300 nM and was similar for all three compartments. Marked differences, however, were found when the [Ca2+]i for half-maximal exocytosis (EC50) was determined. In the absence of cytochalasin B the EC50 was 1,100 +/- 220 nM and 1,600 +/- 510 nM for specific granules and secretory vesicles, respectively, and approximately 6,000 nM for azurophil granules. Cytochalasin B did not affect the threshold [Ca2+]i but decreased the EC50 and enhanced the rate of exocytosis. In the presence of cytochalasin B the EC50 was approximately 600 nM both for secretory vesicles and specific granules, and approximately 2,600 nM for azurophil granules. The addition of the chemotactic peptide N-formyl- methionyl-leucyl-phenylalanine dramatically changed the [Ca2+]i dependency of granule secretion: It decreased the threshold [Ca2+]i to less than 20 and less than 50 nM, and the EC50 to 50 and 200 nM for specific and azurophil granules, respectively, and it significantly increased the rate of exocytosis. Thus, the additional signal(s) provided by receptor activation markedly lower(s) the Ca2+ requirement of the exocytotic process. Furthermore, these results indicate that the secretion from three different granule populations within the same cell type are differently modulated by [Ca2+]i.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker P. F., Knight D. E., Umbach J. A. Calcium clamp of the intracellular environment. Cell Calcium. 1985 Apr;6(1-2):5–14. doi: 10.1016/0143-4160(85)90030-2. [DOI] [PubMed] [Google Scholar]
  2. Bennett J. P., Cockcroft S., Gomperts B. D. Use of cytochalasin B to distinguish between early and late events in neutrophil activation. Biochim Biophys Acta. 1980 Oct 2;601(3):584–591. doi: 10.1016/0005-2736(80)90560-x. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  4. Capponi A. M., Lew P. D., Jornot L., Vallotton M. B. Correlation between cytosolic free Ca2+ and aldosterone production in bovine adrenal glomerulosa cells. Evidence for a difference in the mode of action of angiotensin II and potassium. J Biol Chem. 1984 Jul 25;259(14):8863–8869. [PubMed] [Google Scholar]
  5. Cockcroft S., Bennett J. P., Gomperts B. D. The dependence on Ca2+ of phosphatidylinositol breakdown and enzyme secretion in rabbit neutrophils stimulated by formylmethionyl-leucylphenylalanine or ionomycin. Biochem J. 1981 Dec 15;200(3):501–508. doi: 10.1042/bj2000501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cribbs D. H., Glenney J. R., Jr, Kaulfus P., Weber K., Lin S. Interaction of cytochalasin B with actin filaments nucleated or fragmented by villin. J Biol Chem. 1982 Jan 10;257(1):395–399. [PubMed] [Google Scholar]
  7. Dewald B., Bretz U., Baggiolini M. Release of gelatinase from a novel secretory compartment of human neutrophils. J Clin Invest. 1982 Sep;70(3):518–525. doi: 10.1172/JCI110643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Di Virgilio F., Lew D. P., Pozzan T. Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels. Nature. 1984 Aug 23;310(5979):691–693. doi: 10.1038/310691a0. [DOI] [PubMed] [Google Scholar]
  9. Fumagalli G., Zanini A. In cow anterior pituitary, growth hormone and prolactin can be packed in separate granules of the same cell. J Cell Biol. 1985 Jun;100(6):2019–2024. doi: 10.1083/jcb.100.6.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Knight D. E., Hallam T. J., Scrutton M. C. Agonist selectivity and second messenger concentration in Ca2+-mediated secretion. Nature. 1982 Mar 18;296(5854):256–257. doi: 10.1038/296256a0. [DOI] [PubMed] [Google Scholar]
  11. Kojima I., Lippes H., Kojima K., Rasmussen H. Aldosterone secretion: effect of phorbol ester and A23187. Biochem Biophys Res Commun. 1983 Oct 31;116(2):555–562. doi: 10.1016/0006-291x(83)90559-4. [DOI] [PubMed] [Google Scholar]
  12. Lew P. D., Wollheim C. B., Waldvogel F. A., Pozzan T. Modulation of cytosolic-free calcium transients by changes in intracellular calcium-buffering capacity: correlation with exocytosis and O2-production in human neutrophils. J Cell Biol. 1984 Oct;99(4 Pt 1):1212–1220. doi: 10.1083/jcb.99.4.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lew V. L., Garcia-Sancho J. Use of the ionophore A23187 to measure and control cytoplasmic Ca2+ levels in intact red cells. Cell Calcium. 1985 Apr;6(1-2):15–23. doi: 10.1016/0143-4160(85)90031-4. [DOI] [PubMed] [Google Scholar]
  14. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  15. Pozzan T., Lew D. P., Wollheim C. B., Tsien R. Y. Is cytosolic ionized calcium regulating neutrophil activation? Science. 1983 Sep 30;221(4618):1413–1415. doi: 10.1126/science.6310757. [DOI] [PubMed] [Google Scholar]
  16. Rink T. J., Smith S. W., Tsien R. Y. Cytoplasmic free Ca2+ in human platelets: Ca2+ thresholds and Ca-independent activation for shape-change and secretion. FEBS Lett. 1982 Nov 1;148(1):21–26. doi: 10.1016/0014-5793(82)81234-9. [DOI] [PubMed] [Google Scholar]
  17. Romeo D., Zabucchi G., Miani N., Rossi F. Ion movement across leukocyte plasma membrane and excitation of their metabolism. Nature. 1975 Feb 13;253(5492):542–544. doi: 10.1038/253542a0. [DOI] [PubMed] [Google Scholar]
  18. Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science. 1983 May 6;220(4597):568–575. doi: 10.1126/science.6301011. [DOI] [PubMed] [Google Scholar]
  19. Smolen J. E., Stoehr S. J. Micromolar concentrations of free calcium provoke secretion of lysozyme from human neutrophils permeabilized with saponin. J Immunol. 1985 Mar;134(3):1859–1865. [PubMed] [Google Scholar]
  20. Southwick F. S., Stossel T. P. Contractile proteins in leukocyte function. Semin Hematol. 1983 Oct;20(4):305–321. [PubMed] [Google Scholar]
  21. Wilson S. P., Kirshner N. Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4994–5000. [PubMed] [Google Scholar]
  22. Wright D. G., Gallin J. I. Secretory responses of human neutrophils: exocytosis of specific (secondary) granules by human neutrophils during adherence in vitro and during exudation in vivo. J Immunol. 1979 Jul;123(1):285–294. [PubMed] [Google Scholar]

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