Skip to main content
SpringerLink
Log in

The dual effector system for exocytosis in mast cells: Obligatory requirement for both Ca2+ and GTP

  • Papers
  • Published:
Bioscience Reports

Abstract

The secretory process is a coordinated cellular response, initiated by occupation of surface receptors and comprising an ordered sequence of biochemical steps subject to multiple controls. Conceptually we can divide the sequence into two main sections comprising early, receptor-mediated events leading to generation of intracellular second messengers, and later events leading to membrane fusion and exocytosis. With the discovery that occupation of Ca2+ mobilising receptors leads to activation of polyphosphoinositide phosphodiesterase (PPI-pde) through the mediation of a G-protein (Gp), all the early events can be ascribed to the plasma membrane. Investigation of the exocytotic stage of secretion has been simplified by the use of permeabilised cells in which the composition of the cytosol can be precisely controlled. We have used streptolysin-O, a bacterial cytolysin which generates protein-sized pores in the plasma membrane, to investigate the exocytotic mechanism of rat mast cells. We find that in addition to the activation of PPI-dpe, GTP also acts in concert with Ca2+ at, or close to, the exocytotic site. Exocytosis can occur after substantial depletion of cytosol lactate dehydrogenase and 3-phosphoglycerate kinase indicating that soluble cytosol proteins are unlikely to play any role. There is no absolute requirement for ATP or phosphorylating nucleotide in exocytosis though when present the effective affinities of the two obligatory effectors (i.e. Ca2+ and GTP) are substantially enhanced.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Reed, P. W. and Lardy, H. A. (1972). A23187: A divalent cation ionophore.J. Biol. Chem. 247: 6970–6977.

    Google Scholar 

  2. Liu, C.-M. and Hermann, T. E. (1978). Characterization of ionomycin as a calcium ionophore.J. Biol. Chem. 253:5892–5894.

    Google Scholar 

  3. Foreman, J. C., Mongar, J. L. and Gomperts, B. D. (1973). Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process.Nature 245:249–251.

    Google Scholar 

  4. Prince, W. T., Rasmussen, H. and Berridge, M. J. (1973). The role of Ca2+ in fly salivary gland secretion analysed with the ionophore A23187.Biochim. Biophys. Acta 329:98–107.

    Google Scholar 

  5. Selinger, Z., Eimerl, S. and Schramm, M. (1974). A calcium ionophore stimulating the action of epinephrine on the α-adrenergic receptor.Proc. Nat. Acad. Sci. USA 71:128–131.

    Google Scholar 

  6. Bennett, J. P., Cockcroft, S. and Gomperts, B. D. (1980). Ionomycin stimulates mast cell histamine secretion by forming a lipid soluble calcium complex.Nature 282:851–853.

    Google Scholar 

  7. Douglas, W. W. (1968). Stimulus-secretion coupling: The concept and clues from chromaffin and other cells.Brit. J. Pharmacol. 34:451–474.

    Google Scholar 

  8. Means, A. R. and Dedman, J. R. (1980). Calmodulin—an intracellular calcium receptor.Nature 285:73–77.

    Google Scholar 

  9. Henquin, J.-C. (1981). Effects of trifluoperazine and pimozide on stimulus-secretion coupling in pancreatic β-cells: Suggestion for a role of calmodulin?Biochem. J. 196:771–780.

    Google Scholar 

  10. Douglas, W. and Nemeth, E. (1982). On the calcium receptor activating exocytosis:inhibitory effects of calmodulin-interacting drugs on rat mast cells.J. Physiol. 323:229–244.

    Google Scholar 

  11. Bauduin, H., Stock, C., Vincent, D. and Grenier, J. (1975). Microfilamentous system and secretion of enzymes in the exocrine pancreas: Effect of cytochalasin B.J. Cell. Biol. 66:165–181.

    Google Scholar 

  12. Van Obberghen, E., Somers, G., Devis, G., Ravazzola, M., Malaisse-Lagae, F., Orci, L. and Malaisse, W. (1975). Dynamics of insulin release and microtubular-microfilamentous system: Do microfilaments provide the motive force for the translocation and extrusion of beta granules?Diabetes 24:892–901.

    Google Scholar 

  13. Dormer, R. L. and Ashcroft, S. J. H. (1974). Studies on the role of calcium ions in the stimulation by adrenaline of amylase release from rat parotid.Biochem. J. 144:543–550.

    Google Scholar 

  14. Takemura, H. (1985). Changes in free cytosolic calcium concentration in isolated rat parotid cells by cholinergic and β-adrenergic agonists.Biochem. Biophys. Res. Commun. 131:1048–1055.

    Google Scholar 

  15. Rink, T. and Sanchez, A. (1984). Effects of prostaglandin I2 and forskolin on the secretion from platelets evoked at basal concentrations of cytoplasmic free calcium by thrombin collagen, phorbol ester and exogenous diacylglycerol.Biochem. J. 222:833–836.

    Google Scholar 

  16. Rink, T., Sanchez, A. and Hallam, T. (1983). Diacyglycerol and phorbol ester stimulate secretion without raising cytoplasmic free calcium in human platelets.Nature 305:317–319.

    Google Scholar 

  17. Di Virgilio, F., Lew, D. P. and Pozzan, T. (1984). Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels.Nature 310:691–693.

    Google Scholar 

  18. Arkhammar, P., Nilsson, T. and Berggren, P.-O. (1986). Stimulation of insulin release by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in the clonal cell line RINm5F despite a lowering of the free cytoplasmic Ca2+ concentration.Biochim. Biophys. Acta 887:236–241.

    Google Scholar 

  19. Bruzzone, R., Pozzan, T. and Wollheim, C. B. (1986). Caerulein and carbamoylcholine stimulate pancreatic amylase release at resting free Ca2+.Biochem. J. 235:139–143.

    Google Scholar 

  20. Davis, B. and Lazarus, N. (1976). Anin vitro system for studying insulin release caused by secretory granules-plasma membrane interaction: Definition of the system.J. Physiol. (Lond.) 256:709–729.

    Google Scholar 

  21. Davis, B. and Lazarus, N. (1977). Anin vitro system for studying insulin release: Effects of glucose and glucose-6-phosphate.J. Physiol. 271:273–288.

    Google Scholar 

  22. Konings, F. and De Potter, W. (1981). Calcium-dependentin vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules as a model for exocytosis.FEBS Lett. 126:103–106.

    Google Scholar 

  23. Morris, S. J. and Bradley, D. (1984). Calcium-promoted fusion of isolated chromaffin granules detected by resonance energy transfer between labeled lipids embedded in the membrane bilayer.Biochemistry 23:4642–4650.

    Google Scholar 

  24. Duzgunes, N. and Bentz, J. (1986). Fluorescence assays for membrane fusion. In:Spectroscopic Membrane Probes (L. M. Loew, Ed.), CRC Press (in press).

  25. Konings, F., Majchrowicz, B. and De Potter, W. (1983). Release of chromaffin granular content on interaction with plasma membranes.Amer. J. Physiol. 244:C309-C312.

    Google Scholar 

  26. Izumi, F., Yanagihara, N., Wada, A., Toyohira, Y. and Kobayashi, H. (1986). Lysis of chromaffin granules by phospholipase A2-treated plasma membranes: A cell-free model for exocytosis in adrenal medulla.FEBS Lett. 196:349–352.

    Google Scholar 

  27. Gomperts, B. D. and Fernandez, J. M. (1985). Techniques for membrane permeabilisation.Trends in Biochem. Sci. 10:414–417.

    Google Scholar 

  28. Zimmermann, U., Pilwat, G. and Riemann, F. (1974). Dielectric breakdown of cell membranes.Biophys. J. 14:881–899.

    Google Scholar 

  29. Baker, P. F., Knight, D. E. and Umbach, J. A. (1985). Calcium clamp of the intracellular environment.Cell Calcium. 6:5–14.

    Google Scholar 

  30. Knight, D. E. and Baker, P. F. (1982). Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields.J. Membrane Biol. 68:107–140.

    Google Scholar 

  31. Knight, D. E., Niggli, V. and Scrutton, M. C. (1984). Thrombin and activators of protein kinase C modulate secretory responses of permeabilised human platelets induced by Ca2+.Eur. J. Biochem. 143:437–446.

    Google Scholar 

  32. Yaseen, M. A., Pedley, K. C. and Howell, S. L. (1982). Regulation of insulin secretion from islets of Langerhans rendered permeable by electric discharge.Biochem. J. 206:81–87.

    Google Scholar 

  33. Vallar, L., Biden, T. J. and Wollheim, C. B. (1987). Guanine nucleotides induce Ca2+ independent secretion from permeabilized RINm5F cells.J. Biol. Chem. (in press).

  34. Knight, D. E. and Koh, E. (1984). Ca2+ and cyclic nucleotide dependence of amylase release from isolated rat pancreatic acinar cells rendered permeable by intense electric fields.Cell Calcium. 5:401–418.

    Google Scholar 

  35. Baker, P. F., Knight, D. E. and Whitaker, M. J. (1980). The relation between ionized calcium and cortical granule exocytosis in eggs of the sea urchin Echinus esculentus.Proc. R. Soc. Lond. B. 207:149–161.

    Google Scholar 

  36. Knight, D. E. and Scrutton, M. C. (1986). Gaining access to the cytosol: The technique and some applications of electropermeabilisation.Biochem. J. 234:497–506.

    Google Scholar 

  37. Impraim, C. C., Foster, K. A., Micklem, K. J. and Pasternak, C. A. (1980). Nature of virally mediated changes in membrane permeability to small molecules.Biochem. J. 186:847–860.

    Google Scholar 

  38. Barrowman, M. M., Cockcroft, S. and Gomperts, B. D. (1986). Two roles for guanine nucleotides in stimulus secretion sequence of neutrophils.Nature 319:504–507.

    Google Scholar 

  39. Barrowman, M. M., Cockcroft, S. and Gomperts, B. D. (1987). Differential control of azurophilic and specific granule exocytosis in Sendai virus permeabilised rabbit neutrophils.J. Physiol. 383:115–124.

    Google Scholar 

  40. Gomperts, B. D., Baldwin, J. M. and Micklem, K. J. (1983). Rat mast cells permeabilised with Sendai virus secrete histamine in response to Ca2+ buffered in the micromolar range.Biochem. J. 210:737–745.

    Google Scholar 

  41. Gomperts, B. D. (1985). Manipulation of the cytosolic composition of mast cells: a study of early events in stimulus-secretion coupling. In:Developments in Cell Biology (vol. 1) (R. T. Dean and P. Stahl, Eds.), Butterworths, London, pp. 18–37.

    Google Scholar 

  42. Heppel, L. A., Weisman, G. A. and Friedberg, I. (1985). Permeabilization of transformed cells in culture by external ATP.J. Membrane Biol. 86:189–196.

    Google Scholar 

  43. Cockcroft, S. and Gomperts, B. D. (1979). ATP induces nucleotide permeability in rat mast cells.Nature 279:541–542.

    Google Scholar 

  44. Cockcroft, S. and Gomperts, B. (1979). Activation and inhibition of calcium dependent histamine secretion by ATP ions applied to rat mast cells.J. Physiol. (Lond.) 296:229–243.

    Google Scholar 

  45. Bennett, J. P., Cockcroft, S. and Gomperts, B. D. (1981). Rat mast cells permeabilised with ATP secrete histamine in response to calcium ions buffered in the micromolar range.J. Physiol. (Lond.) 317:335–345.

    Google Scholar 

  46. Sung, S.-S. J., Young, D.-E., Origlio, A. M., Heiple, J. M., Kaback, H. R. and Silverstein, S. C. (1985). Extracellular ATP perturbs transmembrane ion fluxes, elevates cytosolic free [Ca2+], and inhibits phagocytosis in mouse macrophages.J. Biol. Chem. 260:13442–13449.

    Google Scholar 

  47. Mustelin, T. M., Poso, H. and Andersson, L. C. (1986). Role of G-proteins in T cell activation: Nonhydrolysable GTP analogues induce early ornithine decarboxylase activity in human T lymphocytes.EMBO J. 5:3278–3290.

    Google Scholar 

  48. Gomperts, B. D. (1983). Involvement of guanine nucleotide-binding protein in the gating of Ca2+ by receptors.Nature 306:64–66.

    Google Scholar 

  49. Tatham, P. E. R., Cusack, N. J. and Gomperts, B. D. (1987). Characterisation of the ATP4− receptor that mediates permeabilisation of rat mast cells (unpublished experiments).

  50. Neher, E. and Marty, A. (1982). Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.Proc. Nat. Acad. Sci. USA 79:6712–6716.

    Google Scholar 

  51. Fernandez, J. M., Neher, E. and Gomperts, B. D. (1984). Capacitance measurements reveal stepwise fusion events in degranulating mast cells.Nature 312:453–455.

    Google Scholar 

  52. Howell, T. W. and Gomperts, B. D. (1987). Rat mast cells permeabilised with streptolysin-O secrete histamine in response to Ca2+ at concentrations buffered in the micromolar range.Biochim. Biophys. Acta 927:177–183.

    Google Scholar 

  53. Buckingham, L. and Duncan, J. (1983). Approximate dimensions of membrane lesions produced by streptolysin S and streptolysin-O.Biochim. Biophys. Acta 729:115–122.

    Google Scholar 

  54. Bronner, C., Landry, Y., Fonteneau, P. and Kuhry, J.-G. (1986). A fluorescent hydrophobic probe used for monitoring the kinetics of exocytosis phenomena.Biochemistry 25:2149–2154.

    Google Scholar 

  55. Krietsch, W. K. G. and Bucher, T. (1970). 3-Phosphoglycerate kinase from rabbit skeletal muscle and yeast.Eur. J. Biochem. 17:568–580.

    Google Scholar 

  56. Bashford, C. L., Alder, G. M., Menestrina, G., Micklem, K. J., Murphy, J. J. and Pasternak, C. A. (1986). Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents: A common mechanism blocked by divalent cations.J. Biol. Chem. 261:9300–9308.

    Google Scholar 

  57. Howell, T. W., Cockcroft, S. and Gomperts, B. D. (1987). Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilised mast cells.J. Cell Biol. (in press).

  58. Bilezikian, J. P. and Aurbach, G. D. (1974). The effects of nucleotides on the expression of β-adrenergic adenylate cyclase activity in membranes from turkey erythrocytes.J. Biol. Chem. 249:157–161.

    Google Scholar 

  59. Haslam, R. J. and Davidson, M. M. L. (1984). Guanine nucleotides decrease the free [Ca2+] required for secretion of serotonin from permeabilized blood platelets: Evidence of a role for a GTP-binding-protein in platelet activation.FEBS Lett. 174:90–95.

    Google Scholar 

  60. Knight, D. E. and Scrutton, M. C. (1986). Effects of guanine nucleotides on the properties of 5-hydroxytryptamine secretion from electro-permeabilised human platelets.Eur. J. Biochem. 160:183–190.

    Google Scholar 

  61. Cockcroft, S., Howell, T. W. and Gomperts, B. D. (1987). Role for two guanine nucleotide regulatory proteins in mast cell activation: Gp regulates transmembrane signalling and GE regulates exocytosis (unpublished experiments).

  62. Bennett, J. P., Cockcroft, S., Caswell, A. H. and Gomperts, B. D. (1982). Plasma membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionylleucylphenylalanine.Biochem. J. 208:801–808.

    Google Scholar 

  63. Gomperts, B. D., Barrowman, M. M. and Cockcroft, S. (1986). Dual role for guanine nucleotides in stimulus-secretion coupling: an investigation of mast cells and neutrophils.Fed. Proc. 45:2156–2161.

    Google Scholar 

  64. Gomperts, B. D. (1986). Calcium shares the limelight in stimulus-secretion coupling.Trends in Biochem. Sci. 11:290–292.

    Google Scholar 

  65. Hallam, T. J. and Rink, T. J. (1985). Responses to adenosine diphosphate in human platelets loaded with the fluorescent calcium indicator quin2.J. Physiol. (Lond.) 368:131–146.

    Google Scholar 

  66. Hallam, T. J., Daniel, J. L., Kendrick-Jones, J. and Rink, T. J. (1985). Relationship between cytoplasmic free calcium and myosin light chain phosphorylation in intact platelets.Biochem. J. 232:373–377.

    Google Scholar 

  67. Lew, P. D., Monod, A., Waldwogel, F. A., Dewald, B., Baggiolini, M. and Pozzan, T. (1986). Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils.J. Cell Biol. 102:2197–2204.

    Google Scholar 

  68. Pandol, S., Schoeffield, M., Sachs, G. and Muallem, S. (1985). Role of free cytosolic calcium in secretogogue-stimulated amylase release from dispersed acini from guinea pig pancreas.J. Biol. Chem. 260:10081–10086.

    Google Scholar 

  69. Kao, L.-S. and Schneider, A. S. (1986). Calcium mobilisation and catecholamine secretion in adrenal chromaffin cells: A quin-2 fluorescence study.J. Biol. Chem. 261:4881–4888.

    Google Scholar 

  70. Cockcroft, S. and Gomperts, B. D. (1986). Questions concerning the role of calcium in exocytosis. In:Calcium and Drug Action (P. F. Baker Ed.), Springer, Heidelberg, in press.

    Google Scholar 

  71. Johnson, G. S. and Mukku, V. R. (1979). Evidence in intact cells for an involvement of GTP in the activation of adenylate cyclase.J. Biol. Chem. 254:95–100.

    Google Scholar 

  72. Marquardt, D. L., Gruber, H. E. and Walker, L. L. (1987). Ribavirin inhibits mast cell mediator release.J. Pharmacol. Exp. Therap. 240:145–149.

    Google Scholar 

  73. Hamill, O., Marty, A., Neher, E., Sakmann, B. and Sigworth, F. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.Eur. J. Physiol. 391:85–100.

    Google Scholar 

  74. Marty, A. and Neher, E. (1983). Tight seal whole-cell recording. In:Single Channel Recording (B. Sakmann and E. Neher Eds.), Plenum, New York, pp. 107–121.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Experimental Pathology, University College London, University Street, WC1E 6JJ, London, UK

    B. D. Gomperts, S. Cockcroft, T. W. Howell, O. Nüsse & P. E. R. Tatham

Authors
  1. B. D. Gomperts
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. Cockcroft
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. W. Howell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. O. Nüsse
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P. E. R. Tatham
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gomperts, B.D., Cockcroft, S., Howell, T.W. et al. The dual effector system for exocytosis in mast cells: Obligatory requirement for both Ca2+ and GTP. Biosci Rep 7, 369–381 (1987). https://doi.org/10.1007/BF01362501

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01362501

Key Words

Search

Navigation