Abstract
ATP is not required for exocytosis from permeabilised mast cells, and therefore there is no direct role for protein phosphorylation in the late stages of the activation pathway. We have measured the timecourse of exocytosis from permeabilised cells triggered to release hexosaminidase following addition of Ca2+ to cells equilibrated for 2 min with GTP-γ-S. If ATP is included at the time of permeabilisation, then exocytosis commences after a delay, the duration of which depends on the square root of the product [Ca2+][GTP-γ-S], and which may extend to beyond 3 min. When ATP is excluded then the maximal rate of exocytosis is established within 3 secs of completing the effector combination. These results suggest that the achievement of a new steady-state, induced by Ca2+ and GTP-γ-S, and required for exocytosis is inhibited by ATP. From this we conclude that dephosphorylation of an unknown regulator protein may comprise a step in the exocytotic pathway.
References
Baker, P. F., Knight, D. E. and Umbach, J. A. (1985) Calcium clamp of the intracellular environment.Cell Calcium 6:5–14.
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.
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. (Lond) 383:115–124.
Brown, E. M., LeBoff, M. S., Oetting, M., Posillico, J. T. and Chen, C. (1987) Secretory control in normal and abnormal parathyroid tissue.Recent Prog. Hormone Res. 43:337–382.
Cockcroft, S. and Gomperts, B. D. (1979a) Activation and inhibition of calcium dependent histamine secretion by ATP ions applied to rat mast cells.J. Physiol. (Lond) 296:229–243.
Cockcroft, S. and Gomperts, B. D. (1979b) ATP induces nucleotide permeability in rat mast cells.Nature 279:541–542.
Cockcroft, S., Howell, T. W. and Gomperts, B. D. (1987) Two G-proteins act in series to control stimulus-secretion coupling in mast cells: Use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphodiesterase and exocytosis.J. Cell Biol. 105:2745–2750.
Fernandez, J. M., Lindau, M. and Eckstein, F. (1987) Intracellular stimulation of mast cells with guanine nucleotides mimic antigenic stimulation.FEBS Lett. 216:89–93.
Gilligan, D. M. and Satir, B. H. (1982) Protein phosphorylation/dephosphorylation and stimulus-secretion coupling in wild type and mutant Paramecium.J. Biol. Chem. 257:13903–13906.
Gomperts, B. D. (1989) G-proteins in exocytotic secretion. In:G-Proteins (R. Iyengar and L. Birnbaumer, eds.) (in press).
Gomperts, B. D., Cockcroft, S., Howell, T. W., Nüsse, O. and Tatham, P. E. R. (1987) The dual effector system for exocytosis in mast cells: Obligatory requirement for both Ca2+ and GTP.Biosci. Rep. 7:369–381.
Gomperts, B. D. and Fernandez, J. M. (1985) Techniques for membrane permeabilisation.Trends in Biochem. Sci. 10:414–417.
Gomperts, B. D. and Fewtrell, C. M. S. (1985) The mast cell: a paradigm for receptor and exocytotic mechanisms. In:Molecular Mechanisms of Membrane Signalling, Chapter 12, Vol. 4 ofMolecular Aspects of Cellular Regulation (P. Cohen and M. D. Houslay, Eds). Elsevier, Amsterdam, pp. 377–409.
Howell, T. W., Cockroft, S. and Gomperts, B. D. (1987) Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilised mast cells.J. Cell Biol. 105:191–197.
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.
Howell, T. W., Kramer, I. and Gomperts, B. D. (1988) Protein phosphorylation and the dependence on Ca2+ for GTP-γ-S stimulated exocytosis from permeabilised mast cells.Cellular Signalling (in press).
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.
Momayezi, M., Lumpert, C. J., Kerksen, H., Gras, U., Plattner, H., Krinks, M. H. and Klee, C. B. (1987) Exocytosis induction in Paramecium tetraurelia cells by exogenous phosphoprotein phosphtase in vivo and in vitro: Possible involvement of calcineurin in exocytotic membrane fusion.J. Cell Biol. 105:181–189.
Ullrich, S. and Wollheim, C. B. (1988) GTP-dependent inhibition of insulin secretion by epinephrine in permeabilized RINm5F cells: Lack of correlation between insulin secretion and cyclic AMP levels.J. Biol. Chem. 263:8615–8620.
Vallar, L., Biden, T. J. and Wollheim, C. B. (1987) Guanine nucleotides induce Ca2+ independent secretion from permeabilized RINm5F cells.J. Biol. Chem. 262:5049–5056.
Vilmart-Seuwen, J., Kerksen, H., Sturtzl, R. and Plattner, H. (1986) ATP keeps exocytosis sites in a primed state but is not required for membrane fusion: An analysis with Paramecium cells in vivo and in vitro.J. Cell Biol. 103:1279–1288.
Zieseniss, E. and Plattner, H. (1985) Synchronous exocytosis inParamecium cells involves very rapid (≤1 s), reversible dephosphorylation of a 65-kD phosphoprotein in exocytosis-competent strains.J. Cell Biol. 101:2028–2035.
Zimmerberg, J., Curran, M., Cohen, F. S. and Brodwick, M. (1987) Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells.Proc. Nat. Acad. Sci. USA 84:1585–1589.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Tatham, P.E.R., Gomperts, B.D. ATP inhibits onset of exocytosis in permeabilised mast cells. Biosci Rep 9, 99–109 (1989). https://doi.org/10.1007/BF01117516
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01117516