Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Nov 15;288(Pt 1):181–187. doi: 10.1042/bj2880181

Guanine nucleotide is essential and Ca2+ is a modulator in the exocytotic reaction of permeabilized rat mast cells.

T H Lillie 1, B D Gomperts 1
PMCID: PMC1132097  PMID: 1445262

Abstract

Exocytosis from metabolically depleted permeabilized rat mast cells was measured in response to provision of Ca2+ and guanine nucleotide [GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S])]. For cells permeabilized in simple salt solutions (NaCl), both of these effectors were required to induce secretion. Exclusion of Mg2+ caused an increase in both the sensitivity of the system to GTP and the extent of secretion elicited, while having no such effects on secretion induced by GTP[S]. The effect of Mg2+ depletion on the ability of GTP to stimulate secretion is probably due to the dependence on Mg2+ of the GTPase activity of GE (a postulated GTP-binding protein which mediates exocytosis). This argues that a persistent stimulus to the G-protein is required to support secretion. Affinity for both GTP[S] and GTP is enhanced when the cells are permeabilized in zwitterionic electrolytes (glutamate, gamma-aminobutyric acid, glycine) instead of NaCl. Under these conditions, secretion occurs in response to provision of either GTP[S] [in the effective absence of Ca2+ (pCa 9)] or Ca2+ (in the absence of guanine nucleotide). Secretion induced by GTP[S] is strongly promoted by the presence of Mg2+ at concentrations in the millimolar range; this promotion by Mg2+ declines as the concentration of Ca2+ is elevated towards pCa 7. At pCa 6, Mg2+ is without effect. Ca(2+)-induced secretion requires the provision of MgATP. Since this is further enhanced by low concentrations (< 100 microM) and then inhibited by high concentrations of GDP, the essential role of ATP is likely to be in the maintenance of GTP via transphosphorylation by a nucleoside diphosphate kinase reaction. Thus, under conditions of high affinity (glutamate environment), GTP[S] alone is capable of inducing exocytosis. Ca2+ acts in concert with guanine nucleotides: it enhances the rate and extent of secretion and increases the affinity for Mg2+ and guanine nucleotides in the activation of the GTP-binding protein (GE) which regulates exocytosis.

Full text

PDF

Selected References

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

  1. Bacon R. A., Salminen A., Ruohola H., Novick P., Ferro-Novick S. The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants. J Cell Biol. 1989 Sep;109(3):1015–1022. doi: 10.1083/jcb.109.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Balch W. E. Small GTP-binding proteins in vesicular transport. Trends Biochem Sci. 1990 Dec;15(12):473–477. doi: 10.1016/0968-0004(90)90301-q. [DOI] [PubMed] [Google Scholar]
  3. Barrowman M. M., Cockcroft S., Gomperts B. D. Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils. Nature. 1986 Feb 6;319(6053):504–507. doi: 10.1038/319504a0. [DOI] [PubMed] [Google Scholar]
  4. Bennett J. P., Cockcroft S., Gomperts B. D. Ionomycin stimulates mast cell histamine secretion by forming a lipid-soluble calcium complex. Nature. 1979 Dec 20;282(5741):851–853. doi: 10.1038/282851a0. [DOI] [PubMed] [Google Scholar]
  5. Bourne H. R. Do GTPases direct membrane traffic in secretion? Cell. 1988 Jun 3;53(5):669–671. doi: 10.1016/0092-8674(88)90081-5. [DOI] [PubMed] [Google Scholar]
  6. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  7. Chavrier P., Parton R. G., Hauri H. P., Simons K., Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990 Jul 27;62(2):317–329. doi: 10.1016/0092-8674(90)90369-p. [DOI] [PubMed] [Google Scholar]
  8. Churcher Y., Gomperts B. D. ATP-dependent and ATP-independent pathways of exocytosis revealed by interchanging glutamate and chloride as the major anion in permeabilized mast cells. Cell Regul. 1990 Mar;1(4):337–346. doi: 10.1091/mbc.1.4.337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cromwell O., Bennett J. P., Hide I., Kay A. B., Gomperts B. D. Mechanisms of granule enzyme secretion from permeabilized guinea pig eosinophils. Dependence on Ca2+ and guanine nucleotides. J Immunol. 1991 Sep 15;147(6):1905–1911. [PubMed] [Google Scholar]
  10. Fernandez J. M., Neher E., Gomperts B. D. Capacitance measurements reveal stepwise fusion events in degranulating mast cells. 1984 Nov 29-Dec 5Nature. 312(5993):453–455. doi: 10.1038/312453a0. [DOI] [PubMed] [Google Scholar]
  11. Fischer von Mollard G., Mignery G. A., Baumert M., Perin M. S., Hanson T. J., Burger P. M., Jahn R., Südhof T. C. rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1988–1992. doi: 10.1073/pnas.87.5.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fischer von Mollard G., Südhof T. C., Jahn R. A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature. 1991 Jan 3;349(6304):79–81. doi: 10.1038/349079a0. [DOI] [PubMed] [Google Scholar]
  13. Gomperts B. D., Barrowman M. M., Cockcroft S. Dual role for guanine nucleotides in stimulus-secretion coupling. Fed Proc. 1986 Jun;45(7):2156–2161. [PubMed] [Google Scholar]
  14. Gomperts B. D., Tatham P. E. Regulated exocytotic secretion from permeabilized cells. Methods Enzymol. 1992;219:178–189. doi: 10.1016/0076-6879(92)19020-7. [DOI] [PubMed] [Google Scholar]
  15. Goud B., Zahraoui A., Tavitian A., Saraste J. Small GTP-binding protein associated with Golgi cisternae. Nature. 1990 Jun 7;345(6275):553–556. doi: 10.1038/345553a0. [DOI] [PubMed] [Google Scholar]
  16. Haslam R. J., Davidson M. M. Potentiation by thrombin of the secretion of serotonin from permeabilized platelets equilibrated with Ca2+ buffers. Relationship to protein phosphorylation and diacylglycerol formation. Biochem J. 1984 Sep 1;222(2):351–361. doi: 10.1042/bj2220351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Higashijima T., Ferguson K. M., Sternweis P. C. Regulation of hormone-sensitive GTP-dependent regulatory proteins by chloride. J Biol Chem. 1987 Mar 15;262(8):3597–3602. [PubMed] [Google Scholar]
  18. Howell T. W., Cockcroft S., Gomperts B. D. Essential synergy between Ca2+ and guanine nucleotides in exocytotic secretion from permeabilized rat mast cells. J Cell Biol. 1987 Jul;105(1):191–197. doi: 10.1083/jcb.105.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Iyengar R., Birnbaumer L. Hormone receptor modulates the regulatory component of adenylyl cyclase by reducing its requirement for Mg2+ and enhancing its extent of activation by guanine nucleotides. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5179–5183. doi: 10.1073/pnas.79.17.5179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iyengar R., Birnbaumer L. Hysteretic activation of adenylyl cyclases. I. Effect of Mg ion on the rate of activation by guanine nucleotides and fluoride. J Biol Chem. 1981 Nov 10;256(21):11036–11041. [PubMed] [Google Scholar]
  21. Iyengar R. Hysteretic activation of adenylyl cyclases. II. Mg ion regulation of the activation of the regulatory component as analyzed by reconstitution. J Biol Chem. 1981 Nov 10;256(21):11042–11050. [PubMed] [Google Scholar]
  22. Johnson R. A., Pilkis S. J., Hamet P. Liver membrane adenylate cyclase. Synergistic effects of anions on fluoride, glucagon, and guanyl nucleotide stimulation. J Biol Chem. 1975 Aug 25;250(16):6599–6607. [PubMed] [Google Scholar]
  23. Kikkawa S., Takahashi K., Takahashi K., Shimada N., Ui M., Kimura N., Katada T. Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins. J Biol Chem. 1990 Dec 15;265(35):21536–21540. [PubMed] [Google Scholar]
  24. Kimura N., Shimada N. Evidence for complex formation between GTP binding protein(Gs) and membrane-associated nucleoside diphosphate kinase. Biochem Biophys Res Commun. 1990 Apr 16;168(1):99–106. doi: 10.1016/0006-291x(90)91680-q. [DOI] [PubMed] [Google Scholar]
  25. Knight D. E., Baker P. F. The phorbol ester TPA increases the affinity of exocytosis for calcium in 'leaky' adrenal medullary cells. FEBS Lett. 1983 Aug 22;160(1-2):98–100. doi: 10.1016/0014-5793(83)80944-2. [DOI] [PubMed] [Google Scholar]
  26. Koopmann W. R., Jr, Jackson R. C. Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C. Biochem J. 1990 Jan 15;265(2):365–373. doi: 10.1042/bj2650365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lillie T. H., Gomperts B. D. Nucleotides and divalent cations as effectors and modulators of exocytosis in permeabilized rat mast cells. Philos Trans R Soc Lond B Biol Sci. 1992 Apr 29;336(1276):25–34. doi: 10.1098/rstb.1992.0040. [DOI] [PubMed] [Google Scholar]
  28. Lillie T. H., Whalley T. D., Gomperts B. D. Modulation of the exocytotic reaction of permeabilised rat mast cells by ATP, other nucleotides and Mg2+. Biochim Biophys Acta. 1991 Sep 24;1094(3):355–363. doi: 10.1016/0167-4889(91)90097-h. [DOI] [PubMed] [Google Scholar]
  29. Marquardt D. L., Gruber H. E., Walker L. L. Ribavirin inhibits mast cell mediator release. J Pharmacol Exp Ther. 1987 Jan;240(1):145–149. [PubMed] [Google Scholar]
  30. Melançon P., Glick B. S., Malhotra V., Weidman P. J., Serafini T., Gleason M. L., Orci L., Rothman J. E. Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell. 1987 Dec 24;51(6):1053–1062. doi: 10.1016/0092-8674(87)90591-5. [DOI] [PubMed] [Google Scholar]
  31. Miller S. G., Moore H. P. Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium. J Cell Biol. 1991 Jan;112(1):39–54. doi: 10.1083/jcb.112.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Neher E. The influence of intracellular calcium concentration on degranulation of dialysed mast cells from rat peritoneum. J Physiol. 1988 Jan;395:193–214. doi: 10.1113/jphysiol.1988.sp016914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Novick P. J., Goud B., Salminen A., Walworth N. C., Nair J., Potenza M. Regulation of vesicular traffic by a GTP-binding protein on the cytoplasmic surface of secretory vesicles in yeast. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):637–647. doi: 10.1101/sqb.1988.053.01.073. [DOI] [PubMed] [Google Scholar]
  34. Nüsse O., Lindau M., Cromwell O., Kay A. B., Gomperts B. D. Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils. J Exp Med. 1990 Mar 1;171(3):775–786. doi: 10.1084/jem.171.3.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Penner R., Neher E. Secretory responses of rat peritoneal mast cells to high intracellular calcium. FEBS Lett. 1988 Jan 4;226(2):307–313. doi: 10.1016/0014-5793(88)81445-5. [DOI] [PubMed] [Google Scholar]
  36. Penner R., Pusch M., Neher E. Washout phenomena in dialyzed mast cells allow discrimination of different steps in stimulus-secretion coupling. Biosci Rep. 1987 Apr;7(4):313–321. doi: 10.1007/BF01121453. [DOI] [PubMed] [Google Scholar]
  37. Randazzo P. A., Northup J. K., Kahn R. A. Activation of a small GTP-binding protein by nucleoside diphosphate kinase. Science. 1991 Nov 8;254(5033):850–853. doi: 10.1126/science.1658935. [DOI] [PubMed] [Google Scholar]
  38. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  39. Strittmatter S. M., Valenzuela D., Kennedy T. E., Neer E. J., Fishman M. C. G0 is a major growth cone protein subject to regulation by GAP-43. Nature. 1990 Apr 26;344(6269):836–841. doi: 10.1038/344836a0. [DOI] [PubMed] [Google Scholar]
  40. Stutchfield J., Cockcroft S. Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin O. Biochem J. 1988 Mar 1;250(2):375–382. doi: 10.1042/bj2500375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Svoboda M., Christophe J. The monovalent anions chloride and azide as potent activators of NaF- and p(NH)ppG-stimulation of pancreatic adenylate cyclase. FEBS Lett. 1978 Feb 15;86(2):230–234. doi: 10.1016/0014-5793(78)80568-7. [DOI] [PubMed] [Google Scholar]
  42. Südhof T. C., Jahn R. Proteins of synaptic vesicles involved in exocytosis and membrane recycling. Neuron. 1991 May;6(5):665–677. doi: 10.1016/0896-6273(91)90165-v. [DOI] [PubMed] [Google Scholar]
  43. Walworth N. C., Goud B., Kabcenell A. K., Novick P. J. Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 1989 Jun;8(6):1685–1693. doi: 10.1002/j.1460-2075.1989.tb03560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yamada Y., Natsumeda Y., Weber G. Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase. Biochemistry. 1988 Mar 22;27(6):2193–2196. doi: 10.1021/bi00406a057. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES