Abstract
AggregatingDictyostelium cells release protons when stimulated with cAMP. To find out whether the protons are generated by acidic vesicles or in the cytosol, we permeabilized the cells and found that this did not alter the cAMP-response. Proton efflux in intact cells was inhibited by preincubation with the V-type H+ ATPase inhibitor concanamycin A and with the plasma membrane H+ ATPase blocker miconazole. Surprisingly, miconazole also inhibited efflux in permeabilized cells, indicating that this type of H+ ATPase is present on intracellular vesicles as well. Vesicular acidification was inhibited by miconazole and by concanamycin A, suggesting that the acidic vesicles contain both V-type and P-type H+ ATPases. Moreover, concanamycin A and miconazole acted in concert, both in intact cells and in vesicles. The mechanism of cAMP-induced Ca2+-fluxes involves phospholipase A2 activity. Fatty acids circumvent the plasma membrane and stimulate vesicular Ca2+-efflux. Here we show that arachidonic acid elicited H+-efflux not only from intact cells but also from acidic vesicles. The target of regulation by arachidonic acid seemed to be the vesicular Ca2+-relase channel.
Similar content being viewed by others
Abbreviations
- AA:
-
arachidonic acid
- BHQ:
-
2,5-di(tert-butyl)-1,4-hydroquinone
- CMA:
-
concanamycin A
- DMO:
-
5,5-dimethyl-2,4-oxazolidinedione, fura-2 (l-[2-(5-Carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxyl)-ethane-N,N,N′,N′-tetraacetic acid
- IP3 :
-
inositol 1,4,5 trisphosphate
- pHi :
-
intracellular pH
References
Aeckerle S, Wurster B and Malchow D 1985 Oscillations and cyclic AMP-induced changes of the K+ concentration inDictyostelium discoideum;EMBO J. 4 39–43
Aerts R J, De Wit R J W and Van Lookeren Campagne M M 1987 Cyclic AMP induces a transient alkalinization inDictyostelium;FEBS Lett. 220 366–370
Aubry L, Klein G, Martiel J-L and Satre M 1993 Kinetics of endosomal pH evolution inDictyostelium discoideum amoebae;J. Cell Sci. 105 861–866
Bradford M M 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding;Anal. Biochem. 72 255–260
Brdiczka D, Pette D, Brunner G and Miller G 1968 Kompartimentierte Verteilung von Enzymen in Rattenlebermitochondrien;Eur. J. Biochem. 5 294–304
Bumann J, Malchow D and Wurster B 1986 Oscillations of Ca++ concentration during the cell differentiation ofDictyostelium discoideum;Differentiation 31 85–91
Bumann J, Wurster B and Malchow D 1984 Attractant-induced changes and oscillations of the extracellular Ca2+ concentration in suspensions of differentiatingDictyostelium cells;J. Cell Biol. 98 173–178
Chyb S, Raghu P and Hardie R C 1999 Polyunsaturated fatty acids activate theDrosophila light-sensitive channels TRP and TRPL;Nature (London) 397 255–259
Devreotes P N and Zigmond S H 1988 Chemotaxis in eukaryotic cells: a focus on Leukocytes andDictyostelium;Annu. Rev. Cell Biol. 4 649–686
Estabrook R W 1967 Mitochondrial respiratory control and the polarographic measurement of ADP: O ratios;Methods Enzymol. 10 41–47
Edmonds B T, Murray J and Condeelis J 1995 pH regulation of the f-actin binding propertiesof Dictyostelium elongation factor lα;J. Biol. Chem. 270 15222–15230
Ferber E, Munder P G, Fischer H and Gerisch G 1970 High phospholipase activities in amoebae ofDictyostelium discoideum;Eur. J. Biochem. 14 253–257
Flaadt H, Jaworski E, Schlatterer C and Malchow D 1993a Cyclic AMP- and Ins(1,4,5)P3-induced Ca2+ fluxes in permeabilised cells ofDictyostelium discoideum: cGMP regulates Ca2+ entry across the plasma membrane;J. Cell Sci. 105 255–261
Flaadt H, Jaworski E and Malchow D 1993b Evidence for two intracellular calcium pools inDictyostelium: the cAMP-induced calcium influx is directed into a NBD-Cl- and 2,5-di-(tert-butyl)-1,4-hydroquinone-sensitive pool;J. Cell Sci. 105 1131–1135
Gerisch G 1971 Periodische Signale steuern die Musterbildung in Zellverbänden;Naturwissenschaften 58 430–438
Gutknecht J 1988 Proton conductance caused by long-chain fatty acids in phospholipid bilayer membranes;J. Membr. Biol. 106 83–93
Hacker U, Albrecht R and Maniak M 1997 Fluid-phase uptake by macropinocytosis inDictyostelium;J. Cell Sci. 110 105–112
Hanakam F, Albrecht R, Eckerskorn C, Matzner M and Gerisch G 1996 Myristoylated and non-myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein;EMBO J. 15 2935–2943
Heuser J, Zhu Q and Clarke M 1993 Proton pumps populate the contractile vacuoles ofDictyostelium amoebae;J. Cell Biol. 121 1311–1327
Kessin R H 1997 The evolution of the cellular slime molds; inDictyostelium — A model system for cell and developmental biology (eds) Y Maeda, K Inouye and I Takeuchi (Tokyo: Universal Academy Press) pp 3–13
Maeda Y, Inouye K and Takeuchi I 1997Dictyostelium — A model system for cell and developmental biology (Tokyo: Universal Academic Press)
Malchow D, Nanjundiah V and Gerisch G 1978a pH oscillations in cell suspensions ofDictyostelium discoideum: their relation to cyclic-AMP signals;J. Cell Sci. 30 319–330
Malchow D, Nanjundiah V, Wurster B, Eckstein F and Gerisch G 1978b Cyclic AMP-induced pH changes inDictyostelium discoideum and their control by calcium;Biochim. Biophys. Acta 538 473–478
Marx D, Tuckerman M E, Hutter J and Parrinello M 1999 The nature of the hydrated excess proton in water;Nature (London) 397 601–604
Mignen O and Shuttleworth T J 2000 IARC, a novel arachidonate-regulated, noncapacitative Ca2+ entry channel;J. Biol. Chem. 275 9114–9119
Moniakis J, Coukell M B and Janiec A 1999 Involvement of the Ca2+-ATPase PAT1 and the contractile vacuole in calcium regulation inDictyostelium discoideum;J. Cell Sci. 112 405–414
Newell P C, Malchow D and Gross J D 1995 The role of calcium in aggregation and development ofDictyostelium;Experientia 51 1155–1165
Ohsumi Y and Anraku Y 1983 Calcium transport driven by a proton motive force in vacuolar membrane vesicles ofSaccharomyces cerevisiae;J. Biol. Chem. 258 5614–5617
Padh H, Lavasa M and Steck T L 1989 Characterization of a vacuolar proton ATPase inDictyostelium discoideum;Biochim. Biophys. Acta 982 271–278
Padh H and Tanjore S 1995 Localization of cyclic-AMP receptors with acidosomes inDictyostelium discoideum;FEBS Lett. 368 358–362
Pogge-von Strandmann R, Kay R R and Dufour J-P 1984 An electrogenic proton pump in plasma membranes from the cellular slime moldDictyostelium discoideum;FEBS Lett. 175 422–427
Rooney E K and Gross J D 1992 ATP-driven Ca2+/H+ antiport in acid vesicles fromDictyostelium;Proc. Natl. Acad. Sci. USA 89 8025–8029
Rooney E K, Gross J D and Satre M 1994 Characterisation of an intracellular Ca2+ pump inDictyostelium;Cell Calcium 16 509–522
Schaloske R and Malchow D 1997 Mechanism of cAMP-induced Ca2+ influx inDictyostelium: role of phospholipase A2;Biochem. J. 327 233–238
Schaloske R, Sonnemann J, Malchow D and Schlatterer C 1998 Fatty acids induce release of Ca2+ from acidosomal stores and activate capacitative Ca2+ entry inDictyostelium discoideum;Biochem. J. 33 541–548
Simchovitz L and Cragoe E J Jr 1986 Regulation of human neutrophil chemotaxis by intracellular pH;J. Biol. Chem. 261 6492–6500
Loomis W F 1998 Cell-cell signalling duringDictyostelium development;Trends Microbiol. 6 402–406
Temesvari L A, Rodriguez-Paris J M, Bush J M, Zhang L and Cardelli J A 1996 Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system ofDictyostelium discoideum;J. Cell Sci. 109 1479–1495
Troll H, Malchow D, Müller-Taubenberger A, Humbel B, Lottspeich F, Ecke M, Gerisch G, Schmid A and Benz R 1992 Purification, functional characterization, and cDNA sequencing of mitochondrial porin fromDictyostelium discoideum;J. Biol. Chem. 26 21072–21079
Van Duijn B and Inouye K 1991 Regulation of movement speed by intracellular pH duringDictyostelium discoideum chemotaxis.Proc. Natl. Acad. Sci. USA88 4951–4955
Vercesi A E, Moreno S N J and Docampo R 1994 Ca2+/H+ exchange in acidic vacuoles ofTrypanosoma brucei;Biochem. J. 304 227–233
Waldmann R, Champigny G, Bassilana F, Heurteaux, Lazdunski M 1997 A proton-gated cation channel involved in acid-sensing;Nature (London) 386 173–177
Wurster B, Bek F and Butz K 1981 Folic acid and pterin deaminases inDictyostelium discoideum: Kinetic properties and regulation by folic acid, pterin and adenosine 3',5'-phosphate;J. Bacteriol. 148 183–192
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Flaadt, H., Schaloske, R. & Malchow, D. Mechanism of cAMP-induced H+-efflux ofDictyostelium cells: a role for fatty acids. J Biosci 25, 243–252 (2000). https://doi.org/10.1007/BF02703931
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF02703931