Mechanism of cAMP-induced H+-efflux ofDictyostelium cells: a role for fatty acids
- 43 Downloads
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.
KeywordsAcidic vesicles arachidonic acid Ca2+ transport concanamycin A H+-efflux miconazole
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
inositol 1,4,5 trisphosphate
Unable to display preview. Download preview PDF.
- 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. 105 255–261Google Scholar
- 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 2935–2943Google Scholar
- 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 3–13Google Scholar
- Maeda Y, Inouye K and Takeuchi I 1997Dictyostelium — A model system for cell and developmental biology (Tokyo: Universal Academic Press)Google Scholar
- 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. 33 541–548Google Scholar
- Simchovitz L and Cragoe E J Jr 1986 Regulation of human neutrophil chemotaxis by intracellular pH;J. Biol. Chem. 261 6492–6500Google Scholar
- 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 1479–1495Google Scholar
- 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 21072–21079Google Scholar