TY - JOUR A1 - Dames, Petra A1 - Zimmermann, Bernhard A1 - Schmidt, Ruth A1 - Rein, Julia A1 - Voss, Martin A1 - Schewe, Bettina A1 - Walz, Bernd A1 - Baumann, Otto T1 - cAMP regulates plasma membrane vacuolar-type H+-ATPase assembly and activity in blowfly salivary glands N2 - Reversible assembly of the V0V1 holoenzyme from V-0 and V-1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H+-ATPases (V-ATPases) in animal cells. in the blowfly (Calliphora vicina) salivary gland, V- ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the CAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that CAMP, Sp-adenosine-3',5'-cyclic monophosphorothioate, or forskolin, similar to serotonin, cause V-ATPase-dependent luminal acidification. In addition, V-ATPase-dependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V, components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca2+, our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus Y1 - 2006 UR - http://www.pnas.org/ U6 - https://doi.org/10.1073/pnas.0600011103 SN - 0027-8424 ER - TY - GEN A1 - Rein, Julia A1 - Voss, Martin A1 - Blenau, Wolfgang A1 - Walz, Bernd A1 - Baumann, Otto T1 - Hormone-induced assembly and activation of V-ATPase in blowfly salivary glands is mediated by protein kinase A N2 - The vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland cells energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of V-0 and V-1 subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of V1 components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced V-1 translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA. KW - Vacuolar h+-atpase KW - camp binding-sites KW - cyclic-amp KW - plasma-membrane KW - drosophila-melanogaster Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-46126 ER - TY - JOUR A1 - Voss, Martin A1 - Fechner, Lennart A1 - Walz, Bernd A1 - Baumann, Otto T1 - Calcineurin activity augments cAMP/PKA-dependent activation of V-ATPase in blowfly salivary glands N2 - We have examined the role of the Ca2+-dependent protein phosphatase 2B (calcineurin) in the regulation of the vacuolar H+-ATPase (V-ATPase) in blowfly salivary glands. In response to the neurohormone serotonin [5-hydroxytryptamine (5-HT)] and under the mediation of the cAMP/PKA signaling pathway, the secretory cells assemble and activate V-ATPase molecules at the apical membrane. We demonstrate that the inhibition of calcineurin activity by cyclosporin A, by FK- 506, or by prevention of the elevation of Ca2+ diminishes the 5-HT-induced assembly and activation of V-ATPase. The effect of calcineurin on V-ATPase is mediated by the cAMP/PKA signaling pathway, with calcineurin acting upstream of PKA, because 1) cyclosporin A does not influence the 8-(4-chlorophenylthio) adenosine-3',5'-cyclic monophosphate (8-CPT-cAMP)-induced activation of V-ATPase, and 2) the 5-HT-induced rise in cAMP is highly reduced in the presence of cyclosporin A. Moreover, a Ca2+ rise evoked by the sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid leads to an increase in intracellular cAMP concentration and a calcineurin-mediated PKA- dependent activation of V-ATPase. We propose that calcineurin activity mediates cross talk between the inositol 1,4,5- trisphosphate/Ca2+ and the cAMP/PKA signaling pathways, thereby augmenting the 5-HT-induced rise in cAMP and thus the cAMP/PKA-mediated activation of V-ATPase. Y1 - 2010 UR - http://ajpcell.physiology.org/ U6 - https://doi.org/10.1152/ajpcell.00328.2009 SN - 0363-6143 ER - TY - JOUR A1 - Voss, Martin A1 - Schmidt, Ruth A1 - Walz, Bernd A1 - Baumann, Otto T1 - Stimulus-induced translocation of the protein kinase A catalytic subunit to the apical membrane in blowfly salivary glands N2 - Secretion in blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT), which activates the InsP(3)/Ca2+ pathway and the cAMP/protein kinase A (PKA) pathway in the secretory cells. The latter signaling cascade induces the activation of a vacuolar H+-ATPase on the apical membrane. Here, we have determined the distribution of PKA by using antibodies against the PKA regulatory subunit-II (PKA-RII) and the PKA catalytic subunit (PKA-C) of Drosophila. PKA is present in high concentrations within the secretory cells. PKA-RII and PKA-C co-distribute in non-stimulated glands, being enriched in the basal portion of the secretory cells. Exposure to 8-CPT-cAMP or 5-HT induces the translocation of PKA-C to the apical membrane, whereas the PKA-RII distribution remains unchanged. The recruitment of PKA-C to the apical membrane corroborates our hypothesis that vacuolar H+-ATPase, which is enriched in this membrane domain, is a target protein for PKA. Y1 - 2009 UR - http://www.springerlink.com/content/100524 U6 - https://doi.org/10.1007/s00441-008-0673-x SN - 0302-766X ER - TY - GEN A1 - Voss, Martin A1 - Blenau, Wolfgang A1 - Walz, Bernd A1 - Baumann, Otto T1 - V-ATPase deactivation in blowfly salivary glands is mediated by protein phosphatase 2C N2 - The activity of vacuolar H+-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc. KW - vacuolar H+-ATPase KW - assembly KW - regulation KW - protein phosphatise KW - dephosphorylation Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-44360 ER -