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  - 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  -