Hormone-induced assembly and activation of V-ATPase in blowfly salivary glands is mediated by protein kinase A.
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 V(1) 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.
Rein
Julia
J
Institut für Biochemie und Biologie, Universität Potsdam, Potsdam, Germany.
Voss
Martin
M
Blenau
Wolfgang
W
Walz
Bernd
B
Baumann
Otto
O
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004175:Diptera; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000378:metabolism*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D012469:Salivary Glands / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D019476:Insect Proteins; D047428:Protein Kinase Inhibitors; D021122:Protein Subunits; D012701:Serotonin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D025262:Vacuolar Proton-Translocating ATPases