cAMP regulates plasma membrane vacuolar-type H+-ATPase assembly and activity in blowfly salivary glands.
Reversible assembly of the V0V1 holoenzyme from V0 and V1 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 V1 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.
Dames
Petra
P
Institut für Biochemie und Biologie, Universität Potsdam, D-14415 Potsdam, Germany.
Zimmermann
Bernhard
B
Schmidt
Ruth
R
Rein
Julia
J
Voss
Martin
M
Schewe
Bettina
B
Walz
Bernd
B
Baumann
Otto
O
D000818:Animals; D002118:Calcium / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004175:Diptera / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D046912:Multiprotein Complexes; D017434:Protein Structure, Tertiary; D011985:Receptors, Serotonin / Q000378:metabolism; D012469:Salivary Glands / Q000187:drug effects / Q000378:metabolism*; D015290:Second Messenger Systems; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*
D046912:Multiprotein Complexes; D011985:Receptors, Serotonin; D005576:Colforsin; D000242:Cyclic AMP; D025262:Vacuolar Proton-Translocating ATPases; D002118:Calcium