cAMP regulates plasma membrane vacuolar-type H ⁺ -ATPase assembly and activity in blowfly salivary glands

Abstract

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 VATPase-dependent luminal acidification. In addition, V-ATPasedependent 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 Ca 2؉ , our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus.regulation ͉ translocation ͉ secretion T he vacuolar-type H ϩ -ATPases (V-ATPases) are multisubunit heteromeric complexes that are organized into two domains, designated V 0 and V 1 (1-4). V 0 forms a membranespanning proton-translocating complex; in yeast, it is composed of at least five different subunits termed a, c, cЈ, cЉ, and d, of which subunit c binds bafilomycin A 1 , a specific inhibitor of V-ATPases (5-8). The V 1 sector is attached to the cytoplasmic side of the V 0 sector, consists of at least eight different subunits termed A-H, and contains catalytic and noncatalytic ATPbinding sites. V-ATPase is vital for almost every eukaryotic cell and fulfils a variety of functions. On intracellular acidic membrane systems, such as endosomes, lysosomes, and synaptic vesicles, these proton pumps are involved in protein sorting during biosynthetic and endocytotic pathways, zymogen activation, and transmitter uptake, respectively (3, 4). V-ATPase molecules in the plasma membrane of animal cells, especially on the apical plasma membrane of epithelial cells, contribute to intracellular pH homeostasis, extracellular acidification, or alkalinization, or they energize the plasma membrane for secondary transport processes (3, 4).In some cells, V-ATPase requires a considerable amount of energy. For reasons of economy, it is thus favorable if V-ATPase activity is adapted to the physiological needs of the cell. Several regulatory mechanisms have been identified (1,2,4,9). One of these is the reversible dissociation of the V 1 sector from the V 0 sector, as revealed by experiments performed in yeast, midgut epithelial cells of the tobacco hornworm Manduca sexta, mammalian dendritic cells, and renal epithelial cells (10-16). In th...

Development of apical membrane organization and V-ATPase regulation in blowfly salivary glands

“…Cross-reactivity of these antibodies with the homologous proteins in C. vicina has been demonstrated previously (Zimmermann et al, 2003;Dames et al, 2006;Voss et al, 2007;Voss et al, 2009;Baumann and Walz, 2012). Monoclonal anti-actin (clone C4) was obtained from Merck (Darmstadt, Germany), rabbit antiserum (cat.…”

“…The canalicular system of the apical membrane was labelled at a similar intensity as the cytoplasm (supplementary material Fig.S1). V 0 component subunit d and V 1 components subunits B and D thus have a different distribution pattern under these conditions, suggesting that most V-ATPase molecules are disassembled into the inactive V 1 and V 0 complexes (Dames et al, 2006). Upon exposure to 8-CPT-cAMP, both V 1 subunits became enriched on the apical membrane in the salivary glands of freshly eclosed flies and in older imagines.…”

“…Subsequently, glands were chemically fixed, cryosectioned and immunostained as described in detail previously (Dames et al, 2006;Rotte et al, 2008). Fluorescence images were recorded with a Zeiss LSM 510 or a Zeiss LSM 710 confocal laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany).…”

“…The neurohormone 5-HT induces, via the cAMP/PKA signalling pathway, a reversible assembly of functional V 1 V 0 holoenzymes on the apical membrane of the secretory cells and thus boosts V-ATPase activity and H + transport in adult salivary glands (Dames et al, 2006;Rein et al, 2008;Baumann and Walz, 2012). To determine whether developmental differences exist in the ability of secretory cells to assemble V 1 V 0 holoenzymes in a cAMP-dependent mode, we first determined the developmental expression pattern of the catalytic (PKA-C) and regulatory (PKA-RII) subunit of PKA.…”

“…5-HT thus evokes a parallel rise in both [cAMP] and [Ca 2+ ] within the secretory cells (Berridge, 1980a;Heslop and Berridge, 1980;Zimmermann and Walz, 1997). The 5-HT-induced increase in intracellular [cAMP] elicits, via PKA, the assembly and activation of vacuolar-type proton pumps (V-ATPase) in the apical membrane (Dames et al, 2006;Rein et al, 2006;Rein et al, 2008). This ion pump is largely disassembled into a membrane-integral V 0 domain and a cytosolic V 1 domain, and thus is non-functional in unstimulated secretory cells.…”