Hormone-induced assembly and activation of V-ATPase in blowfly salivary glands is mediated by protein kinase A

Abstract

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.

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

“…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 days p.e. of age becomes positive by several tens of millivolts when cAMP or cAMP analogues are applied Prince and Berridge, 1972;Rein et al, 2008). This electrophysiological response is reversible and reflects a hyperpolarization of the apical membrane by the enhanced activity of the electrogenic V-ATPase Baumann and Walz, 2012).…”

“…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.…”

Protein kinase A dependent and independent activation of the V-ATPase in Malpighian tubules ofAedes aegypti.

“…The cAMP effect is implemented by PKA, and inhibitors of PKA abolish the response of the V-ATPase to stimulation with serotonin even though intracellular cAMP and Ca 2+ levels do increase (Rein et al, 2008;Voss et al, 2010). Congruently, only subtle changes in subunit distribution were observed in Drosophila Malpighian tubules that had been diuretically stimulated with the Capa-1 neuropeptide, known to act via Ca that Ca 2+ regulates mitochondrial ATP-production and thereby indirectly influences V-ATPase activity via the ATP supply (Terhzaz et al, 2006).…”

“…In salivary glands of Calliphora vicina, the reversible assembly and activation of the V-ATPase was also shown to depend on cAMP and the activation of protein kinase A in response to the hormone serotonin (Dames et al, 2006;Rein et al, 2008). The V 1 subunit C is one likely target of the PKA in salivary glands since it was shown to be phosphorylated in a cAMP-dependent manner (Voss et al, 2007).…”