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Keywords
- Vacuolar h+-atpase (1)
- cAMP analog (1)
- camp binding-sites (1)
- cyclic-amp (1)
- drosophila-melanogaster (1)
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- salivary gland (1)
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.
Background
Serotonin induces fluid secretion from Calliphora salivary glands by the parallel activation of the InsP3/Ca2+ and cAMP signaling pathways. We investigated whether cAMP affects 5-HT-induced Ca2+ signaling and InsP3-induced Ca2+ release from the endoplasmic reticulum (ER).
Results
Increasing intracellular cAMP level by bath application of forskolin, IBMX or cAMP in the continuous presence of threshold 5-HT concentrations converted oscillatory [Ca2+]i changes into a sustained increase. Intraluminal Ca2+ measurements in the ER of ß-escin-permeabilized glands with mag-fura-2 revealed that cAMP augmented InsP3-induced Ca2+ release in a concentration-dependent manner. This indicated that cAMP sensitized the InsP3 receptor Ca2+ channel for InsP3. By using cAMP analogs that activated either protein kinase A (PKA) or Epac and the application of PKA-inhibitors, we found that cAMP-induced augmentation of InsP3-induced Ca2+ release was mediated by PKA not by Epac. Recordings of the transepithelial potential of the glands suggested that cAMP sensitized the InsP3/Ca2+ signaling pathway for 5-HT, because IBMX potentiated Ca2+-dependent Cl- transport activated by a threshold 5-HT concentration.
Conclusion
This report shows, for the first time for an insect system, that cAMP can potentiate InsP3-induced Ca2+ release from the ER in a PKA-dependent manner, and that this crosstalk between cAMP and InsP3/Ca2+ signaling pathways enhances transepithelial electrolyte transport.