TY - JOUR A1 - Dames, Petra A1 - Zimmermann, Bernhard A1 - Schmidt, Ruth A1 - Rein, Julia A1 - Voss, Martin A1 - Schewe, Bettina A1 - Walz, Bernd A1 - Baumann, Otto T1 - cAMP regulates plasma membrane vacuolar-type H+-ATPase assembly and activity in blowfly salivary glands N2 - Reversible assembly of the V0V1 holoenzyme from V-0 and V-1 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 V, 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 Y1 - 2006 UR - http://www.pnas.org/ U6 - https://doi.org/10.1073/pnas.0600011103 SN - 0027-8424 ER - TY - JOUR A1 - Hille, Carsten A1 - Walz, Bernd T1 - Dopamine-induced graded intracellular Ca2+ elevation via the Na+-Ca2+ exchanger operating in the Ca2+-entry mode in cockroach salivary ducts N2 - Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca2+ concentration ([Ca2+](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca2+ influx from the extracellular space. Additionally, dopamine induces a massive [Na+](i) elevation via the Na+- K+-2Cl(-) cotransporter (NKCC). We have reasoned that Ca2+-entry is mediated by the Na+-Ca2+ exchanger (NCE) operating in the Ca2+-entry mode. To test this hypothesis, [Ca2+](i) and [Na+](i) were measured by using the fluorescent dyes Fura- 2, Fluo-3, and SBFI. Inhibition of Na+-entry from the extracellular space by removal of extracellular Na+ or inhibition of the NKCC by 10 mu M bumetanide did not influence resting [Ca2+]i but completely abolished the dopamine-induced [Ca2+](i) elevation. Simultaneous recordings of [Ca2+](i) and [Na+](i) revealed that the dopamine-induced [Na+](i) elevation preceded the [Ca2+](i) elevation. During dopamine stimulation, the generation of an outward Na+ concentration gradient by removal of extracellular Na+ boosted the [Ca2+](i) elevation. Furthermore, prolonging the dopamine-induced [Na+](i) rise by blocking the Na+/K+-ATPase reduced the recovery from [Ca2+](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na+](i), which reverses the NCE activity into the reverse mode causing a graded [Ca2+](i) elevation in the duct cells. Y1 - 2006 UR - http://www.sciencedirect.com/science/journal/01434160 U6 - https://doi.org/10.1016/j.ceca.2005.11.006 SN - 0143-4160 ER - TY - JOUR A1 - Rein, Julia A1 - Zimmermann, Bernhard A1 - Hille, Carsten A1 - Lang, Ingo A1 - Walz, Bernd A1 - Baumann, Otto T1 - Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina N2 - Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H+- ATPase (V-ATPase) located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N- hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H+ transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H+ transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface Y1 - 2006 UR - http://jeb.biologists.org/ U6 - https://doi.org/10.1242/Jeb.02187 SN - 0022-0949 ER - TY - GEN A1 - Schmälzlin, Elmar A1 - Walz, Bernd A1 - Klimant, Ingo A1 - Schewe, Bettina A1 - Löhmannsröben, Hans-Gerd T1 - Monitoring hormone-induced oxygen consumption in the salivary glands of the blowfly, Calliphora vicina, by use of luminescent microbeads N2 - The salivary glands of the blowfly were injected with luminescent oxygen-sensitive microbeads. The changes in oxygen content within individual gland tubules during hormone-induced secretory activity were quantified. The measurements are based on an upgraded phase-modulation technique, where the phase shift of the sensor phosphorescence is determined independently from concentration and background signals. We show that the combination of a lock-in amplifier with a fluorescence microscope results in a convenient setup to measure oxygen concentrations within living animal tissues at the cellular level. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 006 Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-12206 ER - TY - JOUR A1 - Walz, Bernd A1 - Baumann, Otto A1 - Krach, Christian A1 - Baumann, Arnd A1 - Blenau, Wolfgang T1 - The aminergic control of cockroach salivary glands N2 - The acinar salivary glands of cockroaches receive a dual innervation from the subesophageal ganglion and the stomatogastric nervous system. Acinar cells are surrounded by a plexus of dopaminergic and serotonergic varicose fibers. In addition, seroton-ergic terminals lie deep in the extracellulor spaces between acinar cells. Excitation-secretion coupling in cockroach salivary glands is stimulated by both dopamine and serotonin. These monoamines cause increases in the intracellular concentrations of cAMP and Ca2+. Stimulation of the glands by serotonin results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Thus, two elementary secretary processes, namely electrolyte/water secretion and protein secretion, are triggered by different aminergic transmitters. Because of its simplicity and experimental accessibility, cockroach salivary glands have been used extensively as a model system to study the cellular actions of biogenic amines and to examine the pharmacological properties of biogenic amine receptors. In this review, we summarize current knowledge concerning the aminergic control of cockroach salivary glands and discuss our efforts to characterize Periplaneta biogenic amine receptors molecularly Y1 - 2006 UR - 1960 = Doi 10.1002/Arch.20128 ER -