@article{HeindorffBaumann2014, author = {Heindorff, Kristoffer and Baumann, Otto}, title = {Calcineurin is part of a negative feedback loop in the InsP(3)/Ca2+ signalling pathway in blowfly salivary glands}, series = {Cell calcium}, volume = {56}, journal = {Cell calcium}, number = {3}, publisher = {Churchill Livingstone}, address = {Edinburgh}, issn = {0143-4160}, doi = {10.1016/j.ceca.2014.07.009}, pages = {215 -- 224}, year = {2014}, abstract = {The ubiquitous InsP(3)/Ca2+ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca2+ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca2+] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP(3)-induced Ca2+ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP(3)/Ca2+ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca2+ signals; (2) cyclosporin A and FK506, inhibitors of Ca2+/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca2+ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca2+ responses does not involve Ca2+ entry into the cells; (4) cyclosporin A increases InsP(3)-dependent Ca2+ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca2+ responses, indicating that PKA and calcineurin act antagonistically on the InsP(3)/Ca2+ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP(3)/Ca2+ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations. (C) 2014 Elsevier Ltd. All rights reserved.}, language = {en} } @article{GerhardtWalzBeta2014, author = {Gerhardt, Matthias and Walz, Michael and Beta, Carsten}, title = {Signaling in chemotactic amoebae remains spatially confined to stimulated membrane regions}, series = {Journal of cell science}, volume = {127}, journal = {Journal of cell science}, number = {23}, publisher = {Company of Biologists Limited}, address = {Cambridge}, issn = {0021-9533}, doi = {10.1242/jcs.161133}, pages = {5115 -- 5125}, year = {2014}, abstract = {Recent work has demonstrated that the receptor-mediated signaling system in chemotactic amoeboid cells shows typical properties of an excitable system. Here, we delivered spatially confined stimuli of the chemoattractant cAMP to the membrane of differentiated Dictyostelium discoideum cells to investigate whether localized receptor stimuli can induce the spreading of excitable waves in the G-protein-dependent signal transduction system. By imaging the spatiotemporal dynamics of fluorescent markers for phosphatidylinositol (3,4,5)-trisphosphate (PIP3), PTEN and filamentous actin, we observed that the activity of the signaling pathway remained spatially confined to the stimulated membrane region. Neighboring parts of the membrane were not excited and no receptor-initiated spatial spreading of excitation waves was observed. To generate localized cAMP stimuli, either particles that carried covalently bound cAMP molecules on their surface were brought into contact with the cell or a patch of the cell membrane was aspirated into a glass micropipette to shield this patch against freely diffusing cAMP molecules in the surrounding medium. Additionally, the binding site of the cAMP receptor was probed with different surface-immobilized cAMP molecules, confirming results from earlier ligand-binding studies.}, language = {en} } @misc{BaumannWalz2012, author = {Baumann, Otto and Walz, Bernd}, title = {The blowfly salivary gland - A model system for analyzing the regulation of plasma membrane V-ATPase}, series = {Journal of insect physiology}, volume = {58}, journal = {Journal of insect physiology}, number = {4}, publisher = {Elsevier}, address = {Oxford}, issn = {0022-1910}, doi = {10.1016/j.jinsphys.2011.11.015}, pages = {450 -- 458}, year = {2012}, abstract = {Vacuolar H+-ATPases (V-ATPases) are heteromultimeric proteins that use the energy of ATP hydrolysis for the electrogenic transport of protons across membranes. They are common to all eukaryotic cells and are located in the plasma membrane or in membranes of acid organelles. In many insect epithelia, V-ATPase molecules reside in large numbers in the apical plasma membrane and create an electrochemical proton gradient that is used for the acidification or alkalinization of the extracellular space, the secretion or reabsorption of ions and fluids, the import of nutrients, and diverse other cellular activities. Here, we summarize our results on the functions and regulation of V-ATPase in the tubular salivary gland of the blowfly Calliphora vicina. In this gland, V-ATPase activity energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). Because of particular morphological and physiological features, the blowfly salivary glands are a superior and exemplary system for the analysis of the intracellular signaling pathways and mechanisms that modulate V-ATPase activity and solute transport in an insect epithelium.}, language = {en} } @article{HeindorffBlenauWalzetal.2012, author = {Heindorff, Kristoffer and Blenau, Wolfgang and Walz, Bernd and Baumann, Otto}, title = {Characterization of a Ca2+/calmodulin-dependent AC1 adenylyl cyclase in a non-neuronal tissue, the blowfly salivary gland}, series = {Cell calcium}, volume = {52}, journal = {Cell calcium}, number = {2}, publisher = {Churchill Livingstone}, address = {Edinburgh}, issn = {0143-4160}, doi = {10.1016/j.ceca.2012.04.016}, pages = {103 -- 112}, year = {2012}, abstract = {Crosstalk between intracellular signalling pathways is a functionally important and widespread phenomenon in cell physiology across phyla. In the salivary gland of the blowfly, serotonin induces fluid secretion via parallel activation of both the InsP(3)/Ca2+ and the cAMP/PKA signalling pathways, which interact on multiple levels. We have determined the molecular identity of a link between both pathways that mediates a Ca2+-dependent rise of intracellular cAMP. Whereas hydrolysis of cAMP via phosphodiesterases is largely independent of Ca2+, cAMP synthesis by adenylyl cyclases (AC) is potentiated in a Ca2+/calmodulin (Ca2+/CaM)-dependent manner. The existence of a Ca2+/CaM-dependent AC is supported by physiological data and a molecular approach. We have cloned Cv rutabaga cDNA, encoding the first blowfly AC, and confirmed its expression in the salivary gland via reverse transcription followed by polymerase chain reaction. The putative gene product of Cv rutabaga is a Ca2+/CaM-dependent type I AC and shows highest homology to Rutabaga from Drosophila. Thus, a Ca2+/CaM-dependent AC serves as a link between the InsP(3)/Ca2+ and the cAMP/PKA signalling pathways in the salivary gland of the blowfly and might be important for the amplification and optimization of the secretory response.}, language = {en} } @article{FechnerBaumannWalz2013, author = {Fechner, Lennart and Baumann, Otto and Walz, Bernd}, title = {Activation of the cyclic AMP pathway promotes serotonin-induced Ca2+ oscillations in salivary glands of the blowfly Calliphora vicina}, series = {Cell calcium}, volume = {53}, journal = {Cell calcium}, number = {2}, publisher = {Churchill Livingstone}, address = {Edinburgh}, issn = {0143-4160}, doi = {10.1016/j.ceca.2012.10.004}, pages = {94 -- 101}, year = {2013}, abstract = {Ca2+ and cAMP signalling pathways interact in a complex manner at multiple sites. This crosstalk fine-tunes the spatiotemporal patterns of Ca2+ and cAMP signals. In salivary glands of the blowfly Calliphora vicina fluid secretion is stimulated by serotonin (5-hydroxytryptamine, 5-HT) via activation of two different 5-HT receptors coupled to the InsP(3)/Ca2+ (Cv5-HT2 alpha) or the cAMP pathway (Cv5-HT7), respectively. We have shown recently in permeabilized gland cells that cAMP sensitizes InsP(3)-induced Ca2+ release to InsP(3). Here we study the effects of the CAMP signalling pathway on 5-HT-induced oscillations in transepithelial potential (TEP) and in intracellular [Ca2+]. We show: (1) Blocking the activation of the cAMP pathway by cinanserin suppresses the generation of TEP and Ca2+ oscillations, (2) application of 8-CPT-cAMP in the presence of cinanserin restores 5-HT-induced TEP and Ca2+ oscillations, (3) 8-CPT-cAMP sensitizes the InsP(3)/Ca2+ signalling pathway to 5-HT and the Cv5-HT2 alpha, receptor agonist 5-MeOT, (4) 8-CPT-cAMP induces Ca2+ oscillations in cells loaded with subthreshold concentrations of InsP(3), (5) inhibition of protein kinase A by H-89 abolishes 5-HT-induced TEP and Ca2+ spiking and mimics the effect of cinanserin. These results suggest that activation of the cyclic AMP pathway promotes the generation of 5-HT-induced Ca2+ oscillations in blowfly salivary glands.}, language = {en} } @article{BaumannBauer2013, author = {Baumann, Otto and Bauer, Alexandra}, title = {Development of apical membrane organization and V-ATPase regulation in blowfly salivary glands}, series = {The journal of experimental biology}, volume = {216}, journal = {The journal of experimental biology}, number = {7}, publisher = {Company of Biologists Limited}, address = {Cambridge}, issn = {0022-0949}, doi = {10.1242/jeb.077420}, pages = {1225 -- 1234}, year = {2013}, abstract = {Secretory cells in blowfly salivary gland are specialized via morphological and physiological attributes in order to serve their main function, i.e. the transport of solutes at a high rate in response to a hormonal stimulus, namely serotonin (5-HT). This study examines the way that 5-HT-insensitive precursor cells differentiate into morphologically complex 5-HT-responsive secretory cells. By means of immunofluorescence microscopy, immunoblotting and measurements of the transepithelial potential changes, we show the following. (1) The apical membrane of the secretory cells becomes organized into an elaborate system of canaliculi and is folded into pleats during the last pupal day and the first day of adulthood. (2) The structural reorganization of the apical membrane is accompanied by an enrichment of actin filaments and phosphorylated ERM protein (phospho-moesin) at this membrane domain and by the deployment of the membrane-integral part of vacuolar-type H+-ATPase (V-ATPase). These findings suggest a role for phospho-moesin, a linker between actin filaments and membrane components, in apical membrane morphogenesis. (3) The assembly and activation of V-ATPase can be induced immediately after eclosion by way of 8-CPT-cAMP, a membrane-permeant cAMP analogue. (4) 5-HT, however, produces the assembly and activation of V-ATPase only in flies aged for at least 2 h after eclosion, indicating that, at eclosion, the 5-HT receptor/adenylyl cyclase/cAMP signalling pathway is inoperative upstream of cAMP. (5) 5-HT activates both the Ca2+ signalling pathway and the cAMP signalling cascade in fully differentiated secretory cells. However, the functionality of these signalling cascades does not seem to be established in a tightly coordinated manner during cell differentation.}, language = {en} } @misc{BlenauMustardHamiltonetal.2003, author = {Blenau, Wolfgang and Mustard, Julie A. and Hamilton, Ingrid S. and Ward, Vernon K. and Ebert, Paul R. and Mercer, Alison R.}, title = {Analysis of two D1-like dopamine receptors from the honey bee Apis mellifera reveals agonist-independent activity}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-44378}, year = {2003}, abstract = {Dopamine is found in many invertebrate organisms, including insects, however, the mechanisms through which this amine operates remain unclear. We have expressed two dopamine receptors cloned from honey bee (AmDOP1 and AmDOP2) in insect cells (Spodoptera frugiperda), and compared their pharmacology directly using production of cAMP as a functional assay. In each assay, AmDOP1 receptors required lower concentrations of dopamine and 6,7-ADTN for maximal activation than AmDOP2 receptors. Conversely, butaclamol and cis(Z)-flupentixol were more potent at blocking the cAMP response mediated through AmDOP2 than AmDOP1 receptors. Expression of AmDOP1, but not AmDOP2, receptors significantly increased levels of cAMP even in the absence of ligand. This constitutive activity was blocked by cis(Z)-flupentixol. This work provides the first evidence of a constitutively activated dopamine receptor in invertebrates and suggests that although AmDOP1 and AmDOP2 share much less homology than their vertebrate counterparts, they display a number of functional parallels with the mammalian D1-like dopamine receptors.}, language = {en} } @phdthesis{Schmidt2006, author = {Schmidt, Ruth Maria}, title = {Signalkaskaden und Steuermechanismen in den Speicheldr{\"u}sen von Dipteren}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-7714}, school = {Universit{\"a}t Potsdam}, year = {2006}, abstract = {Fl{\"u}ssigkeitssekretion und Proteinsekretion werden in Speicheldr{\"u}sen von Insekten {\"u}ber Hormone und Neurotransmitter gesteuert. Diese entfalten ihre physiologische Wirkung in den sekretorischen Dr{\"u}senzellen haupts{\"a}chlich {\"u}ber den zyklischen Adenosinmonophosphat (cAMP)-Signalweg und den Inositoltrisphosphat (IP3) / Ca2+-Signalweg. Die Mechanismen m{\"o}glicher Wechselwirkungen zwischen diesen Signalwegen und ihre physiologischen Auswirkungen sind unzureichend bekannt. Im Mittelpunkt dieser Arbeit stand die Frage, ob und wie sich der Ca2+-Signalweg und der cAMP-Signalweg in der Speicheldr{\"u}se der Diptere Calliphora vicina beeinflussen. Substanzen wie 5-Fluoro-α-Methyltryptamin und Histamin wurden in fr{\"u}heren Arbei-ten als Agonisten genutzt, um in den Speicheldr{\"u}sen von C. vicina selektiv den cAMP-Signalweg (getrennt vom IP3/Ca2+-Signalweg) zu aktivieren. Es zeigte sich in transepithelialen Potentialmessungen und mikrofluorometrischen Ca2+-Untersuchungen, dass beide Substanzen sowohl den cAMP-Weg als auch den Ca2+-Signalweg aktivierten. Die physiologischen Ursachen der Histamin-induzierten Ca2+-Erh{\"o}hung wurden genauer untersucht. Zusammengefasst zeigten diese Untersuchungen, dass Histamin wie 5-HT den cAMP-Weg und die Phosphoinositidkaskade aktivierte. Im Gegensatz zu den 5-HT-induzierten Ca2+-Oszillationen, welche durch interzellul{\"a}re Ca2+-Wellen synchronisiert werden, verursachte Histamin bei niedrigen Konzentrationen lokale Ca2+-Oszillationen in einzelnen Zellen (keine Wellen). Bei h{\"o}heren Histamin-Konzentrationen war eine anhaltende Ca2+-Erh{\"o}hung oder ein synchrones Ca2+-beating in der gesamten Dr{\"u}se zu beobachten. Des Weiteren wurde die Frage untersucht, ob eine Erh{\"o}hung der intrazellul{\"a}ren cAMP-Konzentration den IP3 Ca2+-Signalweg in den Epithelzellen der Speicheldr{\"u}se beeinflussen kann. Es zeigte sich, dass cAMP den durch schwellennahe 5-HT-Konzentrationen induzierten Ca2+-Anstieg verst{\"a}rkte. Diese Verst{\"a}rkung wurde durch eine PKA-vermittelte Sensitivierung des IP3-Rezeptor/Ca2+-Kanals f{\"u}r IP3 verursacht. Immunzytochemische Untersuchungen deuten dar-auf hin, dass die Proteinkinase A eng mit dem IP3-Rezeptor/Ca2+-Kanal assoziiert ist. Diese Messungen zeigen erstmals, dass auch bei Invertebraten der Botenstoff cAMP, PKA-vermittelt, den IP3-Rezeptor/Ca2+-Kanal des ER f{\"u}r IP3 sensitiviert.}, subject = {Speichel}, language = {de} }