TY - THES A1 - Hille, Carsten T1 - Charakterisierung von Transportmechanismen in der Speicheldrüse der Schabe Periplaneta americana T1 - Characterisation of transport mechanisms in salivary glands of the cockroach Periplaneta americana N2 - Die Aktivierung der Speichelsekretion erfolgt in der innervierten Speicheldrüse der Schabe Periplaneta americana durch die biogenen Amine Dopamin (DA) und Serotonin (5-HT). Die Acini der Speicheldrüse sezernieren einen Primärspeichel, der in den Ausführgängen modifiziert wird. Die durch DA und 5-HT aktivierten Signalwege sowie die an der Elektrolyt- und Flüssigkeitssekretion bzw. Speichel-modifikation beteiligten Transportmechanismen sind weitgehend unbekannt. Mikrofluorometrische Ca2+-, Na+- und pH-Messungen in Kombination mit pharmakologischen Experimenten, biochemische Messungen der Aktivitäten von Ionentransport-ATPasen sowie videomikroskopische Analysen zu transepithelialen Wasserbewegungen wurden in dieser Arbeit durchgeführt. Sie sollten Informationen über die an der Speichelbildung und -modifikation beteiligten Transportmechanismen und die Signalwege liefern, welche durch DA und/oder 5-HT aktiviert werden. Wesentliche Ergebnisse dieser Arbeit waren: Diese Arbeit trug zur Kenntnis der komplexen Funktionsweise von Speicheldrüsen in Insekten bei und erweiterte das lückenhafte Wissen über die zellulären Wirkungen biogener Amine in Insekten. Zudem wurden in dieser Arbeit viele Parallelen zu Funktionsweisen der Speicheldrüsen in Vertebraten deutlich. N2 - The acinar salivary glands in the cockroach Periplaneta americana are innervated by dopaminergic and serotonergic fibers and secrete a NaCl-rich primary saliva upon stimulation with the biogenic amines dopamine (DA) or serotonin (5-HT). The ducts downstream of the acini are thought to modify the primary saliva by Na+ reabsorption and K+ secretion. The electrolyte and fluid transport processes activated by DA and 5-HT as well as the second messenger pathways mediating between the biogenic amine receptors and the effector transport mechanisms are poorly understood.In this sudy, microfluorometrical Ca2+, Na+ and pH measurements were performed in combination with pharmacological experiments. Furthermore, ATPase activity assays and microscopical analyses of transepithelial fluid transport were done. The aim of this work has been the characterisation of the DA-induced transport mechanisms in the cockroach salivary glands in order to improve our understanding of the cellular actions of biogenic amines in insects. Intracellular pH measurements in duct cells of isolated small lobes of salivary glands consiting of several acini and ducts showed a strong intracellular acidification upon DA or 5-HT stimulation. On the other hand, only a small intracellular acidification could be recognised in isolated ducts without acini. The acini are innervated by dopaminergic and serotonergic fibers, whereas the ducts are innervated only by dopaminergic fibers. Thus, this result demonstrates, that the DA- or 5-HT-induced production of primary saliva in the acini causes the intracellular pH changes in the ducts. Consequently, intracellular pH measurements in ducts are also useful to characterise transport processes in the acini. The Na+-K+-2Cl- cotransport and/or the Cl--HCO3- exchange combined with the Na+ H+ exchange (NHE) were responsible for the NaCl uptake at the basolateral membrane in the peripheral cells of the acini during production of primary saliva. The activity of these transporters was regulated by the CO2/HCO3--availability and was Ca2+-dependent. The activity of the basolateral Na+-K+-ATPase, but not of the apical vacuolar-type proton pump (V-H+-ATPase) in the duct cells was necessary for the strong intracellular acidification in the ducts with acini. Thus, the Na+-K+-ATPase seems to energise the saliva modification in the ducts. In ducts with acini, the V-H+-ATPase and Na+-dependent transporters (e.g. NHE) were responsible for the pH-recovery after a DA- or NH4Cl-induced intracellular acidification in the duct cells. In the regulation of the intracellular resting pH these transporters played a minor role. In addition, DA induced an increase in the intracellular Na+ concentration, followed by an increase in the intracellular Ca2+ concentration in duct cells with acini, but never in duct cells without acini. The Na+ elevation was probably the result of the activity of apical Na+ channels. The DA-induced Na+ elevation and a depolarisation of the basolateral membrane of the duct cells reversed a Na+-Ca2+ exchange activity into the reverse mode causing a graded Ca2+ elevation in duct cells. The Ca2+ elevation is probably involved in the regulation of the Na+ reabsorption during saliva modification. Transepithelial fluid transport in isolated ducts was detected with a fluorescent microscopical method. Already unstimulated isolated ducts reabsorbed fluid from the duct lumen to the bath side. Perhaps unstimulated acini possess a basic secretion rate and this primary saliva is than reabsorbed in the ducts. The fluid reabsorption was ATP-dependent, but the ATP-consuming transport mechanism could not be identified. Neither the basolateral Na+-K+-ATPase, nor the apical V-H+-ATPase were involved in fluid reabsorption. This work extends our knowledge about the complex function of insect salivary glands and about the cellular action of biogenic amines in insects. Additionally, it indicates lots of similarities between the functions of salivary glands in vertebrates and invertebrates. KW - Speicheldrüse KW - Amerikanische Schabe KW - Insekten KW - Speichel KW - epithelialer Transport KW - ratiometric imaging KW - Signalkaskaden KW - biogene Amine KW - Dopamin KW - Serotonin KW - salivary glands KW - epithelial transport KW - biogenic amines KW - dopamine KW - serotonin KW - cockroach KW - insects Y1 - 2006 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-9422 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 - Schulze, Sven A1 - Wehrhold, Michel A1 - Hille, Carsten T1 - Femtosecond-Pulsed laser written and etched fiber bragg gratings for fiber-optical biosensing JF - Sensors N2 - We present the development of a label-free, highly sensitive fiber-optical biosensor for online detection and quantification of biomolecules. Here, the advantages of etched fiber Bragg gratings (eFBG) were used, since they induce a narrowband Bragg wavelength peak in the reflection operation mode. The gratings were fabricated point-by-point via a nonlinear absorption process of a highly focused femtosecond-pulsed laser, without the need of prior coating removal or specific fiber doping. The sensitivity of the Bragg wavelength peak to the surrounding refractive index (SRI), as needed for biochemical sensing, was realized by fiber cladding removal using hydrofluoric acid etching. For evaluation of biosensing capabilities, eFBG fibers were biofunctionalized with a single-stranded DNA aptamer specific for binding the C-reactive protein (CRP). Thus, the CRP-sensitive eFBG fiber-optical biosensor showed a very low limit of detection of 0.82 pg/L, with a dynamic range of CRP detection from approximately 0.8 pg/L to 1.2 mu g/L. The biosensor showed a high specificity to CRP even in the presence of interfering substances. These results suggest that the proposed biosensor is capable for quantification of CRP from trace amounts of clinical samples. In addition, the adaption of this eFBG fiber-optical biosensor for detection of other relevant analytes can be easily realized. KW - fiber Bragg gratings KW - ultra-fast laser inscription KW - fiber etching KW - nanostructure fabrication KW - fiber-optical sensors KW - aptamers KW - C-reactive protein KW - biomarker Y1 - 2018 U6 - https://doi.org/10.3390/s18092844 SN - 1424-8220 VL - 18 IS - 9 PB - MDPI CY - Basel ER - TY - GEN A1 - Schulze, Sven A1 - Wehrhold, Michel A1 - Hille, Carsten T1 - Femtosecond-pulsed laser written and etched fiber bragg gratings for fiber-optical biosensing T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - We present the development of a label-free, highly sensitive fiber-optical biosensor for online detection and quantification of biomolecules. Here, the advantages of etched fiber Bragg gratings (eFBG) were used, since they induce a narrowband Bragg wavelength peak in the reflection operation mode. The gratings were fabricated point-by-point via a nonlinear absorption process of a highly focused femtosecond-pulsed laser, without the need of prior coating removal or specific fiber doping. The sensitivity of the Bragg wavelength peak to the surrounding refractive index (SRI), as needed for biochemical sensing, was realized by fiber cladding removal using hydrofluoric acid etching. For evaluation of biosensing capabilities, eFBG fibers were biofunctionalized with a single-stranded DNA aptamer specific for binding the C-reactive protein (CRP). Thus, the CRP-sensitive eFBG fiber-optical biosensor showed a very low limit of detection of 0.82 pg/L, with a dynamic range of CRP detection from approximately 0.8 pg/L to 1.2 µg/L. The biosensor showed a high specificity to CRP even in the presence of interfering substances. These results suggest that the proposed biosensor is capable for quantification of CRP from trace amounts of clinical samples. In addition, the adaption of this eFBG fiber-optical biosensor for detection of other relevant analytes can be easily realized. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1073 KW - fiber Bragg gratings KW - ultra-fast laser inscription KW - fiber etching KW - nanostructure fabrication KW - fiber-optical sensors KW - aptamers KW - C-reactive Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-472692 SN - 1866-8372 IS - 1073 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 - JOUR A1 - Techen, Anne A1 - Hille, Carsten A1 - Dosche, Carsten A1 - Kumke, Michael Uwe T1 - Fluorescence study of drug-carrier interactions in CTAB/PBS buffer model systems JF - Journal of colloid and interface science N2 - The well-known cationic surfactant hexadecyltrimethylammonium bromide (CTAB) was used as a model carrier to study drug-carrier interactions with fluorescence probes (5-hexadecanoylaminofluorescein (HAF) and 2,10-bis-(3-aminopropyloxy)dibenzo[aj]perylene-8,16-dione (NIR 628) by applying ensemble as well as single molecule fluorescence techniques. The impact of the probes on the micelle parameters (critical micelle concentration, average aggregation number, hydrodynamic radius) was investigated under physiological conditions. In the presence of additional electrolytes, such as buffer, the critical micelle concentration decreased by a factor of about 10. In contrast, no influence of the probes on the critical micelle concentration and on average aggregation number was observed. The results show that HAF does not affect the characteristics of CTAB micelles. Analyzing fluorescence correlation spectroscopy data and time-resolved anisotropy decays in terms of the "two-step" in combination with the "wobbling-in-cone" model, it was proven that HAF and NIR 628 are differently associated with the micelles. Based on ensemble and single molecule fluorescence experiments, intra- and intermicellar energy transfer process between the two dyes were probed and characterized. KW - Hexadecyltrimethylammonium bromide KW - 5-Hexadecanoylaminofluorescein KW - 2,10-Bis-(3-aminopropyloxy)dibenzo[aj]perylene-8,16-dione KW - Fluorescence correlation spectroscopy KW - Fluorescence anisotropy KW - Single-molecule FRET Y1 - 2012 U6 - https://doi.org/10.1016/j.jcis.2012.03.063 SN - 0021-9797 VL - 377 SP - 251 EP - 261 PB - Elsevier CY - San Diego ER - TY - JOUR A1 - Roder, Phillip A1 - Hille, Carsten T1 - Local tissue manipulation via a force- and pressure-controlled AFM micropipette for analysis of cellular processes JF - Scientific reports N2 - Local manipulation of complex tissues at the single-cell level is challenging and requires excellent sealing between the specimen and the micromanipulation device. Here, biological applications for a recently developed loading technique for a force-and pressure-controlled fluidic force microscope micropipette are described. This technique allows for the exact positioning and precise spatiotemporal control of liquid delivery. The feasibility of a local loading technique for tissue applications was investigated using two fluorescent dyes, with which local loading behaviour could be optically visualised. Thus, homogeneous intracellular distribution of CellTracker Red and accumulation of SYTO 9 Green within nuclei was realised in single cells of a tissue preparation. Subsequently, physiological micromanipulation experiments were performed. Salivary gland tissue was pre-incubated with the Ca2+-sensitive dye OGB-1. An intracellular Ca2+ rise was then initiated at the single-cell level by applying dopamine via micropipette. When pre-incubating tissue with the nitric oxide (NO)-sensitive dye DAF-FM, NO release and intercellular NO diffusion was observed after local application of the NO donor SNP. Finally, local micromanipulation of a well-defined area along irregularly shaped cell surfaces of complex biosystems was shown for the first time for the fluidic force microscope micropipette. Thus, this technique is a promising tool for the investigation of the spatiotemporal effects of locally applied substances in complex tissues. Y1 - 2018 U6 - https://doi.org/10.1038/s41598-018-24255-9 SN - 2045-2322 VL - 8 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Jahn, Karolina A1 - Buschmann, Volker A1 - Hille, Carsten T1 - Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells JF - Scientific reports N2 - In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the mu s-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. Y1 - 2015 U6 - https://doi.org/10.1038/srep14334 SN - 2045-2322 VL - 5 PB - Nature Publ. Group CY - London ER - TY - JOUR A1 - Jahn, Karolina A1 - Buschmann, Volker A1 - Hille, Carsten T1 - Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells JF - Scientific Reports N2 - In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. KW - Confocal microscopy KW - Fluorescence imaging KW - Fluorescence spectroscopy KW - Fluorescent probes Y1 - 2015 U6 - https://doi.org/10.1038/srep14334 SN - 2045-2322 IS - 5 PB - Nature Publishing Group CY - London ER - TY - GEN A1 - Jahn, Karolina A1 - Buschmann, Volker A1 - Hille, Carsten T1 - Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells N2 - In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 202 KW - Confocal microscopy KW - Fluorescence imaging KW - Fluorescence spectroscopy KW - Fluorescent probes Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-82156 ER -