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Charakterisierung von Transportmechanismen in der Speicheldrüse der Schabe Periplaneta americana
(2006)
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 Ca<sup>2+-, Na<sup>+- 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: <ul> <li>Messungen des intrazellulären pH (pHi) in Gangzellen zeigten, dass isolierte Ausführgänge mit Acini bei Stimulierung mit DA und 5-HT stark ansäuerten. In isolierten Ausführgängen ohne Acini verursachte nur DA eine schwache Ansäuerung. Da nur die Ausführgänge dopaminerg innerviert sind, die Acini jedoch dopaminerg und serotonerg, zeigt dieses Ergebnis, dass die DA- und/oder 5-HT-induzierte Primärspeichelbildung die Ursache für die pHi-Änderungen in den Gangzellen ist. pHi-Messungen in den Gangzellen geben also auch Hinweise auf Transportvorgänge in den Acini.</li> <li> Der Na<sup>+-K<sup>+-2Cl<sup>--Symporter und der Cl<sup>--HCO3<sup>--Antiporter, gekoppelt mit dem Na<sup>+ H<sup>+-Antiporter (NHE) waren an der NaCl-Aufnahme in die peripheren Zellen der Acini zur Bildung des NaCl-reichen Primärspeichels beteiligt. Die Aktivität dieser Transporter hing von der CO2/HCO3<sup>--Verfügbarkeit ab und war Ca<sup>2+-abhängig.</li> <li>Die starke Ansäuerung in den Gangzellen hing nicht von der Aktivität der apikalen vakuolären Protonen-ATPase (V-H<sup>+-ATPase), aber von der Aktivität der basolateralen Na<sup>+-K<sup>+-ATPase ab, die anscheinend in den Ausführgängen die Speichelmodifikation energetisiert.</li> <li>In isolierten Ausführgängen mit Acini waren die V-H<sup>+-ATPase und Na<sup>+-abhängige Transporter (u. a. NHE) an der Erholung von einer DA-induzierten oder einer NH4Cl-Vorpuls-induzierten Ansäuerung in den Gangzellen beteiligt. Bei der Regulation des pHi in unstimulierten Gangzellen spielten diese Transporter keine Rolle.</li> <li>In isolierten Ausführgängen mit Acini induzierte DA in den Gangzellen einen Anstieg der [Na<sup>+]i und, zeitlich verzögert, auch der [Ca<sup>2+]i. Der [Na<sup>+]i-Anstieg war von der Aktivität der Acini abhängig und erfolgte möglicherweise über apikale Na+-Kanäle. Der [Ca<sup>2+]i-Anstieg war graduiert und tonisch. Der DA-induzierte [Na<sup>+]i-Anstieg in den Gangzellen und deren Depolarisation führten dazu, dass der basolaterale Na<sup>+-Ca<sup>2+-Antiporter in den Ca<sup>2+-Influx-Modus umkehrte. Die daraus resultierende tonische [Ca<sup>2+]i-Erhöhung könnte an der Regulation der Na<sup>+-Rückresorption beteiligt sein.</li> <li>Zum Nachweis transepithelialer Flüssigkeitsbewegungen in isolierten Ausführgängen wurde eine videomikroskopische Methode entwickelt. Isolierte Ausführgänge ohne Acini resorbierten im unstimulierten Zustand Flüssigkeit aus dem Ausführganglumen. Möglicherweise sezernieren die Acini auch im unstimulierten Zustand mit geringerer Rate einen Primärspeichel, der in den Ausführgängen resorbiert wird. Die Resorption war ATP-abhängig. Der ATP-verbrauchende Transportmechanismus konnte nicht identifiziert werden. Weder die Na<sup>+-K<sup>+-ATPase noch die V-H<sup>+-ATPase waren an der Resorption beteiligt.</li> </ul> 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.
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.
Femtosecond-Pulsed laser written and etched fiber bragg gratings for fiber-optical biosensing
(2018)
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.
Femtosecond-pulsed laser written and etched fiber bragg gratings for fiber-optical biosensing
(2018)
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.
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
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.
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.
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.
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.
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.