Wong, Miu Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 140-152). / Abstracts in English and Chinese. / DECLARATION --- p.i / ACKNOWLEDGEMENT --- p.ii / ABBREVIATIONS --- p.iii / ABSTRACT IN ENGLISH --- p.iv / ABSTRACT IN CHINESE --- p.vii / TABLE OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xviii / Chapter CHAPTER I - --- INTRODUCTION / Chapter 1.1 --- Regulation of human airway surface liquid --- p.1 / Chapter 1.2 --- Sodium reabsorption and chloride secretion in airway epithelium --- p.3 / Chapter 1.3 --- Purinergic receptors --- p.7 / Chapter 1.4 --- P2Y receptors in epithelial cells --- p.11 / Chapter 1.5 --- Autocrine or paracrine regulation of ion transport in epithelial cells --- p.13 / Chapter 1.6 --- Signaling pathways underlying the regulation of ion transport by P2Y receptors stimulation --- p.16 / Chapter 1.7 --- The therapeutic potential of P2Y receptors in treating cystic fibrosis --- p.18 / Chapter 1.8 --- Particular interest on P2Y6 receptor as potential target for treatment of cystic fibrosis --- p.21 / Chapter 1.9 --- Properties of 16HBE14o- cell line --- p.23 / Chapter 1.10 --- Objectives of the present experiments --- p.25 / Chapter CHAPTER II - --- MATERIALS AND METHODS / Chapter 2.1 --- Solutions and Chemicals --- p.26 / Chapter 2.2 --- Cell culture --- p.28 / Chapter 2.3 --- Simultaneous measurement of short-circuit current (Isc) and intracellular calcium concentration ([Ca2+ ])i --- p.29 / Chapter 2.3.1 --- Preparation of 16HBE14o- cells for simultaneous measurement of Isc and [Ca2+]i --- p.29 / Chapter 2.3.2 --- Measurement of Isc and transepithelial resistance with Ussing chamber --- p.32 / Chapter 2.3.3 --- Simultaneous measurement of Isc and [Ca2+]i --- p.35 / Chapter 2.4 --- Measurement of protein kinase A activity --- p.38 / Chapter 2.5 --- Data analysis --- p.39 / Chapter CHAPTER III - --- RESULTS / Chapter 3.1 --- Apical and basolateral application of P2Y agonists induced Isc and [Ca2+］i responses in 16HBE14o- cells --- p.40 / Chapter 3.1.1 --- Effect of apical and basolateral application of ATP on Isc and [Ca2+̐]ư --- p.40 / Chapter 3.1.2 --- Effect of apical and basolateral application of UTP on Isc and [Ca2+̐]ư --- p.45 / Chapter 3.1.3 --- Effect of apical and basolateral application of UDP on Isc and [Ca2+̐]ư --- p.50 / Chapter 3.1.4 --- "Summary of the effects of apical and basolateral application of ATP, UTP and UDP on Isc and [Ca2+̐]ư" --- p.55 / Chapter 3.2 --- Ionic mechanisms underlying the effect of apical and basolateral UDP on 16HBE14o- cells --- p.56 / Chapter 3.2.1 --- Differential effect of apical and basolateral UDP on Isc --- p.56 / Chapter 3.2.2 --- Effect of various apical CI－ channel blockers on Isc response induced by apical and basolateral application of UDP --- p.59 / Chapter 3.2.3 --- Effect of various basolateral K+ channel blockers on Isc response induced by apical and basolateral application of UDP --- p.83 / Chapter 3.3 --- Involvement of other signaling molecules or pathways in regulation of the chloride secreting response evoked by apical and basolateral UDP --- p.108 / Chapter 3.3.1 --- Effect of apical and basolateral UDP on PKA activity --- p.109 / Chapter 3.3.2 --- Effect of PKC inhibitors on Isc response induced by apical and basolateral application of UDP --- p.111 / Chapter CHAPTER IV - --- DISCUSSION / Chapter 4.1 --- Simultaneous measurement of Isc and [Ca2+ ̐]ư upon apical and basolateral application of P2Y agonists in 16HBE14o- cells --- p.125 / Chapter 4.2 --- Ionic mechanism underlying the effect of apical and basolateral UDP on 16HBE14o- cells --- p.128 / Chapter 4.2.1 --- Possible ionic mechanism for chloride secretion mediated by apical P2Y6 receptors --- p.131 / Chapter 4.2.2 --- Possible ionic mechanism for chloride secretion mediated by basolateral P2Y6 receptors --- p.133 / Chapter 4.3 --- Involvement of other possible signaling molecules or pathway underlying the action of apical and basolateral UDP --- p.135 / Chapter 4.4 --- Summary --- p.138 / Chapter CHAPTER V - --- REFERENCES --- p.140 / Publications --- p.153

Les protéines synthétisées et sécrétées par les bactéries jouent des rôles importants pour leur survie. Les bactéries à Gram négatif ont développé des voies de sécrétion en tant qu'armes principales pour transporter des facteurs de virulence dans l'environnement extracellulaire ou dans des cellules hôte. L'un de ces systèmes, le T9SS a été principalement étudié chez l'agent pathogène oral Porphyromonas gingivalis et chez la bactérie mobile Flavobacterium johnsoniae. Un autre complexe, le T2SS est le principal déterminant de la virulence de la bactérie Pseudomonas aeruginosa, un agent pathogène de la fibrose kystique. Dans le cadre de ma thèse, j'ai résolu la structure atomique de plusieurs composants centraux du T9SS et du T2SS. Concernant le projet T9SS, j'ai essayé de cristalliser le domaine cytoplasmique de GldL de F. johnsoniae. La co-cristallisation de GldL avec des Nbs a été réalisée sans succès. Néanmoins, les structures cristallines de deux nanobody contre GldL ont été résolues par remplacement moléculaire. De plus, j'ai également travaillé sur la protéine PG1058 de P. gingivalis. J'ai résolu sa structure par diffraction anomale à la longueur d’onde du selenium. Concernant le projet T2SS, je me suis concentré sur la partie N-terminale de XcpQ, une sous-unité de la sécrétine. J'ai résolu la structure cristalline de XcpQN012 seul et en complexe avec le nanobody vhh04 à une résolution de 2,98 Å et de 2,9 Å, respectivement. Enfin, j'ai participé à la détermination structurale de TssK, un composant de plaque de base du système de T6SS et déterminer la structure cristalline d'un nanobody contre le domaine périplasmique de PorM. / Proteins synthesized and secreted by bacteria serve many important roles in their survival. In particular, Gram-negative bacteria have evolved secretion pathways as the main weapons for transporting virulence factors into target cells or into the extracellular environment. One of these systems, the type IX secretion system (T9SS) or the Por secretion system, has been studied mainly in the oral pathogen Porphyromonas gingivalis and the gliding bacterium Flavobacterium johnsoniae. Another complex, the type II secretion system (T2SS) is the main determinant of the virulence of Pseudomonas aeruginosa, a cystic fibrosis pathogen. In my PhD thesis, I solved the atomic structure of several core components of both T9SS and T2SS.For the T9SS project, I tried to crystallize the cytoplasmic domain of GldL from F. johnsoniae. The co-crystallization of GldL with Nbs was unsuccessfull. The crystal structures of two nanobodies against GldL were solved by molecular replacement. I also worked on the PG1058 protein of P. gingivalis. I obtained crystals of the selenomethionine-derivatized PG1058 OmpA_C-like domain that diffracted up to 1.55 Å, and solved its structure by single-wavelength anomalous diffraction. For the T2SS project, I focused on the N-terminal part of XcpQ, a subunit of the secretin. I solved the crystal structure of XcpQN012 alone and in complex with nanobody vhh04 at a resolution of 2.98 Å and 2.9 Å, respectively. In addition, I also took part in the structural determination of the base plate component TssK of the T6SS and determined the crystal structure of one nanobody (vhh19) against the periplasmic domain of PorM.

The development of type 2 diabetes over time involves defects in insulin action and insulin secretion. Defects in insulin action alone can be compensated with appropriate hyperinsulinemia. However, the progressive loss of pancreatic beta-cell function leads eventually to the development of persistent hyperglycemia that characterizes type 2 diabetes. Insulin secretion patterns reflect two phases when beta-cells are exposed to acute and sustained glucose stimulation. Through the study and understanding of the roles of these two phases in the regulation of glucose homeostasis, it is clear that insulin must not only be secreted in sufficient amounts, but also at the right time. In type 2 diabetes, the timing and magnitude of insulin secretion are altered, and an abnormal first-phase release initiates before the onset of the disease. Only a few pharmacokinetic/pharmacodynamic (PK/PD) models have considered the biphasic nature of insulin secretion. This study is aimed at describing the biphasic dynamics of insulin secretion through developing a PK/PD model based on current knowledge of the cellular mechanism of biphasic insulin secretion. The objectives of this work are to 1) evaluate the insulin-glucose kinetics using nonparametric analysis, 2) develop a physiologically based mechanistic PK/PD model to dynamically describe the biphasic insulin secretion, 3) evaluate the impact of ethnicity on insulin secretion kinetics following an intravenous glucose administration using population analysis and 4) extend the proposed model to oral glucose administration and utilize the co-secretion kinetics of insulin and C-peptide in a population PK/PD analysis of the prehepatic insulin secretion. Population analysis was done using a nonlinear mixed-effects model combined with the proposed PK/PD model to estimate population parameters and their variations between- and within-subjects and the covariates' effects on model parameters. The proposed model describes biphasic insulin behavior, accounts for first-phase insulin secretion, and also applies to oral glucose administration for estimating prehepatic insulin secretion in vivo and in liver extraction. This is done by an analysis that simultaneously uses plasma insulin and C-peptide concentrations. A significant higher first-phase insulin secretion was identified in healthy youths of African-American compared to Caucasians. The analysis showed no significant differences in the clearance of insulin from the plasma and the liver extraction of insulin between subjects with various levels of glucose tolerance. Obesity leads to a higher insulin production rate and lower elimination rate from the plasma than normal weight subjects. Also, type 2 diabetes and impaired glucose tolerance were found to reduce insulin production rate and resulted in a delayed insulin secretion from the beta-cells.

Le système immunitaire est un ensemble de mécanismes impliquant différents types de cellules qui produisent des facteurs solubles (cytokines, chimiokines ou molécules cytotoxiques) qui contribuent à la régulation et aux réponses immunitaires. La caractérisation à l'échelle cellulaire de la production de ces facteurs solubles présente un grand intérêt d'une part sur le plan fondamental pour comprendre les cascades d'événements des régulations cellulaires, et d'autre part dans le suivi de la réponse immunitaire (infections, cancers, auto-immunités, greffes, vaccins, etc.). Cependant, la plupart des techniques actuellement disponibles (ELISpot, cytométrie en flux, microarrays, etc.) ne permettent pas d'étudier plusieurs cytokines en rapport avec le phénotype des cellules sécrétrices et/ou sans marquage et d'analyser les secrétions de cytokines en temps réel par des cellules individuelles. Dans cette étude, une « biopuce à cellules » a été développée pour analyser les secrétions de cytokines par les cellules individuelles (lymphocytes T) in vitro. La biopuce est fonctionnalisée par greffage électrochimique des motifs d'anticorps spécifiques aux protéines membranaires de cellules et/ou d'anticorps spécifiques aux cytokines, tous couplés au pyrrole. Ensuite, un traitement de surface est effectué avec du poly (éthylène glycol) thiol (Thiol-PEG) pour empêcher la fixation non spécifique de cellules sur la surface de la biopuce. Un dispositif microfluidique en polydimethyllsiloxane (PDMS) et maintenu à 37°C a aussi été développé afin d'intégrer toutes les opérations d'analyse et de détection dans un seul système. La biopuce développée dans cette étude permet la capture spécifique et stable de lymphocytes T individuels viables et la détection ultérieure de cytokines sécrétées par chaque cellule individuelle. Dans ce travail, la détection des cytokines sécrétées (IL-2 et IFN-γ) a été effectuée par fluorescence dans un format en sandwich. Cette «biopuce à cellules» est également compatible avec l'imagerie par résonance plasmonique de surface (SPRi), ce qui pourrait permettre de réaliser des analyses en temps réel et la détection sans marquage de plusieurs cytokines sécrétées par des cellules individuelles. Cette technique fournit un outil très prometteur pour l'analyse de marqueurs biologiques et de l'activité de cellules et l'étude des réponses immunitaires. / The immune system is a set of mechanisms involving many different cell types which communicate through downstream signals mediated principally by soluble factors (i.e. cytokines) to protect the host against invading organisms and to control adequate immune responses. The identification and characterization at the cellular level of cytokine production has a huge interest for both fundamental research and clinical studies. However, the majority of techniques currently available (ELISpot, flow cytometry, microarrays, etc.) have several shortcomings including notably the assessment of multiple cytokines in relation to secreting cell phenotypic classification and/or label-free and real-time analysis of cytokine secretions at individual cell level. Hence, in this study, we developed a « cell biochip » to analyze the secretion of cytokines by individual cells (T lymphocytes) activated and cultured in vitro. The biochip is functionalized by electrochemically grafting patterns of pyrrole-conjugated cell membrane-specific and cytokine-specific antibodies and treated with Poly(ethylene glycol)thiol (Thiol-PEG) self-assembled monolayers (SAMs) to stably avoid non-specific binding of cells on the surface. A polydimethyllsiloxane (PDMS)-based microfluidic device maintained at 37°C was also developed in order to integrate all the detection assay operations in a single system. The biochip developed here allows specific and stable capture of viable and bioactive individual T cells and subsequent detection of secreted cytokines in the close vicinity of each individual cell. In this work, the detection of secreted cytokines (IL-2 and IFN-γ) was performed by fluorescence in an immunosandwich assay format. This « cell biochip » is also compatible with surface plasmon resonance imaging (SPRi), which could therefore allow expanding its functionality to enable real-time and label-free detection of multiple cytokines from individual cells. Such technique provides a very promising tool for the analysis of biomarkers and cell activity and the monitoring of immune responses.

[Truncated abstract] Intracellular communication between osteoclasts and osteoblasts is imperative to maintain bone integrity. A myriad of molecules are responsible for regulating osteoblast and osteoclast activity. In particular, it is well documented that osteoblast-derived factors are crucial in directly controlling osteoclast formation and function. Since bone formation is coupled to bone resorption, it would be expected that osteoclasts also have some role in regulating the growth and function of osteoblast cells. However, despite extensive research upon osteoclast and osteoblast biology, the mechanisms by which osteoclasts regulate osteoblast growth and function is not well understood. In an attempt to further elucidate the mechanisms by which osteoclasts and osteoblasts communicate, the technique of subtractive hybridisation was used to identify a novel osteoclastderived factor identical to that of mouse Seminal Vesicle Secretion VII (SVS VII). Previous characterisation of the gene in bone demonstrated that SVS VII was abundantly and specifically expressed by mature osteoclasts (Phan, 2004). Additional research hinted that SVS VII acted as a novel osteoclast-derived factor, that by paracrine mechanisms, targeted osteoblast function (Phan, 2004). However, it remained open as to whether the SVS VII molecule did uniquely target the osteoblast, and whether this interaction influenced bone formation in vivo. Therefore, this thesis endeavoured to functionally characterise the role of the SVS VII molecule in the bone environment. ... Further work is needed to identigy a clear consensus binding sequence, to determine the specificity of the interaction between SVS VII protein and each phage clone, and to isolate a specific binding partner for SVS VII. In conclusion, the studies of this thesis sought to characterise the significance of SVS VII expression by mature osteoclasts, relative to its effects on osteoblast behaviour, but failed to conclusively determine a role for SVS VII in bone. Given that the effects of SVS VII on in vitro osteoblast activity and function are minimal, it is doubtful that SVS VII primarily acts as a paracrine factor integral to osteoblast function. Therefore, these findings conflict with those presented previously (Phan, 2004). However, it was demonstrated that SVS VII treatment was associated with in vivo effect on the skeleton, suggesting that SVS VII may target other elements of the bone microenvironment. Via mechanisms not yet understood, which possibly involves additional factors of the bone 11 extracellular matrix, SVS VII may target a subset of osteoprogenitor cells within the bone environment and act to regulate their proliferation. Therefore, SVS VII may enhance osteogenic precursor cell number at sites of bone formation which would increase the pool of cells that can differentiate down the osteoblast linage and contribute to bone formation. In this regard, SVS VII might function in a manner homologous to the Ly-6 molecule Sca-1 and act as an important factor that maintains a balance between the bone formation and resorption process. Clearly, more work focusing on alternative facets of bone biology is needed to identify whether there is a significant role for SVS VII in skeletal tissue.

Bibliography: leaves 171-211 / 211 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, 1988

<p><i>Francisella tularensis</i> is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. <i>F. tularensis</i> can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of <i>F. tularensis</i>: the highly virulent <i>tularensis</i> (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent <i>holarctica</i> (Type B) and <i>mediasiatica</i>, and the essentially avirulent subspecies <i>F. novicida</i>. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between <i>F. tularensis</i> subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of <i>F. tularensis</i>, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the <i>Francisella</i> pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in <i>Neisseria gonorrhoeae. pilA</i> was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to <i>F. tularensis</i> and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (<i>pilA</i>) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them <i>in cis</i> could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia.</p>

<p>In order to establish colonisation of a human host, pathogenic <i>Yersinia</i> use a type III protein secretion system to directly intoxicate host immune cells. Activation of this system requires target cell contact and is a highly regulated process. Both the intoxication and regulation events depend on the <i>lcrGVHyopBD </i>transloco<i>n</i> operon, which is highly conserved in many bacterial pathogens. In this study, the role of individual operon members was analysed and functional domains identified by using the highly homologous <i>pcrGVHpopBD</i> operon of <i>P. aeruginosa</i> as a comparative tool. </p><p><i>Yersinia</i> spp. and<i> P. aeruginosa </i>were shown to form translocation pores of a similar size that promoted equally efficient protein delivery. A strong dependency on interactions between native translocator(s) in protein delivery was revealed, suggesting that each pathogen has delicately fine-tuned this process to suit its own infection niche. In particular, the C-terminus of YopD was shown to possess functional specificity for effector delivery in <i>Yersinia</i> that could not be conferred by the comparable region in homologous PopD. Moreover, a role for LcrV and PcrV in substrate recognition during the protein delivery process was excluded. </p><p>The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in <i>Yersinia</i>, while equivalent complexes with analogous proteins were not formed in <i>P. aeruginosa</i>. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to <i>Yersinia</i>. </p><p>Finally, PcrH was identified as a new member of the translocator class of chaperones, being essential for assembly of a functional PopB/PopD mediated translocon in <i>P. aeruginosa</i>. However, in contrast to the other members of this family, PcrH was dispensable for type III regulation. Moreover, both LcrH and PcrH were shown to possess tetratricopeptide repeats crucial for their chaperone function. One tetratricopeptide repeat mutant in LcrH was even isolated that failed to secrete both YopB and YopD substrates, even though stability was maintained. This demonstrates for the first time that LcrH has a role in substrate secretion in addition to its critical role in promoting substrate stability.</p>

<p>Parathyroid hormone (PTH) is the major physiological regulator of the extracellular Ca²⁺ concentration ([Ca²⁺]_o) in the body. The secretion of this hormone is suppressed at high [Ca²⁺]_o. Previously this was thought to occur by intracellular degradation of the hormone in the secretory pathway of parathyroid (PT) cells but is now believed to result from extracellular Ca²⁺ stimulus-secretion coupling via the calcium sensing receptor (CaR). In contrast to the stimulation of PTH secretion upon inhibition of mature PTH proteolysis, inhibition of PT proteasomes caused the accumulation of PTH precursors and inhibited secretion of PTH. This suggests that PT proteasomes play a quality control function in the maturation of PTH but they do not directly participate in the [Ca²⁺]_o-regulated secretion of the hormone. Treatment of PT cells with 12-O-tetradecanyolphorbol-13-acetate (TPA) blocks the high [Ca²⁺]_o-induced CaR-mediated suppression of PTH secretion. To delineate the role of DAG-responsive protein kinase C (PKC) isoforms in this process, we complemented pharmacological modulation of PKC activity with physiological activation of the enzyme via the CaR. PKC-α was rapidly activated by high [Ca²⁺]_o and was efficiently down-regulated by prolonged TPA treatment. In CaR-transfected HEK293 cells, TPA and high [Ca²⁺]_o induced the activation of ERK1/2 but the TPA effect was CaR- and Ca²⁺-independent. The magnitude of neomycin-induced release of Ca²⁺ from intracellular stores following pharmacological modulation of PKC activity was opposite to that resulting from physiological activation/inhibition of the enzyme via the CaR. Influx of Ca²⁺ following activation of the receptor occurred by store-operated mechanisms. Over-expression of wt or DN PKC-α or-ε in PT cells using the Tet-On adenovirus gene delivery system revealed that the stimulatory effect of TPA on PTH secretion at high [Ca²⁺]_o was enhanced in cells over-expressing wt PKC-α, but the coupling of the extracellular Ca²⁺ signal to PTH secretion was not dependent on the physiological activation of this PKC isoform via the CaR.</p>

Die Speicheldrüsen der Schmeißfliege Calliphora vicina produzieren bei Stimulierung mit dem Neurohormon Serotonin (5-Hydroxytryptamine, 5-HT) einen KCl-reichen Primärspeichel. Der transepitheliale K+-Transport wird durch eine apikal lokalisierte vakuoläre H+-ATPase (V-ATPase) energetisiert. Stimulierung der Speicheldrüsen mit 5-HT aktiviert die apikale V-ATPase, die Protonen aus der Zelle in das Drüsenlumen transportiert. Trotz des auswärts gerichteten Protonentransportes führt die 5-HT-Stimulierung kurioserweise zu einer intrazellulären Ansäuerung. Die Ursachen dieser 5-HT-induzierten Ansäuerung waren unzureichend untersucht. Deshalb war das Ziel dieser Arbeit die Identifikation aller Transporter, die an der intrazellulären pH-(pHi)-Regulation in unstimulierten Speicheldrüsen von Calliphora vicina beteiligt sind und an der Entstehung und Regulation der 5-HT-induzierten pHi-Änderungen mitwirken. Von besonderem Interesse war hierbei die funktionelle Mitwirkung der V-ATPase, deren Beteiligung an der pHi-Regulation in tierischen Zellen bisher wenig untersucht war. Wesentliche Ergebnisse dieser Arbeit waren: • Messungen des pHi-Wertes in der unstimulierten Drüse zeigten, dass vor allem die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter an der Aufrechterhaltung des Ruhe-pHi beteiligt sind. • Zur Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung (NH4Cl-Vorpuls) tragen ebenfalls im Wesentlichen die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter bei. Der Na+/H+-Antiporter hat in der unstimulierten Drüse keinen messbaren Einfluss auf den Ruhe-pHi. • Die Wiederherstellung des Ruhe-pHi nach einer intrazellulären Alkalisierung (Na-acetat-Vorpuls) ist Cl--abhängig, aber auch unter extremen Bedingungen waren die Zellen noch in der Lage sich vollständig von einer intrazellullären Alkalisierung zu erholen. Einen entscheidenden Anteil daran hat offenbar die hohe intrazelluläre Puerkapazität. • Ein Na+-abhängiger Glutamat-Transporter ist per se kein pHi-regulierender Transporter, seine Aktivität hat jedoch Einfluss auf den Ruhe-pHi in der unstimulierten Speicheldrüse von Calliphora vicina. • 10 nM 5-HT induzieren in den Calliphora Speicheldrüsen eine intrazelluläre Ansäuerung. An dieser Ansäuerung ist der Na+/H+-Antiporter entscheidend beteiligt. Auch eine klare Cl--Abhängigkeit der 5-HT-induzierten Ansäuerung konnte beobachtet werden. Wahrscheinlich ist eine gekoppelte Aktivität von Na+/H+-Antiporter und Cl-/HCO3--Antiporter. • Messungen mit einem O2-empndlichen Fluoreszenzfarbstoff zeigten, dass Stimulierung der Speicheldrüsen mit 5-HT die Zellatmung aktivierte. Der cAMP- und der IP3/Ca2+-Weg tragen auf komplexe Weise zu der 5-HT-induzierten Aktivierung der Zellatmung und damit auch zu den 5-HT-induzierten pHi-Änderungen bei. • Mit molekularbiologischen Untersuchungen ist es gelungen den Na+-abhängigen Glutamat-Transporter, den Na+/H+-Antiporter, die Carboanhydrase und die Untereinheit C der V-ATPase in den Calliphora Speicheldrüsen direkt nachzuweisen. Zudem konnte erstmals der direkte Nachweis für die Expression eines nH+/K+-Antiporters in den Speicheldrüsen von Calliphora vicina erbracht werden. Diese Arbeit trug ganz wesentlich zum Verständnis der pHi-Regulation in der unstimulierten und stimulierten Speicheldrüse von Calliphora vicina bei. Mechanismen die zur Aufrechterhaltung und Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung bzw. Alkalisierung beitragen, konnten mit pHi-Messungen und auch molekularbiologisch nachgewiesen werden. Die Mechanismen, welche die 5-HT-induzierte intrazelluläre Ansäuerung verursachen, konnten ebenfalls aufgeklärt werden. Zudem wurde an den Calliphora Speicheldrüsen eine neue optische Methode zur Messung des O2-Verbrauchs in tierischen Geweben etabliert. / The tubular salivary glands of the blowfly Calliphora vicina consist of a single layer of epithelial cells. Stimulation with the neurohormone serotonin (5-hydroxytryptamine,5-HT) induces the secretion of a KCl-rich primary saliva. Transepithelial K+-transport is energized by a vacuolar-type H+-ATPase (V-ATPase) which is located in the apical membrane. 5-HT stimulates the apical V-ATPase which transports protons out of the cells into the lumen of the glands. Despite this outward directed proton transport, 5-HT stimulation leads to an intracellular acidication. The causes of this intracellular acidication were poorly understood. Therefore the aim of this thesis was the identication of all pHi regulating transporters which are involved in pHi regulation in the unstimulated salivary glands of Calliphora vicina and which contribute to the 5-HT-induced pHi changes. Of special interest was the functional role of the V-ATPase,whose contribution to pHi regulation in animal cells is, as yet, not well studied. Key results were: • pHi measurements in unstimulated glands showed that mainly the V-ATPase and at least one Na+-dependent HCO3--transporter are involved in maintenance of resting pHi. • V-ATPase and at least one Na+-dependent HCO3--transporter are also necessary for the recovery from an intracellular acidication (NH4Cl prepulse). • Recovery from an intracellular alkali load (Na-acetate prepulse) is partially Cl--dependent. • A Na+ dependent gluatamate-transporter is present in Calliphora salivary glands and its activity aects the resting pHi. • 10 nM 5-HT induce an intracellular acidication. This acidication is Na+-dependent, EIPA-sensitive and also Cl--dependent. No DIDS-sensitivity was observed. A coupled activity of a Na+/H+-antiporter and a Cl-/HCO3- -antiporter was suggested. • Using O2-sensitive fluorescent microbeads I could show that 5-HT stimulation of the Calliphora salivary glands activates cellular respiration. The cAMP and Ca2+-signalling pathways contribute in a complex manner to the 5-HT-induced activation of cellular respiration and consequently, also to the 5-HT-induced intracellular acidication. • The expression of a Na+ dependent glutamate-transporter, a Na+/H+-antiporter, a carbonic anhydrase, subunit C of the V-ATPase and a nH+/K+-antiporter were determined on mRNA level by RT-PCR. This thesis contributes signicantly to the understanding of pHi regulation in unstimulated and stimulated salivary glands of Calliphora vicina. Mechanisms which contribute to the maintenance and recovery of resting pHi were identied by using pHi measurements and molecular biological techniques. Mechanisms which are responsible for the 5-HT-induced intracellular acidication were also clarified. Furthermore a new optical method for measuring O2 consumption in animals cells was established by using the Calliphora salivary glands as a model.