| Extracellular nucleotides, acting via purinoreceptors, regulate important physiological processes in most body tissues. In the airways, extracellular nucleotides are implicated in the primary defence mechanism by potentiating mucociliary clearance and surfactant secretion. Two different approaches for studying extracellular nucleotides on the lung epithelium are presented and divide this thesis into two parts. The first part focuses on the physiological aspect of nucleotide release, especially ATP, from the alveolar type II cell line A549 and the second on the development of a side-viewing technique for the observation of ATP-dependent airway surface liquid (ASL) height variations: (1) While previous results showed that cell-swelling induced ATP secretion, and that this secretion was tightly correlated with an intracellular Ca 2+ elevation, the present research was designed to establish the mechanism which is responsible for nucleotide release from A549 cells. Moreover, the source and the progress of the intracellular Ca2+ signal were thoroughly analyzed. In this study, nucleotide release from A549 cells was induced by a 50% hypotonic shock in a flow-through chamber at 37°C. Agents, known to interfere with the exocytotic process, intracellular Ca2+ signalling and purinergic signalling, were applied, and the ATP concentration in the perfusate aliquots was determined with a luciferase-luciferin assay. The individual concentration of all nucleotides was determined by HPLC. The inhibition of the exocytotic process significantly reduced ATP secretion and indicated a Ca2+-dependent exocytosis as the principal release mechanism. The study of the Ca2+ signal revealed that ATP release depended entirely on Ca2+ mobilization from intracellular stores, primarily from the thapsigargin-sensitive endoplasmic reticulum and to a lesser extent from an unidentified thapsigargin-insensitive source, which was, however, sensitive to intracellular pH alterations. Finally, cell-swelling induced, Ca2+-dependent ATP release from A549 cells was shown to be amplified by a positive autocrine/paradine feedback of co-released nucleotides. (2) The side-viewing imaging technique was designed and constructed for a potential time-resolved study of ASL height alterations upon stimulation with extracellular ATP. A custom-designed side-viewing chamber was mounted on the microscope stage and was equipped with a temperature/humidity control system to keep an in vitro cell culture in a physiological environment and to prevent the ASL from insensible water loss. The chamber was comprised of a housing including a perforated knob, which accommodated a filter insert with an air/liquid grown cell monolayer and allowed perfusion of the basal side. The housing positioned the filter insert perpendicular to the microscope objective and enabled a direct observation of fluorescently labelled ASL through an epifluorescence microscope.;Keywords. purinergic signalling, extracellular ATP, Ca2+-signalling, release mechanism, exocytosis, airway defence, airway surface liquid, optical system, side-viewing microscopy, epifluorescence microscopy.;These two areas of study aim to contribute to a better understanding of the physiological mechanisms involved in the primary lung defence via extracellular nucleotides. This knowledge is the basis for the development of novel therapies especially against diseases of impaired mucociliary clearance. |
