Harnblase. Weiterhin bestärkt die beobachtete Kopplung die Hypothese, dass das Myofibroblastennetzwerk afferente Signale Verstärken und modulieren kann. Die Verbesserung des Verständnisses der Harnblasenfunktion ist von grundlegendem Interesse für die Behandlung von Miktionsstörungen. Eine Störung von mechanischer Stimulierbarkeit und Kopplung der suburothelialen Myofibroblasten kann eine Störung der Sensorik der Harnblase plausibel erklären und stellt einen neuen Therapieansatz dar.:1 Abkürzungsverzeichnis 2 Einführung 2.1 Hintergrund 2.2 Anatomische und physiologische Grundlagen der Harnblasenfunktion 2.2.1 Aufbau der Harnblasenwand 2.2.2 Terminologie und Klassifikation interstitieller Zellen der Harnblase 2.2.3 Phänotypisierung der suburothelialen Myofibroblasten 2.2.4 Funktionsweise der Myofibroblasten 2.2.5 Innervation des unteren Harntraktes und neuronale Kontrolle der Harnblasenfunktion 2.2.6 Lokale sensorische Netzwerke der Harnblase 3 Aufgabenstellung 4 Material und Methoden 4.1 Gewinnung und Zellkultur suburothelialer Myofibroblasten 4.2 Lösungen und Chemikalien 4.3 Calcium imaging 4.4 Datenanalyse 4.4.1 Erzeugung der FI-Ratio Datensätze 4.4.2 Automatische Fluoreszenz-Signal-Analyse 4.5 Aufbau und Anordnung der Experimente 4.5.1 Spontanaktivität 4.5.2 Mechanische Stimulation mittels Glasmikropipette 4.5.3 Mechanische Stimulation durch Scherstress 4.5.4 Hypoosmolare Stimulation 5 Ergebnisse 5.1 Spontane Kalziumaktivität humaner suburothelialer Myofibroblasten 5.2 Mechanische Stimulierbarkeit durch Druck mittels Glasmikropipette 5.2.1 Intrazelluläre Ausbreitung mechanisch induzierter Kalziumsignale 5.2.2 Interzelluläre Ausbreitung mechanisch induzierter Kalziumsignale in kultivierten humanen suburothelialen Myofibroblasten 5.3 Mechanische Stimulierbarkeit durch Scherstress 5.4 Mechanische Stimulierbarkeit durch osmotischen Stress 6 Diskussion 6.1 Beobachtete Kalziumsignale suburothelialer Myofibroblasten 6.2 Bedeutung der mechanischen Stimulierbarkeit 6.3 Identifikation der stretch-activated channels 6.4 Bedeutung der interzellulären Kopplung 6.5 Konzept zur Rolle der suburothelialen Myofibroblasten 6.6 Methodischer und experimenteller Ansatz 6.6.1 Selbst entwickelte Fluoresence Analysis Software 6.6.2 Methoden der mechanischen Stimulierbarkeit 6.7 Pathophysiologische Aspekte 7 Zusammenfassung der Arbeit 8 Literaturverzeichnis 9 Anlagen 9.1 Selbstständigkeitserklärung 9.2 Lebenslauf 9.3 Publikationen 9.4 Danksagung

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.:1. Introduction............................................................................ 1 1.1. Anatomy and histology of the human urinary bladder..................... 1 1.1.1. Anatomy of the human urinary bladder..................................... 1 1.1.2. Structure of the human urinary bladder wall............................... 2 1.2. Normal bladder function and bladder dysfunction.......................... 3 1.2.1 Normal bladder function......................................................... 3 1.2.2 Sensory aspect.................................................................... 4 1.2.3 Overactivity or hypersensitivity of bladder.................................. 5 1.3 The role of functional cell types and interaction in urinary bladder... 6 1.3.1 The role of urothelium.......................................................... 7 1.3.2Theroleofsuburotheliamyofibroblast...................................... 7 1.3.3Theroleofdetrusorsmoothmusclecells.................................. 9 1.3.4 Possible interactions in urinary bladder cell types........................ 10 1.4 ATP function and Purinergic signalling in bladder........................... 11 1.5 Spontaneous activity of bladder................................................... 13 2. Objective.................................................................................. 15 3. Material and methods............................................................... 16 3.1. Ethics Statement........................................................................ 16 3.2. Cell preparation.......................................................................... 16 3.3. Solutions and chemicals............................................................. 19 3.4. Intracellular calcium measurements............................................. 20 2.4.1. Preparing cells for Calcium Imaging.......................................... 20 2.4.2. Preparing workspace of calcium imaging................................... 20 2.4.3. Calcium imaging recording...................................................... 22 3.5 Data analysis with automated Fluorescence analysis..................... 22 3.6 Confocal Immunofluorescence.................................................... 25 3.7 Statistics................................................................................. 26 4. Results.................................................................................. 27 4.1 Spontaneous calcium activity of sMF........................................... 27 4.2 ATP effects on calcium response in sMF...................................... 27 4.3 Analysis of purinergic receptors involved.................................... 30 3.3.1 Agonist stimulation.............................................................. 30 3.3.2 Signal inhibition by specific antagonists................................... 31 4.4 Confocal immunofluorescence of purinergic receptors.................. 32 5. Discussion............................................................................. 34 5.1 Myofibroblast identification....................................................... 34 5.2 Spontaneous activity in the bladder............................................ 36 5.3 ATP modulated calcium activity in sMF....................................... 37 5.4 purinergic signalling in sMF........................................................ 39 6. Summary................................................................................ 42 7. References.............................................................................. 45 Declaration............................................................................. 50 Acknowledgements................................................................. 51