Durante o processo de reparação tecidual, o fator de transformação de crescimento <font face=\"symbol\">b1 (TGF-<font face=\"symbol\">b1) apresenta um importante papel na regulação da expressão da <font face=\"symbol\">a-actina de músculo liso (<font face=\"symbol\">a-AML) e portanto, na diferenciação miofibroblástica. Como os fibroblastos pulpares apresentam características peculiares, com a expressão de proteínas específicas que os diferem de fibroblastos de outros tecidos conjuntivos, o presente estudo avaliou in vitro se o TGF-<font face=\"symbol\">b1 aumenta a expressão de <font face=\"symbol\">a-AML em fibroblastos pulpares humanos comparando-os com fibroblastos de gengiva. Para tal, diferentes doses de TGF-<font face=\"symbol\">b1 (5 à 10 ng/ml) foram adicionadas às culturas de células, sendo a expressão da <font face=\"symbol\">a-AML analisada por imunofluorescência e western-blotting. Ambos os tipos celulares imunoexpressaram <font face=\"symbol\">a-AML mesmo sem o tratamento com o TGF-<font face=\"symbol\">b1, estando aumentada consideravelmente, quando o TGF-<font face=\"symbol\">b1 foi adicionado às culturas. Os resultados do presente estudo demonstraram que o TGF-<font face=\"symbol\">b1 induz a expressão de <font face=\"symbol\">a-AML, sugerindo a indução do fenótipo miofibroblástico em fibroblastos pulpares. / Transforming growth factor-beta 1 (TGF-<font face=\"symbol\">b1) has been related to induce the expression of <font face=\"symbol\">a-smooth muscle actin (<font face=\"symbol\">a-SMA) in fibroblasts during repair. Since pulpal fibroblasts seem to be somewhat different from other fibroblasts, the present study investigated in vitro whether TGF-<font face=\"symbol\">b1 enhances the expression of <font face=\"symbol\">a-SMA in human pulpal fibroblasts. TGF-<font face=\"symbol\">b1 was added in doses between 5-10 ng/ml to cultures of both dental pulp and gingiva human fibroblasts. The expression of <font face=\"symbol\">a-SMA was analyzed by immunofluorescence and western-blotting. Both cell types were immunoreactive for <font face=\"symbol\">a-SMA even without TGF-<font face=\"symbol\">b1. When TGF-<font face=\"symbol\">b1 was added to cell cultures, the expression of <font face=\"symbol\">a-SMA increased dramatically in pulpal fibroblasts, independent of the concentration used. It was confirmed by the western blot analysis. The present findings showed that TGF-<font face=\"symbol\">b1 up-regulated the expression of <font face=\"symbol\">a-SMA thus inducing pulpal fibroblasts to acquire the myofibroblast phenotype.

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