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Effect of mast cell granules on urinary tract epithelial cells in culture
ICBR-30 Effects of Resiniferatoxin on the Neurogenic Component of Feline Interstitial Cystitis P. March, B. Teng, J. Westropp, and T. Buffington Department of Veterinary Clinical Sciences, Ohio State University, Columbus, Ohio, USA Many of the symptoms and signs of interstitial cystitits (IC) may be due to sensitization of normal afferent pathways that signal bladder distention and pain. We examined the degree of afferent sensitization in a feline model of IC to determine if pain transmission and vesicosympathetic reflexes are upregulated in this disease. We also assessed the relative reduction in afferent transmission and efferent responses after intravesical infusion of resiniferatoxin (RTX) in both normal cats and cats with IC. Two normal cats and 2 cats with IC were placed under various levels of isoflurane anesthesia, and the EEG bispectral index (BIS), respiratory and heart rate, blood pressure, and bladder pressure and volume were monitored during bladder infusion with warmed saline. Similar parameters were monitored after electrical stimulation of the tail base. Response thresholds (MACcortical and MACsubcortical) for cerebrocortical arousal (BIS number ⬎60) and subcortical activation (respiratory and cardiovascular parameter increases), respectively, were recorded. All normal cats and cats with IC then received an intravesical infusion of RTX (0.1 mol/L solution for 30 minutes). After 30 days, response threshold determinations were repeated as described. At baseline, MACsubcortical was greater in cats with IC than in normal cats, and the MACsubcortical was always greater than the MACcorticalin cats with IC. Furthermore, both heart rate and respiratory rate increased in cats with IC just before elevation of the BIS. This was not observed in normal cats. Tail stimulation caused respiratory rate and BIS elevations at comparable levels of anesthesia in both the affected and control groups. Following RTX desensitization, the decrease in MACcortical was slightly greater in the IC cat group than in normal cats. The decrease in MACsubcortical after RTX treatment was of a greater magnitude in cats with IC than in normal cats. Bladder capacity and compliance were reduced post-RTX in cats with IC, but not in normal cats. These results suggest a lower threshold of central nervous system cerebrocortical and autonomic activation in cats with IC. Furthermore, subcortical reflexes may occur before and independently of conscious perception of a painful stimulus in IC. Bladder inflation failed to cause the same degree of sympathetically mediated increases in heart and respiratory rate post-RTX, suggesting that upregulated vesicosympathetic cardiovascular and respiratory reflexes were “normalized” post-RTX. Additional studies are needed to confirm these findings in a larger number of cats and to identify the afferent pathways (pelvic versus hypogastric) that are upregulated in IC.
ICBR-31 Effect of Mast Cell Granules on Urinary Tract Epithelial Cells in Culture A. Rickard, C. Portel, and D. Lagunoff St. Louis University Medical School, St. Louis, Missouri, USA Supported by the Interstitial Cystitis Association 114
Elevated levels of mast cells in the bladder as well as increased mast cell metabolites in the urine of interstitial cystitis (IC) patients have been reported by several investigators, but as yet a mechanism relating these observations and the pathologic changes in the bladder urothelium has not been established. Because heparin is known to inhibit cell proliferation, we considered the possibility that mast cell granules with their high content of heparin proteoglycan could contribute to the epithelial defects seen in IC by interfering with epithelial repair. With MDCK cells (dog kidney– derived epithelial cells) and RT4 cells (human bladder transitional-cell papilloma cells), we have observed that granules inhibit the ability of preconfluent cell cultures to achieve confluency. We further noted that RT4 cell cultures treated with granules stratified into several more layers than control cultures, suggesting a possible failure of the treated cells to migrate over the gelatin substrate. To study the effect of granules on primary isolates of normal urothelial cells, cultures were initiated from surgical and autopsy samples. Epithelial origin of the isolates was confirmed by the presence of the expected cytokeratins, desmosomes, E-cadherin, and characteristic morphologic changes in response to calcium concentration. In these cultures, we have seen a similar failure to achieve confluence in granule-treated cultures compared with controls. The effect was assessed quantitatively by measuring cell-free area. Measurement of the number of cells by the CyQUANT (Molecular Probes, Eugene, Ore) cell proliferation assay technique showed fewer cells in cultures treated with granules (20 g/mL of the heparin proteoglycan) compared with controls. The effect may result from increased cell loss or decreased cell replication. We are pursuing studies to distinguish between these possibilities and to evaluate the contribution of interference with cell spreading and cell migration to the effect.
ICBR-32 cDNA Expression Arrays: The Effects of Lactacystin in Lipopolysaccharide-induced Cystitis X. C. Wang, J. H. Kaysen, R. Saban, P. L. Allen, E. N. Benses, and T. G. Hammond Nephrology Section, Tulane University Medical Center, Tulane/VA Veterans Administration Environmental Astrobiology Center, and VA Veterans Administration Medical Center, New Orleans, Louisiana, USA (XCW, JHK, PLA, ENB, TGH), and Enteric Neuromuscular Diseases Laboratory, Division of Gastroenterology, University of Texas Medical Branch, Galveston, Texas, USA (RS) Our previous study has shown that the proteasome inhibitor lactacystin inhibited nuclear factor (NF)-B mediates lipopolysaccharide (LPS)-induced cystitis. The objective of this study was to quantitate how many gene expression changes in inflammatory processes are NF-B dependent. Bladder inflammation was induced in anesthetized mice by intravesical instillation of LPS with or without pretreatment with lactacystin. Bladder mucosa total RNA (5 g) was used for Atlas mouse cDNA expression arrays containing 588 known genes in 6 functional groups. The Altas Array membranes (Clontech, Palo Alto, Calif) were exposed to the Packard Cyclone Phosphor Screen (Packard Bioscience Company, Downers Grove, Ill) for 24 to 48 hours. The screens were read by Phosphor Imager (Packard Bioscience Company). Each blot radioactive density was quantitated by OptiQuant (Packard Bioscience Company). The gene expression is presented as a percentage of the average expression of housekeeping genes on the same membrane. A gene expression ratio of experimental to UROLOGY 57 (Supplement 6A), June 2001
ICBR-31 Effect of Mast Cell Granules on Urinary Tract Epithelial Cells in Culture A. Rickard, C. Portel, and D. Lagunoff St. Louis University Medical School, St. Louis, Missouri, USA Supported by the Interstitial Cystitis Association 114
Elevated levels of mast cells in the bladder as well as increased mast cell metabolites in the urine of interstitial cystitis (IC) patients have been reported by several investigators, but as yet a mechanism relating these observations and the pathologic changes in the bladder urothelium has not been established. Because heparin is known to inhibit cell proliferation, we considered the possibility that mast cell granules with their high content of heparin proteoglycan could contribute to the epithelial defects seen in IC by interfering with epithelial repair. With MDCK cells (dog kidney– derived epithelial cells) and RT4 cells (human bladder transitional-cell papilloma cells), we have observed that granules inhibit the ability of preconfluent cell cultures to achieve confluency. We further noted that RT4 cell cultures treated with granules stratified into several more layers than control cultures, suggesting a possible failure of the treated cells to migrate over the gelatin substrate. To study the effect of granules on primary isolates of normal urothelial cells, cultures were initiated from surgical and autopsy samples. Epithelial origin of the isolates was confirmed by the presence of the expected cytokeratins, desmosomes, E-cadherin, and characteristic morphologic changes in response to calcium concentration. In these cultures, we have seen a similar failure to achieve confluence in granule-treated cultures compared with controls. The effect was assessed quantitatively by measuring cell-free area. Measurement of the number of cells by the CyQUANT (Molecular Probes, Eugene, Ore) cell proliferation assay technique showed fewer cells in cultures treated with granules (20 g/mL of the heparin proteoglycan) compared with controls. The effect may result from increased cell loss or decreased cell replication. We are pursuing studies to distinguish between these possibilities and to evaluate the contribution of interference with cell spreading and cell migration to the effect.
ICBR-32 cDNA Expression Arrays: The Effects of Lactacystin in Lipopolysaccharide-induced Cystitis X. C. Wang, J. H. Kaysen, R. Saban, P. L. Allen, E. N. Benses, and T. G. Hammond Nephrology Section, Tulane University Medical Center, Tulane/VA Veterans Administration Environmental Astrobiology Center, and VA Veterans Administration Medical Center, New Orleans, Louisiana, USA (XCW, JHK, PLA, ENB, TGH), and Enteric Neuromuscular Diseases Laboratory, Division of Gastroenterology, University of Texas Medical Branch, Galveston, Texas, USA (RS) Our previous study has shown that the proteasome inhibitor lactacystin inhibited nuclear factor (NF)-B mediates lipopolysaccharide (LPS)-induced cystitis. The objective of this study was to quantitate how many gene expression changes in inflammatory processes are NF-B dependent. Bladder inflammation was induced in anesthetized mice by intravesical instillation of LPS with or without pretreatment with lactacystin. Bladder mucosa total RNA (5 g) was used for Atlas mouse cDNA expression arrays containing 588 known genes in 6 functional groups. The Altas Array membranes (Clontech, Palo Alto, Calif) were exposed to the Packard Cyclone Phosphor Screen (Packard Bioscience Company, Downers Grove, Ill) for 24 to 48 hours. The screens were read by Phosphor Imager (Packard Bioscience Company). Each blot radioactive density was quantitated by OptiQuant (Packard Bioscience Company). The gene expression is presented as a percentage of the average expression of housekeeping genes on the same membrane. A gene expression ratio of experimental to UROLOGY 57 (Supplement 6A), June 2001