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493 DEVELOPMENT OF A NEUROGENIC BLADDER IN TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 KNOCKOUT MICE
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THE JOURNAL OF UROLOGY姞
493 DEVELOPMENT OF A NEUROGENIC BLADDER IN TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 KNOCKOUT MICE Qi Lei*, Xiao-Qing Pan, Glenolden, PA; Tirsit Asfaw, New York, NY; Joseph Hypolite, Glenolden, PA; Alan Wein, Philadelphia, PA; Anna Malykhina, Glenolden, PA INTRODUCTION AND OBJECTIVES: Bladder pain of unknown etiology has been associated with co-morbid conditions and functional abnormalities in adjacent pelvic organs. This phenomenon is called “crosssensitization” and occurs predominantly via convergent neural pathways connecting distinct pelvic organs. Our previous studies showed that colonic inflammation can cause detrusor instability via activation of transient receptor potential vanilloid 1 (TRPV1) signaling pathways. In this study, we aimed to determine whether cross-sensitization in the pelvis can develop in the absence of TRPV1 receptors and lead to a neurogenic bladder. METHODS: Four groups of adult male mice (N⫽25) were included in the study: 2 groups of C57BL/6 wild-type (WT) mice with and without acute colitis (induced by intracolonic trinitrobenzene sulfonic acid, TNBS) and 2 groups of TRPV1 knockout mice (TRPV1⫺/⫺) mice with and without acute colitis. Mice were sacrificed at day 3 after the induction of pelvic organ cross-sensitization by acute colitis and the function of the detrusor was evaluated in vitro using isolated detrusor smooth muscle (DSM) strips. The level of inflammation in the distal colon and urinary bladder was evaluated by the myeloperoxidase (MPO) assay. The contractility of DSM strips was studied in response to potassium chloride test, electric field stimulation and relaxation of DSM by Rho kinase inhibitor. RESULTS: In WT mice, acute colitis caused 2-fold up-regulation of MPO levels in the colon whereas in TRPV1⫺/⫺ mice an increase reached 1.5 fold (p⬍0.05 to respective control). MPO levels were unchanged in the bladders from both groups. The contractile response of DSM strips to electric stimulation was significantly increased after colitis in WT mice (85.97⫾12.27 g/g) when compared to control group (44.39⫾10.81 g/g, p⬍0.05). The same changes were observed in the TRPV1⫺/⫺ group with pelvic organ cross-sensitization. The use of Rho kinase inhibitor Y27632 induced DSM relaxation in WT group by 72.87⫾10.40% whereas it was only 39.38⫾6.46% in the TRPV⫺/⫺ group (N⫽6, p⬍0.05 to WT). CONCLUSIONS: Our data provide evidence that the absence of TRPV1 receptors does not eliminate the occurence of cross-sensitization in the pelvis and TRPV⫺/⫺ mice develop a neurogenic bladder. However, DSM from TRPV⫺/⫺ mice showed decreased relaxation in response to a Rho kinase inhibitor suggesting alterations in DSM signaling pathways.
Vol. 187, No. 4S, Supplement, Sunday, May 20, 2012
RESULTS: RMP was significantly more depolarized in hBUC compared to rBUC (-14.3 ⫾ 5.7 mV versus -26.2 ⫾ 4.97 mV, p⫽0.02). The Cm (pF) and Rm (G) were significantly different in rBUC versus hBUC (30 ⫾ 16 pF vs. 102 ⫾ 38 pF and 1.0 ⫾ 0.5 G vs. 0.2 ⫾ 0.1 G, respectively, both comparisons p⬍0.05). ATP, CCh, ACh, KCl and CaCl depolarized both rBUC and hBUC without significant differences in RMP changes between the species. LPS hyperpolarized the cells. Representative tracings showing changes in RMP in response to these agents are shown in the Figure. CONCLUSIONS: This work represents the first detailed description of measurement of RMP in hBUC and rBUC and provides reference data for future study on urothelial cell physiology. The pharmacologic agents CCh, ACh and ATP depolarized RMP as expected. Extracellular KCl and CaCl also depolarized the cells. However, hyperpolarization of urothelial cells with LPS is a novel finding.
Source of Funding: This study was supported by the NIH/ NIDDK grants DK077699 and DK077699-S2 (to AM)
494 CHARACTERIZATION OF RESTING MEMBRANE POTENTIAL IN RAT AND HUMAN BLADDER UROTHELIAL CELLS Nora Laaris*, Yan Sun, Toby Chai, Baltimore, MD INTRODUCTION AND OBJECTIVES: The bladder urothelium is traditionally thought to only provide barrier function. However, accumulating data suggest that the urothelium may serve additional function including sensor-transducer role, urothelial-afferent signaling and immune regulation. The measurement of urothelial cell resting membrane potential (RMP) is important as RMP has been implicated in mediating these functions. Changes in RMP in response to extracellular ATP, carbachol (CCh), acetylcholine (ACh), lipopolysaccharide (LPS), potassium (KCl), and calcium (CaCl2) were also analyzed. METHODS: This study was approved by both the IRB and IUCAC. Primary cultures of hBUC from 8 subjects and rBUC from 4 rats were prepared as previously described. Epithelial origin of the cells was confirmed with cytokeratin AE1/AE3 expression. RMP (in mV), Cm (capacitance in pF), Rm (resistance in G) were measured electrophysiologically with the following solutions: micropipette (in mM) 145 KCl, 1 MgCl2, 0.2 CaCl2, 5 EGTA, 10 HEPES, pH 7.2, 295 mOsm; bath (in mM) 135 NaCl, 10 KCl, 1CaCl2, 1.2 MgCl2, 10 HEPES, 10 glucose, pH 7.2, 300 mOsm. Concentrations used were ATP 10 M, CCh 100 M, ACh 100 M, KCl 30 mM, CaCl2 30 mM, and LPS 0.1 mg/mL.
Source of Funding: NIH R01-DK075728
THE JOURNAL OF UROLOGY姞
493 DEVELOPMENT OF A NEUROGENIC BLADDER IN TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 KNOCKOUT MICE Qi Lei*, Xiao-Qing Pan, Glenolden, PA; Tirsit Asfaw, New York, NY; Joseph Hypolite, Glenolden, PA; Alan Wein, Philadelphia, PA; Anna Malykhina, Glenolden, PA INTRODUCTION AND OBJECTIVES: Bladder pain of unknown etiology has been associated with co-morbid conditions and functional abnormalities in adjacent pelvic organs. This phenomenon is called “crosssensitization” and occurs predominantly via convergent neural pathways connecting distinct pelvic organs. Our previous studies showed that colonic inflammation can cause detrusor instability via activation of transient receptor potential vanilloid 1 (TRPV1) signaling pathways. In this study, we aimed to determine whether cross-sensitization in the pelvis can develop in the absence of TRPV1 receptors and lead to a neurogenic bladder. METHODS: Four groups of adult male mice (N⫽25) were included in the study: 2 groups of C57BL/6 wild-type (WT) mice with and without acute colitis (induced by intracolonic trinitrobenzene sulfonic acid, TNBS) and 2 groups of TRPV1 knockout mice (TRPV1⫺/⫺) mice with and without acute colitis. Mice were sacrificed at day 3 after the induction of pelvic organ cross-sensitization by acute colitis and the function of the detrusor was evaluated in vitro using isolated detrusor smooth muscle (DSM) strips. The level of inflammation in the distal colon and urinary bladder was evaluated by the myeloperoxidase (MPO) assay. The contractility of DSM strips was studied in response to potassium chloride test, electric field stimulation and relaxation of DSM by Rho kinase inhibitor. RESULTS: In WT mice, acute colitis caused 2-fold up-regulation of MPO levels in the colon whereas in TRPV1⫺/⫺ mice an increase reached 1.5 fold (p⬍0.05 to respective control). MPO levels were unchanged in the bladders from both groups. The contractile response of DSM strips to electric stimulation was significantly increased after colitis in WT mice (85.97⫾12.27 g/g) when compared to control group (44.39⫾10.81 g/g, p⬍0.05). The same changes were observed in the TRPV1⫺/⫺ group with pelvic organ cross-sensitization. The use of Rho kinase inhibitor Y27632 induced DSM relaxation in WT group by 72.87⫾10.40% whereas it was only 39.38⫾6.46% in the TRPV⫺/⫺ group (N⫽6, p⬍0.05 to WT). CONCLUSIONS: Our data provide evidence that the absence of TRPV1 receptors does not eliminate the occurence of cross-sensitization in the pelvis and TRPV⫺/⫺ mice develop a neurogenic bladder. However, DSM from TRPV⫺/⫺ mice showed decreased relaxation in response to a Rho kinase inhibitor suggesting alterations in DSM signaling pathways.
Vol. 187, No. 4S, Supplement, Sunday, May 20, 2012
RESULTS: RMP was significantly more depolarized in hBUC compared to rBUC (-14.3 ⫾ 5.7 mV versus -26.2 ⫾ 4.97 mV, p⫽0.02). The Cm (pF) and Rm (G) were significantly different in rBUC versus hBUC (30 ⫾ 16 pF vs. 102 ⫾ 38 pF and 1.0 ⫾ 0.5 G vs. 0.2 ⫾ 0.1 G, respectively, both comparisons p⬍0.05). ATP, CCh, ACh, KCl and CaCl depolarized both rBUC and hBUC without significant differences in RMP changes between the species. LPS hyperpolarized the cells. Representative tracings showing changes in RMP in response to these agents are shown in the Figure. CONCLUSIONS: This work represents the first detailed description of measurement of RMP in hBUC and rBUC and provides reference data for future study on urothelial cell physiology. The pharmacologic agents CCh, ACh and ATP depolarized RMP as expected. Extracellular KCl and CaCl also depolarized the cells. However, hyperpolarization of urothelial cells with LPS is a novel finding.
Source of Funding: This study was supported by the NIH/ NIDDK grants DK077699 and DK077699-S2 (to AM)
494 CHARACTERIZATION OF RESTING MEMBRANE POTENTIAL IN RAT AND HUMAN BLADDER UROTHELIAL CELLS Nora Laaris*, Yan Sun, Toby Chai, Baltimore, MD INTRODUCTION AND OBJECTIVES: The bladder urothelium is traditionally thought to only provide barrier function. However, accumulating data suggest that the urothelium may serve additional function including sensor-transducer role, urothelial-afferent signaling and immune regulation. The measurement of urothelial cell resting membrane potential (RMP) is important as RMP has been implicated in mediating these functions. Changes in RMP in response to extracellular ATP, carbachol (CCh), acetylcholine (ACh), lipopolysaccharide (LPS), potassium (KCl), and calcium (CaCl2) were also analyzed. METHODS: This study was approved by both the IRB and IUCAC. Primary cultures of hBUC from 8 subjects and rBUC from 4 rats were prepared as previously described. Epithelial origin of the cells was confirmed with cytokeratin AE1/AE3 expression. RMP (in mV), Cm (capacitance in pF), Rm (resistance in G) were measured electrophysiologically with the following solutions: micropipette (in mM) 145 KCl, 1 MgCl2, 0.2 CaCl2, 5 EGTA, 10 HEPES, pH 7.2, 295 mOsm; bath (in mM) 135 NaCl, 10 KCl, 1CaCl2, 1.2 MgCl2, 10 HEPES, 10 glucose, pH 7.2, 300 mOsm. Concentrations used were ATP 10 M, CCh 100 M, ACh 100 M, KCl 30 mM, CaCl2 30 mM, and LPS 0.1 mg/mL.
Source of Funding: NIH R01-DK075728