MP30-15 PHARMACOLOGICAL PROFILE OF DA-8010, A NOVEL BLADDER SELECTIVE MUSCARINIC RECEPTOR 3 ANTAGONIST FOR TREATMENT OF OVERACTIVE BLADDER

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respectively (p¼0.05). A significant contractile response of HDSM to PDBu was associated with increased tone in response to muscarinic stimulation but low incidence of spontaneous contractions. CONCLUSIONS: The results of our study provide evidence that the ratio between the peak contractile responses of HDSM contractions to EFS versus KCl is predictive of spontaneous contractile activity present in the bladder wall. When EFS/KCl ratio is higher than 100%, the detrusor tends to be more quiet with no spontaneous contractions but higher sensitivity to muscarinic receptor stimulation. When the EFS/ KCl ratio is lower than 100%, the detrusor shows more spontaneous activity and lower response to muscarinic activation. Source of Funding: The study was supported by the NIH/ NIDDK grant DK095817.

MP30-13 FORMATION OF DOUBLE-STRANDED RNA IN HUMAN BLADDER SMOOTH MUSCLE CELLS REGULATES STRESS KINASES AND CELL STRUCTURE IN OXIDATIVE STRESS Jing-Hua Yang, Zuohui Zhao, Kazem Azadzoi*, Boston, MA INTRODUCTION AND OBJECTIVES: Double-stranded RNA (ds-RNA) is RNA with two complementary strands, which is not readily detected in normal cells but regulates ds-RNA-dependent protein kinase (PKR) in cellular stress conditions. Cells exposed to stress develop defensive responses in which different kinases and proteases are highly regulated. Activation of ds-RNA-dependent stress kinase PKR inhibits protein translation via phosphorylation mechanisms. Our goal was to evaluate ds-RNA formation and determine structural consequences of ds-RNA dependent PKR in cultured human bladder smooth muscle cells (SMC) oxidative stress. METHODS: Cultured primary human bladder SMC were incubated in normoxia (21% oxygen), continuous hypoxia (2% oxygen) and oxidative stress (2% oxygen for 30 minutes followed by reoxygenation with 21% oxygen for one hour) cycling in this manner for 96 hours in a computerized servo-control cell oxycycler system. Cells were then collected and processed for RT-PCR of ds-RNA precursor Alu-RNA, dot blot analysis of ds-RNA formation, western blotting of ds-RNA-dependent PKR, ELISA of oxidative stress markers, and transmission electron microscopy (TEM). RESULTS: Oxidative stress upregulated Alu-RNA expression and led to the formation of ds-RNA in human bladder SMC. Accumulation of ds-RNA resulted in phosphorylation of the stress kinase PKR and produced significant DNA damage. Activation of the ds-RNA/PKR pathway in human bladder SMC was associated with cellular lipid and protein stress markers, cytoskeleton organization and degeneration of the subcellular elements. Ultrastructural consequences of ds-RNA were confirmed by TEM showing disruption of cell structure and widespread organization of subcellular elements. Thickened deformed cell membrane, swollen mitochondria and enlarged ER were found in cells exposed to continuous hypoxia. Markers of cytoskeleton organization characterized by distorted partially lost cell membrane with increased caveolae, enlarged mitochondria with degraded or lost cristae, swollen and splintered ER and increased cytoplasmic lysosomes were evident in cells exposed to oxidative stress. CONCLUSIONS: We report the formation of ds-RNA in oxidatively stressed human bladder SMC. Accumulation of ds-RNA and subsequent phosphorylation of PKR activated the ds-RNA/PKR pathway and led to DNA damage, cytoskeleton organization and degeneration of cellular and subcellular elements. Our data imply ultrastructural consequences of ds-RNA and suggest the role of ds-RNA/ PKR pathway in human bladder SMC cytoskeleton organization and progressive degeneration under the stress conditions. Source of Funding: Supported by Grant BLR&D MERIT 1I01BX001428 from the U.S. Department of Veterans Affairs.

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MP30-14 UROTHELIAL HYPERPLASIA AND REGENERATION AFTER SPINAL CORD INJURY F. Aura Kullmann*, Dennis Clayton, Gerard Apodaca, Irina Zabbarova, Youko Ikeda, Anthony Kanai, Lori Birder, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: The urothelium (UT) normally functions as an efficient barrier to solutes and pathogens and an integral part of a sensory web that can influence bladder function. Spinal cord injury (SCI) is associated with profound changes of the UT including an initial disruption followed by a UT thickening or hyperplasia. In SCI patients, a number of factors including bladder irritation (i.e. chronic infection; use of indwelling catheters) can cause UT hyperplasia, which increases the risk of bladder cancer. Surprisingly, little is known about the temporal order of events that result in UT proliferation following SCI. Here we investigate the time course of UT hyperplasia after SCI as well as changes in structural/junctional proteins necessary for maintaining epithelial integrity. METHODS: Bladders from sham and spinal cord transected C57Bl6 female mice (T8-T9; 1-28 days post SCI) were prepared for histology (hematoxylin and eosin) and immunohistochemistry. Some mice were injected intraperitoneally with bromodeoxyuridine (BrdU; 100mg/kg) prior to sacrificing to assess the extent of UT hyperplasia. RESULTS: Histology showed thicker UT, lamina propria and smooth muscle (SM) starting 2-3 days post SCI. BrdU staining indicated that epithelial proliferation was evident as early as day two post SCI and followed a temporal gradient from the UT to the SM. The number of BrdU+ cells in the UT increased dramatically at day 2 post SCI and declined thereafter. In contrast, the number of BrdU+ cells in the SM was maximal 7 days post SCI and decreased thereafter. P63+ progenitor cells, located in the basal and intermediate layers of the UT, were more numerous in the SCI versus sham at all time points. We also found that claudin-8, a marker for epithelial junctional integrity, was expressed as early as one week post SCI. CONCLUSIONS: The bladder urothelium possesses a remarkable ability to regenerate after SCI. We find that the process of regeneration starts soon after SCI (>2days) and follows a temporal gradient (over approximately two weeks) from the UT to the SM. Though the structural recovery, as indicated by claudin 8 expression, may be achieved in the first two weeks post SCI, the number of progenitor cells was still elevated one month following SCI, suggesting the potential for further remodeling. Future research will investigate factors involved in SCI-associated urothelial hyperplasia. This is an important issue since epithelial hyperplasia may be a predisposition for bladder cancer, which is a serious oncologic complication in SCI patients associated with significant mortality. Source of Funding: P01 DK093424, R37 DK54824, R01 DK54425, P30 DK079307, Winters foundation (FAK), P30 DK079307 Pilot grant(FAK).

MP30-15 PHARMACOLOGICAL PROFILE OF DA-8010, A NOVEL BLADDER SELECTIVE MUSCARINIC RECEPTOR 3 ANTAGONIST FOR TREATMENT OF OVERACTIVE BLADDER Min Jung Lee*, Jun-Hwan Moon, Hyun-Min Park, Hyung Keun Lee, Jong Hwan Cho, Sung Hak Choi, Weonbin Im, Yongin-si, Korea, Republic of INTRODUCTION AND OBJECTIVES: Several antimuscarinics have been commonly used for overactive bladder patients, but dry mouth as a major anticholinergic side effect remains a shortcoming to limit long-term use. DA-8010 is a novel muscarinic receptor 3 antagonist being developed for the treatment of overactive bladder. The objectives of this study were to characterize the pharmacological profile of DA8010 and to compare its bladder selectivity over salivary gland with those of currently used antimuscarinic agents.

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METHODS: The binding affinity of DA-8010 for various receptors was assessed in competitive radioligand binding assays. The antagonistic potency for muscarinic receptors was measured using human M3-overexpressing cells and rat bladder tissues. To assess the functional tissue selectivity in vitro, inhibition on carbachol-induced intracellular calcium increase was measured in bladder smooth muscle cells and salivary gland cells isolated from mice. Inhibitory effects on rhythmic urinary bladder contraction and carbachol-induced salivary secretion were evaluated after intravenous administration of test compounds in anesthetized female SD rats. RESULTS: DA-8010 had a high binding affinity to human muscarinic receptor 3 (hM3) with mean Ki of 1.55 nM and was 30-fold more selective for M3 than for M2 muscarinic receptors. In functional studies, DA-8010 exhibited a great potency for muscarinic receptor antagonism on hM3-overexpressing cells (Ki of 0.25 nM) and rat bladder preparation (KB of 6.8 nM). The inhibitory effect of DA-8010 for bladder smooth muscle cells (Ki¼0.23) was 3.7-fold more potent than that for salivary gland cells (Ki¼0.86 nM) isolated from mice, exhibiting that the rank order of bladder selectivity was DA8010>solifenacin>tolterodine>oxybutynin. In anesthetized female rats, intravenous administration of DA-8010 dose-dependently inhibited rhythmic bladder contractions induced by distension, indicating the most potent activity (ID30 of 0.08 mg/kg) among the antimuscarinics tested. The in vivo functional selectivity of DA-8010 for bladder over salivary gland was 2.4 and 6-fold greater than those observed for solifenacin and darifenacin, respectively. CONCLUSIONS: DA-8010, a novel and potent muscarinic receptor 3 antagonist exerts the superior selectivity for urinary bladder over salivary gland to solifenacin, tolterodine, darifenacin and oxybutynin in preclinical studies. These findings suggest that DA-8010 may be a therapeutic agent having favorable properties with less dry mouth in the treatment of overactive bladder. Source of Funding: none

MP30-16 GENETICALLY MODIFIED HUMAN MUSCLE PRECURSOR CELLS OVEREXPRESSING PGC-1? SUPPORT EARLY MYOFIBER FORMATION FOR BIOENGINEERING OF SLOW TWITCH SPHINCTER MUSCLE €rich, Switzerland; Deana Haralampieva, Souzan Salemi, Zu €rich, Switzerland; Ivana Dinulovic, Basel, Switzerland; Tullio Sulser, Zu €rich, Sweden; Christoph Handschin, Basel, Simon M. Ametamey, Zu €rich, Switzerland Switzerland; Daniel Eberli*, Zu INTRODUCTION AND OBJECTIVES: Muscle precursor cells (MPCs) are quiescent muscle cells capable of muscle fiber reconstruction. However, their quality and quantity gradually decline with age. Therefore, autologous MPC transplantation is envisioned for the treatment of muscle diseases, such as Stress Urinary Incontinence. The goal of this research was to explore the possibility of genetically modifying human MPCs to overexpress PGC-1a, a central factor in muscle exercise adaptation, in order to enhance skeletal muscle formation and quality for application for urinary sphincter regeneration. METHODS: hMPCs were harvested from M. rectus abdominis of patients. After genetically modifying the cells to overexpress PGC-1a (or GFP control), viability, proliferation and myogenic phenotype were evaluated in vitro. The expanded cells were suspended in a collagen carrier and s.c. injected bilaterally on the back of nude mice. One, two and four weeks later the bioengineered skeletal muscle tissues were harvested and further assessed by histology, WB and RTPCR. RESULTS: We were able to confirm the sustained myogenic phenotype of the genetically modified hMPCs. Viability and proliferation potential were not significantly different compared to native cells in vitro. Fiber formation capacity and contractility were enhanced in PGC-1a modified hMPCs in vitro. Subcutaneously injected cell-collagen suspensions were harvested after 1, 2 and 4 weeks and histological analysis confirmed the earlier myotube formation in PGC-1a modified

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samples. Increased contractile protein levels were detected by WB, with a significant initial switch to slow type muscle fibers. CONCLUSIONS: By genetically modifying hMPCs to overexpress PGC-1a we were able to enhance the early myotube formation in vitro and in vivo, thereby developing a novel strategy for improving sphincter muscle tissue engineering for future application. Source of Funding: none

MP30-17 DOWN REGULATION OF BLADDER ENDOCANNABINOID SYSTEM FOLLOWING BLADDER OUTLET OBSTRUCTION IN THE RATS Teng-Lung Lin*, Wei-Ming Cheng, Taipei, Taiwan INTRODUCTION AND OBJECTIVES: Fibrosis is an important factor contributing to bladder dysfunction following bladder outlet obstruction (BOO). Endocannabinoid system has been shown to involve in tissue fibrosis in several organs, such as liver and heart. However, changes of endocannabinoid system and its correlation with fibrosis in BOO are much less investigated previously. So we studied the alteration in the expression of cannabinoid receptor 1 and 2 (CB1 and CB2), as well as fatty acid amide hydrolase (FAAH), an endocannabinoid degradation enzyme following BOO in rats. METHODS: BOO was induced in Adult female Sprague-Dawley rats. Control group received sham operation. The rats were sacrificed four weeks later, and the urinary bladder was removed for further analysis. The degree of bladder fibrosis was quantified by calculating type III to type I collagen ratio with Image software after picosirus red stain under polarized light microscopy. H&E stain and Massons trichrome stain were used to confirm muscle hypertrophy and fibrosis. The urothelium and the detrusor muscle layers were separated for RTPCR analysis of mRNA expression of TGF-b, CB1, CB2, and FAAH. RESULTS: Bladder weight increased significantly four weeks after BOO (BOO 588.9201.4 mg vs. sham 92.515.2 mg, p ¼ 0.002). Profound detrusor muscle hypertrophy was also noticed on H&E and Massons trichrome stain. Fibrosis mainly located at suburothelial layer, and type III/I collagen ratio was increased in BOO group, but it did not reach statistical significance (BOO 57.6  16.4% vs. sham 39.1  21.9%, p ¼ 0.098). Nevertheless, the expression of a fibrotic marker, TGF-b mRNA, was enhanced significantly in urothelial layer. Expression of CB2 and FAAH mRNA was decreased in both urothelial and detrusor layers, while CB1 mRNA declined in urothelial layer only. Expression of CB2 mRNA in both layers, as well as CB1 and FAAH mRNA in urothelial layer were negatively correlated with the bladder weight. CONCLUSIONS: BOO resulted in an increased bladder weight, detrusor muscle hypertrophy and suburothelial fibrosis in female rats. The expression of both CB1 and CB2 was down regulated in urothelial layer, while CB2 was down regulated in detrusor layer. Our results also showed a down regulation of FAAH expression in urothelial and detrusor in BOO-bladders. This study clearly demonstrated the involvement of endocannabinoid system in bladder outlet obstruction. Targeting at endocannabinoid system might be an innovative treatment strategy for BOO-induced bladder dysfunction. Source of Funding: Taipei Veterans General Hospital

MP30-18 ENHANCED ATP RELEASE AND P2X1R EXPRESSION CONTRIBUTE TO BLADDER DYSFUNCTION IN TYPE 2 DIABETES Zongwei Wang*, Vivian Cristofaro, Hongying Cao, Rongbin Ge, Maryrose Sullivian, Aria Olumi, Boston, MA INTRODUCTION AND OBJECTIVES: Diabetes bladder dysfunction (DBD) is one of the major urologic complications associated with type 2 diabetes (DM2). We have shown specific molecular alterations in a mouse model that harbors hepatic insulin receptor substrate