Keiko Nagase

Modulation of Stretch Evoked Adenosine Triphosphate Release From Bladder Epithelium by Prostaglandin E2

PURPOSE We previously reported that cyclooxygenase inhibitors improved storage function in rats with detrusor overactivity caused by cerebral infarction via C-fiber suppression but the precise mechanism underlying this effect remained unclear. In this study we investigated the effects of cyclooxygenase inhibitors on stretch evoked adenosine triphosphate and prostaglandin E(2) release from bladder epithelium. MATERIALS AND METHODS Whole bladders excised from normal rats were fixed vertically in an organ bath filled with Krebs solution. Bladders were infused with 0.3 ml Krebs solution (baseline), followed by 0.9 ml vehicle or 1.5 ml vehicle/drug solution, or 0.3 ml protamine sulfate (Wako Pure Chemical Industries, Osaka, Japan), followed by 0.3 ml prostaglandin E(2) (Nacalai Tesque, Kyoto, Japan). Solutions were allowed to stand for 10 minutes and collected. Adenosine triphosphate and prostaglandin E(2) concentrations were measured by luciferin-luciferase assay and enzyme-linked immunoassay, respectively. RESULTS Adenosine triphosphate and prostaglandin E(2) release from bladder epithelium was increased by distention in volume dependent fashion. A 100 μM dose of the nonselective cyclooxygenase inhibitors FYO-750, ketoprofen and indomethacin significantly suppressed the increased adenosine triphosphate and prostaglandin E(2) release. Inhibition of adenosine triphosphate release by 100 μM FYO-750 and indomethacin was antagonized by prostaglandin E(2) co-injection. Prostaglandin E(2) increased adenosine triphosphate release in a nondistending condition, and the 1 μM of the selective EP1 and EP3 receptor antagonists ONO-8711 and ONO-AE5-599, respectively, significantly suppressed the increased adenosine triphosphate release. CONCLUSIONS Results indicate that cyclooxygenase inhibitors suppress adenosine triphosphate release from bladder epithelium via decreasing prostaglandin E(2). EP1 and/or EP3 receptors appear to participate in this effect.

Long-lasting breaches in the bladder epithelium lead to storage dysfunction with increase in bladder PGE2 levels in the rat

Increase in bladder mucosal permeability can be reproduced by intravesical administration of protamine sulfate (PS); however, the influence of PS once administered into the bladder disappears within several days. We developed a chronic animal model of urothelial injury using PS. Insertion of a polyethylene catheter through the bladder dome was performed in female Wistar rats. The other end of the catheter was connected to an osmotic pump for continuous delivery of PS or vehicle for 2 wk. Urinary frequency (UF) and voided volume (VV) were measured in the metabolic cage. The fifth group of rats received a high dose of PS (10 mg/ml) for 2 wk and were followed for a further 2 wk without PS. The sixth group received a high dose of PS for 2 wk and loxoprofen (0.1 mg.kg(-1).day(-1)) for 4 wk. UF was increased, and VV was reduced in rats treated with a high dose of PS but not changed in rats treated with a vehicle or a low dose of PS (1 mg/ml). UF was further increased in the fifth group, while unchanged in the sixth group. Histological sections in rats treated with a high dose of PS demonstrated a loss of the upper layer of urothelial cells and an increased number of mast cells. PGE2 level in the bladder was significantly elevated in the fifth group. These results indicate that chronic urotherial injury leads to an increase in UF and a decrease in VV. Increased PGE2 level in the bladder is likely to be associated with long-lasting storage dysfunction.

Improvement in Neurogenic Detrusor Overactivity by Peripheral C Fiber's Suppression With Cyclooxygenase Inhibitors

PURPOSE Cyclooxygenase inhibitors decrease micturition frequency in animals with bladder inflammation but to our knowledge the influence of cyclooxygenase inhibitors on detrusor overactivity has not been investigated. We evaluated the effects, and the site and mechanism of action of cyclooxygenase inhibitors on detrusor overactivity induced by cerebral infarction. MATERIALS AND METHODS Cerebral infarcted rats underwent cumulative intravenous administration of the selective cyclooxygenase-1 inhibitor SC-560 (Sigma), the selective cyclooxygenase-2 inhibitor rofecoxib (Kemprotec, Middlesbrough, United Kingdom) or the nonselective cyclooxygenase inhibitor FYO-750 hourly plus a single intravenous administration of SC-560, rofecoxib or SC-560 plus rofecoxib. To evaluate the site of action cerebral infarcted rats underwent single intracerebroventricular or intrathecal administration of FYO-750. To evaluate the mechanism of action FYO-750 was intravenously administered in diuretic rats or cerebral infarcted rats pretreated with resiniferatoxin. RESULTS For cumulative administration SC-560 (0.3 mg/kg), rofecoxib (0.3 mg/kg) and FYO-750 (0.1 to 1 mg/kg) significantly increased bladder capacity. For single administration neither SC-560 (0.03 mg/kg) nor rofecoxib (0.03 mg/kg) affected bladder capacity but SC-560 plus rofecoxib significantly increased bladder capacity vs vehicle. Intracerebroventricular and intrathecal administration of FYO-750 did not affect bladder capacity. FYO-750 did not affect urinary production in diuretic rats and the effects of FYO-750 were blocked by resiniferatoxin except at the highest drug dose. CONCLUSIONS Results indicate that cyclooxygenase inhibitors improve detrusor overactivity caused by cerebral infarction by suppressing peripheral C fibers without affecting urinary production. The nonselective cyclooxygenase inhibitor showed more potent efficiency than the selective cyclooxygenase-1 or the cyclooxygenase-2 inhibitor alone.

Antidiuretic effect of antimuscarinic agents in rat model depends on C‐fibre afferent nerves in the bladder

Antichollnergic agents are anticipated to diminish storage symptoms, as well as nocturia. Nevertheless, the effect of this treatment on polyuria related to nocturia is not clear. By subgroup analysis of the data set from a phase III clinical trial of antimuscarinic agent for OAB patients in Japan, imidafenacin was found to improve nocturia with a reduction in nocturnal polyuria. This study adds the effects and underlying mechanism of antimuscarinic agents decreasing urine production through inhibition of C‐fibre in the bladder of water‐leaded rats.

Effect of Corticotropin‐Releasing Factor Receptor Antagonist on Psychologically Suppressed Masculine Sexual Behavior in Rats

INTRODUCTION Corticotropin-releasing factor (CRF) coordinates various responses of the body to stress, and CRF receptors are important targets of treatment for stress-related disorders. AIM To investigate the effect of a nonselective CRF receptor antagonist, astressin, on suppression of masculine sexual behavior by psychological stress in rats. METHODS First, we investigated the influence of psychological stress, induced 2 hours per day for three consecutive days, on sexual behavior. Then, rats were divided into 4 groups: a control group, an astressin administration group (A), a psychological stress loading group (PS), and a psychological stress loading and astressin administration group (PS + A). The rats were exposed to sham or psychological stress for three consecutive days. After the last stress loading, the rats were injected with vehicle or astressin, and their sexual behavior was observed. We also measured serum levels of adrenocorticotropic hormone (ACTH). MAIN OUTCOME MEASURE The effects of astressin on sexual behavior and serum levels of ACTH in rats affected by psychological stress were determined. RESULTS Sexual behavior was reduced after psychological stress loading. The PS rats had significantly longer mount, intromission, and ejaculation latencies and lower ejaculation frequency than did the control, A, and PS + A rats. The intromission latency and ejaculation frequency in the PS + A rats did not achieve the level observed in the controls. There was no significant difference in these parameters between the control and A rats. Serum ACTH levels were significantly lower in PS + A rats than in PS rats. CONCLUSIONS Psychologically suppressed masculine sexual behavior could be partially recovered with astressin administration in rats. These data provide a rationale for the further study of CRF receptor antagonists as novel agents for treating psychological sexual disorders.

Co-administration of an α1-blocker improves the efficacy and safety of antimuscarinic agents in rats with detrusor overactivity

Objective:  To investigate the efficacy and safety of a combination treatment with α1‐blockers and antimuscarinics for detrusor overactivity in rats.

Monoclonal antibody RM2 as a potential ligand for a new immunotracer for prostate cancer imaging.

OBJECTIVES To investigate the potential of monoclonal antibody (mAb) RM2 as a ligand for a radioimmunotracer for prostate cancer imaging. METHODS Labeling was conducted with mAb RM2 and (125)I using the chloramine-T method. The cell study was conducted with PC-3 and LNCaP, which are prostate cancer cell lines, and MCF-7, which is a breast cancer cell line. The cells were treated or untreated with unlabeled mAb RM2 to block the haptoglobin-β chains expressed on the surface of the prostate cancer cells. (125)I-mAb RM2 was added into the cell culture media and cellular uptake of (125)I-mAb RM2 was evaluated at 1, 3 and 6 hours of incubation. For the in vivo biodistribution study, PC-3 cells were implanted in athymic male mice. The animals were injected intravenously with (125)I-mAb RM2. At 24, 48 and 72 hours after tracer injection, the animals were sacrificed and the activity levels of blood and tissue samples were determined. RESULTS The uptake of (125)I-mAb RM2 in the PC-3 and LNCaP cells increased according to the incubation time, while the uptake of (125)I-mAb RM2 in MCF-7 cells did not show any increase up to 6 hours. The increase of (125)I-RM2 uptake was not observed when the PC-3 and LNCaP cells were pre-treated with unlabeled RM2. In the biodistribution studies, (125)I-mAb RM2 showed marked uptake into the implanted PC-3 cells. In PC-3 tumor-bearing mice, the tumor muscle ratio of (125)I-RM2 was increased for up to 72 hours in a time-dependent manner. CONCLUSIONS (125)I-mAb RM2 showed excellent prostate cancer cell targeting in vitro and in vivo. Therefore, mAb RM2 seems to be a potential candidate for an immunoligand for prostate cancer imaging.

CYCLOOXYGENASE INHIBITORS IMPROVE NONINFLAMMATORY OVERACTIVE BLADDER CAUSED BY CEREBRAL INFARCTION BECAUSE OF SUPPRESSION OF PERIPHERAL C-FIBER

is unclear. This study aims to investigate the direct effects of BoNT/A on sensory afferent firing in an ex vivo mouse bladder preparation. METHODS: Adult male mice were sacrificed humanely according to UK legislation. The whole pelvic region was dissected out and placed in a recording chamber superfused with oxygenated Krebs-bicarbonate solution (95% O2, 5% CO2) at 35°C. The urethra and dome were catheterised. Recording of afferent nerve firing was performed by placing a pelvic sensory nerve bundle into a suction electrode, as previously described (Daly et al, 2007). This preparation enabled simultaneous sampling of intravesical pressure and multiunit afferent nerve firing. Ramp distension (100μl.min-1) of the bladder with saline (NaCl, 0.9%) up to 40 mmHg was repeated at 10 minute intervals. To determine the effect of BoNT/A, 2U (BOTOX®) was applied to the bladder lumen during distension, preceded and followed by the control (saline) distension protocol. The effects of BoNT/A was monitored for over 2 hours post application. Data are presented as normalised to the response prior to application of BoNT/A and presented as mean ± S.E.M. Statistical analysis was carried out using either a 2-way ANOVA followed by a Bonferroni post test or paired Students t-test. RESULTS: Ramp distension of the bladder evoked a graded increase in afferent firing with increasing intravesical pressure, which was reproducible following repeated distensions with saline. Following addition of BoNT/A, the afferent response was significantly attenuated after 1 hour. At 2 hours, the maximum afferent firing at 40 mmHg was reduced by 75.9 ± 12.2% (control: 116 ± 13.6 vs. BoNT/A: 24.1 ± 12.2, P=0.0016, n=5). A 2-way ANOVA revealed statistically different afferent responses > 15 mmHg. Interestingly, bladder compliance was not affected after application of BoNT/A (P=0.38). CONCLUSIONS: These results indicate that intravesical BoNT/A directly attenuated distension evoked sensory afferent nerve firing in vitro. BoNT/A application did not affect bladder compliance. Further studies are required to determine how BoNT/A affects mechano-sensory pathways in the bladder. Daly D et al. J Physiol 2007 Sep;583(2):663-674

MP12-04 DETRUSOR OVERACTIVITY INDUCED BY CEREBRAL INFARCTION MAY BE ASSOCIATED WITH ENHANCED ACTIVITY OF RHO-KINASE IN THE BLADDER

bladder catheter through the dome. In the CMG measurements in a decerebrated condition, mice underwent cystostomy formation and supracollicular decerebration under isoflurane-anesthesia. After recovery from the anesthesia (waiting for at least 2 hours), CMG was carried out with saline-instillation at 10 ml/min. RESULTS: In the FV measurements, in females, mean voided volume and mean flow rate in TRPM2-KO mice were significantly greater than those in WT mice. In males, similar tendencies were observed, but there were no significant differences (n 1⁄4 10, in each group). In the conscious CMG measurements, in females, threshold pressure, micturition pressure, intercontraction interval and mean voided volume in TRPM2-KO mice were significantly greater than those in WT mice. In contrast, in male mice, only threshold pressure in TRPM2-KO mice was significantly higher than that in WT mice (Figure 1A, n 1⁄4 8 in each group). In the CMG measurements in the decerebrated condition, only threshold pressure in TRPM2-KO mice was significantly higher than that in WT mice in both sexes (Figure 1B, male TRPM2-KO n 1⁄4 7, other groups n 1⁄4 8). CONCLUSIONS: The present study suggests that TRPM2 channels may have a role in modulating bladder mechano-sensation in mice, and also that this contribution might be more remarkable in females.