810 EFFECTS BY THE GNRH ANTAGONIST GANIRELIX ON NORMAL MICTURITION AND PGE2-INDUCED DETRUSOR OVERACTIVITY IN CONSCIOUS FEMALE RATS

Amplitude before GA (mg)

533±406

666±493

864±427

1072±613

Amplitude after GA (mg)

458±401

524±427

410±186

541±339

Frequency before Oxy (sec)

0.34±0.041

0.48±0.15

0.69±0.24

0.80±0.24

Frequency after Oxy (sec)

0.46±0.072

0.59±0.20

0.83±0.27

0.91±0.26

Frequency before GA (sec)

0.35±0.054

0.36±0.08

0.46±0.19

0.69±0.25

Frequency after GA (sec)

0.37±0.053

0.41±0.10

0.55±0.21

0.76±0.24

Connexin 43 release (density/ mm2)

99.11±44.33

109.69±25.42

167.08±35.57

127.39±50.80

Oxy: Oxybutinin, GA: 18 alpha GA, BOO: Bladder Outlet Obstruction Conclusions: Although previous studies have shown the effects of estrogen on uterine gap junctions and connexin-43 levels, oophorectomy had no effect in the expression of connexin-43, gap junctions in bladder and bladder overactivity, as well. Therefore obstruction, as the main factor increases the amount of gap junctions so gap junction blockers are more effective in the obstruction groups.

808

2-Aminoethoxydiphenyl borate inhibits calcium sensitization induced by carbachol in contraction of human urinary bladder smooth muscle

Shahab N., Kajioka S., Seki N., Naito S. Kyushu University, Dept. of Urology, Fukuoka, Japan Introduction & Objectives: We investigated the effect of 2-aminoethoxydiphenyl borate (2-APB) in Ca2+ sensitization using α-toxin permeabilized strips obtained from human detrusor smooth muscle (DSM). Materials & Methods: Tissue specimens were obtained from the bladder in males undergoing cystectomy due to bladder cancer. After removing mucosa and connective tissues, the smooth muscle was dissected into small strips (300-400 μM in diameter, 3-4 mm in length) and then mounted between 2 tungsten wires, one of which was attached to a force transducer in a Perspex block with 100 μl physiological saline solution buffered by HEPES-Tris under a resting of 100 mg. Permeabilization was done in relaxing solution containing 5,000 U/ml α-toxin for 1 hour. The effect of 2-APB in carbachol (CCh)-induced Ca2+ sensitization was studied by the application of 100μM 2-APB during sustained contraction induced by 1 μM Ca2+, 100 μM GTP and 10 μM CCh. The relative predominant effect of 2-APB on Rho-kinase (ROK) pathway and protein kinase C (PKC) pathway was further investigated using specific inhibitor of PKC, bisindolylmaleimide I (GF109203X; 5μM) and specific inhibitor of ROK (Y-27632; 5μM). Low molecular weight of IP-3 inhibitor, heparin (5 mg/ml) was used to confirmed the Ca2+ independent effect of 2-APB. The experiment was carried out with cyclopiazonic acid (CPA; 1μM) present in all solutions after permeabilization at room temperature.

than that after pre-application with 5 μM GF-109203X (10.1% ± 0.4%; n=3) with P<0.001. The effect of 2-APB still exist after pre-application of 5 mg/ml heparin. There was no inhibitory effect of 2-APB in the absence of CCh (Fig.1D). Conclusions: 2-APB can relax the CCh-induced DSM contraction of human by decreasing the Ca2+ sensitivity through both PKC and ROK pathway but predominantly through ROK pathway. The inhibition of Ca2+ sensitization pathways might represent an alternative target in the treatment of overactive bladder.

809

Chronic hyperlipidemia causes detrusor overactivity and increase in urinary prostaglandin E2 release in rabbits

Yoshida M.1, Masunaga K.2, Nagata T.3, Miyamoto Y.1, Haba T.1, Kudoh J.1 1 Kumamoto Rosai Hospital of Japan Labor Health and Welfare Organization, Dept. of Urology, Kumamoto, Japan, 2Tokyo Metropolitan Geriatric Hospital, Dept. of Urology, Tokyo, Japan, 3Social Insurance Saitama Hospital, Dept. of Urology, Saitama, Japan Introduction & Objectives: Life style diseases and bladder ischemia has been suggested as important etiological factors in overactive bladder. Hyperlipidemia is a well known risk factor for development and progression for cardiovascular diseases. Recently, we demonstrated that chronic hyperlipidemia caused bladder dysfunction including detrusor overactivity in heritable hyperlipidemic rabbits (myocardial infarction-prone Watanabe Heritable Hyperlipidemic rabbits: WHHLMI rabbits). Prostaglandins are synthesized locally in both bladder smooth muscle and mucosa and the synthesis is initiated by various pathologic conditions. In the present study, to evaluate the effects of hyperlipidemia on lower urinary tract function, we examined the relationship between hyperlipidemia-induced bladder dysfunction and urinary prostaglandin E2 (PGE2) level in WHHLMI rabbits. Materials & Methods: WHHLMI rabbits and the age and sex-matched Japanese white rabbits (control group) were prepared. All rabbits were housed in metabolic cage, and the volume and the frequency of micturition was recorded for three days, and 24-h urine samples of all rabbits were collected. Cystometrograms were performed under anaesthesia using constant infusion of saline into the bladder to elicit voiding, and voided volume, residual urine, micturition pressure, and micturition interval were evaluated. 24-h urinary PGE2 levels were assayed by enzyme immunoassay (EIA) by using PGE2 EIA kit-Monoclonal (Cayman Chemical). In addition, immunohistochemical staining of COX-2 of bladder tissue of WHHLMI and control rabbits was performed, using mouse monoclonal antiCOX-2 antibody. Results: The number of micturition per day was higher in WHHLMI rabbits (4.24±1.07 times/day) than in control rabbits (1.8±0.3 times/day), and the voided volume was lower in WHHLMI rabbits (14.81±4.60 ml) than in control rabbits (43.8±5.1 ml). In cystometrograms, WHHLMI rabbits showed detrusor overactivity, higher frequency of micturition (7.55±1.25 times/20 min) and lower voided volume (6.92±1.5 ml) than in control rabbits (1.44±2.51 times/20 min and 20.8±3.2 ml, respectively). The urinary excretion of PGE2 was significantly higher in WHHL rabbits (6.44±1.87 ng/ml) than in control rabbits (0.91±0.14 ng/ml). Analysis of relationship between parameters of bladder function and urinary PGE2 level in WHHLMI rabbits indicated a significantly negative correlation (r=0.81) between urinary PGE2 level and voided volume, and a significant positive correlation between urinary PGE2 level and the number of micturition (r=0.86). Immunohistochemical staining of COX-2 showed a significant higher immunoreactivity in bladder mucosa of WHHLMI rabbits, as compared to control. Conclusions: The present study suggested that chronic hyperlipidemia caused increase of COX-2 activity in bladder mucosa, which might cause the enhanced PGE2 release, resulting in activation of sensory input and detrusor overactivity.

810

Effects by the GnRH antagonist ganirelix on normal micturition and PGE2-induced detrusor overactivity in conscious female rats

Russo A.1, Castiglione F.1, Salonia A.1, Benigni F.1, Andersson K.E.2, Hedlund P.1 1 Urological Research Institute, Dept. of Urology, Milan, Italy, 2Wake Forest University, Dept. of Regenerative Medicine, Winston Salem, United States of America

Results: The application of 100 μM 2-APB during sustained contraction-induced by 10 μM CCh in the presence of 100 μM GTP at constant 1 μM [Ca2+]i caused marked relaxation (40.9 ± 2.5%; n=4) compared to time matched control (16.3 ± 0.9%; n=3) with P<0.001 (Fig.1A). This relaxation was decreased by 5 μM GF109203X or 5 μM Y-27632 (Fig 1B). The relaxation effect of 100 μM 2-APB after pre-application with 5 μM Y-27632 was significantly smaller (3.5 ± 0.5%; n=5)

Eur Urol Suppl 2011;10(2):256

Introduction & Objectives: The gonadotropin-releasing hormone (GnRH) may be involved in the control of micturition. The aim was to study effects of ganirelix, a GnRH receptor antagonist, on bladder function and experimental detrusor overactivity (DO) in female rats. Materials & Methods: After ethical approval, Sprague-Dawley rats were given ganirelix (0.1 mg/kg; n=12) or saline vehicle (controls; n=9) subcutaneously once daily. At two weeks, bladder function and responses to intravesical (ives) PGE2 (50µM) were studied with cystometry in conscious rats (Table). Separately, 5 rats were given ganirelix (1.2mg/L) ives. Effects by carbachol (0.01µM-100µM), ganirelix (1nm-1µM) or activation of nerves were studied in isolated detrusor.

elicited contraction by all chloride transport inhibitors and chloride channel blockers (ED80 vs. IC50) were plotted in Figure 1. Conclusions: This study demonstrates that alteration of ECl or substitution of extracellular Cl(-) by Br(-) or I(-) can inhibit the contractility of rat bladder SM induced by KCl. Interference with either the distribution of chloride across the membrane or the ability of chloride channels to open markedly suppresses contractile responses of rat bladder SM to norepinephrine. These results imply in advance that both CLC-3 chloride channel and calcium-dependent chloride channel are of functional importance in the regulation of bladder smooth muscle tone. Results: Bladder and body weights were similar in both groups. Plasma levels of LH were 1.71±0.49ng/ml (controls) and 0.48±0.16ng/ml (ganirelix; p<0.05). Baseline micturition interval (MI), micturition volume (MV), residual volume (RV) bladder capacity (BC), pressure parameters or area under the curve (AUC) were similar in both groups. PGE2 (ives) reduced MI, MV, and BC, and increased basal pressure (BP), threshold pressure (TP), flow pressure (FP), maximum pressure (MP) and AUC of all rats, but changes were larger in controls. For MI, MV, and BC, reductions of 43±4%, 50±4%, and 43±4% (controls) vs. 22±3%, 23±3%, and 21±3% (ganirelix; p<0.001) were recorded. TP and FP increased by 38±8%, and 30±4% in controls and by 16±7% and 16±5% in ganirelix rats (p<0.05). The AUC was 46±7 and 27±6 cmH2O/sec for controls and ganirelix rats (p<0.05) after intravesical PGE2. Compliance was unaffected by ives PGE2 in ganirelix rats but reduced 163±54 % in controls (p<0.05). The Emax to carbachol (10µM) was 231±14% of K+ in detrusor from ganirelix rats compared to 177±24% of K+ for controls (p<0.05) but nerve-contractions were similar. Ives ganirelix increased MI, MV and BC but decreased BP, TP, FP, and MP. Ganirelix had no effect on isolated detrusor contractions by carbachol or by activation of nerves. Conclusions: Systemic treatment with ganirelix counteracted experimental DO in female rats. As bladder preparations from these rats exhibited larger contractions to carbachol and as intravesical ganirelix affected both micturition intervals and urodynamic pressure profiles, a site of action of ganirelix in the urinary bladder may be considered.

811

In vitro functional study of chloride channels on rat bladder smooth muscle

812

Phosphodiesterase type 5 distribution in the guinea pig urinary bladder

Rahnama'i M.S.1, Ona S.2, Van Kerrebroeck Ph.E.V.1, Gillespie J.I.3, Van Koeveringe G.A.1 1 Maastricht University Medical Centre, Dept. of Urology, Maastricht, The Netherlands, 2The Buckman College, Dept. of City University, New York, United States of America, 3The Urophysiology Research Group, Dept. of Newcastle University, Newcastle upon Tyne, United Kingdom Introduction & Objectives: The human bladder is known to express phosphodiesterase type 5 (PDE 5). Recent studies have suggested PDE5 inhibitors, such as Sildenafil and Vardenafil, to improve lower urinary tract symptoms in men. How PDE5 inhibitors effect micturition remains unclear. The current study of the PDE5 distribution in the guinea pig urinary bladder aims to find a clue on the possible site of action of PDE5 in the bladder. Materials & Methods: Six male guinea pig bladders were dissected and treated in 2 ml Krebs’ solution and 10 μM of the specific PDE5 inhibitor, Vardenafil at 36°C for 30 minutes. After stimulating tissues with 100 μM of the NO donor diethylamineNONOate for 10 min, the tissues were snap frozen and cut in 10 μm sections. Then, sections were examined for cGMP immuno-reactivity, co-stained with either Vimentin, a marker for interstitial cells, or the nerve markers, neurofilament (NF), synaptic vesicle protein 2 (SV2), calcitonin gene related peptide (CGRP) and protein gene product 9.5 (PGP 9.5), using the two-step indirect immunohistochemistry technique.

Kuo Y.C.1, Hsieh J.T.2 Taipei City Hospital, Dept. of Urology, Taipei, Taiwan, 2National Taiwan University Hospital and School of Medicine, National Taiwan University, Dept. of Urology, Taipei, Taiwan 1

Introduction & Objectives: Ion channels have been proved to be of functional importance in the regulation of bladder smooth muscle (SM) tone. The role of chloride channels on the bladder SM tissue has not been elucidated. We investigated the physiological role of Cl(-) currents on the maintenance of bladder SM tone in isolated rat bladder tissues. Materials & Methods: Bladder smooth muscle tissue strips (2 x 2 x 10 mm) were suspended in tissue bath chambers for isometric tension experiments. Contractions elicited by administration of KCl were examined in the condition of changing concentration of extracellular chloride (ECl) from 138 mM to 8 mM and substitution of extracellular Cl(-) to Br(-) or I(-). Contractions elicited by administration of norepinephrine (NE) were examined in the presence of chloride transport inhibitors: bumetanide (BUM), 4-(2-hydroxyethyl)-1-1- piperazine ethanesulphonic acid (HEPES) without bicarbonate, ethacrynic acid (ETH), and chloride channel blockers: 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), anthracene-9-carboxylic acid (A9C) and niflumic acid (NFA) (All concentration: 10^-8M~1M).

Results: In bladder SM strips the KCl induced contractility was significantly lowered as the concentration of ECl changed from 138 to 8 mM (p<0.01). The KCl elicited contractile response was also significantly lowered as the extracellular Cl(-) was substituted by Br(-) or I(-) (p<0.01). In addition, pre-treatment with BUM, HEPES without bicarbonate, or ETH could significantly suppress the NE induced contractility in a concentration dependent manner (all p<0.01). Pre-treatment with DIDS, A9C or NFA could also significantly reduce the NE elicited contractile response in a concentration dependent manner (all p<0.01). The inhibitions of NE

Results: After PDE5 inhibition, umbrella cells of the urothelium stain strongly positive for cGMP (red) as shown in figure A. Moreover, a population of Vimentin positive interstitial cells (green) are also positive for cGMP (red). in figures C and D, a few blood vessels are visible expressing strong cGMP signal (red) after PDE5 inhibition. Double stainings of cGMP with the nerve marker SV2, the motor neuron marker NF, afferent nerve marker CGRP and the general neuronal marker PGP 9.5, reveal that all the nerve fibers are negative for cGMP after PDE5 inhibition (figures B-D). Conclusions: Our data reveal expression of cGMP in the urothelium, suburothelial interstitial cells and blood vessels after PDE5 inhibition. This implies that PDE5 is present in these regions of the bladder wall. Furthermore, by comparing cGMP staining with several afferent and efferent neuronal markers as well as Vimentin, it was found that cGMP is only expressed in the urothelium and in the majority of interstitial cells and not in the sensory and motor nerves. This implies that PDE5 inhibition would have its effect through the NO mediated cGMP system in the interstitial cells, rather than a direct effect on neuronal activity.

813

Contribution of non-neuronal adenosine triphosphate release from bladder mucosa to detrusor overactivity in hydrochloric acid (HCl)-induced cystitis rats

Yoshida M.1, Nagata T.2, Masunaga K.3, Inadome A.4, Miyamoto Y.1, Haba T.1, Kudoh J.1

Eur Urol Suppl 2011;10(2):257