495 VNUT (VESICULAR NUCLEOTIDE TRANSPORTER) PLAYS A CRUCIAL ROLE IN STRETCH-EVOKED ATP RELEASE FROM UROTHELIUM Hiroshi Nakagomi*, Tsutomu Mochizuki, Tatsuya Miyamoto, Mitsuharu Yoshiyama, Yamanashi, Japan; Isao Araki, Shiga, Japan; Yoshinori Moriyama, Okayama, Japan; Schuichi Koizumi, Masayuki Takeda, Yamanashi, Japan INTRODUCTION AND OBJECTIVES: ATP from urothelium plays an essential role for afferent nerve stimulation via activation of P2X receptors in suburothelial afferent nerve plexus. However, molec- ular mechanisms and pathways of ATP release are largely unknown. We confirmed VNUT (Vesicular Nucleotide Transporter; PNAS 2008), plays an important role in vesicular storage of ATP in ATP-secreting cells, localized in Human and Mouse bladder urothelium. Here we have investigated the relationship between VNUT and Human clinical symp- toms and urodynamic parameters, and the function of VNUT in mouse urothelial cells upon stretch stimulation. METHODS: Human bladder specimen (15 male patients) and C57BL mice were used. (1) RT-PCR (2) Immunostaining;using an anti-VNUT antibody in bladder specimen and primary culture cells. (3) Visualization of the real-time dynamics of ATP containing vesicles like exocytosis. (4) Photon imaging of ATP release;counting released ATP from control siRNA and VNUT siRNA treated urothelial cells upon mechanical stretch stimulation. (5) Com- parison examination between symptomatic/urodynamic parameters and mRNA expression of VNUT in human urothelium. RESULTS: VNUT was highly expressed in the epithelium of human (fig.1) and mouse bladder tissue and primary urothelial cell culture. We also visualized the ATP-vesicles and their real-time exo- cytosis. Knockdown of VNUT in primary culture cells markedly reduced ATP release upon mechanical stretch stimulation (fig.2). VNUT RNA expression was negative correlation with FDV (First Desire to Void) (fig.3). CONCLUSIONS: VNUT plays an important role in stretch-elicited ATP release from urothelium. These findings suggest that urothelium should transmit ATP to afferent neurons by a mechanism of exocytosis. Source of Funding: Grant-in-Aid for Scientific Research, Grant-in-Aid for Young Scientists(B) from JSPS 496 THE ROLE OF SOMATOSTATIN RECEPTOR SUBTYPE-4 IN MICTURITION REFLEX IN URETHANE-ANESTHETIZED RATS Masashi Honda*, Seiya Inoue, Nobuyuki Hinata, Tadahiro Isoyama, Takehiro Sejima, Atsushi Takenaka, Yonago, Japan; Michael Chancellor, Royal Oak, MI; Naoki Yoshimura, Pittsburgh, PA INTRODUCTION AND OBJECTIVES: Somatostatin binds to G-protein-coupled membrane receptors (sst1-sst5). Recent studies have demonstrated that somatostatin has an important role in neuro- modulations such as pain control, presumably via somatostatin sub- type-4 (sst4) receptor. However, it is not known whether sst4 receptor has a role in the control of the micturition reflex. The therapeutic value of native somatostatin is limited by its broad range of effects, mediated by five different receptor subtypes and its short plasma half life. However, potent and stable somatostatin receptor agonists acting selectively on sst4 receptor have been synthesized and investigated. Therefore, we examined the effects of activation of sst4 receptor on the micturition reflex in rats. METHODS: Continuous cystometrograms (CMG, 0.04ml/min infusion rate) were performed in female Sprague-Dawley rats (242-265 g) under urethane anesthesia. After stable micturition cycles were established, a selective sst4 receptor agonist, NNC 26-9100, was administered intravenously in normal rats or rats pretreated with cap- saicin 4 days before the experiments. Micturition parameters were recorded and compared before and after drug administration. RESULTS: Intravenous administration of NNC 26-9100 at 10, 30, 100 and 300 g/kg (n=6 per dose) increased intercontraction intervals at doses of 30 g/kg or higher in a dose-dependent fashion to 100.8  1.1%, 116.4  9.1%, 137.4  7.5% and 165.1  17.9% of the control value, respectively (at 30, 100 and 300 g/kg, p0.01). Intra- venous administration of NNC 26-9100 at 10, 30, 100 and 300 g/kg (n=6 per dose) also increased pressure threshold at doses of 30 g/kg or higher in a dose-dependent fashion to 7.16  1.23 cm H 2 O, 10.49  1.41 cm H 2 O, 12.61  1.17 cm H 2 O and 17.34  1.95 cm H 2 O, Vol. 187, No. 4S, Supplement, Sunday, May 20, 2012 THE JOURNAL OF UROLOGY e203