1129 PROTEIN KINASE-A DEPENDENT PHOSPHORYLATION OF NEURONAL NITRIC OXIDE SYNTHASE: NOVEL MECHANISM FOR REGULATION OF NEURONAL NITRIC OXIDE SYNTHASE ACTIVATED PENILE ERECTION Sena F. Sezen*, K. Joseph Hurt, Biljana Musicki, Gwen Lagoda, Solomon H. Snyder, Arthur L. Burnett, Baltimore, MD INTRODUCTION AND OBJECTIVES: Penile erection is medi- ated by nitric oxide (NO) that is formed by both neuronal NO synthase (nNOS) and endothelial NOS (eNOS) in the penis. We have previously shown that eNOS is phosphorylated by protein kinase (PK)B/Akt in response to increased blood flow in the penis, leading to activation of eNOS and maintenance of erection. This study investigates the regu- lation and mechanism of nNOS activation by phosphorylation using rodent models of penile erection. METHODS: Male Sprague-Dawley rats (300 –375g) or 8 –10 weeks old C57BL6/J (wild type, wt) and nNOS knockout (ko) mice were used. Penile erection was monitored as intracavernous pressure (ICP) via a needle inserted to right corpus cavernosum. For electrically- induced penile erection studies, cavernous nerve (CN) or major pelvic ganglion (MPG) was stimulated at various stimulation parameters and durations. Inhibition of nNOS phosphorylation was tested after perig- angliar injection of Akt inhibitors [LY294002 (LY, 50 uM) and wortman- nin (WT, 1uM)] and PKA inhibitors [H-89 (30 uM) and PKA inhibitor peptide (PKA-I, 60uM)]. For pharmacologically-induced erections, pa- paverine (1.5mg), forskolin (FSK, 0.25-5ug) or deoxy-FSK (dFSK, 0.25-5ug) were injected intracavernosally (ic) or directly beneath the MPG. Some mice were pretreated with L-NAME (100mg/kg, ip) 30 min prior to FSK injection. MPG and penis from various experiments were collected and processed for phospho(P)-protein analysis with western blot or for immunohistochemistry. RESULTS: P-specific antibody to nNOS-S1412 (homologous to S1177 in eNOS) revealed a 5–10 fold increase in P-nNOS-S1412 after CN electrical stimulation (ES) in rat MPG (p0.05). nNOS-S1412 phosphorylation was voltage- and time-dependent and sustained for 3–5 min after ES. Papaverine, a direct vasorelaxant, injected ic stimu- lated P-eNOS-S1177 in the rat penis, but did not increase P-nNOS- S1412. Perigangliar injection of FSK, an activator of PKA, increased P-nNOS-S1412, while Akt inhibitors, WT and LY, had no effect. Fur- thermore, ic injection of FSK dose-dependently increased ICP in wt mice (p0.05) while d-FSK, an inactive analog of FSK, had no effect. Pretreatment with L-NAME (100mg/kg, ip) abolished the effect of FSK in wt. In nNOS-ko mice, the ICP increase after FSK injection was 30 –50% of the wt response. CONCLUSIONS: Our data suggests a novel mechanism of nNOS activation which involves PKA-dependent phosphorylation and reveals differentially regulated pools of NOS activity mediating penile erection. Source of Funding: RO1DK067223 1130 STREPTOZOTOCIN-INDUCED TYPE 1 DIABETES IN RAT ALTERS SMOOTH MUSCLE MYOSIN ISOFORM COMPOSITION TO A MORE CONTRACTILE PHENOTYPE AND INCREASES SENSITIVITY TO THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN Xinhua Zhang*, Camden, NJ; Arnold Melman, Bronx, NY; Michael DiSanto, Camden, NJ INTRODUCTION AND OBJECTIVES: The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and the 17 kDa essential light chains (LC17) exist as several different isoforms with distinct contractile properties, produced via alternative splicing mech- anisms. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed both rat and human corpus cavernosum smooth muscle (CCSM)(J Sexual Med, 2009). In this study, we determine if diabetes alters SMM expression, alternative splicing and/or functional activities including sensitivity to BLEB. METHODS: Fisher 344 male streptozotocin (STZ)-induced di- abetic rats were kept for 2 months. Functional activities were then tested in vivo by intracavernous pressure (ICP) recording and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin (a tran- scriptional regulator of SMM) and embryonic SMM (SMemb) expres- sion by Real-time RT-PCR. RESULTS: Average blood glucose levels of STZ rats were 407 vs 129.5 mg/dl in rats injected with saline (SHAM). STZ rats showed severe erectile dysfunction (ED) confirmed by decreased ICP response to electrical stimulation of cavernous nerve. For STZ rats, LC17a and SM-B isoforms (both associated with increased contractility and faster shortening velocity) and total SMM and myocardin expression in- creased while LC17b and SM-A isoforms (both associated with de- creased contractility and slower shortening velocity) were decreased with SM1/2 SMM MHC isoforms and SMemb unchanged. Interestingly, BLEB was more effective in relaxing STZ CCSM both in vitro and in vivo. CONCLUSIONS: We show for the first time to our knowledge a diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype is further supported by total SMM expression increase. Moreover, we provide novel data that the change in CCSM phenotype is associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED with specificity to the faster type myosin isoforms found in the diabetic rat CCSM. Source of Funding: None 1131 IN-VITRO STUDIES INVESTIGATING THE ROLE OF PERIPHERAL SEROTONERGIC PATHWAY ON THE NORMAL AND DIABETIC HUMAN ERECTILE PROCESS David Hua, Wu Lau*, Singapore, Singapore; Faiz Mumtaz, London, United Kingdom; Dimitri Mikhailidis, london, United Kingdom; Cecil Thompson, London, United Kingdom INTRODUCTION AND OBJECTIVES: We have previously demonstrated serotonin(5-HT)-mediated normal human corpus caver- nosal (CC) contraction via 5-HT 1A, 2A & 4 receptor subtypes. We aim to study the effect of serotonin and its antagonists on diabetic CC tissue using organ bath and to further investigate the presence of the recep- tors in normal and diabetic CC tissue using immunohistochemistry and western blotting techniques. METHODS: The effect of 5-HT, distilled water and 5-HT-medi- ated CC contractile inhibition by ketanserin (10 –5 M; 5-HT 2A receptor antagonist) on the diabetic human CC strips were assessed using organ bath. Receptology studies to determine the presence of 5-HT receptor subtypes (1A, 2A, 1B & 4) on both normal and diabetic CC tissue were performed using immunohistochemistry and western blot- ting techniques. RESULTS: Organ bath study on diabetic human CC strips (n=10) revealed similar 5-HT mediated contraction (median 32.2 mg/ mg, range 8.3–163.5 mg/mg; p  0.05) and ketanserin induced inhibi- tion of the 5-HT response (by 83.2%, p  0.001; median 5.4 mg/mg, range 1.5–32.9 mg/mg; p  0.05) as compared with those of normal human CC strips, which were previously demonstrated. The presence of 5-HT 1A, 2A,1B & 4 receptor subtypes in normal and diabetic human CC tissue were demonstrated using immu- nohistochemistry and western blotting techniques. CONCLUSIONS: 5-HT mediated contraction is similar in both normal and diabetic human CC tissue via 5-HT 1A, 2A, 1B & 4 receptor subtypes. The 5-HT inhibitory response with ketanserin on the diabetic CC strips is also similar to that on the normal human CC strips. This e454 THE JOURNAL OF UROLOGY Vol. 185, No. 4S, Supplement, Monday, May 16, 2011