1129 PROTEIN KINASE-A DEPENDENT PHOSPHORYLATION OF NEURONAL NITRIC OXIDE SYNTHASE: NOVEL MECHANISM FOR REGULATION OF NEURONAL NITRIC OXIDE SYNTHASE ACTIVATED PENILE ERECTION

Abstract

INTRODUCTION AND OBJECTIVES: Penile erection is mediated 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 regulation 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 electricallyinduced 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 perigangliar injection of Akt inhibitors [LY294002 (LY, 50 uM) and wortmannin (WT, 1uM)] and PKA inhibitors [H-89 (30 uM) and PKA inhibitor peptide (PKA-I, 60uM)]. For pharmacologically-induced erections, papaverine (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 voltageand time-dependent and sustained for 3–5 min after ES. Papaverine, a direct vasorelaxant, injected ic stimulated P-eNOS-S1177 in the rat penis, but did not increase P-nNOSS1412. Perigangliar injection of FSK, an activator of PKA, increased P-nNOS-S1412, while Akt inhibitors, WT and LY, had no effect. Furthermore, 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.