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Priapism is a vasculopathy that occurs in approximately 40% of patients with sickle cell disease. Mouse models suggest that dysregulated nitric oxide synthase and RhoA/ROCK signaling as well as increased oxidative stress may contribute to the mechanisms of sickle cell disease associated priapism. We examined changes in the protein expression of nitric oxide synthase and ROCK signaling pathways, and a source of oxidative stress, NADPH oxidase, in penile erectile tissue from patients with a priapism history etiologically related and unrelated to sickle cell disease.Human penile erectile tissue was obtained from 5 patients with sickle cell disease associated priapism and from 6 with priapism of other etiologies during nonemergent penile prosthesis surgery for erectile dysfunction or priapism management and urethroplasty. Tissue was also obtained from 5 control patients without a priapism history during penectomy for penile cancer. Samples were collected, immediately placed in cold buffer and then frozen in liquid nitrogen. The expression of phosphodiesterase 5, endothelial nitric oxide synthase, neuronal nitric oxide synthase, inducible nitric oxide synthase, RhoA, ROCK1, ROCK2, p47(phox), p67(phox), gp91(phox) and β-actin were determined by Western blot analysis. Nitric oxide was measured using the Griess reaction.In the sickle cell disease group phosphodiesterase 5 (p0.05), endothelial nitric oxide synthase (p0.01) and RhoA (p0.01) expression was significantly decreased, while gp91(phox) expression (p0.05) was significantly increased compared to control values. In the nonsickle cell disease group endothelial nitric oxide synthase, ROCK1 and p47(phox) expression (each p0.05) was significantly decreased compared to control values. Total nitric oxide levels were not significantly different between the study groups.Mechanisms of sickle cell disease associated priapism in the human penis may involve dysfunctional nitric oxide synthase and ROCK signaling, and increased oxidative stress associated with NADPH oxidase mediated signaling.

Nitric oxide is the major neuronal mediator of penile erection but its role in erectile function status after cavernous nerve injury is uncertain. We determined the function of neuronal nitric oxide signaling in the pathobiology of erectile function recovery after partial cavernous nerve injury using genetic and pharmacological mouse experimental paradigms.Erectile function was evaluated in 5 to 7 wild-type and neuronal nitric oxide synthase-α knockout mice per group 1, 3 and 7 days after unilateral crush or sham injury, at day 7 in wild-type mice treated with the nitric oxide synthase inhibitor L-NAME (l-nitro arginine methyl ester) (Sigma-Aldrich®) at baseline and for 6 days after unilateral crush injury. Apoptosis in the penis was evaluated by Western blot analysis of p-Akt-S473, 3-nitrotyrosine and caspase-3 after bilateral crush injury.Intracavernous pressure was significantly decreased at 1, 3 and 7 days in wild-type mice but only at day 1 in knockout mice after unilateral crush injury compared with sham treatment values (p0.05). L-NAME treated wild-type mice had improved erectile function compared with the vehicle treated group at day 7 after unilateral crush injury (p0.05). In penes p-Akt-S473 was significantly decreased in vehicle treated (p0.05) but not in L-NAME treated wild-type mice. In penes 3-nitrotyrosine was significantly decreased in L-NAME treated wild-type and vehicle treated knockout mice (p0.05). Caspase-3 in penes was significantly increased in vehicle treated (p0.05) but not in L-NAME treated wild-type mice and vehicle treated knockout mice.Neuronal nitric oxide signaling regulates erectile function recovery early after partial cavernous nerve injury, exerting an inhibitory role via the induction of apoptotic change in penile tissue. Therapeutic strategies to improve erectile function recovery after radical prostatectomy may consider targeting pathogenic sites of nitric oxide neurobiology.

Conflicting information exists regarding the long-term efficacy of phosphodiesterase-5 (PDE5) inhibitor therapy for erectile dysfunction, particularly in regard to whether therapeutic resistance occurs. We investigated the erectile response, and cavernous PDE5 expression and activity after continuous long-term administration of sildenafil at verified therapeutic plasma concentrations, applying an in vivo rat model of age related erectile dysfunction.Male Fisher 344 young (4 months old) and aged (19 months old) rats (National Institute of Aging, Bethesda, Maryland) were injected with sildenafil mesylate (20 mg/kg) or saline subcutaneously every 8 hours for 3 weeks. After a 10 to 18-hour washout period electrical stimulation of the cavernous nerve was performed to assess penile erection. Penes were excised to measure PDE5 protein expression and activity, and blood was collected for sildenafil measurement. Responses were compared with those determined 30 minutes after a single sildenafil injection.Chronic sildenafil treatment increased the detumescence phase in young and aged rats (p0.05), although aged rats showed a greater increase than young rats. Baseline cavernous PDE5 expression and activity were greater in aged vs young rats (p0.05). After chronic sildenafil treatment cavernous PDE5 expression was increased in young (p0.05) but not in aged rats. Chronic and acute sildenafil treatment similarly inhibited PDE5 activity in the penis of young and aged rats (p0.05), coincident with its free plasma concentrations equivalent to clinically therapeutic ranges.Pharmacological PDE5 inhibitor therapy with sildenafil chronically does not result in treatment resistance. Rather, therapeutic efficacy is maintained and apparently more pronounced with erectile impairment than with normal erectile ability.

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.