Sildenafil citrate-restored eNOS and PDE5 regulation in sickle cell mouse penis prevents priapism via control of oxidative/nitrosative stress

Abstract

Sildenafil citrate revolutionized the practice of sexual medicine upon its federal regulatory agency approval approximately 15 years ago as the prototypical phosphodiesterase type 5 inhibitor indicated for the treatment of male erectile dysfunction. We now provide scientific support for its alternative use in the management of priapism, a clinical disorder of prolonged and uncontrolled penile erection. Sildenafil administered continuously to sickle cell mice, which show a priapism phenotype, reverses oxidative/nitrosative stress effects in the penis, mainly via reversion of uncoupled endothelial nitric oxide synthase to the functional coupled state of the enzyme, which in turn corrects aberrant signaling and function of the nitric oxide/cyclic GMP/protein kinase G/phosphodiesterase type 5 cascade. Priapism tendencies in these mice are reverted partially toward normal neurostimulated erection frequencies and durations after sildenafil treatment in association with normalized cyclic GMP concentration, protein kinase G activity and phosphodiesterase type 5 activity in the penis. Thus, sildenafil exerts pleiotropic effects in the penis that extend to diverse erection disorders.

Figure 1. Downregulation of NO/cGMP/PKG pathway in the penis of Sickle mice.

Baseline calcium-dependent (constitutive) NOS activity (a), cGMP production (b), PDE5 activity (c), and PKG activity (d) are reduced in the penis of Sickle compared to WT and Hemi mice. Each bar represents the mean ± SEM of 5 mice using ANOVA post-hoc Tukey test. *P<0.05 vs. WT and Hemi.

Figure 2. Calcium-independent NOS activity is not affected by SCD (a) or continuous sildenafil treatment (b).

Each bar represents the mean ± SEM of 5 mice using ANOVA post-hoc Tukey test.

Figure 3. Penes of Sickle mice exhibit increased production of ROS compared to WT and Hemi mice.

Superoxide production was measured by DHE fluorescence (a). Arrows indicate smooth muscle cells; arrowheads indicate endothelial cells; asterisks indicate neurons. ROS production was measured by luminol-dependant chemiluminescence (b), and protein tyrosine nitration by nitrotyrosine, a marker of peroxynitrite formation (c). Each bar represents the mean ± SEM of 6 WT and Sickle mice penes samples and 3 Hemi mice penes samples using ANOVA post-hoc Tukey test. *P<0.05 vs. WT and Hemi.

Figure 4. ROS production is decreased in the presence of NOS, NADPH oxidase, and xanthine oxidase inhibitors.

All 3 inhibitors (L-NAME 1 mM, apocynin 10 µM, oxypurinol 1 mM, respectively) decrease the production of ROS from penes of Sickle mice, indicating that NOS, NADPH oxidase, and xanthine oxidase are sources of ROS in penes of Sickle mice. ROS production was measured by luminol activity. Each bar represents the mean ± SEM of 6 mice using ANOVA post-hoc Tukey test. *P<0.05 vs. WT; **P<0.05 vs. Sickle.

Figure 6. Priapism in Sickle mice is due to unrestrained cGMP/PDE5 function.

Intracavernous pressure (ICP) is increased in response to intracavernosal (i.c) injection of the NO donor DEA/NO (0.3 µg/kg) (a) and is decreased in response to i.c. injection of sildenafil citrate (30 nmol/kg) (b) in Sickle compared to WT and Hemi mice. Basal cGMP levels are decreased (c), while neurostimulated cGMP levels are increased (d) in the penis of Sickle compared to WT mice. Each bar represents the mean ± SEM of 8 WT and Sickle mice and 6 Hemi mice; unpaired t-test were used to determine statistical significance. *P<0.05 vs. WT and Hemi.

Figure 7. Normalized penile erection by sildenafil treatment.

Continuous sildenafil citrate treatment (daily for 3 weeks, 100 mg/kg orally) decreases the frequency of spontaneous erections (erections/hr) before and after cavernous nerve stimulation (CNS; 2 volts) in Sickle mice. Each bar represents the mean ± SEM of 6 WT and WT+sildenafil and 7 Sickle and Sickle+sildenafil treated mice; ANOVA post-hoc Tukey test and repeated measures of ANOVA were used to obtain statistical significance. *P<0.05 vs. WT, **P<0.05 vs. Sickle.

Figure 8. Normalized NO signal transduction pathway in the penis of Sickle mice by sildenafil treatment.

Continuous sildenafil citrate treatment increases basal calcium-dependent NOS activity (a; n = 6), cGMP levels (b; n = 7), PDE5 activity (d; n = 6), and PKG activity (e; n = 6), and decreases neurostimulated cGMP levels (c; n = 7) in the penis of Sickle mice. Each bar represents the mean ± SEM using ANOVA post-hoc Tukey test. *P<0.05 vs. WT, **P<0.05 vs. Sickle.

Figure 5. Reduced ROS production and eNOS uncoupling in the penis of Sickle mice by sildenafil treatment.

ROS production was measured by luminol activity (a; n = 4–6) and nitrotyrosine activity (b; n = 7). Representative Western blots (c) and densitometric analysis (d; n = 5) of eNOS dimers and monomers in the penis after low-temperature SDS-PAGE. eNOS is uncoupled in the penis of Sickle mice compared to WT mice, while continuous treatment of Sickle mice with sildenafil citrate recouples eNOS. The results are expressed as dimer/monomer ratio. Each bar represents the mean ± SEM using ANOVA post-hoc Tukey test. *P<0.05 vs. WT; **P<0.05 vs. Sickle.