Up-Regulation of NO/cGMP Signaling Pathway in Corpus Cavernosum of Sickle Cell Disease Transgenic Mice

Abstract

Priapism is defined as prolonged and persistent penile erection, unassociated with sexual interest or stimulation. This condition is common among males with sickle cell disease (SCD), however the molecular mechanisms involved in this syndrome are not well understood. Penile nitric oxide (NO), originating from both neurons and endothelium, modulates penile vascular and smooth muscle relaxation. NO, by binding sGC, converts cyclic guanosine triphosphate (cGTP) to cyclic guanosine monophosphate (cGMP), relaxing vascular smooth muscle and causing penile erection. The Berkeley mouse model is a well-accepted animal model of severe anemia that displays features of priapism similar to those seen in humans. These animals have a decreased activity of the enzyme responsible for cGMP hydrolysis, phosphodiesterase type 5A (PDE5) in the penis, suggesting that dysregulated NO signaling may lead to priapism in this mice model. Thus, the aim of this study was to evaluate the cavernosum smooth muscle relaxation mediated by the NO-cGMP signaling pathway in the SCD transgenic mouse. SS mice and normal C57BL/6 mice (control) penile tissues were removed at the level of attachment of the corporeal body; tunica albuginea was then carefully opened from its proximal extremity toward the penile shaft, and the erectile tissue within the corpus cavernosum (CC) was surgically dissected free. The strips were mounted in 10 ml organ baths containing Krebs solution at 37°C that was continuously bubbled with a mixture of 95% oxygen and 5% carbon dioxide (pH 7.4), and vertically suspended between two metal hooks. The tissues were stretched to a resting tension of 2.5 mN and allowed to equilibrate for 60 min. Changes in isometric force were recorded using a PowerLab 400 Data Acquisition System (Software Chart, version 5.2, AD Instruments, Colorado Springs, Colo). In a first set of experiments, cumulative concentration-response curves were constructed for a muscarinic agonist, acetylcholine (ACh; 0.001–1 μM), the NO donor compound, sodium nitroprusside (SNP; 0.001–10 μM) and the NO-independent stimulator of soluble guanylyl cyclase (sGC), BAY 41–2272 (0.001–10 μM), in corpus cavernosum (CC) precontracted with phenylephrine (PE; 10 μM). In a second set of experiments, to evaluate the nitrergic cavernosal relaxations, an electrical field stimulation (EFS; 1–32 Hz; 50 V; 10-s trains) was used. EFS were applied in strips placed between two platinum ring electrodes connected to a Grass S88 stimulator (Astro-Med Industrial Park, RI, U.S.A.). The endothelium-dependent NO-mediated relaxing responses to ACh induced concentration-dependent CC relaxations, and both potency (pEC50; 7.27 ± 0.09) and maximal responses (Emax; 78 ± 5%) were significantly higher in SS mice, compared with control mice (6.85 ± 0.07 and 60 ± 3%, respectively). EFS-induced relaxations in SS mice CC were significantly increased in all frequency studies compared to control mice. The endothelium-independent NOmediated relaxing responses to SNP produced concentration-dependent relaxation in isolated mice CC and both pEC50 (6.30 ± 0.09) and Emax (113 ± 6%) were significantly increased in the SS group when compared to the control group (5.80 ± 0.15 and 83 ± 4%, respectively). The NO-independent sGC stimulator, BAY 41–2272, induced significant increases in both pEC50 (7.07 ± 0.05) and Emax (124 ± 5 %) in the SS group when compared to the control group (6.78 ± 0.06 and 99 ± 2 %). In summary, these data demonstrate that SS mice exhibit an excessive relaxation response in CC, possibly by up-regulation of the NO-cGMP signaling pathway, reflecting in uncontrolled erection function. As such this dysregulated NO-pathway may constitute a potential therapeutic target to treat priapism in SCD individuals.