Sustained nitric oxide (NO)‐releasing compound reverses dysregulated NO signal transduction in priapism

Abstract

We evaluated the therapeutic potential of a sustained nitric oxide (NO)-releasing compound to correct the molecular hallmarks and pathophysiology of priapism, an important but poorly characterized erectile disorder. 1,5-Bis-(dihexyl-N-nitrosoamino)-2,4-dinitrobenzene (C6') and an inactive form of the compound [1,5-bis-(dihexylamino)-2,4-dinitrobenzene (C6)] were tested in neuronal cell cultures and penile lysates for NO release (Griess assay) and biological activity (cGMP production). The effect of local depot C6' or C6 was evaluated in mice with a priapic phenotype due to double neuronal and endothelial NO synthase deletion (dNOS(-/-)) or human sickle hemoglobin transgenic expression (Sickle). Changes in NO signaling molecules and reactive oxygen species (ROS) surrogates were assessed by Western blot. The physiological response after C6' treatment was assessed using an established model of electrically stimulated penile erection. C6' generated NO, increased cGMP, and dose dependently increased NO metabolites. C6' treatment reversed abnormalities in key penile erection signaling molecules, including phosphodiesterase type 5, phosphorylated endothelial nitric oxide synthase, and phosphorylated vasodilator-stimulated phosphoprotein. In Sickle mice, C6' also attenuated the increased ROS markers gp91(phox), 4-hydroxynonenal, and 3-nitrotyrosine. Finally, C6' corrected the excessive priapic erection response of dNOS(-/-) mice. Exogenous sustained NO release from C6' corrects pathological erectile signaling in mouse models of priapism and suggests novel approaches to human therapy.

Beneficial Effect of the Nitric Oxide Donor Compound 3-(1,3-Dioxoisoindolin-2-yl)Benzyl Nitrate on Dysregulated Phosphodiesterase 5, NADPH Oxidase, and Nitrosative Stress in the Sickle Cell Mouse Penis: Implication for Priapism Treatment

“…Mice were anesthetized with isoflurane (1-1.5%) inhalation. The right carotid artery was cannulated with PE-10 tubing and attached to a pressure transducer to measure mean arterial pressure (MAP) (Lagoda et al, 2014). Surgical procedures required freeing the shaft of the penis of skin and fascia and removal of part of the ischiocavernous muscle to expose the crus.…”

“…A midline abdominal incision exposed the prostate, where the right major pelvic ganglion and cavernous nerve were identified posterolateral to the prostate. For electrically stimulated penile erections, a bipolar electrode attached to an S48 stimulator (Grass Instruments, Quincy, MA) was placed around the cavernous nerve, and stimulation parameters were 4 V at a frequency of 16 Hz with square-wave duration of 5 ms. ICP was recorded using the DI-190 system (Dataq Instruments, Akron, OH) from the beginning of the experiment when the cannula was inserted into the crura continuously until the animal was euthanized (Lagoda et al, 2014). Erectile function was represented by the maximal ICP response during electrical stimulation (ICP at the plateau), ICP response after stimulation [poststimulated area under the curve (AUC)], as well as half detumescence time, as recorded using the MATLAB program (Mathworks, Natick, MA), and parameters were normalized per MAP.…”

“…Penile tissue was homogenized as described (Hurt et al, 2002). Total penile homogenates (50 mg total protein) were run on 4-20% Tris-HCl gels (Bio-Rad Laboratories, Hercules, CA), transferred to a polyvinylidene fluoride membrane, and incubated overnight at 4°C with the following antibodies: polyclonal anti-PDE5 (1:500; Abcam, Cambridge, MA), polyclonal anti-vasodilator-stimulated phosphoprotein (VASP; 1:1000; Cell Signaling Technology, Danvers, MA), polyclonal anti-VASP phosphorylated at Ser 239 (1:1000; Cell Signaling Technology), monoclonal anti-3-nitrotyrosine (1:1000; Abcam), monoclonal anti-gp91 phox (1:1000; BD Transduction Laboratories, San Diego, CA), and monoclonal anti-b-actin (1:7000; SigmaAldrich, St. Louis, MO) (Lagoda et al, 2014). Quantified densitometry results were set as a ratio to b-actin and normalized to the control group (WT) probed on the same gel, making the control group 1 arbitrary unit.…”

“…Thus, when an erectile stimulus occurs in vivo, cGMP accumulates in cavernosal smooth muscle cells, rendering the penile vasculature uncontrollably dilated, and penile erection persists (i.e., priapism) as cGMP is not degraded as a consequence of PDE5 dysregulation. Endothelial nitric oxide synthase (eNOS) gene-deficient mice and combined eNOS and neuronal NO synthase (nNOS) double genedeficient (dNOS 2/2 ) mice also display a priapism phenotype associated with downregulation of PDE5 protein expression (Champion et al, 2005;Lagoda et al, 2014) and exhibit exacerbated cavernosal relaxations induced by the NO donor sodium nitroprusside (Nangle et al, 2004).…”

“…Excess superoxide anion reacts rapidly with NO, decreasing NO bioavailability and forming peroxynitrite, which is a powerful oxidant and cytotoxic agent (Pacher et al, 2007). Recently, upregulation of nitrotyrosine, a product of protein tyrosine nitration mediated by reactive nitrogen species, was shown to be associated with priapism in SCD mice (Bivalacqua et al, 2013;Lagoda et al, 2014).…”