Transnitrosylation: A Factor in Nitric Oxide-Mediated Penile Erection.

INTRODUCTION: Nitric oxide (NO) signaling can be mediated not only through classic 3',5'-cyclic guanosine monophosphate but also through S-nitrosylation. However, the impact of S-nitrosylation on erectile function and in NO regulation and oxidative stress in the penis remains poorly understood. AIMS: To characterize the role of S-nitrosoglutathione reductase (GSNOR), a major regulator of S-nitrosylation homeostasis, on erection physiology and on endothelial NO synthase (eNOS) function and oxidative-nitrosative stress in the penis. METHODS: Adult GSNOR-deficient and wild-type (WT) mice were used. Erectile function was assessed in response to electrical stimulation of the cavernous nerve. Total NO in penile homogenates was measured by Griess reaction. Protein S-nitrosylation, eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS uncoupling, and markers of oxidative stress (4-hydroxy-2-nonenal, malondialdehyde, and nitrotyrosine) in the penis were measured by western blot. MAIN OUTCOME MEASURES: Erectile function, eNOS function, and oxidative stress in the penis of GSNOR-deficient mice. RESULTS: Erectile function was intact in GSNOR-deficient mice. Total S-nitrosylated proteins were increased (P < .05) in the GSNOR(-/-) compared with WT mouse penis. Although eNOS phosphorylation on Ser-1177 did not differ between the GSNOR(-/-) and WT mouse penises at baseline, electrical stimulation of the cavernous nerve increased (P < .05) phosphorylated eNOS in the WT mouse penis but failed to increase phosphorylated eNOS in the GSNOR(-/-) mouse penis. Total NO production was decreased (P < .05), whereas eNOS uncoupling, 4-hydroxy-2-nonenal, malondialdehyde, and nitrotyrosine were increased (P < .05) in the GSNOR-deficient mouse penis compared with the WT mouse penis. CONCLUSION: Transnitrosylation mechanisms play an important role in regulating NO bioactivity in the penis. Deficiency of GSNOR leads to eNOS dysfunction and increased oxidative damage, suggesting that homeostatic eNOS function in the penis is governed by transnitrosylation.

GGF2 is neuroprotective in a rat model of cavernous nerve injury-induced erectile dysfunction.

INTRODUCTION: Erectile dysfunction is a major complication of radical prostatectomy, commonly associated with penile neuropathy. In animal models of peripheral nerve injury, glial growth factor-2 (GGF2), a member of the neuregulin family of growth factors, has neuroprotective and neurorestorative properties, but this potential has not been established after cavernous nerve (CN) injury. AIMS: The effectiveness of GGF2 in preserving axonal integrity and recovering erectile function in a rat model of radical prostatectomy-associated CN injury. METHODS: Adult male Sprague-Dawley rats underwent bilateral CN crush injury (BCNI) or sham surgery. Rats were administered GGF2 (0.5, 5, or 15 mg/kg) or vehicle subcutaneously 24 hour pre and 24-hour post-BCNI, and once weekly for 5 weeks. Erectile function was assessed in response to electrical stimulation of the CN. CN survival was assessed by fluorogold retrograde axonal tracing in major pelvic ganglia (MPG). Unmyelinated axons in the CNs were quantitated by electron microscopy. MAIN OUTCOME MEASURES: Erectile function recovery, CN survival, and unmyelinated CN axon preservation in response to GGF2 treatment following BCNI. RESULTS: Erectile function was decreased (P < 0.05) after BCNI, and it was improved (P < 0.05) by all doses of GGF2. The number of fluorogold-labeled cells in the MPG was reduced (P < 0.05) by BCNI and was increased (P < 0.05) by GGF2 (0.5 and 5 mg/kg). The percentage of denervated Schwann cells in the BCNI group was higher (P < 0.05) than that in the sham-treated group and was decreased (P < 0.05) in the GGF2-treated (5 mg/kg) BCNI group. In the BCNI + GGF2 (5 mg/kg) group, the unmyelinated fiber histogram demonstrated a rightward shift, indicating an increased number of unmyelinated axons per Schwann cell compared with the BCNI group. CONCLUSIONS: GGF2 promotes erectile function recovery following CN injury in conjunction with preserving unmyelinated CN fibers. Our findings suggest the clinical opportunity to develop GGF2 as a neuroprotective therapy for radical prostatectomy.

Hydroxyl fasudil, an inhibitor of Rho signaling, improves erectile function in diabetic rats: a role for neuronal ROCK.

INTRODUCTION: The pathogenesis of diabetic erectile dysfunction (ED) includes neuropathy, but the molecular basis for neurogenic ED is incompletely understood. The RhoA/ROCK pathway has been implicated in diabetic neuropathy and in ED, but its role in diabetic neurogenic ED is not known. AIMS: The aim of this study was to determine whether hydroxyl fasudil, a ROCK inhibitor, affects diabetic neuropathy-related ED. METHODS: Type 1 diabetes mellitus was induced in male rats by streptozotocin (75 mg/kg, intraperitoneally). After 8 weeks, diabetic rats were administered hydroxyl fasudil, a selective ROCK inhibitor (10 mg/kg/day, intraperitoneally) or vehicle, for 4 weeks. Age-matched control, nondiabetic, rats were treated intraperitoneally for 4 weeks with saline. At week 12, after a 2 day washout, neuro-stimulated erectile function was evaluated. Major pelvic ganglia (MPG) were collected for Western blot analysis of RhoA, ROCK-1, ROCK-2, phospho (P)-AKT (Ser(473) ), and P-phosphatase and tensin homolog (P-PTEN) (Ser(380) /Thr(382/383) ). MAIN OUTCOME MEASURES: Effect of ROCK inhibitor hydroxyl fasudil on erectile function and ROCK/P-AKT/P-PTEN pathway in the MPG of diabetic rats. RESULTS: Erectile response was significantly (P < 0.05) reduced in diabetic rats compared with nondiabetic rats and was preserved (P < 0.05) in diabetic rats treated with hydroxyl fasudil. In diabetic rats, RhoA and ROCK-2 protein expressions in MPG were increased (P < 0.05) and remained increased in hydroxyl fasudil-treated rats. P-AKT (Ser(473) ) expression was decreased (P < 0.05), whereas P-PTEN (Ser(380) /Thr(382/383) ) expression was increased (P < 0.05) in MPG of diabetic rats compared with nondiabetic rats, and both were reversed (P < 0.05) in diabetic rats treated with hydroxyl fasudil. CONCLUSION: Improved erectile function and restored P-AKT and P-PTEN in the MPG with hydroxyl fasudil treatment suggest the role of Rho signaling via PTEN/AKT pathway in neurogenic diabetic ED.

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

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.

Fibrotic protein expression profiles in penile tissue of patients with erectile dysfunction.

OBJECTIVE: To characterize transforming growth factor beta 1 (TGFß1) and related signaling pathway proteins in a large cohort of human penile tissue (HPT) samples. METHODS: HPT was collected from patients undergoing penile prosthesis implantation for erectile dysfunction (ED) and divided into the following 2 groups: postradical prostatectomy ED (RP-ED; n = 57) and organic ED (O-ED; n = 30). HPT from patients undergoing partial penectomy without ED was used as controls (CON; n = 6). Western blot analysis was performed to investigate the protein expressions of TGFß1, thrombospondin 1 (TSP1; an activator of TGFß1), fibronectin (an extracellular matrix glycoprotein induced by TGFß1), and a family of transcriptional factors activated by TGFß1 (Smad2, phospho-Smad2-serine-465/467 [pSmad2], Smad3, phospho-Smad3-serine-423/425 [pSmad3]). RESULTS: Expressions of TGFß1 and TSP1 were significantly higher in RP-ED (P <.05) and O-ED (P <.05) groups compared with that of the CON group and were not different between either ED groups. Expressions of Smad2, pSmad2, Smad3, pSmad3, and fibronectin were similar among all groups. Within the RP-ED group, a subgroup analysis showed that time from RP to penile prosthesis implantation was related to increased expression of pSmad2 (P <.05), and previous history of intracavernosal injection was related to increased expression of TGFß1 (P <.05). CONCLUSION: Our results demonstrate that TSP1- and TGFß1-dependent fibrotic changes occur in penile tissue in patients with ED regardless of etiology. The unchanged expression of the Smad transcriptional factors may be reconciled by a Smad-independent downstream signaling pathway transmitting TGFß1 signals.

Continuous-wave infrared subsurface optical stimulation of the rat prostate cavernous nerves using a 1490-nm diode laser.

OBJECTIVE: To optimize the infrared laser wavelength and optical nerve stimulation (ONS) parameters for both deep and rapid subsurface cavernous nerve (CN) stimulation in a rat model, in vivo. MATERIALS AND METHODS: A 150-mW, 1490-nm diode laser providing an optical penetration depth (OPD) of 518 µm in water was operated in continuous-wave mode during stimulation of the CNs in 8 rats for 15 seconds irradiation time through a custom-built, single-mode fiber optic probe capable of producing a collimated, 1-mm diameter laser beam. Successful ONS was judged by an intracavernous pressure response in the rat penis. Subsurface ONS at 1490 nm was also compared with previous studies using 1455 nm and 1550 nm near-infrared diode laser wavelengths. RESULTS: Subsurface ONS of the rat CN was successful through fascia layers with a thickness up to 380 µm using an incident laser power of ∼50 mW. Intracavernous pressure response times as short as 4.6 ± 0.2 seconds were recorded using higher laser powers below the nerve damage threshold. CONCLUSION: The 1490-nm diode laser represents a compact, low cost, high power, and high quality infrared light source for use in ONS. This wavelength provides deeper penetration than 1455-nm diode laser and more rapid and efficient nerve stimulation than 1550-nm diode laser.

Temperature-controlled optical stimulation of the rat prostate cavernous nerves.

Optical nerve stimulation (ONS) may be useful as a diagnostic tool for intraoperative identification and preservation of the prostate cavernous nerves (CN), responsible for erectile function, during prostate cancer surgery. Successful ONS requires elevating the nerve temperature to within a narrow range (~42 to 47°C) for nerve activation without thermal damage to the nerve. This preliminary study explores a prototype temperature-controlled optical nerve stimulation (TC-ONS) system for maintaining a constant (±1°C) nerve temperature during short-term ONS of the rat prostate CNs. A 150-mW, 1455-nm diode laser was operated in continuous-wave mode, with and without temperature control, during stimulation of the rat CNs for 15 to 30 s through a fiber optic probe with a 1-mm-diameter spot. A microcontroller opened and closed an in-line mechanical shutter in response to an infrared sensor, with a predetermined temperature set point. With TC-ONS, higher laser power settings were used to rapidly and safely elevate the CNs to a temperature necessary for a fast intracavernous pressure response, while also preventing excessive temperatures that would otherwise cause thermal damage to the nerve. With further development, TC-ONS may provide a rapid, stable, and safe method for intraoperative identification and preservation of the prostate CNs.

Molecular analysis of erection regulatory factors in sickle cell disease associated priapism in the human penis.

PURPOSE: 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. MATERIALS AND METHODS: 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 ß-actin were determined by Western blot analysis. Nitric oxide was measured using the Griess reaction. RESULTS: In the sickle cell disease group phosphodiesterase 5 (p <0.05), endothelial nitric oxide synthase (p <0.01) and RhoA (p <0.01) expression was significantly decreased, while gp91(phox) expression (p <0.05) was significantly increased compared to control values. In the nonsickle cell disease group endothelial nitric oxide synthase, ROCK1 and p47(phox) expression (each p <0.05) was significantly decreased compared to control values. Total nitric oxide levels were not significantly different between the study groups. CONCLUSIONS: 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.

Cyclic AMP-dependent phosphorylation of neuronal nitric oxide synthase mediates penile erection.

Nitric oxide (NO) generated by neuronal NO synthase (nNOS) initiates penile erection, but has not been thought to participate in the sustained erection required for normal sexual performance. We now show that cAMP-dependent phosphorylation of nNOS mediates erectile physiology, including sustained erection. nNOS is phosphorylated by cAMP-dependent protein kinase (PKA) at serine(S)1412. Electrical stimulation of the penile innervation increases S1412 phosphorylation that is blocked by PKA inhibitors but not by PI3-kinase/Akt inhibitors. Stimulation of cAMP formation by forskolin also activates nNOS phosphorylation. Sustained penile erection elicited by either intracavernous forskolin injection, or augmented by forskolin during cavernous nerve electrical stimulation, is prevented by the NOS inhibitor L-NAME or in nNOS-deleted mice. Thus, nNOS mediates both initiation and maintenance of penile erection, implying unique approaches for treating erectile dysfunction.

An NMDA antagonist in the MPOA impairs copulation and stimulus sensitization in male rats.

Systemic injections of an NMDA antagonist have been shown to impair mating in male rats. One site where glutamate and its NMDA receptors may contribute to mating is the medial preoptic area (MPOA), which is vital for male sexual behavior. Glutamate is released in the MPOA during copulation, and especially at the time of ejaculation. We report here that the NMDA antagonist MK-801, microinjected into the MPOA, impaired copulatory behavior in sexually naïve as well as experienced males. In rats tested both as naïve and after sexual experience, drug treatment produced more profound impairment in naïve males. In addition, MK-801, microinjected into the MPOA before each of 7 noncopulatory exposures to receptive female rats, resulted in copulatory impairments on a drug-free test on Day 8, relative to aCSF-treated rats; their behavior was similar to that of males that had not been preexposed to females. Therefore, NMDA receptors in the MPOA contribute to the control of copulation and stimulus sensitization. Glutamate, acting via NMDA receptors, regulates many neural functions, including neuronal plasticity. This is the first demonstration that a similar mechanism in the MPOA sensitizes male rats to the stimuli from a receptive female, and thereby enhances their behavior.

Subsurface near-infrared laser stimulation of the periprostatic cavernous nerves.

Successful identification and preservation of the cavernous nerves (CN), which are responsible for sexual function and vulnerable to damage during prostate cancer surgery, will require subsurface detection of the CN's beneath a thin fascia layer. This study explores the feasibility of optical nerve stimulation (ONS) in the rat with a fascia layer placed over the CN. Two near-infrared diode lasers with wavelengths of 1455 and 1550 nm were operated in continuous-wave mode for stimulation of the CN in 8 rats, in vivo. Successful ONS was confirmed by an intracavernous pressure (ICP) response in the rat penis at 1455 nm through fascia with a thickness up to 110 µm and at 1550 nm through fascia with a thickness up to 450 µm. Higher incident laser power was required to produce an ICP response as fascia thickness was increased. Also, weaker and slower ICP responses were observed as fascia thickness was increased. Subsurface ONS of the rat CN at a depth of 450 µm using a 1550 nm laser is feasible as an intermediate step towards developing ONS as an intra-operative diagnostic tool for identification and preservation of the cavernous nerves during prostate cancer surgery.

Neuronal nitric oxide signaling regulates erection recovery after cavernous nerve injury.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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 (p <0.05). L-NAME treated wild-type mice had improved erectile function compared with the vehicle treated group at day 7 after unilateral crush injury (p <0.05). In penes p-Akt-S473 was significantly decreased in vehicle treated (p <0.05) 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 (p <0.05). Caspase-3 in penes was significantly increased in vehicle treated (p <0.05) but not in L-NAME treated wild-type mice and vehicle treated knockout mice. CONCLUSIONS: 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.

FK506 neuroprotection after cavernous nerve injury is mediated by thioredoxin and glutathione redox systems.

INTRODUCTION: Immunophilin ligands such as FK506 (FK) preserve erectile function (EF) following cavernous nerve injury (CNI), although the precise mechanisms are unclear. We examined whether the thioredoxin (Trx) and glutathione (GSH) redox systems mediate this effect after CNI. AIM: To investigate the roles of Trx reductase 2 (TrxR2) and S-Nitrosoglutathione reductase (GSNOR) as antioxidative/nitrosative and antiapoptotic mediators of the neuroprotective effect of FK in the penis after CNI. METHODS: Adult male rats, wild-type (WT) mice, and GSNOR deficient (GSNOR -/-) mice were divided into four groups: sham surgery (CN [cavernous nerves] exposure only) + vehicle; sham surgery + FK (5 mg/kg/day/rat or 2 mg/kg/day/mouse, for 2 days, subcutaneous); CNI + vehicle; and CNI + FK. At day 4 after injury, electrically stimulated changes in intracavernosal pressure (ICP) were measured. Penises were collected for Western blot analysis of TrxR2, GSNOR, and Bcl-2, and for immunolocalization of TrxR2 and GSNOR. MAIN OUTCOME MEASURES: EF assessment represented by maximal ICP and total ICP in response to electrical stimulation. Evaluation of protein expression levels and distribution patterns of antioxidative/nitrosative and antiapoptotic factors in penile tissue. RESULTS: EF decreased after CNI compared with sham surgery values in both rats (P < 0.01) and WT and GSNOR -/- mice (P < 0.05). FK treatment preserved EF after CNI compared with vehicle treatment in rats (P < 0.01) and WT mice (P < 0.05) but not in GSNOR -/- mice. In rats, GSNOR (P < 0.01) and Bcl-2 (P < 0.05) expressions were significantly decreased after CNI. FK treatment in CN-injured rats restored expression of GSNOR and upregulated TrxR2 (P < 0.001) and Bcl-2 (P < 0.001) expressions compared with vehicle treatment. Localizations of proteins in the penis were observed for TrxR2 (endothelium, smooth muscle) and for GSNOR (nerves, endothelium, smooth muscle). CONCLUSIONS: The neuroprotective effect of FK in preserving EF after CNI involves antioxidative/nitrosative and antiapoptotic mechanisms mediated, to some extent, by Trx and GSH systems.

Continuous-wave laser stimulation of the rat prostate cavernous nerves using a compact and inexpensive all single mode optical fiber system.

BACKGROUND AND PURPOSE: Laser stimulation of the rat cavernous nerve (CN) recently has been demonstrated as an alternative to electrical stimulation for potential application in nerve mapping during nerve-sparing radical prostatectomy. Advantages include noncontact stimulation and improved spatial selectivity. Previous studies, however, have used large and/or expensive laser sources for stimulation. This study demonstrates the feasibility of optical stimulation of the rat CN, in vivo, using a compact, inexpensive all-single-mode fiberoptic system. MATERIALS AND METHODS: A 1455-nm wavelength infrared diode laser beam was coupled into a 9-µm-core single-mode fiber for delivery through a 10F laparoscopic probe and used for laser stimulation of the CN in a total of eight rats, in vivo. RESULTS: Laser stimulation of the CN was observed at threshold temperatures of 41°C, with intracavernous pressure response times as short as 4 s, and magnitudes up to 50 mm Hg, compared with baselines of 10 mm Hg. CONCLUSION: This novel, all-single-mode-fiber laser nerve stimulation system introduces several advantages including: (1) lower cost laser; (2) more robust fiberoptic design, eliminating alignment and cleaning of bulk optical components; and (3) improved Gaussian spatial beam profile for simplified alignment of the laser beam with the nerve. With further development, laser nerve stimulation may be useful for identification and preservation of the CN during prostate cancer surgery.

Continuous-wave infrared optical nerve stimulation for potential diagnostic applications.

Optical nerve stimulation using infrared laser radiation has recently been developed as a potential alternative to electrical nerve stimulation. However, recent studies have focused primarily on pulsed delivery of the laser radiation and at relatively low pulse rates. The objective of this study is to demonstrate faster optical stimulation of the prostate cavernous nerves using continuous-wave (cw) infrared laser radiation for potential diagnostic applications. A thulium fiber laser (λ=1870 nm) is used for noncontact optical stimulation of the rat prostate cavernous nerves in vivo. Optical nerve stimulation, as measured by an intracavernous pressure (ICP) response in the penis, is achieved with the laser operating in either cw mode, or with a 5-ms pulse duration at 10, 20, 30, 40, 50, and 100 Hz. Successful optical stimulation is observed to be primarily dependent on a threshold nerve temperature (42 to 45 °C), rather than an incident fluence, as previously reported. cw optical nerve stimulation provides a significantly faster ICP response time using a lower power (and also less expensive) laser than pulsed stimulation. cw optical nerve stimulation may therefore represent an alternative mode of stimulation for intraoperative diagnostic applications where a rapid response is critical, such as identification of the cavernous nerves during prostate cancer surgery.

Hypercholesterolemia-induced erectile dysfunction: endothelial nitric oxide synthase (eNOS) uncoupling in the mouse penis by NAD(P)H oxidase.

INTRODUCTION: Hypercholesterolemia induces erectile dysfunction (ED) mostly by increasing oxidative stress and impairing endothelial function in the penis, but the mechanisms regulating reactive oxygen species (ROS) production in the penis are not understood. AIMS: We evaluated whether hypercholesterolemia activates nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the penis, providing an initial source of ROS to induce endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction resulting in ED. METHODS: Low-density-lipoprotein receptor (LDLR)-null mice were fed Western diet for 4 weeks to induce early-stage hyperlipidemia. Wild type (WT) mice fed regular chow served as controls. Mice received NAD(P)H oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Erectile function was assessed in response to cavernous nerve electrical stimulation. Markers of endothelial function (phospho [P]-vasodilator-stimulated-protein [VASP]-Ser-239), oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NAD[P]H oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), P-eNOS-Ser-1177, and eNOS were measured by Western blot in penes. MAIN OUTCOME MEASURES: The main outcome measures are the molecular mechanisms of ROS generation and endothelial dysfunction in hypercholesterolemia-induced ED. RESULTS: Erectile response was significantly (P<0.05) reduced in hypercholesterolemic LDLR-null mice compared with WT mice. Relative to WT mice, hypercholesterolemia increased (P<0.05) protein expressions of NAD(P)H oxidase subunits p67(phox) , p47(phox) and gp91(phox) , eNOS uncoupling, and 4-HNE-modified proteins, and reduced (P<0.05) P-VASP-Ser-239 expression in the penis. Apocynin treatment of LDLR-null mice preserved (P<0.05) maximal intracavernosal pressure, and reversed (P<0.05) the abnormalities in protein expressions of gp67(phox) and gp47(phox) , 4-HNE, P-VASP-Ser-239, and eNOS uncoupling in the penis. Apocynin treatment of WT mice did not affect any of these parameters. Protein expressions of P-eNOS-Ser-1177 and total eNOS were unaffected by hypercholesterolemia. CONCLUSION: Activated NAD(P)H oxidase in the penis is an initial source of oxidative stress resulting in eNOS uncoupling, thus providing a mechanism of eNOS uncoupling and endothelial dysfunction in hypercholesterolemia-induced ED.

Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina.

INTRODUCTION: Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown. AIMS: Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression. METHODS: We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17beta (15 microg). MAIN OUTCOME MEASURES: Molecular mechanisms of eNOS regulation by estrogen in the rat vagina. RESULTS: We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina. CONCLUSIONS: These results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS-caveolin-1 interaction.

FK506 and rapamycin neuroprotect erection and involve different immunophilins in a rat model of cavernous nerve injury.

INTRODUCTION: Immunophilin ligands function by binding to receptor proteins such as FK506 binding proteins (FKBPs). FKBPs are studied for their roles in neuroprotection. AIM: Compare the effect of FK506 (FK) and rapamycin (RAP) on erectile function (EF) recovery and FKBP expressions in penis and major pelvic ganglion (MPG) after cavernous nerve (CN) injury. METHODS: Adult male rats were divided into four groups: sham surgery (CN exposure only) + vehicle; bilateral CN injury (BCNI; bilateral crush, 3 minutes with hemostat clamp) + vehicle; BCNI + FK (5 mg/kg/day, 5 days, sc); and BCNI + RAP (2 mg/kg/day, 5 days, sc). At both 24 hours (Day 1) or 1 week (Day 7) after BCNI, EF was assessed by intracavernosal pressure measurement and FKBPs 12, 38, 52, and 65 expressions were evaluated by Western blot analysis in collected penises and MPGs. MAIN OUTCOME MEASURES: EF and change in protein expressions of FKBPs in the rat penis and MPG after BCNI with and without immunophilin ligand treatment. RESULTS: Both FK- and RAP-treated rats had preserved EF compared with vehicle-treated rats after BCNI. FKBPs changed variably following injury and treatment. In particular, in the penis at Day 1, FKBP 38 expression was decreased after BCNI and both FK and RAP attenuated this decrease. In MPG at Day 1, FKBP 38 expression was also decreased after BCNI and FK attenuated the decrease, while at Day 7, FKBP 38 expression was still decreased and RAP attenuated the decrease. Also, in the penis at Day 1, FKBP 65 expression decreased after BCNI and FK attenuated the decrease. In the MPG, FKBP 65 expression increased at both Days 1 and 7 with FK treatment. CONCLUSIONS: Improved EF after BCNI, as shown with RAP, further suggests a role of immunophilin ligands as a protective therapy of CN injury associated erectile dysfunction. Our findings also suggest that select FKBPs, such as FKBP 38 and FKBP 65, may mediate these effects.

Losartan preserves erectile function after bilateral cavernous nerve injury via antifibrotic mechanisms in male rats.

PURPOSE: Angiotensin II is a known mediator of smooth muscle vasoconstriction and fibrosis. It up-regulates thrombospondin-1, a major activator of latent transforming growth factor-beta. Transforming growth factor-beta induces vascular fibrosis via intracellular SMAD signaling pathways. We evaluated the effect of treatment with the angiotensin II type 1 receptor antagonist losartan on erectile function in the rat following bilateral cavernous nerve injury. MATERIALS AND METHODS: A total of 36 adult male rats were divided equally into 6 groups, including group 1-sham surgery with cavernous nerve exposure only plus vehicle, group 2-sham surgery plus oral low dose losartan (10 mg/kg per day), group 3-sham surgery plus high dose losartan (40 mg/kg per day), group 4-bilateral cavernous nerve injury (3-minute crush using a hemostat clamp) plus vehicle, group 5-bilateral cavernous nerve injury plus low dose losartan and group 6-bilateral cavernous nerve injury plus high dose losartan. Seven days following surgery erectile function was measured by electrically stimulating the cavernous nerves and monitoring intracavernous pressure. Penile tissue was collected for Western blot analysis of fibronectin, transforming growth factor-beta, thrombospondin-1, alpha-actin, and phosphorylated and total SMAD2 and SMAD3 expression. RESULTS: Erectile function was significantly decreased after bilateral cavernous nerve injury compared with that after sham surgery (p <0.01). Low and high dose losartan preserved erectile function after bilateral cavernous nerve injury compared to that in vehicle controls (p <0.01 and <0.05, respectively). Fibronectin, pSMAD2, pSMAD3, transforming growth factor-beta-1, thrombospondin-1 and alpha-actin expression was up-regulated, and total SMAD2 and SMAD3 expression was down-regulated in the penis after bilateral cavernous nerve injury. Each dose of losartan after bilateral cavernous nerve injury significantly attenuated the up-regulated expression of fibronectin (p <0.01), pSMAD2 (p <0.05) and thrombospondin-1 (p <0.05), and up-regulated total SMAD2 (p <0.05). CONCLUSIONS: These data suggest that fibrotic activators in the penis may cause decreased erectile function after bilateral cavernous nerve injury. Angiotensin II type 1 receptor antagonism may counteract this effect and promote erectile function preservation for conditions associated with penile fibrosis.

Role of immunophilins in recovery of erectile function after cavernous nerve injury.

INTRODUCTION: Immunophilin ligands provide potentially new alternatives for the treatment of erectile dysfunction (ED), which occurs after injury of the cavernous nerves (CNs). AIM: To review and update current knowledge of the neurotrophic effects and likely mechanism of action of immunophilin proteins with emphasis on the FK506-binding protein (FKBP) subfamily and the role of immunophilin ligands for the treatment of CN injury-induced ED. METHODS: Review of available reports of studies investigating the effects and neurotrophic mechanisms of immunophilin ligands involved in erectile function recovery in rodent models of CN injury. MAIN OUTCOME MEASURES: Erection parameters and molecular correlations associated with CN injury and functional recovery. RESULTS: Treatment with prototype immunosuppressive immunophilin ligands FK506 (FK) and rapamycin (Rapa) improve erectile function in animal models of CN injury. Similarly, non-immunosuppressive analogs such as GPI-1046 and FK1706 are effective in recovery of erections after CN injury. Neuronal nitric oxide may influence the erection recovery effects of immunophilin ligands after CN injury. FKBPs 38 and 65 expression changes in the penis and its innervation coincide with the neurotrophic effects of immunophilin ligands. Antioxidative actions of immunophilin ligands contribute to their neurotrophic effects. Immunophilins are localized to nerves coursing in human prostate and penile tissue. CONCLUSIONS: The findings support the hypothesis that immunophilin ligands, working through specific receptor mechanisms that are specific to injured CN, are potentially useful to sustain erectile function in men following radical prostatectomy.