Corpus Cavernosum Strips Research Articles

A novel reuptake inhibitor, IP2015, induces erection by increasing central dopamine and peripheral nitric oxide release.

An estimated 40% of patients with erectile dysfunction have a poor prognosis for improvement with currently available treatments. The present study investigated whether a newly developed monoamine transport inhibitor, IP2015, improves erectile function. We investigated the effects of IP2015 on monoamine uptake and binding, erectile function in rats and diabetic mice and the effect on corpus cavernosum contractility. IP2015 inhibited the uptake of 5-HT, noradrenaline and dopamine by human monoamine transporters expressed in cells and in rat brain synaptosomes. Intracavernosal pressure measurement in anaesthetized rats revealed that IP2015 dose-dependently increased the number and the duration of spontaneous erections. Whereas pretreatment with the dopamine D2-like receptor antagonists, clozapine and (-)-sulpiride, or cutting the cavernosal nerve inhibited IP2015-induced erectile responses, the phosphodiesterase type 5 inhibitor sildenafil further enhanced the IP2015-mediated increase in intracavernosal pressure. IP2015 also increased the number of erections in type 2 diabetic db/db mice. Direct intracavernosal injection of IP2015 increased penile pressure, and in corpus cavernosum strips, IP2015 induced concentration-dependent relaxations. These relaxations were enhanced by sildenafil and blunted by endothelial cell removal, a nitric oxide synthase inhibitor, NG-nitro-l-arginine and a D1-like receptor antagonist, SCH23390. Quantitative polymerase chain reaction (qPCR) showed the expression of the dopamine transporter in the rat corpus cavernosum. Our findings suggest that IP2015 stimulates erectile function by a central mechanism involving dopamine reuptake inhibition and direct NO-mediated relaxation of the erectile tissue. This novel multi-modal mechanism of action could offer a new treatment approach to erectile dysfunction.

(247) A Rat Model for Bipolar Androgen Therapy is Evaluated for Its Effects on Erectile Dysfunction after Castration

Abstract Introduction Prostate cancer (PCa), an androgen-dependent tumor and one of the most prevalent cancers in males, has traditionally been treated with androgen suppression. In recent years, bipolar androgen treatment (BAT) has emerged as a potential therapeutic approach for men with metastatic castrate-resistant PCa. The control of testosterone (T) between castration and supraphysiological levels inhibits the proliferation of cancer cells and slows the progression of cancer. Furthermore, while there has been prior research between T and erectile function, there is no evidence linking BAT and erectile dysfunction (ED). Objective The purpose of the current investigation was to determine if BAT therapy was effective in treating ED in a castration-prone rat model. Methods Sprague-Dawley rats (n=30) were randomly divided into four groups: 1) Control, 2) Control rats-received BAT treatment, 3) castrated Rats, and 4) castrated rats-received BAT treatment. The rats were castrated by making bilateral scrotal incisions. A subcutaneous injection of testosterone propionate (250 mg/kg/week, intramuscular) was administered four weeks following castration. Four weeks after the initial dose, the second dose of the medication was given in the same manner. By stimulating the cavernous nerve, the intracavernosal pressure (ICP) to mean arterial pressure (MAP) ratio and total ICP were determined. Corpus cavernosum (CC) strips were used as in vitro preparations for evaluating the relaxant and contractile responses. Nitric oxide synthase (nNOS) and endothelial NOS (eNOS) protein expression was analyzed using Western blotting. Using Masson trichrome staining, the location of the smooth muscle in relation to the collagen was identified. Results Castrated rats' prostate weight was reduced when compared to uncastrated rats (P< 0.001). Compared to controls and castrated rats, BAT marginally boosted prostate weight reduction. The ICP/MAP ratio and total ICP for 7.5 V were lowest in castrated rats (P< 0.001 vs. controls). BAT enhanced ICP/MAP and total ICP responses (P<0.01 vs. castrated rats). In vitro, endothelium-dependent acetylcholine-induced relaxation at 1 nM (P 0.05 vs controls), endothelium-independent sodium nitroprusside-induced relaxation at 1 nM (P 0.01 vs controls), electrical field stimulation-induced relaxant response at 20 Hz (P<0.05 vs. controls) and sildenafil-induced relaxation at 10 M (P 0.05 vs controls) were all reduced in the CC strips from the castrated group. Additionally, the castrated group showed increased neurogenic contractile response at 40 Hz and the contractile response to phenylephrine at 1 mM (P 0.001 vs. controls). The variations in the relaxation and contractile responses were returned by BAT. The smooth muscle cell-to-collagen ratio and nNOS and eNOS protein expression were both markedly reduced in castrated rats, though BAT reversed these modifications. Conclusions The results of the study show that BAT improves neurogenic relaxation, endothelium relaxation, sildenafil-induced relaxing of the CC, and changes in penile morphology after castration. BAT might be a cutting-edge therapeutic approach for patients with castration-resistant prostate cancer who have an ED route. Disclosure No.

Hydroxyurea does not reverse functional alterations of the nitric oxide-cGMP pathway associated with priapism phenotype in corpus cavernosum from sickle cell mouse.

Sickle cell disease (SCD) is a genetic disorder that has been associated with priapism. The role of hydroxyurea, a common SCD therapy, in influencing the nitric oxide (NO)-cGMP pathway and its effect on priapism is unclear. To investigate the effect of hydroxyurea treatment on smooth muscle relaxation of corpus cavernosum induced by stimulation of the NO-cGMP pathway in SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice, which are used as model of priapism associated with the low bioavailability of endothelial NO. Four-month-old wild-type (WT, C57BL/6), SCD transgenic, and eNOS-/- male mice were treated with hydroxyurea (100 mg/Kg/day) or its vehicle (saline) daily for three weeks via intraperitoneal injections. Concentration-response curves for acetylcholine (ACh), sodium nitroprusside (SNP), and electrical field stimulation (EFS) were generated using strips of mice corpus cavernosum. The SCD mice demonstrated an amplified CC relaxation response triggered by ACh, EFS, and SNP. The corpus cavernosum relaxation responses to SNP and EFS were found to be heightened in the eNOS-/- group. However, the hydroxyurea treatment did not alter these escalated relaxation responses to ACh, EFS, and SNP in the corpus cavernosum of the SCD group, nor the relaxation responses to EFS and SNP in the eNOS-/- group. In conclusion, hydroxyurea is not effective in treating priapism associated with SCD. It is likely that excess plasma hemoglobin and reactive oxygen species, which are reported in SCD, are reacting with NO before it binds to GCs in the smooth muscle of the corpus cavernosum, thus preventing the restoration of baseline NO/cGMP levels. Furthermore, the downregulation of eNOS in the penis may impair the pharmacological action of hydroxyurea at the endothelial level in SCD mice. This study emphasize the urgency for exploring alternative therapeutic avenues for priapism in SCD that are not hindered by high plasma hemoglobin and ROS levels.

Mechanism of Social Stress-Related Erectile Dysfunction in Mice: Impaired Parasympathetic Neurotransmission and Ketamine.

This study aimed to investigate the mechanism underlying social stress (SS)-induced erectile dysfunction (ED) and evaluate the effects of a single subanesthetic dose of ketamine on SS-related ED. Male FVB mice were exposed to retired male C57BL/6 mice for 60 min daily over a 4-week period. In the third week, these FVB mice received intraperitoneal injections of either saline (SSS group) or ketamine (SSK group). Erectile function was assessed by measuring the intracavernosal pressure (ICP) during electrical stimulation of the major pelvic ganglia. Corpus cavernosum (CC) strips were utilized for wire myography to assess their reactivity. Both SSS and SSK mice exhibited significantly lower ICP in response to electrical stimulation than control mice. SS mice showed increased contractility of the CC induced by phenylephrine. Acetylcholine-induced relaxation was significantly reduced in SSS and SSK mice. Sodium nitroprusside-induced relaxation was higher in SSS mice compared to control and SSK mice. Nicotine-induced neurogenic and nitric oxide-dependent relaxation was significantly impaired in both SSS and SSK mice. An immunohistochemical analysis revealed co-localization of tyrosine hydroxylase and neuronal nitric oxide synthase-immunoreactive fibers in the CC. These findings highlight the complex nature of SS-related ED and suggest the limited efficacy of ketamine as a therapeutic intervention.

(099) Dual Effect of a Serotonin and Dopamine Reuptake Inhibitor (IP2018), and the Search for a Depressive Rat Model with Erectile Dysfunction

Abstract Introduction In patients with major depressive disorder, up to 68% present with comorbid sexual dysfunction, which resolves in only 5% to 30% of patients treated with antidepressants. Several groups of antidepressants negatively affect sexual function. Objective The present study investigated whether a newly developed amine transport inhibitor, IP2018, improves depression and erectile function in the same dose range. Methods The dose-dependent effect of IP2018 in two different mouse models for depression, the mouse forced swim test (mFST) and mouse tail suspension test (mTST), is reported. In male Wistar, Sprague Dawley and the depressive rats (Flinders Sensitive Line (FSL) and Flinders Resistant Line (FRL)), increases in intracavernosal pressure (ICP) and mean arterial pressure (MAP) were measured, and the ratio ICP/MAP was used as a measure of erectile function. We investigated whether IP2018 induces changes in ICP or flow and whether IP2018 facilitates suboptimal increases in ICP induced by cavernosal nerve electrical stimulation. The late IP2018 procedure was performed in the rest of the rats to evaluate their erectile function. Results We found that infusion of IP2018 dose-dependently improved mFST and was more potent than citalopram. At the same time, higher doses of IP2018 were required to obtain effect in the mTST model, and IP2018 was equipotent to citalopram. Infusion of low doses of IP2018 dose-dependently increased the number and the duration of spontaneous erections. At the highest dose of IP2018, the increases in the number and duration of spontaneous erections were similar to the effect of the lowest dose, and the increases in flow were 5 times higher. IP2018 (1-30 μM) caused small relaxations in isolated corpus cavernosum strips. FSL and FRL rats showed a diminished ICP/MAP compared to Sprague Dawley control rats. Conclusions IP2018 is more potent or has a similar effect to the well-known antidepressant drug citalopram in mouse models for depression. The potent effect of IP2018 on erectile function in anaesthetized rats suggests that IP2018 in the same dose range has positive effects on both depression and erectile function. FSL/FRL rats appear to allow the study of drug effects on both depression and sexual function in one animal model. Disclosure Yes, this is sponsored by industry/sponsor: Initiator Pharma A/S Clarification Industry funding only - investigator initiated and executed study Any of the authors act as a consultant, employee or shareholder of an industry for: Initiator Pharma A/S

Combined levothyroxine and testosterone treatment for restoring erectile dysfunction in propylthiouracil-induced hypothyroid rats.

Sexual dysfunction may indicate severe endocrine diseases. Recent research has suggested a link between hypothyroidism, low testosterone (T) levels, and erectile dysfunction (ED); however, the exact cause is unknown. We sought to investigate possible beneficial effects of levothyroxine and T alone or in combination on ED in propylthiouracil (PTU)-induced hypothyroid rats. Adult Wistar rats (n = 35) were divided into 5 groups: control, PTU-induced hypothyroidism, PTU + levothyroxine, PTU + Sustanon (a mixture of 4 types of T: propionate, phenylpropionate, isocaproate, and decanoate) and PTU + levothyroxine + Sustanon. PTU was given in drinking water for 6 weeks. Four weeks after PTU administration, levothyroxine (20μg microgram kg/day, oral) and Sustanon (10mg/kg/week, intramuscular) were given for 2weeks. Serum levels of total T, triiodothyronine (T3), and thyroxine (T4) were determined. In vivo erectile response and in vitro relaxant responses were measured. Localization of neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), and phosphodiesterase type 5 (PDE5) were determined using immunohistochemical analysis. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Outcome variables included in vivo erectile function, in vitro relaxant and contractile responses of corpus cavernosum (CC) strips; protein localization of eNOS, nNOS, and PDE5; and smooth muscle content in penile tissue. The rat model of hypothyroidism showed a significant decline in serum levels of total T, T3, and T4. Levothyroxine increased T3 and T4 levels, whereas Sustanon normalized only total T levels. Combined treatment enhanced all hormone levels. Rats with hypothyroidism displayed the lowest erectile response (P < 0.001 vs controls). Combined treatment returned reduced responses, while partial amelioration was observed after levothyroxine and Sustanon treatment alone. Acetylcholine (P < 0.01 vs controls), electrical field stimulation (P < 0.001 vs controls), and sildenafil-induced relaxant responses (P < 0.05 vs controls) were decreased in the CC strips from hypothyroid rats. The combined treatment increased the reduction in relaxation responses. Levothyroxine and Sustanon restored decreases in eNOS and nNOS expression in the hypothyroid group. There was no significant difference in PDE5 expression among groups. Monotreatment partially enhanced reduced smooth muscle mass, while combined therapy completely recovered. The combination of thyroid hormones and T is likely to be a therapeutic approach for treatment of hypothyroidism-induced ED in men. Beneficial effects of levothyroxine and Sustanon treatment were shown in vitro and in vivo in PTU-induced hypothyroid rats. The main limitation of the study was the lack of measurement of androgen-sensitive organ weights and luteinizing hormone, follicle-stimulating hormone, and prolactin levels. These findings demonstrate that neurogenic and endothelium-dependent relaxation responses are reduced by hypothyroidism, which is detrimental to T levels and erectile responses. Levothyroxine and Sustanon combination medication was able to counteract this effect.

The Acute Effects and Mechanism of Ketamine on Nicotine-Induced Neurogenic Relaxation of the Corpus Cavernosum in Mice.

The present study aimed to investigate the acute effects and the mechanism of ketamine on nicotine-induced relaxation of the corpus cavernosum (CC) in mice. This study measured the intra-cavernosal pressure (ICP) of male C57BL/6 mice and the CC muscle activities using an organ bath wire myograph. Various drugs were used to investigate the mechanism of ketamine on nicotine-induced relaxation. Direct ketamine injection into the major pelvic ganglion (MPG) inhibited MPG-induced increases in ICP. D-serine/L-glutamate-induced relaxation of the CC was inhibited by MK-801 (N-methyl-D-aspartate (NMDA) receptor inhibitor), and nicotine-induced relaxation was enhanced by D-serine/L-glutamate. NMDA had no effect on CC relaxation. Nicotine-induced relaxation of the CC was suppressed by mecamylamine (a non-selective nicotinic acetylcholine receptor antagonist), lidocaine, guanethidine (an adrenergic neuronal blocker), Nw-nitro-L-arginine (a non-selective nitric oxide synthase inhibitor), MK-801, and ketamine. This relaxation was almost completely inhibited in CC strips pretreated with 6-hydroxydopamine (a neurotoxic synthetic organic compound). Ketamine inhibited cavernosal nerve neurotransmission via direct action on the ganglion and impaired nicotine-induced CC relaxation. The relaxation of the CC was dependent on the interaction of the sympathetic and parasympathetic nerves, which may be mediated by the NMDA receptor.

In vitro effect of relaxin in the rat corpus cavernosum under hyperglycemic and normoglycemic conditions.

Relaxin, an endogenous peptide hormone, elicits vascular relaxation by its direct effect or by modulating the endothelium-dependent relaxation response and is clinically evaluated for the treatment of coronary artery disease. However, its effect on penile tissue has not been explored yet. This study aimed to investigate the effect of serelaxin, recombinant human relaxin-2, on rat corpus cavernosum (CC) under healthy and hyperglycemic conditions. Strips of CC obtained from thirty-nine male Wistar rats weighing 300-350 g were used in organ baths for isometric tension studies to investigate the serelaxin-mediated relaxation (10-12-10-7 M) under normoglycemic conditions and the effect of serelaxin on endothelium-dependent [nitric oxide (NO)- and prostacyclin-mediated] relaxation responses under hyperglycemic conditions. The in vitro hyperglycemia model was created by 3 h of incubation with 44 mM glucose monohydrate +120 μM methylglyoxal. NO-dependent relaxation responses were evaluated by cumulative acetylcholine (10-9-10-4 M) administration in the presence of indomethacin (10-6 M). Prostacyclin-mediated relaxation was evaluated by cumulative administration of iloprost (10-9-10-6 M), a prostacyclin analog. Maximum relaxation responses to serelaxin were not significantly different compared to the time-control (p = 0.480). Three hours of incubation of rat CC in hyperglycemic conditions impaired NO- and prostacyclin-mediated relaxation responses (p = 0.032 and p = 0.047, respectively). Serelaxin coincubation worsened NO-mediated relaxation responses (p = 0.016) but did not affect prostacyclin-mediated responses (p = 0.425). Together, our results demonstrate that in vitro administration of serelaxin does not cause relaxation in penile tissue and short-term in vitro serelaxin treatment in hyperglycemic conditions mimicked diabetes modulates endothelium-dependent responses by worsening NO-mediated responses. Serelaxin exerts different effects via different mechanism on endothelium-dependent responses depending on the dose and duration of exposure. Therefore, proper timing and dosing of serelaxin administration in the penile tissue need to be investigated in further studies in diabetic animal models.

Treatment with Resveratrol Reverses Priapism in the Sickle Cell Mouse

Introduction: Sickle cell disease (SCD) men display priapism. Townes and Berkeley SCD mice models are widely studied in basic sciences because they express 100% HbS in their circulating erythrocytes and exhibit features of priapism, and, as in SCD humans, this appears to be due to decreased nitric oxide (NO) signaling and increased oxidative stress, which leads to lower PDE5 in the penis. Thus, when an erectile stimulus occurs in vivo, cGMP accumulates into the 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 downregulation. Moreover, oxidative/nitrosative stress has been associated with priapism in SCD mice.Resveratrol (trans-3,5,4′-trihydroxystilbene) is a natural phytoalexin product with potent antioxidant activity found in peanuts, berries, and grapes. In this study, since decreased NO bioavailability and increased oxidative stress leads to lower PDE5 in the penis, we aimed to evaluate the effects of resveratrol on functional and molecular alterations of erectile function in SCD mice. We have focused on the dysregulated NO-cGMP-PDE5 pathway and oxidative/nitrosative stress markers (4-HNE and 3-nitrotyrosine) in the erectile tissue of SCD. Methods: Wild type (WT, C57BL/6) and Townes SCD transgenic male mice (3-5 mo old) were treated with resveratrol (100 mg /kg/day) or its vehicle daily for two weeks via gavage. Corpus cavernosum (CC) strips were mounted in isolated organ baths, and the relaxing responses to acetylcholine (ACh; endothelium-dependent response) and sodium nitroprusside (SNP; endothelium-independent response), as well as electrical-field stimulation (EFS; nitrergic relaxation), were obtained in CC strips precontracted with the α1-adrenergic receptor agonist phenylephrine (3-10 µM). Results: The cumulative addition of ACh (0.001-10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but the ACh potency (pEC50) value was higher (P<0.05) in CC of SCD compared to WT mice, which was reversed by resveratrol. Likewise, the nitrergic relaxation induced by EFS was also higher (P<0.05) in SCD mice compared to control mice (4 Hz: 30 ± 8 and 80 ± 5 %, respectively; n=5), which was reversed by resveratrol treatment. Similarly, SNP (0.01- 10 µM) produced concentration-dependent CC relaxations in WT and SCD groups, but, again, the maximal relaxations elicited by this agent were higher (P<0.05) in SCD (90 ± 1%) group compared to the WT mice (65 ± 6%), which were fully restored to WT values with resveratrol treatment (n=6). The mRNA expression for the PDE5 was reduced (P<0.05) by approximately 57% in penile tissues from SCD compared to the WT group, respectively. Resveratrol treatment restored (P<0.05) the mRNA expression of PDE5 in the penises from the SCD group (n=5-6). The protein expressions for 4-HNE and 3-nitrotyrosine (3-NT) were significantly higher (P<0.05) in erectile tissues from SCD compared to the WT group, which were reduced (P<0.05) by resveratrol treatment (n=6). In WT mice, PDE5, 4-HNE, and 3-NT protein expressions were not affected by resveratrol, as well as CC relaxations induced by ACh, SNP, and EFS. Conclusion: Resveratrol treatment reduced enhanced NO-mediated CC relaxations along with normalization of PDE5 expression and reduced oxidative stress markers expressions (3-NT and 4-HNE) in the penises from SCD. Therefore, resveratrol may constitute an additional strategy to prevent priapism in SCD. Financial Support: FAPESP.

STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses

Stromal interaction molecule (STIM)/Orai calcium entry system appears to have a role in erectile dysfunction (ED) pathophysiology but its specific contribution to diabetic ED was not elucidated. To evaluate STIM/Orai inhibition on functional alterations associated with diabetic ED in rat and human penile tissues and on in vivo erectile responses in diabetic rats. Rat corpus cavernosum (RCC) strips from nondiabetic (No DM) and streptozotocin-induced diabetic (DM) rats and human penile resistance arteries (HPRA) and corpus cavernosum (HCC) from ED patients undergoing penile prosthesis insertion were functionally evaluated in organ chambers and wire myographs. Erectile function in vivo in rats was assessed by intracavernosal pressure (ICP) responses to cavernous nerve electrical stimulation (CNES). Expression of STIM/Orai elements in HCC was determined by immunofluorescence and immunoblot. Functional responses in RCC, HCC and HPRA and STIM/Orai protein expression in HCC. In vivo erectile responses to CNES. Inhibition of Orai channels with YM-58483 (20 µM) significantly reduced adrenergic contractions in RCC but more effectively in DM. Thromboxane-induced and neurogenic contractions were reduced by STIM/Orai inhibition while defective endothelial, neurogenic and PDE5 inhibitor-induced relaxations were enhanced by YM-58483 (10 µM) in RCC from DM rats. In vivo, YM-58483 caused erections and attenuated diabetes-related impairment of erectile responses. YM-58483 potentiated the effects of PDE5 inhibition. In human tissues, STIM/Orai inhibition depressed adrenergic and thromboxane-induced contractions in ED patients more effectively in those with type 2 diabetes. Diabetes was associated with increased expression of Orai1 and Orai3 in ED patients. Targeting STIM/Orai to alleviate diabetes-related functional alterations of penile vascular tissue could improve erectile function and potentiate therapeutic effects of PDE5 inhibitors in diabetic ED. Improving effects of STIM/Orai inhibition on diabetes-related functional impairment was evidenced in vitro and in vivo in an animal model and validated in human tissues from ED patients. Functional findings were complemented with expression results. Main limitation was low numbers of human experiments due to limited human tissue availability. STIM/Orai inhibition alleviated alterations of functional responses in vitro and improved erectile responses in vivo in diabetic rats, potentiating the effects of PDE5 inhibition. STIM/Orai inhibition was validated as a target to modulate functional alterations of human penile vascular tissue in diabetic ED where Orai1 and Orai3 channels were upregulated. STIM/Orai inhibition could be a potential therapeutic strategy to overcome poor response to conventional ED therapy in diabetic patients. Sevilleja-Ortiz A, El Assar M, García-Gómez B, et al. STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses. J Sex Med 2022;19:1733-1749.

Haptoglobin treatment contributes to regulating nitric oxide signal and reduces oxidative stress in the penis: A preventive treatment for priapism in sickle cell disease.

Background: In sickle cell disease (SCD), reduced bioavailability of endothelial NO and cGMP results in reduced expression of phosphodiesterase type 5 (PDE5), thus impairing the penile erection control mechanism and resulting in prolonged penile erection (priapism). In SCD, reduced NO bioavailability is associated with excess plasma hemoglobin due to intravascular hemolysis and increased oxidative stress. Haptoglobin is the plasma protein responsible for reducing plasma hemoglobin levels, but in SCD, haptoglobin levels are reduced, which favors the accumulation of hemoglobin in plasma. Therefore, we aimed to evaluate the effects of haptoglobin treatment on functional and molecular alterations of erectile function, focusing on the contractile and relaxant mechanisms of corpus cavernosum (CC), as well as oxidative stress. Methods: SCD mice were treated with haptoglobin (400 mg/kg, subcutaneous) or vehicle of Monday, Wednesday and Friday for a period of 1 month. Corpus cavernosum strips were dissected free and placed in organ baths. Cumulative concentration-response curves to the acetylcholine, sodium nitroprusside, phenylephrine and KCL, as well as to electrical field stimulation (EFS), were obtained in CC. Protein expressions of eNOS, phosphorylation of eNOS at Ser-1177, nNOS, PDE5, ROCK1, ROCK2, gp91phox, 3-nitrotyrosine, and 4-HNE were measured by western blot in CC. Results: Increased CC relaxant responses to acetylcholine, sodium nitroprusside and electrical-field stimulation were reduced by haptoglobin in SCD mice. Reduced CC contractile responses to phenylephrine and KCl were increased by haptoglobin in SCD mice. Haptoglobin prevented downregulated eNOS, p-eNOS (Ser-1177), PDE5, and ROCK2 protein expressions and reduced protein expressions of reactive oxygen species markers, NADPH oxidase subunit gp91phox, 3-nitrotyrosine and 4-HNE in penises from SCD mice. Haptoglobin treatment did not affect ROCK1 and nNOS protein expressions in penises from SCD mice. Basal cGMP production was lower in the SCD group, which was normalized by haptoglobin treatment. Conclusion: Treatment with haptoglobin improved erectile function due to up-regulation of eNOS-PDE5 expression and down-regulation of the gp91phox subunit of NADPH oxidase and oxidative/nitrosative stress in the penises of SCD mice. Treatment with haptoglobin also increased contractile activity due to up-regulation of ROCK2. Therefore, haptoglobin treatment may be an additional strategy to prevent priapism in SCD.

Intravascular hemolysis leads to exaggerated corpus cavernosum relaxation: Implication for priapism in sickle cell disease

Patients with sickle cell disease (SCD) display priapism. Clinical studies have shown a strong positive correlation between priapism and high levels of intravascular hemolysis in men with SCD. However, there are no experimental studies that show that intravascular hemolysis promotes alterations in erectile function. Therefore, we aimed to evaluate the corpus cavernosum smooth muscle relaxant function in a murine model that displays intravascular hemolysis induced by phenylhydrazine (PHZ), as well as the role of intravascular hemolysis in increasing the stress oxidative in the penis. Corpus cavernosum strips were dissected free and placed in organ baths. Acetylcholine and electrical field stimulation (EFS)-induced corpus cavernosum relaxations in vitro were obtained. Increased corpus cavernosum relaxant responses to acetylcholine and EFS were observed in the PHZ group. Protein expression of heme oxygenase-1 increased in the corpus cavernosum of the PHZ group, but PDE5 protein expression was not modified. Preincubation with the heme oxygenase inhibitor 1J completely reversed the increased relaxant responses to acetylcholine and EFS in PHZ mice. Protein expression of NADPH oxidase subunit gp91phox, 3-nitrotyrosine, and 4-hydroxynonenal increased in the corpus cavernosum of the PHZ group, suggesting a state of oxidative stress. Basal cGMP production was lower in the PHZ group. Our results show that intravascular hemolysis promotes increased corpus cavernosum smooth muscle relaxation associated with increased HO-1 expression, as well as increased oxidative stress associated with upregulation of gp91phox expression. Moreover, our study supports clinical studies that point to a strong positive correlation between priapism and high levels of intravascular hemolysis in men with SCD.

Resveratrol-nitric oxide donor hybrid effect on priapism in sickle cell and nitric oxide-deficient mouse.

BackgroundChildren and adult with sickle cell disease (SCD) display priapism associated with low nitric oxide (NO) bioavailability and oxidative stress in penis.AimThis study aimed to evaluate the effects of hybrid compound RVT-FxMe, derived from resveratrol bearing a NO-donor subunit, on two murine model that display priapism phenotype, SCD transgenic mice and endothelial NO synthase gene-deficient (eNOS-/-) mice.MethodsWild-type, SCD, and eNOS-/- mice were treated with RVT-FxMe (25 mg/kg/d, 2 weeks).OutcomesHematological parameters, concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), as well as to electrical field stimulation (EFS), were obtained in mice corpus cavernosum strips.ResultsCorpus cavernosum relaxations to SNP and EFS were increased in eNOS-/- group, which were normalized by RVT-FxMe treatment. SCD mice exhibited an excessive CC relaxant response induced by ACh, EFS and SNP RVT-FxMe treatment did not change the increased relaxant responses to ACh, EFS and SNP in corpus cavernosum from SCD group.Clinical translationExcess of plasma hemoglobin in SCD may interfere in pharmacological activity of NO donors compounds.Strength/LimitationsWhile mechanistic data with promising potential is showed, the current study is not without limitations. RVT-FxMe effects in the mid- and long-term warrant complementary studies.ConclusionTreatment with RVT-FxMe reversed the enhanced NO-cGMP-mediated CC relaxations in eNOS-/- mice, but not in SCD mice; it is likely that excess of plasma hemoglobin in SCD mice act to inactivate NO before it reaches soluble guanylyl cyclase, avoiding restoration of NO bioavailability in penis.

Treatment with levothyroxine and testosterone ameliorated erectile dysfunction in a rat model of propylthiouracil-induced hypothyroidism

ABSTRACT Introduction The endocrine disease frequently interrupts sexual function, and sexual dysfunction may signal serious endocrine disease. Recent studies have claimed an association with hypothyroidism, low testosterone (T) levels and erectile dysfunction (ED), but causality is unclear. There is no study on the experimental model of hypothyroidism that was induced in rats by administration of propylthiouracil (PTU). Objective The purpose of this study was to investigate the deleterious effects of PTU-induced hypothyroidism on ED and T levels. In addition, Levothyroxine is known worldwide as the most common and least effective drug for controlling hypothyroidism. Methods Adult Sprague-Dawley rats (n = 35) were divided into five groups; control, PTU-induced hypothyroidism, PTU+ Levothyroxine, PTU+ Sustanon (a mixture of 4 types of T: propionate, phenylpropionate, isocaproate, decanoate) and PTU+ Levothyroxine + Sustanon. PTU was given in drinking water (0.05%) for 6 weeks. 4 weeks after the beginning of PTU treatment, Levothyroxine (20 mg/kg/day, oral) and Sustanon (10 mg/kg, I.M., once/week) were given for 2 weeks. Serum levels of total T, triiodothyronine (T3) and thyroxine (T4) were determined. The ratio of intracavernosal pressure (ICP) to mean arterial pressure (MAP) and total ICP were measured by cavernous nerve stimulation. The relaxant and contractile responses were evaluated on in vitro preparations of corpus cavernosum (CC) strips. The expression and localization of neuronal (nNOS), endothelial NOS (eNOS) and phosphodiesterase type 5 (PDE5​) were determined using Western blotting and immunohistochemistry. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Results The rat model of hypothyroidism showed a significant decline in serum levels of total T, T3 and T4. Levothyroxine increased T3 and T4 levels while Sustanon normalized only total T levels. In addition, combined treatment of Levothyroxine and Sustanon enhanced all hormone levels. Rats with hypothyroidism displayed the lowest ICP/MAP ratio and total ICP for 7.5 V (P &amp;lt; .001 vs controls). Combined treatment returned reduced ICP/MAP and total ICP responses, while partial amelioration was observed after Levothyroxine and Sustanon treatment alone. In vitro endothelium-dependent acetylcholine-caused relaxation at 1 nM (P &amp;lt; .001 vs controls), electrical field stimulation-induced relaxant response at 20 Hz (P &amp;lt; .001 vs controls) and sildenafil-induced relaxation at 10 µM (P &amp;lt; .05 vs controls) were decreased in the CC strips from hypothyroid rats. The treatment with Levothyroxine and Sustanon increased the reduction in relaxation responses. Decreased in the expressions of eNOS and nNOS and increased in the expressions of PDE5 in the hypothyroid group were restored by Levothyroxine and Sustanon. Mono-treatment partially enhanced reduced smooth muscle mass while combined therapy completely recovered. Conclusions These results show that hypothyroidism adversely affects T levels and erectile responses via decreasing neurogenic and endothelium-dependent relaxation responses which were reversed by the combination therapy with Levothyroxine and Sustanon. Even though the associated alterations in T levels, ED and its reversibility indicate an exact role of thyroid hormones in the erection physiology. Disclosure Work supported by industry: no.

Treatment with levothyroxine and testosterone ameliorated erectile dysfunction in rat model of propylthiouracil-induced hypothyroidism

ABSTRACT Introduction The endocrine disease frequently interrupts sexual function, and sexual dysfunction may signal serious endocrine disease. Recent studies have claimed an association with hypothyroidism, low testosterone (T) levels and erectile dysfunction (ED), but causality is unclear. There is no study on the experimental model of hypothyroidism that was induced in rats by administration of propylthiouracil (PTU). Objective The purpose of this study was to investigate the deleterious effects of PTU-induced hypothyroidism on ED and T levels. In addition, Levothyroxine is known worldwide as the most common and least effective drug for controlling hypothyroidism. Methods Adult Sprague-Dawley rats (n = 35) were divided into five groups; control, PTU-induced hypothyroidism, PTU+ Levothyroxine, PTU+ Sustanon (a mixture of 4 types of T: propionate, phenylpropionate, isocaproate, decanoate) and PTU+ Levothyroxine + Sustanon. PTU was given in drinking water (0.05%) for 6 weeks. 4 weeks after the beginning of PTU treatment, Levothyroxine (20 mg/kg/day, oral) and Sustanon (10 mg/kg, I.M., once/week) were given for 2 weeks. Serum levels of total T, triiodothyronine (T3) and thyroxine (T4) were determined. The ratio of intracavernosal pressure (ICP) to mean arterial pressure (MAP) and total ICP were measured by cavernous nerve stimulation. The relaxant and contractile responses were evaluated on in vitro preparations of corpus cavernosum (CC) strips. The expression and localization of neuronal (nNOS), endothelial NOS (eNOS) and phosphodiesterase type 5 (PDE5) were determined using Western blotting and immunohistochemistry. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Results The rat model of hypothyroidism showed a significant decline in serum levels of total T, T3 and T4. Levothyroxine increased T3 and T4 levels while Sustanon normalized only total T levels. In addition, combined treatment of Levothyroxine and Sustanon enhanced all hormone levels. Rats with hypothyroidism displayed the lowest ICP/MAP ratio and total ICP for 7.5 V (P &amp;lt; .001 vs controls). Combined treatment returned reduced ICP/MAP and total ICP responses, while partial amelioration was observed after Levothyroxine and Sustanon treatment alone. In vitro endothelium-dependent acetylcholine-caused relaxation at 1 nM (P &amp;lt; .001 vs controls), electrical field stimulation-induced relaxant response at 20 Hz (P &amp;lt; .001 vs controls) and sildenafil-induced relaxation at 10 µM (P &amp;lt; .05 vs controls) were decreased in the CC strips from hypothyroid rats. The treatment with Levothyroxine and Sustanon increased the reduction in relaxation responses. Decreased in the expressions of eNOS and nNOS and increased in the expressions of PDE5 in the hypothyroid group were restored by Levothyroxine and Sustanon. Mono-treatment partially enhanced reduced smooth muscle mass while combined therapy completely recovered. Conclusions These results show that hypothyroidism adversely affects T levels and erectile responses via decreasing neurogenic and endothelium-dependent relaxation responses which were reversed by the combination therapy with Levothyroxine and Sustanon. Even though the associated alterations in T levels, ED and its reversibility indicate an exact role of thyroid hormones in the erection physiology. Disclosure No

Dual Strategy with Intracavernosal Implantation of Adipose-derived Stem Cells and Daily Oral L-arginine Optimize Recovery of and Cavernosal Functions after Cavernous Nerve Injury in Rats

ABSTRACT Introduction Radical prostatectomy for prostate cancer has a significant potential role in inducing cavernous nerve injury (CNI). Increasing hypoxia and fibrosis induces destructive changes within cavernous vasculature. Adipose-derived stem cells (ADSCs) can maintain self-renewal and enhanced multi-differentiation potential through the release of a variety of paracrine factors and extracellular vesicles, allowing to repair damaged tissues. Considerable attention has increasingly been paid to their application in tissue engineering and regeneration. In addition, ADSCs can be obtained with a minimally invasive procedure and easily expanded in vitro with low immunogenicity. Nitric oxide (NO) is synthesized from the amino acid L-arginine by a family of enzymes, the nitric oxide synthases (NOS). Orally administered L-arginine enhances the erectile response presumably by stimulating NO production by the corporal tissues increasing cGMP production. Objective To evaluate the efficacy and tolerability of ADSCs and daily oral L-arginine alone or combination in treating rats with bilateral CNI (BCNI)-induced ED. Methods Adult Sprague-Dawley rats (n = 35) were divided into five groups: 1) control; 2) BCNI (4-wk); 3) BCNI+ADSCs (1 × 106 cells by intracavernosal injection); 4) BCNI+ L-arginine (4wk,10 mg/kg/day, oral); and 5) combination treatment with ADSCs and L-arginine in BCNI. Rats in the ADSCs treatment groups were subjected to intracavernosal injection after BCNI. In vivo erectile response was measured by CN stimulation. The relaxant and contractile responses were also detected on in vitro preparations of corpus cavernosum (CC) strips. The expression and localization of neuronal (nNOS), endothelial NOS (eNOS) as well as hypoxia (hypoxia-inducible factor-1α, HIF-1α), fibrosis [transforming growth factor-beta 1 (TGF- β1)]and apoptosis (Bax and Bcl-2) markers were determined using Western blotting and immunohistochemistry. The relative area of smooth muscle to collagen was measured using Masson trichrome staining. Results Rats with CNI had the lowest ICP/MAP ratio at 63% and total ICP at 55% for 7.5 V (P &amp;lt; .001 vs controls) as well as in vitro electrical field stimulation-induced relaxant response at 10% (P &amp;lt; .05 vs controls) and endothelium-dependent acetylcholine-caused relaxation at 56% (P &amp;lt; .001 vs controls). The combined treatment showed more significantly improved erectile and relaxant responses more effectively than ADSCs or L-arginine treatment alone. Increased in the expressions of nNOS, HIF-1α, Bax, and Bcl-2 in the BCNI group were restored by all treatment regiments. eNOS and TGF- β1 protein expressions were similar among groups. Mono-treatment partially improved decreased smooth muscle mass while combined therapy completely recovered. Conclusions The intracavernous injection of ADSCs and oral administration of L-arginine, and, importantly, their combination could effectively protect against hypoxia-induced neurotoxicity, via their increasing endothelial cell function and smooth muscle content, anti-inflammatory, anti-apoptotic, anti-fibrotic properties in addition to reversing erectile function in the BCNI rat model. HIF-1α overexpression by the inhibition of the apoptotic pathway with the Bcl-2 family may have protective effects on the survival of cavernosal neurons. Disclosure No

Erectile Dysfunction and Altered Contribution of KCa1.1 and KCa2.3 Channels in the Penile Tissue of Type-2 Diabetic db/db Mice

BackgroundActivation of endothelial small conductance calcium-activated K+ channels (KCa2.3) and intermediate conductance calcium-activated K+ channels (KCa3.1) leads to vascular relaxation. We found endothelial KCa2.3 down-regulation in the corpus cavernosum diminishes erectile function. AimWe hypothesized that in type-2 diabetic mice, the function of KCa2.3 and KCa1.1 channels is impaired in erectile tissue. MethodsErectile function was measured, and corpus cavernosum strips were mounted for functional studies and processed for qPCR and immunoblotting. OutcomesEffects of type 2 diabetes on erectile function, expression and function of calcium-activated potassium channels. ResultsIn anesthetized diabetic db/db mice, erectile function was markedly decreased compared to non-diabetic heterozygous db/+ mice, and the impairment was even more pronounced compared to normal C57BL/6 mice. qPCR revealed KCa2.3 and KCa1.1α channel expressions were upregulated in corpus cavernosum from db/db mice. Immunoblotting showed down-regulation of KCa2.3 channels in the corpus cavernosum from db/db mice. Acetylcholine relaxations were impaired while relaxations induced by the nitric oxide, donor SNP were unaltered in corpus cavernosum from db/db compared to C57BL/6 and db/+ mice. Apamin, a blocker of KCa2 channels, inhibited acetylcholine relaxation in corpus cavernosum from all experimental groups. In the presence of apamin, acetylcholine relaxation was markedly decreased in corpus cavernosum from db/db vs C57BL/6 and db/+ mice. An opener of KCa2 and KCa3.1 channels, NS309, potentiated acetylcholine relaxations in corpus cavernosum from db/+ and db/db mice. Iberiotoxin, a blocker of KCa1.1 channels, inhibited acetylcholine relaxation in corpus cavernosum from db/+ mice, while there was no effect in tissue from db/db mice. Clinical TranslationErectile function in diabetic db/db mice was severely affected compared to heterozygous and control mice, findings suggesting the non-diabetic db/+ and diabetic db/db mice for translational purpose can be used for drug testing on, respectively, moderate and severe erectile dysfunction. The altered expressions and impaired acetylcholine relaxation in the presence of apamin compared to C57BL/6 mice may suggest decreased KCa1.1 channel function may underpin impaired endothelium-dependent relaxation and erectile dysfunction in diabetic db/db mice. Strengths & LimitationsThe present study provides a mouse model for type 2 diabetes to test moderate and severe erectile dysfunction drugs. Decreased KCa1.1 channel function contributes to erectile dysfunction, and it is a limitation that it is not supported by electrophysiological measurements. ConclusionOur results suggest that the contribution of iberiotoxin-sensitive KCa1.1 channels to relaxation is reduced in the corpus cavernosum, while apamin-sensitive KCa2.3 channels appear upregulated. The impaired KCa1.1 channel function may contribute to the impaired erectile function in diabetic db/db mice.Comerma-Steffensen S, Prat-Duran J, Mogensen S, et al. Erectile Dysfunction and Altered Contribution of KCa1.1 and KCa2.3 Channels in the Penile Tissue of Type-2 Diabetic db/db Mice. J Sex Med 2022;19:697–710.

Possible mechanisms in Ficus exasperata ethanol root fraction amelioration of lead-induced corpus cavernosal contractile impairments in male Wistar rats.

Background:Lead exposure is reported to cause impaired male reproductive tract functions often due to elevated oxidative species. F. exasperata leaf extract contains potent phyto-antioxidants with beneficial endothelial functions. Objectives: This study investigates the effect of ethanolic extract of F. exasperata (EEFE) treatment on the corpus cavernosal contractile activity in lead-exposed adult male Wistar rats. Materials &amp; Methods: Corpus cavernosum strips (CS) were excised from control, EEFE only treated (400 mg/kg), lead + EEFE treated and lead only treated (100 mg/kg) male Wistar rats. Cumulative contractile responses of CS to acetylcholine, potassium chloride, and calcium influx were determined. Furthermore, cumulative dose responses to acetylcholine after incubation in LNAME, indomethacin, nifedipine, and acetovanillone were also determined by measuring isometric contractions using Ugo Basile data capsule acquisition system. Results: Relaxation(%) response to cumulative doses of acetylcholine (10-9–10-5 M) was enhanced in EEFE only treated (59%) compared to lead only (45%). Contractile response to KCl (10-60 M) was restrained in control and EEFE cotreated when compared to lead only treated. Preincubation of the CS with nifedipine (10-4 M) suppressed the relaxation(%) in the lead + EEFE co-treated. Preincubation of the CS in indomethacin improved the relaxant response(%) of the CS in EEFE only and lead + EEFE co-treated (53.53% ±2.80 &amp; 54.65%± 2.95 respectively) compared with the lead only treated (45.40%±1.79). Preincubation with L-NAME significantly reduced relaxation response(%) in EEFE only treated group. Relaxation response(%) of the CS was enhanced in the lead + EEFE group after preincubation with acetovanillone. Conclusion: The study suggests that L-type and voltage-gated calcium channels, NO, and potent antioxidant activity, are important in EEFE amelioration of lead-induced contractile impairments.

Oxaliplatin, an Anticancer Agent, Causes Erectile Dysfunction in Rats due to Endothelial Dysfunction

Chemotherapeutics, one of the standard treatment options for cancer worldwide, have various adverse effects, including erectile dysfunction (ED). To investigate erectile function in an animal model after administration of the anticancer agent oxaliplatin (L-OHP). Male Wistar/ST rats were divided into 2 groups: L-OHP rats (n = 21), which were intravenously administered L-OHP (4 mg/kg; twice a week for 4 weeks), and Control rats (n = 21), which were injected with the same volume of 5% glucose solution, using the same dosing schedule. At the end of the study period, erectile function was evaluated by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP) after cavernous nerve stimulation (n = 9-10). Endothelial function was evaluated with an isometric tension study using corpus cavernosum strips (n = 11). Western blot analysis was used to assess neuronal nitric oxide (nNOS) and endothelial NO synthase (eNOS) protein levels (n = 7). Real-time quantitative polymerase chain reaction (qRT-PCR) was used to assess the expression of inflammation- and oxidative stress-related markers (nicotinamide adenine dinucleotide phosphate oxidase-1, p22phox, interleukin [IL]-6, and nuclear factor-kappa B) (n = 6). Statistical significance was determined using the Student's t-test. The L-OHP group had a significantly lower ICP:MAP ratio than the control group (P < .05). Compared to the Control group, the L-OHP group exhibited significantly lower responses to ACh and eNOS protein levels and significantly higher inflammatory biomarker levels. The results based on this animal model indicate that use of the anticancer agent L-OHP should be considered as a risk factor for ED occurring via reduction of NO bioavailability in humans; our results provide possible treatment strategies for maintaining the erectile function of cancer survivors. Our study showed that the anticancer agent L-OHP has the propensity to cause ED in rats. A major limitation of this study is the lack of an established cure for ED associated with L-OHP and the lack of clinical evidence. L-OHP causes ED in rats via reduction of NO bioavailability caused by endothelial dysfunction.

Mechanisms in Tridax procumbens leaf extract reversal of paroxetine-induced erectile dysfunction in corpus cavernosum of male Wistar rats

Introduction: Aqueous leave extract of Tridax procumbens (AETPL) is reported to improve erectile functions; however, the mechanism is unclear. This study investigates the mechanism involved in the contractile activity of the corpus cavernosum after AETPL treatment of paroxetine-induced erectile dysfunctional adult male Wistar rats.&#x0D; Methods: A total of 20 male Wistar rats were categorized into four groups of five and treated orally for four weeks: Group 1 (distilled water), Group 2 (paroxetine 10 mg/kg), Group 3 (paroxetine + AETPL 100 mg/kg), and Group 4 (paroxetine + Viagra 0.5 mg/kg). Contractile responses of excised corpus cavernosum strips (CS) were determined in response to acetylcholine (ACh), phenylephrine (PHE), potassium chloride (KCl), and calcium chloride (CaCl2), and after incubation in L-NAME, indomethacin, nifedipine, adenosine, caffeine, nicorandil, and acetovanillone.&#x0D; Results: The relaxation response (%) of CS to ACh was significantly inhibited in the paroxetine group compared to the AETPL- and the Viagra-co-treated group. Pre-incubation in L-NAME considerably enhanced the percentage relaxation in groups co-treated with AETPL and Viagra. Groups co-treated with AETPL and Viagra significantly inhibited contraction in response to cumulative doses of CaCl2. Contractile responses of CS to cumulative doses of PHE after incubation in caffeine and adenosine were considerably inhibited in groups co-treated with AETPL and Viagra. Similarly, nicorandil (10-4 M) enhanced the percentage relaxation to cumulative doses of ACh (10-9 — 10-5 M) in groups co-treated with AETPL and Viagra. The pre-incubation of CS with acetovanillone (10-4 M) enhanced the percentage relaxation to ACh across groups.&#x0D; Conclusion: Erectile dysfunction was reversed by AETPL-induced antioxidant/NADPH oxidase inhibitor activity, reduced calcium sensitivity, activation of ATP-sensitive K+ channel, and endothelial Nitric Oxide (NO) release.&#x0D;