Novel nitric oxide signaling mechanisms regulate the erectile response

Nitric oxide (NO) is a physiologic signal essential to penile erection, and disorders that reduce NO synthesis or release in the erectile tissue are commonly associated with erectile dysfunction. NO synthase (NOS) catalyzes production of NO from L-arginine. While both constitutively expressed neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms mediate penile erection, nNOS is widely perceived to predominate in this role. Demonstration that blood-flow-dependent generation of NO involves phosphorylative activation of penile eNOS challenges conventional understanding of NO-dependent erectile mechanisms. Regulation of erectile function may not be mediated exclusively by neurally derived NO: Blood-flow-induced fluid shear stress in the penile vasculature stimulates phosphatidyl-inositol 3-kinase to phosphorylate protein kinase B, which in turn phosphorylates eNOS to generate NO. Thus, nNOS may initiate cavernosal tissue relaxation, while activated eNOS may facilitate attainment and maintenance of full erection.     

Therapeutic strategies for optimizing PDE-5 inhibitor therapy in patients with erectile dysfunction considered difficult or challenging to treat

Phosphodiesterase 5 (PDE5) inhibitors prevent the normal hydrolysis of cGMP. As the resulting cGMP accumulation facilitates penile smooth muscle relaxation, PDE5 inhibitors can partially reverse deficiencies in the nitric oxide (NO)/cGMP pathway to treat erectile dysfunction (ED). However, approximately 30-40% of men with ED do not respond to drug therapy. Patients with severe neurologic damage, diabetes mellitus, or severe vascular disease may be resistant to PDE5 inhibitors. Decreased expression or activity of neuronal or endothelial NO synthase (NOS), impaired NO release, or NO destruction will preclude sufficient cGMP formation to permit PDE5 inhibitor efficacy. This article discusses the possible reasons for unresponsiveness and strategies to overcome it. Therapeutic approaches proposed to increase available NO in penile tissue include facilitating NO release by using alpha-2 antagonists, enhancing NO synthesis by providing more substrate for the reaction, and using antioxidants to inhibit NO breakdown by reactive oxygen species.     

Sildenafil citrate, a selective phosphodiesterase type 5 inhibitor: urologic and cardiovascular implications

Erectile dysfunction (ED) occurs in varying degrees in an estimated 20 to 30 million American men and is associated with adverse effects on quality of life; particularly personal well-being, family and social interrelationships. Research into ED has focused primarily on the physiologic mechanisms of corpus cavernosum smooth muscle relaxation, and penile erection as the end result of smooth muscle relaxation. These processes are mediated by cholinergic, nonadrenergic, noncholinergic (NANC, e.g., nitric oxide), vasoactive intestinal peptide (VIP), and potentially calcitonin gene-related peptide (CGRP) containing nerves. Release of nitric oxide following sexual stimulation from non-adrenergic, non cholinergic nerves and vascular endothelium activates guanylyl cyclase and induces intracellular cGMP synthesis. In turn, cGMP results in lowering intracellular concentrations, inhibits contractility of the penile smooth muscle, and induces an erectile response. Phosphodiesterase type 5 (PDE 5) is the predominant enzyme responsible for cGMP hydrolysis in trabecular smooth muscle. Activation of PDE 5 terminates NO-induced, cGMP-mediated smooth muscle relaxation, and subsequent penile flaccidity. Sildenafil citrate is a potent PDE type 5 reversible and selective inhibitor which blocks cGMP hydrolysis effectively. FDA approval of sildenafil citrate as the first oral agent for ED in males has resulted in significant interest. We discuss the clinical and pharmacologic properties of sildenafil citrate as well as the urologic and cardiac implications.     

Immunocytochemical distribution of nitric oxide synthase in the human corpus cavernosum: an electron microscopical study using the tyramide signal amplification technique

Nitric oxide has proven to be an important mediator in the relaxation of human cavernosal smooth muscle. Nevertheless, there are many inconsistencies in the literature regarding the cellular and subcellular distribution of endothelial nitric oxide synthase in the human penis. The purpose of this study was to reexamine the localization of eNOS and nNOS in the cellular anatomy of the human cavernous body by means of electron microscopical immunocytochemistry in combination with the tyramide signal amplification technique (TSA). Using specific antibodies against eNOS and nNOS, the NAPDH-diaphorase reaction and advanced protocols for fixation and staining procederes, the occurrence of NOS isoenzymes eNOS and nNOS were examined in cavernosal specimens of ten male patients who were subjected to surgery for penile deviation. eNOS immunoreactivity and NADPH-d staining was seen to be significantly present in the endothelial cells covering the cavernous spaces and in the endothelium of helicine arteries. In endothelial cells, the NADPH-d reaction product BSPT-formazan was abundantly detectable attached to membranes of the endoplasmatic reticulum and the mitochondria whereas posititve eNOS immunostaining was seen in the endothelial cells throughout their cytoplasm without any particular relation to organelles. No considerable eNOS immunoreactivity was detectable in the trabecular smooth muscle cells. nNOS staining was found in nerve fibers innervating the cavernous body and cavernosal arteries. Our results counteract the hypothesis of the cavernous smooth muscle as a local source of NO and underline the importance of an intact endothelial function for penile erection and the contribution of eNOS to this process. Received: 14 September 2000 / Accepted: 9 February 2001     

Plasma levels of cyclic guanosine-3′,5′-monophosphate in the cavernous and systemic blood of healthy males during different functional conditions of the penis

The relaxation of cavernous arterial and trabecular smooth muscle is dependent upon the stimulation of guanylyl cyclase activity by nitric oxide (NO), which is released from nerve terminals and endothelial cells within the cavernous tissue, and the subsequent accumulation of cyclic guanosine-3',5'-monophosphate (cGMP) in the intracellular space. The present study was undertaken to determine whether or not plasma levels of cGMP in the systemic and cavernous blood of healthy male subjects change from penile flaccidity to tumescence, rigidity and detumescence. Fifteen adult healthy males were exposed to visual and tactile erotic stimuli to elicit penile tumescence and rigidity. Whole blood was simultaneously aspirated from the corpus cavernosum and the cubital vein in the respective penile stages, and cGMP was determined in plasma aliquots by means of a radioimmunoassay. Mean systemic and cavernous plasma levels of cGMP in the blood samples obtained from the healthy volunteers ranged from 1.2-1.7 pmol/ ml. cGMP levels in the systemic circulation and in the cavernous blood did not change during developing erection, rigidity and detumescence. No significant differences were found between cGMP plasma levels in the systemic and cavernous blood in the different penile stages. Our results may reflect the fact that the stimulation of NO production in healthy males during sexual arousal and developing penile erection either does not yield substantial quantities of cGMP or that the rate of cGMP-extrusion from cavernous smooth muscle cells into the extracellular space accounts only for a minor fraction of plasma cGMP. Moreover, basal levels of cGMP in the blood flushing the lacunar spaces of the cavernous body in the state of developing erection may conceal any local release of cGMP that may occur within the penile erectile tissue. Thus, we conclude that the quantification of cGMP is of no use in the evaluation of the physiologic mechanisms of penile erection in vivo.     

Chronic inhibition of nitric‐oxide synthase induces hypertension and erectile dysfunction in the rat that is not reversed by sildenafil

Study Type – Aetiology (case control)
Level of Evidence 3b

OBJECTIVE

To evaluate the effect of N(G)‐nitro‐l ‐arginine methyl ester (L‐NAME)‐induced hypertension (HT) on erectile function in the rat and determine if the phosphodiesterase (PDE)‐5 inhibitor, sildenafil, can reverse the effects of nitric oxide (NO) deficiency, as HT is a risk factor for erectile dysfunction (ED) and the NO synthase (NOS) inhibitor L‐NAME induces NO‐deficient HT.

MATERIALS AND METHODS

Thirty‐six adult Sprague‐Dawley male rats were divided into three groups, i.e. a control, L‐NAME‐HT (40 mg/rat/day in the drinking water for 4 weeks), and sildenafil‐treated L‐NAME‐HT (1.5 mg/rat/day sildenafil, by oral gavage concomitantly with L‐NAME). The erectile response expressed as a ratio of intracavernosal pressure (ICP)/mean arterial pressure (MAP), evaluated after electrical stimulation of the right cavernous nerve. The isometric tension of corpus cavernosum smooth muscle (CCSM) was measured in organ‐bath experiments. NOS expression was determined immunohistochemically for neuronal (n)NOS and by Western blot analysis for endothelial (e) and inducible (i) NOS protein. cGMP levels were evaluated by enzyme‐linked immunosorbent assay.

RESULTS

The erectile response was diminished in the HT group. Nitrergic and endothelium‐dependent relaxation was reduced, while the relaxation response to sodium nitroprusside and contractile response to phenylephrine were not altered in CCSM from L‐NAME‐treated rats. HT rats showed decreased expression of nNOS, whereas eNOS and iNOS protein expression was increased. Sildenafil partly restored endothelial and molecular changes in CCSM from HT rats, but did not reverse the decreased erectile response, even as cGMP levels returned to normal levels.

CONCLUSIONS

Sildenafil treatment did not correct the ED in L‐NAME‐treated HT rats. Under sustained high blood pressure, up‐regulation of PDE5 expression failed to reverse the depletion of neuronal NO and/or impaired nNOS activity. However, endothelium‐dependent relaxation was restored. Drug targeting of neuronal dysfunction might delay the onset of ED in HT.     

Cyclic AMP-specific and cyclic GMP-specific phosphodiesterase isoenzymes in human cavernous arteries—immunohistochemical distribution and functional significance

Objectives: It has been well established that male erectile dysfunction is frequently associated with vascular diseases. The normal function of cavernous arteries is considered a prerequisite to maintain sufficient blood flow to the trabecular spaces in order to enable penile erection. Contractility of cavernous arteries is regulated by the peripheral autonomic nervous system and endogenous factors released from the endothelial cell layer. A significant increase of blood flow in the central cavernous arteries is the initial event leading to penile tumescence and rigidity. Besides the significance of the nitric oxide/cyclic guanosine monophosphate (cGMP)-mediated mechanisms, the cyclic AMP (cAMP) signalling pathway is also involved in the regulation of tone of the erectile tissue, and interactions between cGMP- and cAMP-mediated mechanisms have been demonstrated. The aim of the present study was to investigate by means of immunohistochemistry the presence of the phosphodiesterase (PDE) isoenzymes 3, 4 (cAMP-specific PDEs) and 5 (cGMP-specific PDE) in thin sections of human central cavernous arteries (HCA) and their functional significance in the mechanism of vessel tone regulation. Methods: Functional experiments were performed using circular segments of HCA and strip preparations of the human corpus cavernosum (HCC). Relaxant effects induced by the cumulative addition of the PDE inhibitors milrinone (PDE3 inhibitor), rolipram (PDE4 inhibitor) and sildenafil (PDE5 inhibitor; 0.01, 0.1, 1 and 10 M) were studied in preparations of HCA and HCC challenged by 1 M norepinephrine (NE). Moreover, immunohistochemistry was carried out in order to evaluate the expression of PDE3, PDE4 and PDE5 in thin sections of HCA. Results: Milrinone, rolipram and sildenafil dose-dependently reversed the NE-induced tension of the isolated vascular segments and HCC strips with sildenafil being the most effective drug. Neither rolipram nor milrinone reached an EC₅₀ value. Abundant immunoreactivities specific for PDE3, PDE4 and PDE5 were observed in the entire smooth musculature of the wall of HCA and resistance arteries. In addition, immunoreactivity for PDE4 was also detected in the cytoplasm of endothelial cells lining the cavernous arteries. Conclusions: The cGMP-dependent relaxation of cavernous arteries is not only dependent on the normal function of the peripheral autonomic nervous system but also on the functional integrity of the vascular endothelium. The expression of the cGMP-specific PDE5 and the ability of the PDE5 inhibitor sildenafil to reverse the adrenergic tension of isolated segments of HCA underline the important role of the NO/cGMP pathway in the control of smooth muscle tone of human trabecular smooth musculature and penile cavernous arteries. Our results also suggest a significance of the cAMP-dependent signaling mechanisms in the regulation of tension of central HCAs. The present findings are also in support of the hypothesis of interactions between the cGMP- and cAMP-mediated signaling pathways in HCAs. Further investigations are indicated in order to outline potential differences between central HCAs and helicine resistance arteries. This may help to understand better the relations between structural and functional changes in the penile erectile tissue in patients with cardiovascular diseases and endothelial dysfunction.     

The phosphodiesterase inhibitory selectivity and the in vitro and in vivo potency of the new PDE5 inhibitor vardenafil

We investigated the potency and the selectivity profile of vardenafil on phosphodiesterase (PDEs) enzymes, its ability to modify cGMP metabolism and cause relaxation of penile smooth muscle and its effect on erections in vivo under conditions of exogenous nitric oxide (NO) stimulation. PDE isozymes were extracted and purified from human platelets (PDE5) or bovine sources (PDEs 1, 2, 3, 4 and 6). The inhibition of these PDEs and of human recombinant PDEs by vardenafil was determined. The ability to potentiate NO-mediated relaxation and influence cGMP levels in human corpus cavernosum strips was measured in vitro, and erection-inducing activity was demonstrated in conscious rabbits after oral administration together with intravenous doses of sodium nitroprusside (SNP). The effects of vardenafil were compared with those of the well-recognized PDE5 inhibitor, sildenafil (values for sildenafil in brackets). Vardenafil specifically inhibited the hydrolysis of cGMP by PDE5 with an IC50 of 0.7 nM (6.6 nM). In contrast, the IC50 of vardenafil for PDE1 was 180 nM; for PDE6, 11 nM; for PDE2, PDE3 and PDE4, more than 1000 nM. Relative to PDE5, the ratios of the IC50 for PDE1 were 257 (60), for PDE6 16 (7.4). Vardenafil significantly enhanced the SNP-induced relaxation of human trabecular smooth muscle at 3 nM (10 nM). Vardenafil also significantly potentiated both ACh-induced and transmural electrical stimulation-induced relaxation of trabecular smooth muscle. The minimum concentration of vardenafil that significantly potentiated SNP-induced cGMP accumulation was 3 nM (30 nM). In vivo studies in rabbits showed that orally administered vardenafil dose-dependently potentiated erectile responses to intravenously administered SNP. The minimal effective dose that significantly potentiated erection was 0.1 mg/kg (1 mg/kg). The selectivity for PDE5, the potentiation of NO-induced relaxation and cGMP accumulation in human trabecular smooth muscle and the ability to enhance NO-induced erection in vivo indicate that vardenafil has the appropriate properties to be a potential compound for the treatment of erectile dysfunction. Vardenafil was more potent and selective than sildenafil on its inhibitory activity on PDE5.     

Mechanisms of action of PDE5 inhibition in erectile dysfunction

A spinal reflex and the L-arginine-nitric oxide-guanylyl cyclase-cyclic guanosine monophosphate (cGMP) pathway mediate smooth muscle relaxation that results in penile erection. Nerves and endothelial cells directly release nitric oxide in the penis, where it stimulates guanylyl cyclase to produce cGMP and lowers intracellular calcium levels. This triggers relaxation of arterial and trabecular smooth muscle, leading to arterial dilatation, venous constriction, and erection. Phosphodiesterase 5 (PDE5) is the predominant phosphodiesterase in the corpus cavernosum. The catalytic site of PDE5 normally degrades cGMP, and PDE5 inhibitors such as sildenafil potentiate endogenous increases in cGMP by inhibiting its breakdown at the catalytic site. Phosphorylation of PDE5 increases its enzymatic activity as well as the affinity of its allosteric (noncatalytic/GAF domains) sites for cGMP. Binding of cGMP to the allosteric site further stimulates enzymatic activity. Thus phosphorylation of PDE5 and binding of cGMP to the noncatalytic sites mediate negative feedback regulation of the cGMP pathway.     

全面康复:勃起功能障碍治疗的新目标

5型磷酸二酯酶(PDE5)抑制剂有效改善勃起功能障碍(ED)患者的勃起功能。枸橼酸西地那非的应用范围不断扩展,肺动脉高压已成为新的适应证。临床研究发现,西地那非能改善多种血管性疾病患者的内皮功能。在ED领域的研究进展包括:动物实验发现,西地那非可以改善海绵体内皮功能,增强磷酸化内皮型一氧化氮合酶(eNOS)蛋白表达,逆转缺血或缺氧导致的海绵体内压(ICP)降低。临床研究证实,西地那非可以使50%以上ED患者阴茎勃起恢复到最充分的硬度(4级勃起);使50%以上保留神经的根治性前列腺切除术后患者勃起功能康复,自发产生足以性交的勃起;使ED患者的自尊心、自信心和性关系满意度等社会心理功能恢复正常。从勃起功能到社会心理功能的全面恢复可能成为今后ED治疗的新目标。     

硫化氢与阴茎勃起功能关系研究进展

硫化氢(H2S)是继NO和CO之后第3种被发现的具有内源性活性的气体信号分子,在哺乳动物体内H2S主要由两种蛋白酶———胱硫醚-β-合酶(CBS)和胱硫醚-γ-裂合酶(CSE)合成产生。H2S在体内具有重要的生理调节功能,它可作用于ATP敏感性钾离子通道(K+-ATP)舒张血管平滑肌,与睾酮及NO协同作用舒张阴茎海绵体平滑肌,促进阴茎勃起等。目前,治疗勃起功能障碍(ED)主要应用选择性的5-型磷酸二酯酶(PDE5)抑制剂,而临床发现PDE5抑制剂对部分ED患者治疗无效,因此,进一步研究H2S在阴茎勃起过程中的调节机制及作用,可能为ED提供了新的治疗途径。     

Oxidative stress in arteriogenic erectile dysfunction: prophylactic role of antioxidants

PURPOSE: We searched for markers of oxidative stress in cavernous ischemia and examined the effect of long-term antioxidant intake on arteriogenic erectile dysfunction (ED) in the rabbit. MATERIALS AND METHODS: Antioxidant activity of known antioxidant beverages, such as pomegranate juice (PJ), red wine, blueberry juice, cranberry juice, orange juice and green tea, was examined spectrophotometrically. PJ demonstrated the highest free radical scavenging capacity. The effect of long-term PJ intake on intracavernous blood flow and penile erection was then examined in the rabbit model. Erectile tissues were processed to assess oxidative stress and smooth muscle relaxation, immunohistochemical staining of nitric oxide synthase (NOS) and histomorphometry. RESULTS: On spectrophotometric analysis PJ showed the highest capacity to decrease low density lipoprotein oxidation and inhibit cellular oxidative stress in macrophages. The rabbit model of arteriogenic ED demonstrated decreased intracavernous blood flow, erectile dysfunction, loss of smooth muscle relaxation, decreased endothelial NOS and neuronal NOS, increased inducible NOS expression, diffused cavernous fibrosis and increased cavernous levels of the oxidative product isoprostane 8-epi-prostaglandin F2alpha. Long-term PJ intake increased intracavernous blood flow, improved erectile response and smooth muscle relaxation in ED and control groups while having no significant effect on NOS expression. PJ intake prevented erectile tissue fibrosis in the ED group. CONCLUSIONS: Arteriogenic ED accumulates oxidative products in erectile tissue, possibly via an intrinsic mechanism. Oxidative stress may be of great importance in the pathophysiology of arteriogenic ED. Antioxidant therapy may be a useful prophylactic tool for preventing smooth muscle dysfunction and fibrosis in ED.     

Effect of chronic ischemia on constitutive and inducible nitric oxide synthase expression in erectile tissue

Arterial occlusive disease is one of the leading causes of organic erectile dysfunction (ED). Recent studies have shown that the incidence of cardiovascular disease closely correlates with the prevalence of ED. Also, ED is thought to be an early signal of impending cardiovascular problems. We previously found that the atherosclerosis of iliohypogastric arteries in the rabbit causes ED, down-regulates cavernosal neuronal nitric oxide synthase (nNOS) gene expression, and impairs NO synthesis. The goal of this study was to determine the effect of atherosclerosis-induced ischemia on cavernosal nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS) expression and NO-mediated smooth muscle relaxation in the rabbit. Our study showed that iliac artery blood flow, intracavernosal blood flow, and intracavernosal oxygen tension were unchanged 4 weeks after the induction of arterial atherosclerosis, whereas they were significantly diminished at weeks 8 and 16. Erectile responses to nerve stimulation and cavernosal smooth muscle relaxation were unchanged at week 4 and were significantly diminished at weeks 8 and 16 after the induction of atherosclerosis. Western blotting showed that cavernosal nNOS and eNOS protein levels were unaffected at week 4 but were significantly decreased at weeks 8 and 16 after the induction of atherosclerosis. iNOS protein, however, markedly increased during the course of the induced arterial disease. Immunohistochemical staining showed no change in cavernosal eNOS or nNOS expression at week 4. A dramatic decrease in both was evident at 8 and 16 weeks. iNOS expression progressively increased between 4 and 16 weeks of atherosclerosis. Down-regulation of nNOS and eNOS, along with up-regulation of iNOS, may explain ischemic cavernosal smooth muscle relaxation impairment in the rabbit. Ischemically altered NOS expression may be of great pathophysiologic importance in atherosclerosis-induced ED. These data may provide further insight into the mechanism of arteriogenic ED.     

Increased cavernosal relaxation by Phoneutria nigriventer toxin, PnTx2-6, via activation at NO/cGMP signaling

Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10(-5)?M) and relaxed by electrical field stimulation (20?V, 1-32?Hz) in the presence or absence of PnTx2-6 (10(-8)?M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout (KO) mice, besides nNOS inhibitor (10(-5)?M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10(-4)?M) and ω-conotoxin GVIA (10(-6)?M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production.     

Gene transfer of endothelial nitric oxide synthase partially restores nitric oxide synthesis and erectile function in streptozotocin diabetic rats

PURPOSE: We determined whether adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis of streptozotocin induced diabetic rats could improve the impaired erectile response. MATERIALS AND METHODS: Two experimental groups of animals were transfected with adenoviruses, including streptozotocin (Sigma Chemical Company, St. Louis, Missouri) diabetic rats with AdCMVbetagal and streptozotocin diabetic rats with AdCMVeNOS. At 1 to 2 days after transfection these study animals underwent cavernous nerve stimulation to assess erectile function and their responses were compared with those of age matched control rats. In control and transfected streptozotocin diabetic rats eNOS and neuronal NOS (nNOS) were examined by Western blot analysis. Constitutive and inducible NOS activities were evaluated in the presence and absence of calcium by L-arginine to L-citrulline conversion and nitrate plus nitrite levels were measured. In control and streptozotocin diabetic penes beta-galactosidase activity and localization were determined. RESULTS: After transfection with AdCMVbetagal beta-galactosidase was localized to the endothelium and smooth muscle cells of the streptozotocin diabetic rat penis. Streptozotocin diabetic rats had a significant decrease in erectile function, as determined by peak and total intracavernous pressure (area under the curve) after cavernous nerve stimulation compared with control rats. Streptozotocin diabetic rats transfected with AdCMVeNOS had peak intracavernous pressure and area under the curve similar to those in control animals. This change in erectile function was a result of eNOS over expression with an increase in eNOS protein expression and constitutive NOS activity as well as an increase in nitric oxide biosynthesis, as reflected by an increase in cavernous nitrate plus nitrite formation. There was no change in nNOS protein expression or calcium independent conversion of NOS (inducible NOS activity). CONCLUSIONS: Adenoviral gene transfer of eNOS significantly increased peak and total intracavernous pressure to cavernous nerve stimulation in streptozotocin diabetic rats to a value similar to the response observed in control rats. Our results suggest that eNOS contributes significantly to the physiology of penile erection. These data demonstrate that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the streptozotocin diabetic rat.     

Molecular pharmacotherapeutic targeting of PDE5 for preservation of penile health

The molecular science of erection physiology has established that phosphodiesterase 5 (PDE5) serves an important biological role in the penis. Current research in the field has revealed this molecular effector to be relevant for penile erection, controlling the erectile response by degrading the second messenger product of the erection mediatory nitric oxide (NO) signaling pathway, 3', 5'-cyclic guanosine monophosphate. Accordingly, PDE5 has been targeted for sexual medicine purposes, and orally administered PDE5 inhibitors such as sildenafil, tadalafil, and vardenafil comprise a foremost intervention for erectile dysfunction (ED). New investigation of PDE5 regulation in the penis has suggested alternative roles for the enzyme and new therapeutic opportunities involving its molecular interactions. In particular, PDE5 function is altered under derangements of androgen deficiency, decreased NO bioactivity, and oxidative stress-associated inflammatory changes, thus contributing to an assortment of erectile disorders including hypogonadism-associated ED, recurrent ischemic priapism, penile vasculopathy, and penile fibrosis. This review provides a critical examination of the multifaceted role of the PDE5 regulatory system in the penis and its relevance for applying existing and emerging therapeutic strategies for erectile disorders.     

Molecular mechanisms that could contribute to prolonged effectiveness of PDE5 inhibitors to improve erectile function

Cyclic guanosine monophosphate (cGMP) in penile vascular smooth muscle cells (VSMC) plays a key role in promoting penile erection. Phosphodiesterase-5 (PDE5) in VSMC breaks down cGMP to counter this effect. Sildenafil (Viagra), vardenafil (Levitra) and tadalafil (Cialis), treatments for erectile dysfunction, inhibit PDE5 action. Many men with erectile dysfunction have improved erectile function after plasma inhibitor concentration falls below therapeutic levels. Maximum effect plus onset and duration of action of inhibitor determines its efficacy. The rate and extent of cellular drug accumulation and efflux of drug from smooth muscle cells plus persistence of drug effects in these cell impact these parameters. We propose possible molecular mechanisms that could account for prolonged action of PDE5 inhibitors including (1) persistence of biochemical effects after inhibitor is cleared from cells, and (2) retention of drug in VSMC beyond plasma clearance.     

Interactions between cGMP- and cAMP-pathways are involved in the regulation of penile smooth muscle tone

Nitric oxide (NO)/cyclic GMP (cGMP)-mediated mechanisms have a pivotal function in reducing the tone of the penile smooth musculature during normal erectile responses. The cyclic AMP (cAMP) signaling pathway is also involved in the adjustment of smooth muscle contractility, and suggestions for interactions between cGMP- and cAMP-mediated mechanisms have been presented. Using activators of the cGMP- or the cAMP-pathway, as well as inhibitors of protein kinase A (PKA; cAMP-dependent kinase) and protein kinase G (PKG; cGMP-dependent kinase), the present study was undertaken to further delineate the functional relation between these pathways in the penis. In addition, the distribution of PKA and some cAMP-binding phosphodiesterases (cAMP-PDEs) were investigated in human erectile tissue. Functional experiments were performed on isolated human corpus cavernosum (HCC). The effects of an inhibitor of the PKA, Rp-8CPT-cAMPS (10 µM), or the PKG, Rp-8-pCPT-cGMPS (10 µM), on relaxation induced by the cumulative administration of sodium nitroprusside (SNP), forskolin, sildenafil or tadalafil (IC351) were studied in preparations of HCC precontracted with 1 µM norepinephrine (NE). Using immunohistochemical procedures, the presence of immunoreactivity for cAMP-PDEs PDE3, PDE4, and PDE4A, as well as for PKA was investigated in specimens of HCC from which preparations were also used in the functional experiments. Forskolin, SNP, sildenafil, and IC 351 dose-dependently reversed NE-induced tension of isolated HCC preparations. The relaxing effects of SNP were significantly attenuated by Rp-8-pCPT-cGMPS, but not by Rp-8CPT-cAMPS. In contrast, relaxation induced by forskolin, sildenafil and tadalafil were significantly reversed by both Rp-8-pCPT-cGMPS and Rp-8CPT-cAMPS. Abundant immunoreactivity for PDE3 and PKA was observed in the corpus cavernosum smooth muscle cells. Immunoreactivity for PDE4 was also detected in the smooth musculature and in the cytoplasm of endothelial cells lining the cavernous sinusoids, as well as in nerve fibres interspersing the trabecular stroma. The present results support the hypothesis of interactions between cGMP- and cAMP-mediated signals in the HCC, and suggest that the effects of inhibitors of PDE5 on isolated erectile tissue may also partly or indirectly include actions of the cAMP second messenger system. The exact mechanism by which such an interaction occurs is not clear, but it may involve altered activity of the cGMP-inhibited PDE3 brought about by a change in the intracellular levels of cGMP by the inhibition of PDE5. This will in turn lead to increasing levels of cAMP, facilitating the interaction of cAMP with the PKA. The immunoreactivity specific for PDE3, PDE4, PDE4A and PKA registered in HCC section is also in support of an important role for the cAMP/PKA-system for penile smooth muscle function.     

Evaluation of tetrahydrobiopterin （BH4） as a potential therapeutic agent to treat erectile dysfunction

Aim： Nitric oxide （NO）-mediated smooth muscle relaxation causes penile erections. The endothelial NO synthase （eNOS） coenzyme tetrahydrobiopterin （BH4） converts eNOS-mediated catalytic activity from oxygen radical to NO production, improving endothelial function and vascular smooth muscle relaxation. Methods： Using quantitative immunohistochemistry, 8-isoprostane and nitrotyrosine concentrations were compared in cavernosal tissue from 17 potent and 7 impotent men, and the effect of single oral doses of BH4 on penile rigidity and tumescence was investigated. The pharmacodynamic effect of single oral doses of BH4 on penile rigidity and tumescence was investigated in a randomized, placebo-controlled, double-blind cross-over fashion in 18 patients with erectile dysfunction （ED） while receiving visual sexual stimulation. Results： 8-isoprostane content in endothelium and smooth muscle was signifi- cantly higher in impotent patient samples; the level of nitrotyrosine was unchanged in ED patients. Relative to placebo, a single dose of 200 mg BH4 led to a mean increase in duration of 〉 60% penile rigidity （33.5 rain [95% confidence interval （CI）： 13.1-49.3] at base and 29.4 rain [95% CI： 8.9-42,2] at tip）. A 500-mg dose increased the relative duration of 〉 60% penile rigidity by 36. I rain （95% CI： 16.3-51.8） at the base and 33.7 rain （95% CI： 11.4-43.9） at the tip. Treatments were well tolerated. Conclusion： BH4 treatment is suggested to switch eNOS catalytic activity from super-oxide to NO formation, leading to a reduced formation of free radical reaction product 8-isoprostane without alteration of nitrotyrosine. The observed results make BH4 a suitable candidate as an ED treatment through reconstitution of altered catalytic activity of the eNOS. （Asian JAndro12006 Mar; 8： 159-167）