Effect of dual mTOR inhibitor on TGFß1-induced fibrosis in primary human urethral scar fibroblasts.

BACKGROUND: TGFß1 and mTOR are considered to play important roles in fibrotic diseases. Rapamycin has been reported to inhibit urethral stricture formation in a rabbit model of urethral fibrosis. AIM: To evaluate if dual mTOR inhibitor has a superior efficacy compared with rapamycin on inhibiting cell proliferation and collagen expression in human urethral scar fibroblasts (HUSFs). METHODS: We established HUSF cultures from fresh surgical specimen. The HUSFs were identified with typical fibroblast markers using immunofluorescence. Then we examined the effect of TGFß1 on HUSFs using Cell Counting Kit-8 and Western blot. The inhibiting effects of OSI-027 (a dual mTOR inhibitor) on cell proliferation and collagen expression in TGFß1-induced HUSFs were compared with rapamycin using Cell Counting Kit-8, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: HUSFs were stained positive for vimentin, collagen I, and collagen III. TGFß1 had no effect on cell proliferation but increased collagen I and collagen III expressions in HUSFs. OSI-027 was more effective inhibiting cell proliferation and collagen expression compared with rapamycin in TGFß1-induced HUSFs. OSI-027 played a more important role in inhibiting TGFß1-induced mTOR pathway and phosphorylation of Smad2 compared with rapamycin in HUSFs. CONCLUSION: OSI-027 can inhibit the pro-fibrotic effects of TGFß1 significantly compared with rapamycin in HUSFs. These findings may provide a new therapy in the adjunctive treatment of urethral stricture disease.

Expression and distribution of phosphodiesterase isoenzymes in the human male urethra.

OBJECTIVE: To investigate the expression and distribution of phosphodiesterase (PDE) isoenzymes PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A in human urethral tissue. METHODS: Specimens of penile urethra were obtained from male subjects who had undergone male-to-female sex reassignment surgery. Using immunohistochemistry (immunofluorescence), the occurrence of PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A, the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide was examined in urethral sections. Cytosolic supernatants prepared from isolated human urethral tissue were subjected to Western blot analysis using specific anti-PDE antibodies. RESULTS: Immunosignals specific for PDE1A, 4A, 4B, and 5A were observed in the urethral smooth musculature. The smooth muscle bundles were seen innervated by slender nerve fibers, characterized by the expression of the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide. The expression of the PDE isoenzymes mentioned was confirmed by Western blotting. CONCLUSION: The results provide evidence for a significance of both the cyclic adenosine monophosphate and cyclic guanosine monophosphate signaling in the control of human urethral smooth muscle. The selective inhibition of PDE isoenzymes might represent a pharmacologic option to influence the function of smooth musculature in the human outflow region.

Phosphodiesterase isoenzymes in the human urethra: a molecular biology and functional study.

Experimental and clinical studies have suggested a role for phosphodiesterase (PDE) isoenzymes in the control of the human lower urinary tract. This study aimed to investigate the expression of PDE isoenzymes and the effects of PDE inhibitors (PDE-Is) in isolated human urethral smooth muscle (USM). The expression of messenger ribonucleic acid (mRNA) specifically encoding for PDE isoenzymes and isoforms (1A, 1B, 1C, 2A, 4A, 4B, 4C, 4D, 5A and 11A) was analyzed by means of reverse transcriptase polymerase chain reaction (RT-PCR). Using a tissue bath technique, the effects of vinpocetine (PDE1-I), erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA-HCl=MEP1) (PDE2-I), rolipram (PDE4-I), sildenafil, vardenafil and tadalafil (PDE5-Is) (0.01-10µM) on the tension of USM induced by norepinephrine were investigated. The production of cyclic guanosine monophosphate (cyclic GMP) and cyclic adenosine monophosphate (cyclic AMP) was measured by means of radioimmunoassays. RT-PCR analysis revealed the expression of PDE1B, PDE1C, PDE4A, PDE4C, PDE4D, PDE5A and PDE11A. The tension induced by norepinephrine (NE) was reversed by the PDE inhibitors with the following rank order of efficacy: rolipram (mean: -39%)≥sildenafil (-35%)>vardenafil (-26%)>tadalafil (-20%)>vinpocetine (-16%)>MEP1 (-2%). The relaxing effects of the drugs were paralleled by an elevation in tissue levels of cyclic AMP and cyclic GMP. Selective inhibitors of PDE4 and PDE5 can antagonize the tension induced by alpha-adrenergic stimulation of USM. PDE inhibition might represent an interesting option to facilitate the relaxation of the human outflow region.

Urethral sphincter fatigue after robot-assisted radical prostatectomy: descriptive questionnaire-based study and anatomic basis.

OBJECTIVE: To investigate the hypothesis that preservation of the neurovascular bundle (NVB) contributes to the recovery from sphincter fatigue symptoms after robot-assisted radical prostatectomy (RARP) and to examine the sarcolemmal localization of neuronal nitric oxide synthase (nNOS) and nNOS-positive nerves supplying striated muscles in the pelvic floor. METHODS: Whether preservation of the NVB influences early continence or sphincter fatigue symptoms was examined in 211 consecutive patients undergoing RARP. Continence and sphincter fatigue symptoms were assessed at 1, 3, and 6 months after surgery. An anatomic study was performed using semiserial sections obtained from 14 male cadavers. The association of continence rate and sphincter fatigue symptoms with preservation of the NVB was assessed by the chi-square test. RESULTS: There was a significant difference across the bilateral, unilateral, and non-nerve-sparing groups with regard to sphincter fatigue symptoms at 1 month (P=.0004) and 3 months (P=.0326) postoperatively. Sarcolemmal nNOS was detected in the rhabdosphincter (mean, 0.57 per 10 muscle fibers) and levator ani (mean, 1.13 per 10 fibers), with fibers originating from periprostatic nNOS-positive nerves. CONCLUSION: Postoperative sphincter fatigue was reduced by NVB preservation, suggesting that decreased sphincter fatigue may contribute to improvement of continence after RARP. As a background, existence of sarcolemmal nNOS and nNOS-positive nerve terminals arising from the NVB was confirmed in male pelvic floor striated muscles.

Soluble guanylyl cyclase (sGC) degradation and impairment of nitric oxide-mediated responses in urethra from obese mice: reversal by the sGC activator BAY 60-2770.

Obesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study investigated the USM relaxant machinery in obese mice and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 [acid 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4-(trifluoromethyl) biphenyl-4-yl] methoxy} phenyl) ethyl] amino} methyl) benzoic] rescues the urethral reactivity through improvement of sGC-cGMP (cyclic guanosine monophosphate) signaling. Male C57BL/6 mice were fed for 12 weeks with a high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg per day for 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS), and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO donors (S-nitrosoglutathione and glyceryl trinitrate), and BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine] (sGC stimulator) were markedly reduced in obese compared with lean mice. In contrast, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice (P < 0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (10 µM) potentiated BAY 60-2770-induced USM responses in the lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactive-oxygen species (ROS) production was enhanced, but protein expression of ß1 second guanylate cyclase subunit was reduced in USM from obese mice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and down-regulation of sGC-cGMP signaling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese mice.

[Nitric oxide pathway and female lower urinary tract. Physiological and pathophysiological role]. / Voie du monoxyde d'azote et bas appareil urinaire féminin. Rôles physiologique et physiopathologique.

GOAL: The aim was to review the literature on nitric oxide and female lower urinary tract. MATERIAL: A literature review through the PubMed library until December, 31 2012 was carried out using the following keywords: lower urinary tract, bladder, urethra, nervous central system, innervation, female, women, nitric oxide, phosphodiesterase, bladder outlet obstruction, urinary incontinence, overactive bladder, urinary tract infection. RESULTS: Two nitric oxide synthase isoforms, the neuronal (nNOS) and the endothelial (eNOS), are constitutively expressed in the lower urinary tract. Nevertheless, nNOS is mainly expressed in the bladder neck and the urethra. In the bladder, NO modulates the afferent neurons activity. In pathological condition, inducible NOS expression induces an increase in detrusor contractility and bladder wall thickness and eNOS facilitates Escherichia coli bladder wall invasion inducing recurrent urinary tract infections. In the urethra, NO play a major role in smooth muscle cells relaxation. CONCLUSION: The NO pathway plays a major role in the female lower urinary tract physiology and physiopathology. While it acts mainly on bladder outlet, in pathological condition, it is involved in bladder dysfunction occurrence.

Bmp7 functions via a polarity mechanism to promote cloacal septation.

BACKGROUND: During normal development in human and other placental mammals, the embryonic cloacal cavity separates along the axial longitudinal plane to give rise to the urethral system, ventrally, and the rectum, dorsally. Defects in cloacal development are very common and present clinically as a rectourethral fistula in about 1 in 5,000 live human births. Yet, the cellular mechanisms of cloacal septation remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We previously detected Bone morphogenetic protein 7 (Bmp7) expression in the urorectal mesenchyme (URM), and have shown that loss of Bmp7 function results in the arrest of cloacal septation. Here, we present evidence that cloacal partitioning is driven by Bmp7 signaling in the cloacal endoderm. We performed TUNEL and immunofluorescent analysis on cloacal sections from Bmp7 null and control littermate embryos. We found that loss of Bmp7 results in a dramatic decrease in the endoderm survival and a delay in differentiation. We used immunological methods to show that Bmp7 functions by activating the c-Jun N-terminal kinase (JNK) pathway. We carried out confocal and 3D imaging analysis of mitotic chromosome bundles to show that during normal septation cells in the cloacal endoderm divide predominantly in the apical-basal direction. Loss of Bmp7/JNK signaling results in randomization of mitotic angles in the cloacal endoderm. We also conducted immunohistochemical analysis of human fetal sections to show that BMP/phospho-SMAD and JNK pathways function in the human cloacal region similar as in the mouse. CONCLUSION/SIGNIFICANCE: Our results strongly indicate that Bmp7/JNK signaling regulates remodeling of the cloacal endoderm resulting in a topological separation of the urinary and digestive systems. Our study points to the importance of Bmp and JNK signaling in cloacal development and rectourethral malformations.

RhoA/Rho-kinase as a therapeutic target for the male urogenital tract.

INTRODUCTION: Rho-kinase (ROCK) is a serine/threonine kinase and is one of the major downstream effectors of the small guanosine triphosphatase Rho. In the past few years, evidence has been accumulating to suggest that the RhoA/ROCK system may play an important role in the pathogenesis of a number of cardiovascular and urogenital disorders. AIM: The aim of this study is to review the literature pertaining to the role of the RhoA/ROCK system in male urogenital function. METHODS: Comprehensive literature review was performed using PubMed. MAIN OUTCOME MEASURES: Inhibitors of ROCK may have potential therapeutic applications, as derived from preclinical and a few clinical studies. RESULTS: Published reports suggest that elevated RhoA/Rho-kinase signaling plays a role in the development of benign prostatic hyperplasia, erectile dysfunction, kidney failure, ejaculation disorders, prostate and bladder cancer initiation, and eventual metastasis. CONCLUSIONS: This review focuses on our current understanding of the role of the RhoA/Rho-kinase pathway in the regulation of the male urogenital system. Rho-kinase inhibitors may evolve into an important pharmacologic option in the future treatment of urogenital system disorders.

Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP).

OBJECTIVE: To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra. MATERIALS AND METHODS: Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague-Dawley rats. They were analysed by real-time polymerase chain reaction for the mRNA expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1, a subunit of MLCP). Levels of MLCK and MYPT1 mRNA expression were determined as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The tissues were also analysed by Western blotting for MLCK and MYPT1 protein expression as a ratio to the expression of ß-actin. A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity. RESULTS: MLCK mRNA expression was higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 0.26 (0.17) vs 0.14 (0.12); P = 0.09]. MYPT1 mRNA expression was significantly higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 2.31 (1.04) vs 0.56 (0.36); P = 0.001]. Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared with the urethra [mean (sd) ratio to ß-actin: 1.63 (0.25) vs 0.91 (0.29) and 0.97 (0.10) vs 0.37 (0.29), respectively; both P < 0.001]. Enzymatic assay identified significantly greater MLCK activity in the bladder than in the urethra. While, MLCP activity was lower in the bladder than in the urethra. CONCLUSION: In healthy young female rats, MLCK activity is higher and MLCP activity is lower in the bladder relative to the urethra. These differences probably play a role in modulating the functional differences between bladder and urethral smooth muscle tone.

Prominent expression of phosphodiesterase 5 in striated muscle of the rat urethra and levator ani.

PURPOSE: We investigated phosphodiesterase 5 distribution and activity in the urethra. MATERIALS AND METHODS: Rat tissues were examined for phosphodiesterase 5 and alpha-smooth muscle actin expression. Urethral phosphodiesterase 5 activity was examined by tissue bath in the presence of sildenafil (Pfizer, New York, New York). RESULTS: Anti-alpha-smooth muscle actin antibody (Abcam) stained all known smooth muscles in all tested tissues and revealed a few smooth muscle fibers in the levator ani muscle. Anti-phosphodiesterase 5 antibody (Abcam) stained smooth muscle in the penis and bladder but not striated leg muscle. However, it stained predominantly striated muscle in the urethra and the levator ani muscle. In the urethra the amount of phosphodiesterase 5 in striated muscle was 6 times that in smooth muscle. In urethral striated muscle phosphodiesterase 5 expression was localized to Z-band striations. Smooth and striated muscle intermingling was clearly visible on the inner and outer rims of the circularly arranged striated muscle layer. Relaxation of precontracted urethral tissues by sodium nitroprusside (Sigma-Aldrich) was enhanced by sildenafil, indicating phosphodiesterase 5 activity, which was primarily located in the striated muscle according to phosphodiesterase 5 staining. CONCLUSIONS: Despite its presumed smooth muscle specificity phosphodiesterase 5 was predominantly expressed in the striated muscle of the urethra and in the levator ani muscle. Results are consistent with earlier studies in which these striated muscles were developmentally related to smooth muscle. They also suggest that these striated muscles are possibly regulated by phosphodiesterase 5.

Relaxation effect of phosphodiesterase-5 inhibitor on the animal bladder and prostatic urethra: in vitro and in vivo study.

AIM: To assess the relaxation effect of the phosphodiesterase-5 inhibitor udenafil on the bladder and prostatic urethra and its therapeutic potentials for benign prostatic hyperplasia (BPH)/lower urinary tract symptoms (LUTS). METHODS: For the in vitro study, muscle strips from urinary bladder and urethra were prepared from male New Zealand rabbits. The strips were mounted in organ baths and connected to force transducers. After stabilization, maximal tissue contractions were obtained by the addition of phenylepinephrine for urethra strips and carbachol for bladder strips. When the contraction was stabilized, a dose-response curve of udenafil was constructed. For the in vivo study using adult male Sprague-Dawley rats, changes of intravesical pressure and urethral perfusion pressure after intraarterial administration of udenafil were monitored. RESULTS: Udenafil significantly relaxed the bladder and urethra strips in a dose-dependent manner. At 10(-3) M, udenafil induced a significant relaxation of the bladder strips by 37.3% and of the urethra strips by 44.0%. In the in vivo study, the intercontraction interval was significantly prolonged (p < 0.01) and the duration of urethral relaxation with high-frequency oscillations was significantly prolonged (p < 0.01) after udenafil. CONCLUSIONS: Udenafil had relaxant effects on the bladder and prostatic urethral smooth muscle. Clinically, udenafil could be applied as an effective treatment for BPH/LUTS.

Estradiol increases urethral tone through the local inhibition of neuronal nitric oxide synthase expression.

Estrogens are known to modulate lower urinary tract (LUT) trophicity and neuronal nitric oxide synthase (nNOS) expression in several organs. The aim of this study was to explore the effects of endogenous and supraestrus levels of 17beta-estradiol (E2) on LUT and urethral nNOS expression and function. LUT function and histology and urethral nNOS expression were studied in adult female mice subjected either to sham surgery, surgical castration, or castration plus chronic E2 supplementation (80 microg.kg(-1).day(-1), i.e., pregnancy level). The micturition pattern was profoundly altered by long-term supraestrus levels of E2 with decreased frequency paralleled by increased residual volumes higher than those of ovariectomized mice. Urethral resistance was increased twofold in E2-treated mice, with no structural changes in urethra, supporting a pure tonic mechanism. Acute nNOS inhibition by 7-nitroindazole decreased frequency and increased residual volumes in ovariectomized mice but had no additive effect on the micturition pattern of long-term supraestrus mice, showing that long-term supraestrus E2 levels and acute inhibition of nNOS activity had similar functional effects. Finally, E2 decreased urethral nNOS expression in ovariectomized mice. Long-term supraestrus levels of E2 increased urethral tone through inhibition of nNOS expression, whereas physiological levels of E2 had no effect.

Properties of spontaneous Ca2+ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ.

Interstitial cells of Cajal-like cells (ICC-LCs) in the urethra may act as electrical pacemakers of spontaneous contractions. However, their properties in situ and their interaction with neighbouring urethral smooth muscle cells (USMCs) remain to be elucidated. To further explore the physiological role of ICC-LCs, spontaneous changes in [Ca(2+)](i) (Ca(2+) transients) were visualized in fluo-4 loaded preparations of rabbit urethral smooth muscle. ICC-LCs were sparsely distributed, rather than forming an extensive network. Ca(2+) transients in ICC-LCs had a lower frequency and a longer half-width than those of USMCs. ICC-LCs often exhibited Ca(2+) transients synchronously with each other, but did not often show a close temporal relationship with Ca(2+) transients in USMCs. Nicardipine (1 microm) suppressed Ca(2+) transients in USMCs but not in ICC-LCs. Ca(2+) transients in ICC-LCs were abolished by cyclopiazonic acid (10 microm), ryanodine (50 microm) and caffeine (10 mm) or by removing extracellular Ca(2+), and inhibited by 2-aminoethoxydiphenyl borate (50 microm) and 3-morpholino-sydnonimine (SIN-1; 10 microm), but facilitated by increasing extracellular Ca(2+) or phenylephrine (1-10 microm). These results indicated that Ca(2+) transients in urethral ICC-LCs in situ rely on both Ca(2+) release from intracellular Ca(2+) stores and Ca(2+) influx through non-L-type Ca(2+) channel pathways. ICC-LCs may not act as a coordinated pacemaker electrical network as do ICC in the gastrointestinal (GI) tract. Rather they may randomly increase excitability of USMCs to maintain the tone of urethral smooth muscles.

Phosphodiesterase 5 in the female pig and human urethra: morphological and functional aspects.

OBJECTIVE: To characterize the distribution of phosphodiesterase 5 (PDE-5), cGMP and cGMP-dependent protein kinase I (PKG1), and to evaluate the effect of pharmacological inhibition of PDE-5 in isolated preparations of pig and human urethra, as the nitric oxide (NO)/cGMP pathway generates the main inhibitory signals to reduce resistance in the bladder outlet and urethra during emptying of the bladder. MATERIALS AND METHODS: After obtaining ethics committee approval, urethral specimens were obtained from three female patients during cystectomy, and from young female pigs. The specimens were prepared for immunohistochemical investigations and for functional studies in organ baths. Effects of sildenafil, vardenafil and tadalafil (1 nm to 30 microm) were studied in l-noradrenaline (1 microm)-activated or spontaneously contracted preparations, and on relaxations induced by electrical-field stimulation (EFS). Levels of cGMP were determined by radioimmunoassay. RESULTS: After stimulation with the NO donor, DETA NONO-ate (1 mm), there was greater cGMP-immunoreactivity (IR) in urethral and vascular smooth muscles. There was a wide distribution of cGMP- and vimentin-positive interstitial cells between pig urethral smooth muscle bundles. There was also cGMP-IR within NO-synthase-IR endothelium. There was PDE-5 IR within the urethral and vascular smooth muscle cells, but also in vascular endothelial cells that expressed cGMP-IR. In pig and human sections, there was strong PKG1-IR in alpha-actin-IR urethral smooth muscle cells that also contained IR for cGMP. Sildenafil, vardenafil and tadalafil caused mean (sem) concentration-dependent relaxations of the pig urethra which, at 30 microm, were 80 (3)% (11 samples), 81 (5)% (12 samples) and 64 (4)% (10 samples) of the spontaneous tone. The relaxation of L-noradrenaline-contracted female human urethra was 100% in response to 10 microm sildenafil, and 85 (15)% and 47 (13)% for 30 microm of vardenafil and tadalafil, respectively (three samples). Vardenafil or sildenafil (30 microm) doubled cGMP levels in pig specimens. There were no effects on cGMP levels with tadalafil. EFS (1-32 Hz) caused l-NG-nitroarginine-sensitive relaxations of pig urethral muscle that were increased in amplitude and duration by PDE-5 inhibition. At 0.1 microm, sildenafil, vardenafil or tadalafil significantly prolonged the mean (sem) duration of the relaxation at 4 Hz by 55 (19)%, 45 (14)% and 51 (12)%, respectively. CONCLUSIONS: PDE-5-, cGMP- and PKG1-IR is widely distributed in human and pig urethral tissues. Nerve-induced relaxations of urethral preparations were enhanced at low concentrations of sildenafil, vardenafil and tadalafil, whereas there were direct smooth muscle-relaxant actions of the PDE-5 inhibitors at high concentrations. Inhibition of PDE-5 might be an interesting option to facilitate cGMP-mediated relaxation of the outflow region.

Distribution and functional significance of phosphodiesterase isoenzymes in the human lower urinary tract.

To date, it is widely accepted that several disorders of the male and female urogenital tract, such as erectile dysfunction, bladder overactivity, urinary stone disease and the benign prostatic syndrome, can be therapeutically approached by influencing the function of the smooth musculature of the respective organs. In order to achieve a pronounced drug effect without significant adverse events, especially on the cardiovascular system, a certain degree of tissue selectivity is mandatory. Selective intervention in intracellular pathways regulating smooth muscle tone has become a promising strategy to modulate tissue function. Since the concept of taking a pill as a cure for an illness or the relief of symptoms has become widely accepted by the consumers, the pharmacological treatment of urological diseases has focused on selective, orally available drugs, acting via influencing intracellular regulatory mechanisms, thus combining a high response rate and the advantage of an on-demand intake. PDEs play a central role in controlling the levels of cyclic nucleotides (i.e. cAMP and/or cGMP), which are important second messengers in many transmitter pathways involved in the regulation of biological processes of urogenital tissues. Specifically, the use of isoenzyme selective phosphodiesterase (PDE) inhibitors offers great hope in the medical treatment of various genitourinary diseases. These agents are regarded efficacious, having a fast onset of drug action in the target tissue and an improved effect-to-side-effect ratio. The growing experience with the use of this class of compounds in urology is mainly based on basic research efforts and this field will remain the most exciting and innovative subject in genitourinary physiology and pharmacology for the next few years. These tremendous research efforts may lead to a vast pharmacological armamentarium of possible new treatment options. The purpose of this review is to summarize the current knowledge on the distribution and potential functional significance of PDE isoenzymes in the human lower urinary tract.

Nitric oxide/cGMP-mediated effects in the outflow region of the lower urinary tract--is there a basis for pharmacological targeting of cGMP?

Treatment with alpha-adrenoceptor antagonists that reduce the tone of prostatic stromal and urethral smooth muscle has beneficial effects in patients with benign prostatic hyperplasia (BPH) and lower urinary tracts symptoms (LUTS) and has brought attention to regulatory mechanisms of smooth muscle contractility of the outflow region. The prostate, urethra and bladder neck are densely supplied by nitric oxide (NO)-synthase-containing nerves that cause relaxation upon activation. In various experimental models, altered function or activity of the NO/cGMP pathway of the bladder neck and urethra may be related to inappropriate or un-coordinated functions of the bladder outlet and detrusor, but causal connections between alterations in this signaling system, a dysfunctional bladder outlet, and the development of LUTS are not established for humans. The present review focuses on regulatory functions of smooth muscle contractility by the NO/cGMP-pathway in the bladder neck, urethra, and prostate. Disease-related alterations in the NO/cGMP-pathway, and putative options for pharmacological modification of this signaling pathway in the out-flow region are briefly discussed.

Physiologic role of nitric oxide and nitric oxide synthase in female lower urinary tract.

PURPOSE OF REVIEW: In recent years nitric oxide (NO) has gained increasing recognition as an important neurotransmitter and cell signaling molecule with a broad range of functions in the lower urinary tract. This review discusses recently published data related to the physiologic and pathophysiologic roles of NO and nitric oxide synthase (NOS) in the female lower urinary tract. RECENT FINDINGS: Expression of three isoforms of NOS, namely endothelial NOS, neuronal NOS and inducible NOS, has been identified in various tissues of the lower urinary tract in animals and humans. In addition to its relaxation effects on bladder and urethra, NO also serves as a neurotransmitter in the lower urinary tract. The physiologic roles of NO in overactive bladder, bladder outlet obstruction, diabetic cystopathy, interstitial cystitis, and bladder inflammation have been demonstrated. SUMMARY: NO plays an important role in the micturition process and in disorders of the lower urinary tract. Improved understanding of the pathophysiologic role of NO/NOS system and of the L-arginine-NO-cGMP pathway may allow us to identify suitable therapeutic targets for lower urinary tract disorders. However, there is a need for further investigation to determine the precise function of NO in human lower urinary tract because most work thus far has been done in animal models.

Accumulated endogenous nitric oxide synthase inhibitors in inhibiting urethral relaxation following estrogen supplementation in ovariectomized rabbits.

PURPOSE: We investigated the possible role of the endogenous nitric oxide (NO) synthase (NOS) inhibitors N-monomethyl-L-arginine (L-NMMA) and asymmetrical N, N-dimethyl-L-arginine (ADMA) in inhibiting urethral relaxation following estrogen supplementation in ovariectomized rabbits. MATERIALS AND METHODS: A total of 16 mature Japanese White female rabbits were divided into 2 groups. In the control group rabbits were sacrificed 2 weeks after bilateral ovariectomy. In the estrogen group estradiol was administered subcutaneously for 2 weeks with the aid of sustained release pellet from 2 weeks after ovariectomy until sacrifice. Isolated urethra was cut into transverse strips for functional study and processed to determine endogenous NOS inhibitors, NOS activity, dimethylarginine dimethylaminohydrolase (DDAH) activity as a metabolizing enzyme of endogenous NOS inhibitors and cyclic guanosine monophosphate production. RESULTS: Electrical field stimulation produced NO mediated and neurogenic relaxation of the urethral strip in the presence of guanethidine and atropine under contraction with phenylephrine. Relaxation was significantly decreased in the estrogen group and accompanied by decreased cyclic guanosine monophosphate production. Sodium nitroprusside induced relaxation was not different between the 2 groups. The content of L-NMMA plus ADMA in the urethra was significantly increased in the estrogen group. Ca dependent NOS activity in the urethra remained unaffected. DDAH activity was significantly lower in the estrogen group. CONCLUSIONS: Estrogen supplementation leads to decreased NO mediated and neurogenic urethral relaxation through the accumulation of L-NMMA and ADMA in the urethra. The accumulation of NOS inhibitors is possibly brought about by impaired DDAH activity.

Female pig urethral tone is dependent on Rho guanosine triphosphatases and Rho-associated kinase.

PURPOSE: Circular smooth muscle of the urethra generates spontaneous myogenic tone of relevance for the maintenance of continence. We tested if Rho guanosine triphosphatases (GTPases) and Rho-associated kinase (ROK) are involved in the generation of urethral tone. MATERIALS AND METHODS: Small circular strips of female pig urethra were dissected out and mounted for recording isometric force. The effect of pharmacological agents known to modulate the activity of Rho GTPases or ROK was examined. The intracellular calcium concentration was measured using fura-2. RESULTS: Urethral tone was abolished by removing extracellular calcium or by adding the calcium antagonist felodipine. The decrease in force was closely related to a decrease in intracellular calcium concentration, indicating that tone depends on membrane associated mechanisms. Toxin B, which inactivates Rho GTPases, and Y 27632, which inhibits ROK, completely abolished tone in the female pig urethra. The latter effect occurred without any change in the intracellular calcium concentration. CONCLUSIONS: The results suggests that urethral tone depends on activity in G-protein coupled pathways and inhibition of this activity is sufficient for urethral tone relaxation. Thus, to our knowledge a new pathway in the generation of urethral tone, which might be acted on by autonomic nerves during micturition, has been identified.