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.

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.

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.

Ejaculatory abnormalities in mice with targeted disruption of the gene for heme oxygenase-2.

Nitric oxide (NO) is well established as a neurotransmitter in the central and peripheral nervous systems. More recently, another gas, carbon monoxide (CO) has also been implicated in neurotransmission. In the nervous system CO is formed by a subtype of heme oxygenase (HO) designated HO2. HO2 is localized to discrete neuronal populations in the brain resembling localizations of soluble guanylyl cyclase, which is activated by CO. CO may also function in the peripheral autonomic nervous system, in conjunction with NO. The majority of ganglia in the myenteric plexus possess both HO2 and neuronal NO synthase (NOS). Defects in myenteric plexus neurotransmission occur both in mice with targeted deletion of genes for HO2 and neuronal NOS. HO2 also occurs in other autonomic ganglia including the petrosal, superior cervical and nodose ganglia. Neuronal NOS is localized to neurons regulating male reproductive behavior, such as penile erection, and NOS inhibitors prevent erection. Because of the other parallels between NO and CO, we speculated that CO may play a role in male reproductive behavior. In the present study we describe HO2 localization in neuronal structures regulating copulatory reflexes. Reflex activity of the bulbospongiosus muscle, which mediates ejaculation and ejaculatory behavior, is markedly diminished in mice with targeted deletion of the gene for HO2 (HO2-).

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.

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.

Localization of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the male reproductive tract.

The action of glucocorticoids in target tissues is dependent on the local expression of glucocorticoid receptors and two 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes, 11beta-HSD1 and 11beta-HSD2, which interconvert active and inactive glucocorticoids. This study examined expression of the 11beta-HSD enzymes in the male reproductive tract of the adult rat. 11beta-HSD1 was immunolocalized to the apical region of principal epithelial cells of the caput epididymis, with the less numerous clear cells devoid of signal. Epididymal 11beta-HSD1 expression was confirmed by Western blot analysis, with immunoreactive species identified at 34 kDa (the expected size for 11beta-HSD1) and at approximately 48 kDa. 11beta-HSD bioactivity was readily detectable in the epididymis, with 11-oxoreductase activity clearly the favored reaction (as observed in liver), consistent with 11beta-HSD1 expression. The epithelium of the vas deferens, seminal vesicle, and penile urethra were also immunopositive for 11beta-HSD1, as were smooth muscle cells of the vas deferens and penile blood vessels. 11beta-HSD2 was also immunolocalized to the epididymal epithelium, but its distribution was complementary to that of 11beta-HSD1 (i.e. clear cells showing intense 11beta-HSD2 staining but principal cells devoid of signal). 11beta-HSD2 was also present in the corpora cavernosa of the penis but not in other tissues. In conclusion, the differential expression of 11beta-HSD1 and 11beta-HSD2 throughout the male reproductive tract suggests that these enzymes locally modulate glucocorticoid and mineralocorticoid actions, particularly in the epididymis and penile vasculature.

Distribution of nitric oxide synthase-immunoreactive nerves and identification of the cellular targets of nitric oxide in guinea-pig and human urinary bladder by cGMP immunohistochemistry.

The distribution of nerves with the potential to synthesize nitric oxide was examined within the urinary bladder and proximal urethra of humans and guinea-pigs, using an antibody to nitric oxide synthase. Further experiments identified cells in which cGMP-immunoreactivity was induced following exposure to the nitric oxide donor, sodium nitroprusside. These cells represent the potential physiological targets of neuronally released nitric oxide, since activation of soluble guanylate cyclase, and a consequent rise in intracellular cGMP, mediate many of the effects of this transmitter. Nitric oxide synthase-immunoreactivity was widely distributed in the lower urinary tract. In guinea-pigs, 50-68% of all intrinsic vesical neurons expressed nitric oxide synthase-immunoreactivity, while in humans 72-96% of neurons in the wall of the bladder contained nitric oxide synthase. In both humans and guinea-pigs, varicose nitric oxide synthase-immunoreactive nerve terminals provided a moderate innervation to the detrusor muscle of the bladder body, and a denser innervation to the urethral muscle. Immunoreactive nerves also projected to the subepithelium and around blood vessels, but were rarely observed encircling intramural vesical ganglia. Following stimulation with sodium nitroprusside, smooth muscle cells of the urethra expressed strong cGMP-immunoreactivity, but detrusor muscle cells remained uniformly negative. Although the detrusor muscle fibres did not express cGMP, numerous interstitial cells throughout the bladder body demonstrated an intense induction of cGMP-immunoreactivity by sodium nitroprusside. These cells had long dendritic processes extending parallel to the smooth muscle fibres, and contained vimentin, an intermediate filament expressed by cells of mesenchymal origin. Other cell types in which sodium nitroprusside exposure induced cGMP-immunoreactivity were the uroepithelial cells, vascular smooth muscle cells and pericytes, and a small number of varicose nerve terminals. In the guinea-pig, a minor proportion (less than 10%) of intrinsic neurons in the wall of the bladder also expressed cGMP. No intrinsic neurons were observed in specimens of human bladder processed for cGMP immunohistochemistry. The results provide anatomical evidence that nitric oxide may function as a neurotransmitter in the lower urinary tract. Although nerves with the capacity to produce nitric oxide supply both the detrusor muscle and the urethra, distinct regional differences exist in the effects of nitric oxide on the induction of cGMP. If the nitric oxide-mediated induction of cGMP is a reliable indicator of the physiological responsiveness of a cell to nitric oxide, then smooth muscle cells appear to be the predominant targets of nitric oxide in the urethra, while in the bladder body, interstitial cells may serve this role. These findings support previous studies which have implicated nitric oxide as an inhibitory transmitter involved in the relaxation of the bladder neck. Our experiments further indicate that a number of cell types within the lower urinary tract could potentially mediate the effects of endogenously released nitric oxide.

Characterization of nitric oxide synthase activity in sheep urinary tract: functional implications.

1. To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO-mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L-[14C]-arginine to L-[14C]-citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography. 2. NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg-1 protein min-1): urethra (33 +/- 3.3), detrusor (13.1 +/- 1.1) and ureter (1.5 +/- 0.2). No activity was detected in the particulate fraction of any region. 3. NOS activity was dependent on Ca(2+)-calmodulin and required exogenously added NADPH and tetrahydrobyoptein (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity. 4. NOS activity was inhibited to a greater extent by NG-nitro-L-arginine (L-NOARG) and its methyl ester (L-NAME) than by NG-monomethyl-L-arginine (L-NMMA), while 7-nitroindazole (7-NI) was a weak inhibitor and L-cannavine had no effect. 5. Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin-sensitive manner, suggesting feedback inhibition of NOS by NO. 6. EFS induced prominent NO-mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L-NOARG = L-NAME > 7-NI > L-NMMA. 7. In conclusion, we have demonstrated the presence of NO-synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NO-mediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely.

A pharmacological and histochemical study of hamster urethra and the role of urothelium.

1. Electrical field stimulation (EFS) of circular strips of hamster proximal urethra caused frequency-dependent relaxations at raised tone. Phentolamine (10(-6) M), propranolol (10(-6) M) and atropine (10(-6) M) were present throughout the experiment. Neurogenic relaxation was attenuated by L-NG-nitroarginine methyl ester (L-NAME) (10(-4) M), was restored by L-arginine (3 x 10(-3) M) but not by D-arginine (3 x 10(-3) M) and completely blocked by tetrodotoxin (10(-6) M). Neurogenic relaxation was also reduced by suramin (10(-4) M) and totally blocked by suramin together with L-NAME. Strips of hamster urethra devoid of urothelium showed little, if any, relaxant response to EFS. 2. An immunohistochemical study showed nitric oxide synthase-immunoreactive nerves in the smooth muscle layers and in the lamina propria, just beneath the urothelium, but no nitric oxide synthase (NOS) staining in the urothelial layer. 3. Noradrenaline elicited a significantly greater contraction in strips without urothelium than in control strips. L-NAME (10(-4) M) did not affect noradrenaline-induced contraction in both control and urothelium-free strips. The contractile response to acetylcholine was not dependent on the presence or absence of urothelium. Nevertheless the response induced by exogenous acetylcholine (10(-3) M) was increased by L-NAME (10(-4) M), both in intact and in urothelium-free strips. 4. Prostaglandin E2 (10(-8)-5 x 10(-6) M) and 2-methyl-thio-ATP (10(-9)-10(-5) M) relaxed proximal urethra. Suramin (10(-4) M) significantly inhibited the relaxation induced by 2-methyl-thio-ATP. The amplitude of these responses was not significantly different between intact and urothelium-free strips and was not blocked by L-NAME (10(-4) M). 5. These results suggest that nitric oxide (NO) is the principal transmitter involved in the non-adrenergic, non-cholinergic (NANC) relaxation of hamster proximal urethra possibly together with another inhibitory transmitter released from nerves. NO can be released from nerves located in the circular smooth muscle layer and in the lamina propria rather than in the urothelium. The reduced neurogenic relaxation in urothelium-free preparations suggests that a NO-dependent inhibitory factor is released from the urothelium. In addition, ATP and prostaglandin E2 may be involved, together with NO, in the urethra during micturition.

Carbon monoxide-induced relaxation and distribution of haem oxygenase isoenzymes in the pig urethra and lower oesophagogastric junction.

1. The distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO)-1 and -2 was studied by immunohistochemistry in the pig's lower urinary tract, including bladder extramural arteries, and the oesophagogastric junction (OGJ). In isolated smooth muscle from the urethra and the OGJ, the mechanisms for CO-induced relaxations were characterized by measurement of cyclic nucleotide levels and by responses to the guanylate cyclase inhibitor methylene blue and some K+ channel inhibitors. 2. HO-2 immunoreactivity was observed in coarse nerve trunks within the smooth muscle of the urethra and OGJ, and in nerve cell bodies of the enteric plexuses of the OGJ. Furthermore, the vascular endothelium of the intramural vessels of the urethra, bladder and OGJ, and the extramural vessels of the bladder, displayed HO-2 immunoreactivity. Two different antisera against HO-1 were used, but only one displayed immunoreactivity in neuronal structures. HO-1 immunoreactivity, as displayed by this antiserum, was seen in nerve cells, coarse nerve trunks and varicose nerve fibres in the smooth muscle of the urethra and OGJ. Some HO-2 and/or HO-1 (as displayed by both HO-1 antisera) immunoreactive cells with a non-neuronal appearance were observed within the smooth muscle of the OGJ, bladder and urethra. 3. In the urethral preparations, exogenously applied CO (72 microM) evoked a relaxation amounting to 76 +/- 6%. The relaxation was associated with an increase in cyclic GMP, but not cyclic AMP, content. CO-evoked relaxations were not significantly reduced by treatment with methylene blue, or by inhibitors of voltage-dependent (4-aminopyridine), high (iberiotoxin, charybdotoxin) and low (apamin) conductance Ca(2+)-activated, and ATP-sensitive (glibenclamide) K+ channels. Bladder strips, and ring preparations from the extramural arteries of the bladder, did not respond to exogenously administered CO (12-72 microM). 4. In the OGJ, exogenously applied CO evoked a relaxation of 86 +/- 6%, which was associated with an increase in cyclic GMP, but not cyclic AMP, content. Treatment with 30 microM methylene blue raised the spontaneously developed muscle tone, and reduced the maximum relaxation evoked by CO to 33 +/- 9%. Addition of 4-aminopyridine, apamin, glibenclamide, iberiotoxin, charybdotoxin or glibenclamide had no effect on the relaxations. 4-aminopyridine (0.1-1 mM), iberiotoxin (0.1 microM) and charybdotoxin (0.1 microM) increased the spontaneously developed tone, and a combination of charybdotoxin and apamin reduced CO-induced (24 microM CO) relaxations. 5. The present findings demonstrate the presence of HO in both neuronal and non-neuronal cells in the pig OGJ and lower urinary tract. CO produces relaxation of the smooth muscle in the OGJ and urethra, associated with a small increase in cyclic GMP concentration in both regions. Relaxations evoked by CO in the urethra do not seem to involve voltage-dependent, low and high conductance, or ATP-dependent K+ channels. However, in the OGJ relaxations evoked by CO can be attenuated by methylene blue and a combination of charybdotoxin and apamin.

Nitric oxide synthase in pig lower urinary tract: immunohistochemistry, NADPH diaphorase histochemistry and functional effects.

1. The distribution and colocalization of nitric oxide synthase (NOS)-like immunoreactivity and NADPH diaphorase activity in the pig lower urinary tract were investigated by immunohistochemical and histochemical staining techniques. Functional in vitro studies were performed to correlate the presence of NOS-immunoreactivity/NADPH diaphorase staining with smooth muscle responses involving the L-arginine/nitric oxide (NO) pathway. 2. NOS-immunoreactivity and NADPH diaphorase activity were expressed in nerve trunks and fine nerve fibres in and/or around muscular bundles in the detrusor, trigone and urethra. Thin nerve fibres that dispersed within the muscle bundles were mainly found in the urethral/trigonal area, whereas such fibres were less common in the detrusor. 3. Almost all neuronal structures that were NOS-immunolabeled were also stained for NADPH diaphorase. In contrast, the urothelium, which was intensively stained by the NADPH diaphorase technique, remained unstained by immunohistochemistry. 4. Electrical field stimulation of pig isolated trigonal and urethral preparations induced relaxations, which were inhibited by tetrodotoxin (1 microM) and NG-nitro-L-arginine (L-NOARG, 10 microM). 5. L-Arginine (1 mM), but not D-arginine, inhibited (25-30%) electrically evoked detrusor contractions. This inhibition was reversed by L-NOARG (0.1 mM). L-Arginine did not inhibit detrusor contractions in the presence of scopolamine (1 microM) and had no direct smooth muscle effects per se. 6. Acetylcholine (1 nM-10 microM) caused concentration-dependent relaxations of noradrenaline-induced contractions in pig vesical arteries. Removal of the endothelium practically abolished the acetylcholine-induced relaxation. Pretreatment with L-NOARG (0.1 mM and 0.3 mM) caused a rightward shift of the concentration-response curves to acetylcholine, but the maximal relaxation obtained was significantly reduced (to 65 +/- 12%; n = 6; P < 0.05) only at 0.3 mM L-NOARG. 7. In vessel segments contracted with K+ (60 mM), acetylcholine induced concentration-dependent relaxations. When the vessels were incubated with 0.3 mM L-NOARG and then contracted with K+ (60 mM) all relaxant responses to acetylcholine were abolished. 8. The presence of NO synthesizing enzyme in nerve fibres and the pharmacological evidence for NO-mediated relaxation of the trigone and urethra suggest that NO or a NO-related substance may have a role in inhibitory neurotransmission in these regions. In the detrusor, the presence of NO-synthesizing enzyme in nerves can be demonstrated, but its functional importance is unclear. NO, as well as other endothelium-derived factors seem to be involved in the endothelium-dependent acetylcholine-induced relaxation of pig vesical arteries.

Nitric oxide synthase in dog urethra: a histochemical and pharmacological analysis.

1. To examine the presence of nitric oxide synthase (NOS) activity in female dog urethra, pharmacological experiments were performed using electrical field stimulation (EFS), guanethidine, atropine, NG-nitro-L-arginine methyl ester and L-arginine, NOS immunohistochemistry using specific anti-NOS antibody, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase staining were also performed. 2. EFS caused frequency-dependent contractions in all urethral preparations, but in the presence of guanethidine and atropine, EFS caused significant relaxation in the proximal urethra and was without effect on the distal urethra. 3. In the presence of guanethidine, atropine, and NG-nitro-L-arginine methyl ester, small contractions to EFS were re-established in the proximal urethra, but not in the distal urethra. NG-nitro-D-arginine methyl ester had no such effect. 4. In the presence of guanethidine, atropine, and NG-nitro-L-arginine methyl ester, the addition of L-arginine, restored the EFS-elicited relaxant responses previously seen with guanethidine and atropine alone in the proximal urethra (at 30 Hz; 12.89 +/- 5.27% to -2.44 +/- 4.43%, mean +/- s.e., P < 0.05). D-Arginine had no such effect. 5. In the distal urethra, the addition of NG-nitro-L-arginine methyl ester and then L-arginine had no effect on responses to EFS in preparations treated with guanethidine and atropine. 6. Sodium nitroprusside caused relaxation in both the proximal and distal urethra. The relaxant responses per cm2 cross sectional area in the proximal and distal urethra were 1.23 +/- 0.29, and 2.02 +/- 0.54 g cm-2 cross sectional area (mean +/- s.e.), respectively: there was no significant difference between them. 7. Both NOS and NADPH diaphorase-positive neurones were present in dog urethra, the densities of both being higher in the proximal urethra than in the distal urethra. 8. These results show that female dog urethra possesses NOS nerves and that endogenous NO may play a role in relaxation in the proximal but not the distal urethra.

Involvement of accumulated endogenous NOS inhibitors and decreased NOS activity in the impaired neurogenic relaxation of the rabbit proximal urethra with ischaemia.

1. We examined the effect of ischaemia on the neurogenic and nitric oxide (NO)-mediated urethral relaxation. 2. Rabbits were divided into control and urethral ischaemia (UI) groups, which was prepared by the partial occlusion of bilateral iliac arteries using blood vessel occluders. 3. Neurogenic and NO-mediated proximal urethral relaxation induced by electrical field stimulation (EFS) was greatly impaired in the UI group, while relaxation by sodium nitroprusside (SNP) as a NO donor showed no difference between the two groups. Pretreatment with L-arginine significantly improved but did not normalize the impaired relaxation in the UI group. Not only basal level, but also stimulated production of cyclic GMP with EFS, were significantly decreased in the UI group. 4. The tissue contents of N(G)-methyl-L-arginine (L-NMA) and asymmetric N(G), N(G)-dimethyl-L-arginine (ADMA) in the proximal urethra were increased following ischaemia. While L-arginine and symmetric N(G), N'(G)-dimethyl-L-arginine (SDMA) contents remained unchanged. Exogenously applied authentic L-NMA and ADMA (1 -- 100 microM) concentration-dependently inhibited the EFS-induced urethral relaxation in the control group. The inhibition with L-NMA and ADMA was undetectable in the presence of 3 mM L-arginine. 5. The Ca(2+)-dependent NOS activity in the urethra from the UI group was significantly lower than that from the control group and was not restored by an addition of 3 mM L-arginine. 6. These results suggest that the impaired neurogenic and NO-mediated urethral relaxation with ischaemia is closely related to the increased accumulation of L-NMA and ADMA and decreased NOS activity, which would result in an accelerated reduction in NO production/release.

Effects of superoxide anion generators and thiol modulators on nitrergic transmission and relaxation to exogenous nitric oxide in the sheep urethra.

The effects of superoxide anion generators, the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoine-1-oxyl 3-oxide (carboxy-PTIO), the specific guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ), and thiol modulating agents were investigated on relaxations induced by nitrergic stimulation and exogenous NO addition in the sheep urethra. Methylene blue (MB, 10 microM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml(-1)) inhibited NO-mediated relaxations, without affecting those induced by nitrergic stimulation. This resistance was not diminished following inhibition of endogenous Cu/Zn superoxide dismutase (Cu/Zn SOD) with diethyldithiocarbamic acid (DETCA, 3 mM), which almost abolished tissue SOD activity. Carboxy-PTIO (0.1 - 0.5 mM) inhibited NO-mediated relaxations but had no effect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml(-1)). Relaxations to NO were reduced, but not abolished, by ODQ (10 microM), while nitrergic responses were completely blocked. The thiol modulators, ethacrynic acid (0.1 mM), diamide (1.5 mM), or 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB, 0. 5 mM), and subsequent treatment with dithiothreitol (DTT, 2 mM) had no effect on responses to nitrergic stimulation or NO. In contrast, N-ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. L-cysteine (L-cys, 0.1 mM) had no effect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD-independent manner. Our results do not support the view that the urethral nitrergic transmitter is free NO, and the possibility that another compound is acting as mediator still remains open. British Journal of Pharmacology (2000) 129, 53 - 62

Localization of nitric oxide synthase activity in the human lower urinary tract and its correlation with neuroeffector responses.

OBJECTIVES: The present study was designed to correlate the localization of nitric oxide synthase (NOS) activity to nerve-induced smooth muscle responses in the human lower urinary tract. METHODS: Nerve-induced smooth muscle activity was studied in the human lower urogenital tract. NOS activity was studied by measurement of citrulline formation and guanylate cyclase activity. RESULTS: Nerve-induced contractions in the human detrusor muscle, bladder neck, and prostatic urethra were not significantly enhanced by the NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). In the prostatic urethra, relaxations to transmural nerve stimulation were obtained after increase in tension. The relaxations were abolished by L-NAME and restored by L-arginine. Nerve-induced relaxations were occasionally obtained in the bladder neck, whereas nerve-induced relaxations were never obtained in the detrusor muscle. Citrulline formation was highest in the prostatic urethra, it was intermediate in the bladder neck, and it was less pronounced in the detrusor muscle. Guanylate cyclase activity was also highest in the prostatic urethra, whereas there was no significant difference in guanylate cyclase activity in the bladder neck and detrusor muscle. CONCLUSIONS: The nerve-induced smooth muscle responses and the localization of NOS activity were in good agreement. Thus, in areas where marked relaxations to nerve stimulation were obtained, there was also a high NOS activity. The data suggest that nitric oxide is a mediator for the neurogenic dilation of the bladder neck and urethra during the micturition reflex.