N.P. Wiklund

Localization of nitric oxide synthaseactivity 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.

Elevated nitric oxide in the urinary bladder in infectious and noninfectious cystitis

OBJECTIVES A role for nitric oxide (NO) has been suggested in inflammation and host defense. At higher concentrations, this gas shows cytotoxic effects that may be directed against microorganisms, tumor cells as well as host cells. The aim of the present study was to study the relationship between bladder mucosal inflammation and local production of NO. METHODS We measured NO directly in the urinary bladder in patients with infectious cystitis, interstitial cystitis, irradiation cystitis, and cystitis induced by antitumor treatment with bacillus Calmette-Guérin. NO-free air was introduced into the bladder during cystoscopy. The air was aspirated after 5 minutes of incubation and injected into a chemiluminescence NO analyzer. RESULTS NO levels were 30 to 50 times higher in all varieties of cystitis as compared to controls. CONCLUSIONS NO may contribute to host-defense mechanisms in the bladder during bacterial infection and antitumor treatment. Direct measurement of gaseous NO in the urinary bladder seems to be an attractive diagnostic method for detection of mucosal inflammation.

Urinary nitrite: More than a marker of infection

OBJECTIVES The bacteriostatic gas nitric oxide (NO) is formed when nitrite is acidified. Infected urine may contain considerable amounts of nitrite as a result of bacterial nitrate reductase activity, and detection of nitrite in urine is routinely used in the diagnosis of bacterial cystitis. We sought to determine whether NO was generated from acidified nitrite-containing urine. Furthermore, we also studied the growth of the urinary pathogen Escherichia coll in acidified nitrite-containing urine. METHODS Urine, collected from healthy control subjects or from patients with infected nitrite-containing urine, was acidified and incubated in a closed syringe with varying amounts of nitrite added. After 30 minutes, the headspace gas was removed and immediately injected into a chemiluminescence NO analyzer. In addition, NO was measured in urine collected from healthy control subjects after ingestion of vitamin C. Bacterial growth was measured continuously in control urine for 10 hours after incubation for 2 hours in acidic urine with varying concentrations of nitrite added. RESULTS Large amounts of NO were released from infected nitrite-containing urine after mild acidification. NO was also released from acidified control urine if nitrite was added, and this release was greatly potentiated in the presence of vitamin C. Furthermore, the growth of E. coli was markedly reduced by the addition of nitrite to acidified urine. CONCLUSIONS We propose that nitrite-producing bacteria induce their own death in acidic urine by supplying substrate for generation of bacteriostatic compounds such as NO. This mechanism might explain why urinary acidification and vitamin C may be effective in the treatment of bacteriuria.

Nitric oxide synthase activity in the human urogenital tract

Nitric oxide (NO) has been suggested as a non-adrenergic non-cholinergic neurotransmitter in the urogenital tract and has previously been shown to have a smooth muscle relaxing effect in the urogenital organs both in various animals and in humans. It has been shown that NO is a mediator of the erection and the dilatation of the bladder neck and urethra. The aim of the study was to analyse nitric oxide synthase (NOS) activity in the human urogenital tract. NOS activity was measured by the conversion of l-[U-14C] arginine to l-[U-14C] citrulline. In the upper urinary tract there was Ca2+-dependent NOS activity in the renal pelvis, but no significant NOS activity could be found in the ureter. In the lower urinary tract we found high Ca2+-dependent NOS activity in the urethra, intermediate activity in the bladder neck and comparatively low activity in the detrusor muscle. In the male genital tract the testis and epididymis had no significant NOS activity. The vas deferens, prostate, seminal vesicle and corpus cavernosum were found to have high levels of Ca2+-dependent NOS activity. Ca2+-independent NOS activity was not obtained in the urogenital tract. Our results correspond well with previous functional studies indicating NO to be an important nerve-induced mediator of erection and in the micturition reflex, but also suggest that NO may be involved in several other functions in the human urogenital tract.

Localization and Expression of Inducible Nitric Oxide Synthase in Biopsies From Patients With Interstitial Cystitis

PURPOSE Interstitial cystitis is a chronic inflammatory disease of the bladder and luminal nitric oxide has been shown to be increased in the bladder in patients with interstitial cystitis. We analyzed endogenous nitric oxide formation and inducible nitric oxide synthase gene expression in the bladder of patients with interstitial cystitis to obtain further knowledge of the localization of inducible nitric oxide synthase in the bladder mucosa. MATERIALS AND METHODS Six patients with interstitial cystitis and 8 controls were studied. In these 2 groups endogenous nitric oxide formation was measured and inducible nitric oxide synthase expression in bladder biopsies was analyzed at the transcriptional and protein levels by real-time polymerase chain reaction and Western blot, respectively. Immunohistochemistry for inducible nitric oxide synthase was also performed. RESULTS Patients with interstitial cystitis had higher inducible nitric oxide synthase mRNA expression and nitric oxide formation than controls (p <0.01 and <0.001, respectively). Inducible nitric oxide synthase protein expression was up-regulated in the interstitial cystitis group. Immunohistochemistry showed that inducible nitric oxide synthase was predominantly localized to the urothelium in patients with interstitial cystitis but inducible nitric oxide synthase-like immunoreactivity was also found in macrophages in the bladder mucosa. CONCLUSIONS The increased levels of endogenously formed nitric oxide in patients with interstitial cystitis correspond to increased inducible nitric oxide synthase mRNA expression and protein levels in these patients. Furthermore, inducible nitric oxide synthase was found to be localized to the urothelium but it was also found in macrophages in the bladder mucosa. Whether high levels of endogenously formed nitric oxide are a part of the pathogenesis in interstitial cystitis and whether it has a protective or damaging role remain to be elucidated.

The role of nitric oxide in bacillus Calmette-Guérin mediated anti-tumour effects in human bladder cancer

Bacillus Calmette-Guérin (BCG) has been used for many years to treat cancer of the urinary bladder. It constitutes effective intravesical therapy of carcinoma in situ and recurrent superficial bladder cancer. Although the mechanism of action is unknown, most evidence suggests an immune-mediated mechanism. BCG treatment is known to increase cytokine production in the urinary bladder. As cytokines may induce nitric oxide synthase (NOS) activity and as nitric oxide (NO) exerts cytotoxic effects on tumour cells, we investigated the role of NO in BCG-mediated anti-tumour activity. Here we demonstrate a marked induction of both calcium-dependent and calcium-independent NOS activity in the human urinary bladder after BCG treatment. The presence of NOS in the urothelial cells was also demonstrated by the use of immunohistochemistry. Furthermore, patients treated with BCG showed a 30 times higher production of gaseous NO as measured in the urinary bladder by chemiluminescence. Finally, NO donors exerted cytotoxic effects on bladder cancer cell lines. These findings suggest that NO synthesis may be an important mechanism in BCG-mediated anti-tumour therapy.

In Vitro Evaluation of a New Treatment for Urinary Tract Infections Caused by Nitrate-Reducing Bacteria

ABSTRACT Dietary and endogenous nitrates are excreted in urine, and during infection with nitrate-reducing bacteria they are reduced to nitrite. At a low pH nitrite is converted to a variety of nitrogen oxides that are toxic to bacteria. We hypothesized that acidification of nitrite-rich infected urine would result in the killing of the nitrate-reducing bacteria. An Escherichia coli control strain and a mutant lacking nitrate reductase activity were preincubated in urine supplemented with sodium nitrate (0 to 10 mM) at pH 7.0. Then, the nitrite-containing bacterial culture was transferred (and diluted 1/10) to slightly acidic urine (pH 5 and 5.5) containing ascorbic acid (10 mM) and growth was monitored. The control strain produced nitrite in amounts related to the amount of nitrate added. This strain was killed when the culture was transferred to acidic urine. In contrast, the mutant that did not produce nitrite retained full viability. When control bacteria were grown in acidic urine with nitrate and ascorbic acid present from the start of the experiment, no inhibition of growth was noted. The MICs and minimal bactericidal concentrations of sodium nitrite-ascorbic acid in acidic urine were comparable to those of conventional antibiotics. Preincubation of nitrate-reducing E. coli in nitrate-rich urine leads to the accumulation of nitrite. Subsequent acidification of the urine results in generation of nitrogen oxides that are bactericidal. Killing, however, requires a sequential procedure in which the bacteria are first allowed to grow in a nitrate-rich neutral environment, later followed by acidification. We speculate that ingestion of nitrate followed some hours later by acidification of urine could be a new therapeutic strategy for the treatment of urinary tract infections.

Thromboembolic Complications in 3,544 Patients Undergoing Radical Prostatectomy with or without Lymph Node Dissection

PURPOSE Lymph node dissection in patients with prostate cancer may increase complications. An association of lymph node dissection with thromboembolic events was suggested. We compared the incidence and investigated predictors of deep venous thrombosis and pulmonary embolism among other complications in patients who did or did not undergo lymph node dissection during open and robot-assisted laparoscopic radical prostatectomy. MATERIALS AND METHODS Included in study were 3,544 patients between 2008 and 2011. The cohort was derived from LAPPRO, a multicenter, prospective, controlled trial. Data on adverse events were extracted from patient completed questionnaires. Our primary study outcome was the prevalence of deep venous thrombosis and/or pulmonary embolism. Secondary outcomes were other types of 90-day adverse events and causes of hospital readmission. RESULTS Lymph node dissection was performed in 547 patients (15.4%). It was associated with eightfold and sixfold greater risk of deep venous thrombosis and pulmonary embolism events compared to that in patients without lymph node dissection (RR 7.80, 95% CI 3.51-17.32 and 6.29, 95% CI 2.11-18.73, respectively). Factors predictive of thromboembolic events included a history of thrombosis, pT4 stage and Gleason score 8 or greater. Open radical prostatectomy and lymph node dissection carried a higher risk of deep venous thrombosis and/or pulmonary embolism than robot-assisted laparoscopic radical prostatectomy (RR 12.67, 95% CI 5.05-31.77 vs 7.52, 95% CI 2.84-19.88). In patients without lymph node dissection open radical prostatectomy increased the thromboembolic risk 3.8-fold (95% CI 1.42-9.99) compared to robot-assisted laparoscopic radical prostatectomy. Lymph node dissection induced more wound, respiratory, cardiovascular and neuromusculoskeletal events. It also caused more readmissions than no lymph node dissection (14.6% vs 6.3%). CONCLUSIONS Among other adverse events we found that lymph node dissection during radical prostatectomy increased the incidence of deep venous thrombosis and pulmonary embolism. Open surgery increased the risks more than robot-assisted surgery. This was most prominent in patients who were not treated with lymph node dissection.

Measurement of luminal nitric oxide in bladder inflammation using a silicon balloon catheter: a novel minimally invasive method.

OBJECTIVES Nitric oxide (NO) measured in the gaseous phase has been shown to be a marker of inflammation in the urinary bladder. The NO content of air incubated in the bladder can be measured in an NO analyzer. The aim of our study was threefold: to evaluate whether NO can be measured in air incubated in a catheter balloon, to determine the optimal time of incubation, and to find the most suitable type of catheter. METHODS The NO concentration in air introduced directly into the bladder and into the catheter balloon was measured in patients with and without bladder infections. The air was incubated for 5 to 60 minutes. NO concentration in the bladder of patients with interstitial cystitis was also analyzed. The diffusion rate of NO through silicon and latex catheters was studied. RESULTS Elevated NO levels were detected in the urinary bladder in patients with bladder inflammation due to infection or interstitial cystitis. A marked increase in NO concentration was found after just 5 minutes of incubation and continued to rise for up to 20 minutes, both in air taken directly from the bladder and from the catheter balloon. The NO diffusion rate into the balloons of silicon catheters was high; the recovery rate in latex catheters was poor. CONCLUSIONS Measurement of NO concentration in a silicon balloon catheter inserted into the urinary bladder is a fast, convenient, and reliable method to detect inflammation.

Is glandular formation of nitric oxide a prerequisite for muscarinic secretion of fructose in the guinea-pig seminal vesicle?

The significance of nitric oxide (NO) formation in seminal secretion was studied in guinea-pig seminal vesicles. The nitric oxide synthase (NOS) activity was estimated and reduced nicotinamide-adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry was performed. Furthermore, cyclic guanosine 3,5-monophosphate (cGMP) concentration as well as fructose secretion from isolated vesicles was estimated. High Ca2+-dependent NOS activity as well as prominent glandular NADPH-diaphorase staining was found in the secretory epithelium. The NOS inhibitorsNG-nitrol-arginine methyl ester (L-NAME) andNG-nitrol-ar-ginine (L-NNA) inhibited carbachol-induced fructose secretion but the D-isomer to L-NAME had no effect. Whenl-arginine was administered together with L-NAME, no inhibitory effect on the carbachol-induced fructose secretion could be seen. Nerve-induced fructose secretion was also inhibited by L-NAME. The NO donor glyceryl trinitrate (GTN) increased the fructose secretion. Carbachol or GTN did not increase cGMP levels, nor was fructose secretion inhibited by a guanylate cyclase inhibitor (ODQ). Our results suggests that glandular NO production is a prerequisite for muscarinic fructose secretion in the seminal vesicle via a cGMP-independent pathway.