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.

C-peptide stimulates ERK1/2 and JNK MAP kinases via activation of protein kinase C in human renal tubular cells.

AIMS/HYPOTHESIS: Accumulating evidence indicates that replacement of C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, but the molecular mechanisms involved are incompletely understood. C-peptide shows specific binding to a G-protein-coupled membrane binding site, resulting in Ca(2+) influx, activation of mitogen-activated protein kinase signalling pathways, and stimulation of Na(+), K(+)-ATPase and endothelial nitric oxide synthase. This study examines the intracellular signalling pathways activated by C-peptide in human renal tubular cells. METHODS: Human renal tubular cells were cultured from the outer cortex of renal tissue obtained from patients undergoing elective nephrectomy. Extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and Akt/protein kinase B (PKB) activation was determined using phospho-specific antibodies. Protein kinase C (PKC) and RhoA activation was determined by measuring their translocation to the cell membrane fraction using isoform-specific antibodies. RESULTS: Human C-peptide increases phosphorylation of ERK1/2 and Akt/PKB in a concentration- and time-dependent manner in renal tubular cells. The C-terminal pentapeptide of C-peptide is equipotent with the full-length C-peptide, whereas scrambled C-peptide has no effect. C-peptide stimulation also results in phosphorylation of JNK, but not of p38 mitogen-activated protein kinase. MEK1/2 inhibitor PD98059 blocks the C-peptide effect on ERK1/2 phosphorylation. C-peptide causes specific translocation of PKC isoforms delta and epsilon to the membrane fraction in tubular cells. All stimulatory effects of C-peptide were abolished by pertussis toxin. The isoform-specific PKC-delta inhibitor rottlerin and the broad-spectrum PKC inhibitor GF109203X both abolish the C-peptide effect on ERK1/2 phosphorylation. C-peptide stimulation also causes translocation of the small GTPase RhoA from the cytosol to the cell membrane. Inhibition of phospholipase C abolished the stimulatory effect of C-peptide on phosphorylation of ERK1/2, JNK and PKC-delta. CONCLUSIONS/INTERPRETATION: C-peptide signal transduction in human renal tubular cells involves the activation of phospholipase C and PKC-delta and PKC-epsilon, as well as RhoA, followed by phosphorylation of ERK1/2 and JNK, and a parallel activation of Akt.

C-peptide stimulates Na+, K+-ATPase via activation of ERK1/2 MAP kinases in human renal tubular cells.

Proinsulin-connecting peptide (C-peptide) exerts physiological effects partially via stimulation of Na(+), K(+)-ATPase. We determined the molecular mechanism by which C-peptide stimulates Na(+), K(+)-ATPase in primary human renal tubular cells (HRTCs). Incubation of the cells with 5 nM human C-peptide at 37 degrees C for 10 min stimulated (86)Rb(+) uptake by 40% (p<0.01). The carboxy-terminal pentapeptide was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive (86)Rb(+) uptake, C-peptide increased alpha subunit phosphorylation and basolateral membrane (BLM) abundance of the Na(+), K(+)-ATPase alpha(1) and beta(1) subunits. The increase in BLM abundance of the Na(+), K(+)-ATPase alpha(1) and beta(1) subunits was accompanied by depletion of alpha(1) and beta(1) subunits from the endosomal compartments. C-peptide action on Na(+), K(+)-ATPase was ERK1/2-dependent in HRTCs. C-peptide-stimulated Na(+), K(+)-ATPase activation, phosphorylation of alpha(1)-subunit and translocation of alpha(1) and beta(1) subunits to the BLM were abolished by a MEK1/2 inhibitor (20 muM PD98059). C-peptide stimulation of (86)Rb(+) uptake was also abolished by preincubation of HRTCs with an inhibitor of PKC (1 muM GF109203X). C-peptide stimulated phosphorylation of human Na(+), K(+)-ATPase alpha subunit on Thr-Pro amino acid motifs, which form specific ERK substrates. In conclusion, C-peptide stimulates sodium pump activity via ERK1/2-induced phosphorylation of Thr residues on the alpha subunit of Na(+), K(+)-ATPase.

Does nitric oxide act as a cellular messenger in muscarinic endometrial secretion in the guinea-pig?

Uterine secretory cells receive a sympathetic cholinergic secremotor innervation. Nitric oxide (NO) has been suggested to be a second messenger of neurogenic modulated glandular secretion of the seminal vesicle. Thus a similar pattern for nervous induced carbohydrate secretion of the endometrium was assumed. The nitric oxide synthase (NOS) activity was estimated via formation of L-citrulline from L-arginine and histochemically with the nicotinamide-adenine dinucleotide phosphate diaphorase (NADPH-d) nitro blue technique. The carbohydrate secretion from everted uterine horns placed in organ baths was estimated. A calcium dependent formation of citrulline was found in the uterine horn suggesting an NOS activity. Strong NADPH staining cells were found in the glandular ducts of the endometrium and in the epithelial linings of the oviduct. Carbachol induced carbohydrate secretion of the endometrium while N-nitro L-arginin (L-NNA) and N-nitro L-arginin methyl ester (L-NAME) inhibited the carbachol induced secretion. The isomer D-NAME had no effect on carbachol induced secretion. When L-arginine was administered together with L-NNA no inhibitory effect on carbachol induced secretion was seen. L-arginine only had no effect on carbohydrate secretion. The NO donor glyceryl tritrate increased carbohydrate secretion but no synergistic effect was seen in combination with carbachol. The results suggest that glandular NO production is a prerequisite for muscarinic carbohydrate secretion of the endometrium.

Bacillus Calmette-Guerin induces long-term local formation of nitric oxide in the bladder via the induction of nitric oxide synthase activity in urothelial cells.

PURPOSE: Bladder instillation of bacillus Calmette-Guerin (BCG) is effective therapy for recurrent superficial bladder cancer and carcinoma in situ. BCG induces nitric oxide synthase activity in the bladder. Nitric oxide is formed from L-arginine by nitric oxide synthase. We investigated nitric oxide formation and its localization in bladder cancer patients treated with intravesical BCG instillation. MATERIALS AND METHODS: The L-citrulline conversion assay was done to assess nitric oxide synthase activity in BCG treated T24 human bladder cancer cells and cultured normal human urothelial cells. Nitrite and nitrate in cell culture medium, urine and plasma were measured by capillary electrophoresis. Nitric oxide formation in the bladder was measured by chemiluminescence. RESULTS: A 24-hour treatment with BCG induced calcium independent nitric oxide synthase activity in T24 cells in a dose dependent manner. Nitrite and nitrate production by T24 cells also increased in a dose dependent manner after 24-hour BCG treatment. BCG treatment of cultured normal human urothelial cells resulted in the induction of calcium dependent and independent nitric oxide synthase activity. Nitrite in the urine of patients receiving BCG for the first time was increased 5-fold 24 hours after instillation. Furthermore, BCG increased luminal nitric oxide in the bladder. The increase was noted after a single treatment and sustained for 6 months. No changes in plasma nitrite or nitrate were observed after BCG treatment. CONCLUSIONS: BCG induces the local formation of nitric oxide in the bladder, whereas no evidence for systemic nitric oxide formation was noted. Increased nitric oxide production in the bladder is likely due to the induction of nitric oxide synthase activity in urothelial cells.

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.

Nitric oxide: a useful gas in the detection of lower urinary tract inflammation.

PURPOSE: Luminal nitric oxide has been shown to be elevated in the bladder of patients with cystitis of various etiologies. We determine whether luminal nitric oxide can be used as a marker to differentiate inflammation, that is interstitial cystitis, from urgency, frequency, nocturia and pain due to noninflammatory disorders, such as outflow obstruction and neurogenic dysfunction. MATERIALS AND METHODS: We measured luminal nitric oxide in the bladder of patients with urgency due to detrusor instability (6), outflow obstruction (7), sensory urge (19) and interstitial cystitis (8), and controls without urgency symptoms (11). Nitric oxide-free air was incubated in the bladder for 5 minutes and analyzed in a chemiluminescence nitric oxide analyzer. RESULTS: There was a nearly 20-fold increase in mean bladder nitric oxide concentration in patients with interstitial cystitis (234+/-67 parts per billion) compared to those with detrusor instability (11+/-1), outflow obstruction (9+/-1) and sensory urgency (10+/-1), and controls (13+/-2). CONCLUSIONS: Measurement of nitric oxide in air from the bladder is a simple, safe and fast method to differentiate urgency due to inflammation from neurogenic disorders or outflow obstruction. The simplicity of this method makes it potentially useful as a screening method for office use.

Effects of L-arginine treatment on symptoms and bladder nitric oxide levels in patients with interstitial cystitis.

OBJECTIVES: Nitric oxide (NO) is involved in host defense reactions, and NO production is elevated in various inflammatory disorders. We have found very high levels of luminal NO in the urinary bladder of patients with interstitial cystitis. Oral treatment with low doses of L-arginine, the substrate for NO production, has been reported to alleviate symptoms in patients with interstitial cystitis. The aim of our investigation was to evaluate the effect of higher doses of L-arginine in patients with interstitial cystitis and to study the effects of L-arginine on NO production in the bladder. METHODS: Nine women (age 69+/-3 years) with interstitial cystitis were treated daily with 3 or 10 g of L-arginine for 5 weeks. Symptoms were evaluated with an interstitial cystitis symptom score index, and NO production was measured. Patients with stress incontinence (n=18) were used as control subjects for measurement of NO levels. RESULTS: NO concentration in the urinary bladder was markedly elevated in the patients with interstitial cystitis (239+/-60 ppb) compared with the control patients (15+/-2 ppb). NO levels did not change in the patients with interstitial cystitis after oral treatment with L-arginine (189+/-72 ppb). There was no significant change in the symptom scores at either dose after 5 weeks of L-arginine treatment. CONCLUSIONS: L-arginine treatment in the doses used in this study did not change NO production in the urinary bladder in patients with interstitial cystitis. Furthermore, the patients in our study did not notice any relief of their symptoms.

NADPH-diaphorase in glandular cells and nerves and its relation to acetylcholinesterase-positive nerves in the male reproductive tract of man and guinea-pig.

The presence of NADPH-diaphorase activity and acetylcholinesterase in the testis, epididymis, vas deferens, seminal vesicle, pelvic plexus, prostate and urethra of man and guinea-pig was investigated with the nitro blue NADPH technique and the thiocholine method, respectively. In human material NADPH-diaphorase activity was found in the Leydig cells, Sertoli cells and the epithelial linings of the rete testis, the excretory ducts, seminal vesicle, prostate and urethra. The guinea-pig material showed staining of the Leydig cells and spermatozoa and similar epithelial staining of the tract as man. Nerves beneath the epithelium and in the muscle layers of cauda epididymis, vas deferens, seminal vesicle, prostate and urethra were also stained. NADPH-diaphorase-positive nerve cells were seen in the pelvic plexus. Some cells also displayed acetylcholinesterase activity but others showed activity for only one of the enzymes or no activity for either enzyme. In the cauda epididymis, vas deferens, seminal vesicle, prostate and urethra acetylcholinesterase-positive nerve fibres formed a plexus beneath the secretory cells. It is concluded that NADPH-diaphorase, generally accepted as a nitric oxide synthase, is present in glandular cells of the male genital tract. The enzyme is also present in nerves, where it is partly co-localized with acetylcholinesterase.

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 inhibitors N(G)-nitro L-arginine methyl ester (L-NAME) and N(G)-nitro L-arginine (L-NNA) inhibited carbachol-induced fructose secretion but the D-isomer to L-NAME had no effect. When L-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.

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.

Modulation of smooth muscle activity by nitric oxide in the human upper urinary tract.

The aim of the study was to ascertain whether nitric oxide (NO) might regulate motility in the human upper urinary tract. Smooth muscle activity in the human renal pelvis and proximal ureter was studied in vitro in organ baths, and nitric oxide synthase (NOS) activity was studied by measurement of citrulline formation. NO, glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) significantly reduced the frequency of spontaneous rhythmic contractions in renal pelvis and proximal ureter. Exogenously applied NO elicited relaxations in pre-contracted renal pelvis. Calcium-dependent NOS activity was significant in the renal pelvis but undetectable in the ureter. Also, NOS activity was absent in hydronephrotic renal pelvis. NO, SNP and GTN inhibited smooth muscle activity in the human upper urinary tract. NOS activity was obtained in normal renal pelvis but not in hydronephrotic renal pelvis. Regulation of urinary tract NO concentrations might offer a strategy for treatment of renal colic and disturbances in upper urinary tract motility.

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.

Renal and bladder functions in patients after spinal cord injuries.

Thirty-eight patients with spinal cord lesions, 22 recent and 16 sustained more than three years previously, were investigated with intravenous pyelography, chrome EDTA clearance, cystoscopy and urodynamic studies. Analyses of bladder biopsies for tissue concentrations of nor-adrenaline and occurrence of acetylcholinesterase staining of nerves were also performed. Despite high incidence of fairly mild infections and trabeculation of the bladder, renal function was normal in most patients. Apart from incontinence, stone formation and recurrent urinary tract infections were the most common complications. The concentrations of noradrenaline and the numbers of acetylcholinesterase-stained nerves in bladder tissue specimens did not differ from control findings. The organization of the nerve structures did not vary with time after the injury, suggesting unchanged adrenergic and cholinergic innervation.

Nitric oxide synthase activity in the human urogenital tract.

Nitric oxide (NO) has been suggested as a nonadrenergic 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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-dependent NOS activity. Ca(2+)-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.

Urethral stricture complicating transurethral prostatic surgery.

The incidence of urethral stricture was 9 per cent among 336 patients after transurethral prostatic resection for benign hyperplasia. The strictures were mostly in the penile part of the urethra and could be treated with regular dilation, internal urethrotomy or meatal incision. Bacteriuria or indwelling catheter preoperatively did not seem to predispose to stricture formation. When there is an indication of a narrow urethra before TUR, a fine-calibre resectoscope should be used and a preliminary internal urethrotomy is advisable.

Benign and malignant ovarian tumors in children and adolescents. A review of 63 cases.

Sixty-three patients (15 months to 17 years of age) with ovarian tumors were seen. Fifty-six patients had germ cell tumors and 7 epithelial tumors, 6 patients had bilateral tumors, 47 patients had teratomas (41 benign, 3 with embryonic tissue, and 3 malignant), 6 germinomas, 1 endodermal sinus tumor, and 2 mixed germ cell tumors. Of the patients with epithelial tumors, six had cystadenomas and one cystadenocarcinoma. Abdominal pain was the most common symptom, and an abdominal mass the most common sign. A calcification on abdominal films was seen in 29 patients with benign teratoma. At surgery, the opposite ovary was bivalved or biopsied in 31 patients with benign tumors. Two patients had teratomas burried in the ovarian tissue. Among 47 patients with benign tumors, 37 have remained well after the operation and 10 were lost to follow-up. Two girls with embryonic teratoma are well 71 and 19 months after diagnosis but the third died with embryonal carcinoma. Of the 13 patients with malignant tumors, 8 are alive disease free 4 1/2 to 17 years after diagnosis, 4 are dead, and 1 is lost to follow-up (disease free 15 months after diagnosis).