Urinary exosome as a potential biomarker for urinary tract infection.

Unlike urinary tract infection (UTI), asymptomatic bacteriuria (ABU) should not be treated, with some exceptions such as pregnant women and patients who will undergo traumatic urologic interventions. However, there has been no clinically available marker for their differential diagnosis. Exosomes or small extracellular vesicles carry proteins contained in cells from which they are derived, thus having the potential as a biomarker of several diseases. On the basis of the hypothesis that the molecular signature of exosomes in urine may differ between UTI and ABU patients, we examined if urinary exosomes could serve as a marker for their differential diagnosis. Exosomes were isolated by ultracentrifugation or affinity-based method from cell culture medium of monocytic THP-1 and uroepithelial SV-HUC-1 cells and human urine. Protein expression was examined by Western blot analysis, ELISA, and CLEIA. The results showed that the levels of intracellular signalling molecules Akt and ERK and transcription factor NF-κB increased in exosomes isolated from THP-1 and SV-HUC-1 cells cocultured with Escherichia coli and/or treated with lipopolysaccharide. In urinary exosomes of UTI patients, Akt significantly diminished, and an exosomal marker CD9 showed a trend to decrease after treatment with antimicrobial agents. More importantly, Akt and CD9 levels in urinary exosomes were higher in UTI patients than in ABU patients, which was also observed after correction by urine creatinine. Collectively, these results suggest that Akt and CD9 in urinary exosomes could be useful markers for differential diagnosis of UTI and ABU.

Gamma-glutamyltransferase activity in exosomes as a potential marker for prostate cancer.

BACKGROUND: Exosomes or extracellular vesicles have the potential as a diagnostic marker for various diseases including cancer. In order to identify novel exosomal markers for prostate cancer (PC), we performed proteomic analysis of exosomes isolated from PC cell lines and examined the usefulness of the marker in patients. METHODS: Exosomes isolated by differential centrifugation from the culture medium of androgen-dependent LNCaP prostate cancer cell line and its sublines of partially androgen-independent C4, androgen-independent C4-2 and bone metastatic C4-2B were subjected to iTRAQ-based proteomic analysis. Exosomes were also isolated by immunocapture and separated by size exclusion chromatography and density gradient centrifugation. Protein expression was determined by Western blot analysis. GGT activity was measured using a fluorescent probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG). Immunohistochemical analysis of tissues was performed using anti-GGT1 antibody. RESULTS: Among proteins upregulated in C4-2 and C4-2B cells than in LNCaP cells, we focused on gamma-glutamyltransferase 1 (GGT1), a cell-surface enzyme that regulates the catabolism of extracellular glutathione. The levels of both GGT1 large and small subunits were elevated in exosomes isolated from C4-2 and C4-2B cells by differential centrifugation and by immunocapture with anti-CD9 or -prostate-specific membrane antigen (PSMA) antibody. In cell lysates and exosomes, GGT1 expression correlated with GGT activity. Size exclusion chromatography of human serum demonstrated the presence of GGT activity and GGT1 subunits in fractions positive for CD9. Density gradient centrifugation revealed the co-presence of GGT1 subunits with CD9 in exosomes isolated by differential centrifugation from human serum. Since GGT activity correlated with GGT1 expression in serum exosomes isolated by differential centrifugation, we measured serum exosomal GGT activity in patients. Unexpectedly, we found that serum exosomal GGT activity was significantly higher in PC patients than in benign prostatic hyperplasia (BPH) patients. In support of this finding, immunohistochemical analysis showed increased GGT1 expression in PC tissues compared with BPH tissues. CONCLUSIONS: Our results suggest that serum exosomal GGT activity could be a useful biomarker for PC.

Effects of a Gentle, Self-Administered Stimulation of Perineal Skin for Nocturia in Elderly Women: A Randomized, Placebo-Controlled, Double-Blind Crossover Trial.

BACKGROUND: Somatic afferent nerve stimuli are used for treating an overactive bladder (OAB), a major cause of nocturia in the elderly. Clinical evidence for this treatment is insufficient because of the lack of appropriate control stimuli. Recent studies on anesthetized animals show that gentle stimuli applied to perineal skin with a roller could inhibit micturition contractions depending on the roller's surface material. We examined the efficacy of gentle skin stimuli for treating nocturia. METHODS: The study was a cross-over, placebo-controlled, double-blind randomized clinical study using two rollers with different effects on micturition contractions. Participants were elderly women (79-89 years) with nocturia. Active (soft elastomer roller) or placebo (hard polystyrene roller) stimuli were applied to perineal skin by participants for 1 min at bedtime. A 3-day baseline assessment period was followed by 3-day stimulation and 4-day resting periods, after which the participants were subjected to other stimuli for another 3 days. The primary outcome was change in the frequency of nighttime urination, for which charts were maintained during each 3-day period. RESULTS: Twenty-four participants were randomized, of which 22 completed all study protocols. One participant discontinued treatment because of an adverse event (abdominal discomfort). In participants with OAB (n = 9), change from baseline in the mean frequency of urination per night during the active stimuli period (mean ± standard deviation, -0.74 ± 0.7 times) was significantly greater than that during placebo stimuli periods (-0.15 ± 0.8 times [p < 0.05]). In contrast, this difference was not observed in participants without OAB (n = 13). CONCLUSIONS: These results suggest that gentle perineal stimulation with an elastomer roller is effective for treating OAB-associated nocturia in elderly women. Here the limitation was a study period too short to assess changes in the quality of sleep and life. TRIAL REGISTRATION: UMIN Clinical Trial Registry (CTR) UMIN000015809.

Effects of Chronic Hyperlipidemia on Lower Urinary Tract Function-Bladder Dysfunction in Myocardial Infarction-Prone Watanabe Heritable Hyperlipidemic Rabbits.

To evaluate the effects of chronic hyperlipidemia on bladder function, we examined the functional and histological changes of the bladder in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHL-MI) rabbits. Two age groups of WHHL-MI rabbits (6-12 months old, young WHHL-MI rabbits; and 20-24 months old, old WHHL-MI rabbits group) and the sex- and age-matched control rabbits were prepared. Bladder functions were evaluated using frequency volume charts and cystometrograms, and functional experiments using isolated bladder specimens. Histological studies of bladder were performed with HE staining and immunohistochemical staining with mouse monoclonal S-100 protein antibodies and sheep polyclonal calcitonin gene-related peptide (CGRP) antibodies. In cystometrograms, it has been demonstrated that WHHL-MI rabbits showed significantly shorter micturition interval, smaller voided volume with non-voiding contractions compared to control. There was no significant difference in voiding pressure between young WHHL-MI and control rabbits. However, old WHHL-MI rabbits showed a lower voiding pressure than control rabbits. The functional experiments revealed that carbachol- and electrical field stimulation (EFS)-induced contractile responses of isolated bladder strips were significantly increased in young WHHL-MI rabbits than in control rabbits. However, in the bladder strips of old WHHL-MI rabbits, decreased responses to carbachol and EFS were observed. In WHHL-MI rabbits, bladder urothelium became thinner, smooth muscle area decreased and connective tissue area increased gradually with aging. A significant decrease in S-100 protein-positive neurons, and an increased number of CGRP-positive neurons were observed in both young and old WHHL-MI rabbits. The data demonstrated that there were differences in bladder dysfunction between young and old WHHL-MI rabbits. Old WHHL-MI rabbits showed detrusor hyperactivity with impaired contraction. This study may demonstrate the developmental mechanism of bladder dysfunction in chronic hyperlipidemia.

A gentle mechanical skin stimulation technique for inhibition of micturition contractions of the urinary bladder.

Effects of gentle skin stimulation of various segmental areas on the micturition contractions of the urinary bladder were examined in anesthetized male rats. The bladder was expanded by infusing saline via urethral cannula until the bladder produced rhythmic micturition contractions as a consequence of rhythmic burst discharges of vesical pelvic efferent nerves. Gentle stimulation was applied for 1 min by slowly rolling on top of skin with an elastomer "roller". Rolling on the perineal area inhibited both micturition contractions and pelvic efferent discharges during and after stimulation. Stimulation of the hindlimb, abdomen and forelimb inhibited micturition contractions after stimulation ended, in this order of effectiveness. During stimulation of the perineal skin, the reflex increase in pelvic efferent discharges in response to bladder distension to a constant pressure was also inhibited up to 45% of its control response. The inhibition of the micturition contractions induced by perineal stimulation was abolished, to a large extent by the opioid receptor antagonist naloxone and completely by severing cutaneous nerves innervating the perineal skin. We recorded unitary afferent activity from cutaneous branches of the pudendal nerve and found that the fibers excited by stimulation were low-threshold mechanoreceptive Aß, Aδ and C fibers. Discharge rates of afferent C fibers (7.9 Hz) were significantly higher than those of Aß (2.2 Hz) and Aδ (2.9 Hz) afferents. The results suggest that low frequency excitation of low threshold cutaneous mechanoreceptive myelinated and unmyelinated fibers inhibits a vesico-pelvic parasympathetic reflex, mainly via release of opioids, leading to inhibition of micturition contraction.

Effectiveness of tamsulosin hydrochloride and its mechanism in improving nocturia associated with lower urinary tract symptoms/benign prostatic hyperplasia.

AIMS: To evaluate the action mechanism of α(1)-receptor blockers in improving nocturia, we have studied effectiveness of tamsulosin hydrochloride (TAM) in the patients with nocturia associated with lower urinary tract symptoms/benign prostatic hyperplasia (LUTS/BPH). METHODS: LUTS/BPH patients with nocturia (nocturnal frequency ≥2 times per day) were administered TAM (0.2 mg/day) for 8 weeks. A frequency volume chart (FVC), the International Prostate Symptom Score (I-PSS), quality of life (QOL) index, post-void residual, and uroflowmetry were recorded before and after TAM administration for the patients. The parameters affected by TAM were examined. RESULTS: The FVC and I-PSS of the 160 patients analyzed revealed significant clinical improvements in the nocturnal frequency. On the basis of the FVC, the patients were divided into two groups: the responder group comprising 97 patients with significantly improved nocturnal frequency and the non-responder group comprising 63 patients with less improvement in the nocturnal frequency. Significant differences between groups were observed in the following parameters: the hours of undisturbed sleep (HUS), the interval between the time of sleeping and the first instance of nocturnal voiding, the volume of urine in the first nocturnal voiding episode, nocturnal urine volume, nocturnal polyuria index, daytime urine volume, maximum and average flow rates, and post-void residual. CONCLUSIONS: TAM improved the QOL of LUTS/BPH patients by significantly reducing the nocturnal frequency and increasing HUS; moreover, it improved nocturia by decreasing the nocturnal urine volume.

The effects of chronic hyperlipidemia on bladder function in myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits.

AIMS: Lower urinary tract symptoms (LUTS) are common in the aging population. LUTS cause profoundly negative impacts on their quality of life. Pathophysiology of LUTS is multifactorial, and recently, bladder ischemia and metabolic syndrome have been suggested as etiological factors. To evaluate chronic hyperlipidemia on bladder function, we examined the functional and histological changes of the bladder in myocardial infarction-prone Watanabe Heritable Hyperlipidemic (WHHLMI) rabbits. METHODS: 20- to 24-month-old WHHLMI rabbits and age- and sex-matched control rabbits were prepared. Bladder functions were evaluated using cystometrograms and functional experiments with isolated bladder specimens. Histological studies of bladder and internal iliac arteries were performed with hematoxylin and eosin staining. The bladder was also stained immunohistochemically with mouse monoclonal S-100 antibodies and sheep polyclonal calcitonin gene-related peptide (CGRP) antibodies. RESULTS: In cystometric examination, WHHLMI rabbits showed significantly shorter micturition interval, smaller voided volume with non-voiding contractions, and lower micturition pressure, as compared to control. The functional experiments showed that carbachol- and electrical field stimulation-induced contractions were significantly decreased in WHHLMI rabbits than those in control. In WHHLMI rabbits, cross-sections of internal iliac arteries showed significant atherosclerosis and thickening of media. Bladder showed thinner urothelium and decreased smooth muscle area in WHHLMI rabbits, as compared to control. WHHLMI rabbits showed a significant decrease in S-100 protein positive neurons, and an increased number of CGRP positive neurons. CONCLUSIONS: This study demonstrated that WHHLMI rabbits showed detrusor overactivity with decreased detrusor contraction. It is suggested that chronic hyperlipidemia contributes to the bladder dysfunction.

The forefront for novel therapeutic agents based on the pathophysiology of lower urinary tract dysfunction: pathophysiology and pharmacotherapy of overactive bladder.

Overactive bladder (OAB) syndrome, which is characterized by a complex of storage symptoms (urinary urgency, frequency, nocturia, and urgency incontinence) is highly prevalent within the general population, causing major distress to patients in terms of their psychosocial and physical functioning. Muscarinic receptors of bladder smooth muscles are involved in both normal and disturbed bladder contraction. The muscarinic receptor functions may change in bladder disorders associated with OAB, implying that mechanisms, which normally have little clinical importance, may be up-regulated and contribute to the pathophysiology of OAB. In addition, several reports have suggested that various stimulations release many substances, including adenosine triphosphate, prostaglandins, nitric oxide, and acetylcholine, from bladder urothelium, which contribute to pathophysiology of the increased bladder sensation, OAB symptoms, and detrusor overactivity. Bladder urothelium possesses a non-neuronal cholinergic system and high density of muscarinic receptors. The roles and functions of the non-neuronal cholinergic system in OAB are now being evaluated. In the pharmacotherapy of OAB, antimuscarinic agents are the first choice drugs. Furthermore, new therapeutic targets at the levels of the urothelium, detrusor muscles, autonomic and afferent pathways, spinal cord, and brain are proposed. In this review, the pathophysiology of OAB, especially the role of non-neuronal acetylcholine, is discussed. In addition, new drugs with new action mechanisms will be introduced.

The ß3-adrenoceptor mediates the inhibitory effects of ß-adrenoceptor agonists via the urothelium in pig bladder dome.

AIMS: Relaxation of detrusor muscle via ß-adrenoceptors may contribute to urine storage during bladder filling. Thus there is increasing interest in ß-adrenoceptor agonists as a potential treatment for detrusor overactivity. The role of the urothelium in bladder responses to ß-adrenoceptor agonists is not yet clear, although we have shown that these agonists have a greater inhibitory effect on detrusor contraction when the urothelium is intact. The aim was to determine which ß-adrenoceptor subtype is involved in this effect. METHODS: Paired strips of pig bladder dome mucosa-intact and mucosa-denuded, were mounted in tissue baths. Relaxation responses were obtained to ß-adrenoceptor agonists (isoprenaline, dobutamine, salbutamol or BRL37344) in carbachol pre-contracted tissues. Inhibitory effects were studied by obtaining concentration-response curves (CRCs) to carbachol in the presence and absence of ß-adrenoceptor agonists. The inhibitory effects of isoprenaline were also studied following incubation with ß-adrenoceptor antagonists (propranolol, CGP20712, ICI-118, 551 or SR 59230A; non selected, ß(1), ß(2) and ß(3) selective respectively). RESULTS: isoprenaline, dobutamine, salbutamol and BRL37344 all relaxed carbachol pre-contracted tissues and responses were similar in intact and denuded strips. In inhibition experiments, ß-adrenoceptor agonists caused rightward shifts of carbachol CRCs. In intact strips the shift was greater with isoprenaline and BRL37344, but not with dobutamine or salbutamol. This increased shift was still observed in tissues pre-incubated with propranolo, CGP20712 and ICI-118, 551, but not with SR 59230A. CONCLUSIONS: ß(3)-adrenoceptors are involved in mediating inhibitory effects of ß-adrenoceptor agonists on detrusor contractions via the urothelium in pig bladder dome.

Neuronal Nitric Oxide Synthase Gene Transfer into the Rat Prostate Using In Vivo Electroporation.

OBJECTIVES: It has been reported that nitric oxide (NO) mainly contributes to prostate or urethral smooth muscles relaxation, and that nitrergic innervation and neuronal NO synthase (nNOS) levels are decreased in benign prostatic hyperplasia. The purpose of the present study was to evaluate the feasibility to gene therapy for benign prostatic hyperplasia by transferring nNOS gene into the rat prostate with in vivo electroporation (EP) procedure. METHODS: Male Sprague-Dawley rats were divided into four groups (sham, only EP, only nNOS injection, and nNOS gene injection with EP groups). Fifty micrograms of luciferase gene and nNOS expression vectors in 50 µL of K-PBS (potassium-phosphate buffered saline) were injected into the prostate. Immediately after the injection of these vectors, the vector injection points were electroporated by the two-square parallel electrodes. Two days after gene transfer, luciferase analysis and an immunohistochemical staining for nNOS were performed, and NO2 (-) /NO3 (-) (NOX ) release was measured using high-performance liquid chromatography coupled with the microdialysis procedure. RESULTS: The optimal electric pulse conditions were 50 V, 1 Hz and 10 msec. In vivo EP with these conditions showed the increase in the luciferase gene expression approximately 300-fold of the control group. In the nNOS gene injection with EP group, the marked nNOS immunoreactivity was observed, and NOX release was significantly higher, as compared to other groups. CONCLUSION: The results suggest that EP is a feasible technique for in vivo gene transfer into the rat prostate, and that the transferred nNOS gene functionally expresses and contributes to NO production.

Pharmacological effects of solifenacin on human isolated urinary bladder.

To investigate the effects of solifenacin on human detrusor smooth muscles, we evaluate the effects of solifenacin on the contractions induced by carbachol, KCl, CaCl2 and electrical field stimulation (EFS), and the EFS-induced acetylcholine release from detrusor smooth muscle strips by using the muscle bath and microdialysis technique. The effects of solifenacin were also compared with effects of other antimuscarinic agents (atropine, oxybutynin and propiverine). Pretreatment with various antimuscarinic agents caused parallel shifts to the right of the concentration-response curves to carbachol. The pA2 value of the Schild plots for solifenacin was similar to that for oxybutynin. Atropine did not inhibit the KCl- and CaCl2-induced contractions, while solifenacin, oxybutynin and propiverine significantly inhibited these contractions. EFS-induced contractions were inhibited by various antimuscarinic drugs in a concentration-dependent manner. In the presence of atropine, solifenacin tended to inhibit the residual atropine-resistant contractions induced by EFS, but it was not significant. However, oxybutynin and propiverine inhibited them under the same conditions. Although pretreatment with atropine and propiverine did not cause significant changes in EFS-induced acetylcholine release, solifenacin and oxybutynin caused significant decreases in acetylcholine release. The present results suggest that solifenacin inhibits contractions of human detrusor smooth muscles mainly by the antimuscarinic action and that the high concentration of solifenacin has Ca2+ channel antagonist action. Moreover, solifenacin may block not only postjunctional receptors, but also prejunctional receptors to modulate acetylcholine releases in cholinergic nerve endings in human detrusor smooth muscles. The findings support that muscarinic-receptor-inhibitory actions in human bladder mainly contribute to the usefulness of solifenacin as a therapeutic drug for overactive bladder.

Delivery of DNA into bladder via electroporation.

The possibility of in vivo gene transfer into the rat bladder by electroporation (EP) was evaluated. The bladder was exposed through an abdominal midline incision in 8-week-old male rats. Plasmid DNA of marker genes, green fluorescent protein (GFP) and luciferase, and the neuronal nitric oxide synthase (nNOS) gene were then injected into the subserosal space of the bladder and EP was applied. At 72 h after gene transfer, GFP and luciferase were assayed in the isolated bladder, and immunohistochemical staining was used to detect nNOS. NOx released from isolated bladder strips was also assessed using microdialysis procedure. From the luciferase assay, 45 V, 1 Hz, 50 ms, and 8 pulses were selected as the optimum conditions for EP. Bladder specimens with GFP genes injected by EP showed numerous bright sites of GFP expression in the smooth-muscle layer. In rats with the nNOS gene injected by EP, there was marked nNOS immunoreactivity, and NOx released from bladder strips was significantly greater than that in the control groups. These results suggest that EP is a useful technique for in vivo gene transfer into rat bladder smooth muscles, and that the nNOS gene transferred by this procedure functionally expresses and contributes to NO production.

Basic and clinical aspects of non-neuronal acetylcholine: expression of non-neuronal acetylcholine in urothelium and its clinical significance.

Recently, several reports demonstrate that non-neuronal acetylcholine (ACh) release may contribute to various pathophysiological conditions. In this review, we presented our experiments designed to evaluate the non-neuronal cholinergic system in human bladder. After insertion of the microdialysis probe, human bladder strips were suspended in an organ bath filled with Krebs-Henseleit solution, and Ringer solution was perfused into the probe. ACh release was measured by microdialysis and HPLC. The contribution of urothelium and the effects of age and stretch of bladder strips on non-neuronal ACh release were evaluated. Choline acetyltransferase (ChAT) immunohistochemical staining of bladder was also performed. Immunohistochemistry showed marked ChAT-positive staining in the urothelium. There was tetrodotoxin-insensitive non-neuronal ACh release and this was significantly higher in strips with urothelium than in strips without urothelium. The non-neuronal ACh release was increased with age. Stretch of bladder strips caused increases in non-neuronal ACh release. The stretch-induced release of non-neuronal ACh was increased with age. Our data demonstrate that there is a non-neuronal cholinergic system in human bladder and that urothelium contributes to non-neuronal ACh release. There was significant age-related and stretch-induced increase in non-neuronal ACh release. It is suggested that the non-neuronal cholinergic system may contribute to the physiology and pathophysiology of human bladder. We also discussed the clinical significance of the non-neuronal cholinergic system in human bladder.

Pharmacological effects of propiverine and its active metabolite, M-1, on isolated human urinary bladder smooth muscle, and on bladder contraction in rats.

OBJECTIVE: To investigate the effects of M-1, a major active metabolite of propiverine on the bladder. METHODS: We have evaluated the effects of M-1 on the contractions induced by carbachol, KCl, CaCl(2), and electrical field stimulation (EFS) in human detrusor smooth muscles, and pelvic nerve stimulation-induced bladder contractions in rats. The effects of M-1 were also compared with the effects of propiverine and tolterodine. RESULTS: Pretreatment with propiverine and tolterodine caused parallel shifts to the right of the concentration-response curves to carbachol. M-1 caused concentration-dependent reduction in the maximum contractile responses induced by carbachol. Although tolterodine did not inhibit the KCl- and CaCl(2)-induced contractions, M-1 and propiverine significantly inhibited these contractions. In the presence of atropine, M-1 and propiverine significantly inhibited the atropine resistant part of the contraction induced by EFS. On the other hand, tolterodine did not have significant inhibitory effects on atropine resistant contractions. Pelvic nerve stimulation induced bimodal phasic and tonic contractions in the rat bladder. M-1 mainly inhibited the phasic contraction. Tolterodine caused a significant inhibition in the tonic contraction, and propiverine had inhibitory effects on both contractions. CONCLUSIONS: The present results suggest that M-1 has inhibitory effects on the bladder smooth muscles through calcium antagonistic action. It is possible that the clinical effects of propiverine on the human bladder are based not only on the action of propiverine itself but also on one of its active metabolites, M-1.

Change in acetylcholine release from rat bladder with partial outlet obstruction.

OBJECTIVE: To investigate changes in acetylcholine release from the bladder of rats with partial bladder outlet obstruction (BOO), as partial BOO leads to hypertrophy and an alteration in the contractions of the detrusor smooth muscle, and acetylcholine plays an important role in urinary bladder contractions but there is little available information on acetylcholine release after BOO. MATERIAL AND METHODS: Partial BOO was induced in adult female rats by ligating the proximal urethra over a 1 mm angiocatheter; sham-operated rats served as controls. The rats were killed 2 weeks, 3 and 6 months after induction of BOO. We investigated the contractions induced by carbachol, KCl (80 mm), ATP and electrical-field stimulation (EFS, 2.5-40 Hz), and collected the dialysate obtained from a microdialysis probe inserted into the muscle strips during EFS, and measured the amount of acetylcholine in the dialysate fraction by high-performance liquid chromatography with electro-chemical detection. S-100 immunohistochemical staining of the bladder preparations was used for histological examination in BOO and control rats. RESULTS: The bladder weight gradually increased after BOO. There were no significant changes in KCl-induced contractions throughout the experimental period in either group. There were no significant changes in carbachol-induced contractions until 3 months after BOO but there was a significant reduction at 6 months. ATP-induced contractions were significantly increased 2 weeks and 3 months after BOO. EFS-induced contractions were gradually reduced after BOO. Acetylcholine release from the bladder strips was not significantly different between the groups until 2 weeks after BOO. However, acetylcholine release in BOO rats was significantly decreased 3-6 months after BOO, being significantly lower than that of the control rats. In the histological study, the number of nerve fibres in the BOO rats was significantly lower than in the control rats. CONCLUSIONS: We suggest that the prolonged BOO caused a decrease in EFS-induced acetylcholine release and the number of nerves in the rat urinary bladder, which might contribute to bladder underactivity in BOO.

[Bilateral pheochromocytomas with von Hippel-Lindau disease: a case report].

A case of bilateral pheochromocytomas with von Hippel Lindau disease (VHL) is reported. A 32-year-old man visited Kumamoto Red Cross Hospital for further examination of hypertension. Computed tomography revealed bilateral adrenal tumors and noradrenalin levels in serum and urine were elevated. Suspecting bilateral pheochromocytoma, he was reffered to our hospital for further examination and treatment. 131I-MIBG scintigraphy showed accumulation in bilateral adrenal glands. Moreover, he had cerebellar and spinal hemangioblastomas. Bilateral adrenalectomies and left nephrectomy were performed because tumor thrombus extended into the left renal vein, and pathological diagnosis was pheochromocytoma. His sister had been diagnosed as VHL disease. We diagnosed the patient as VHL disease because of the existence of cerebellar and spinal hemangioblastomas, bilateral pheochromocytomas, missense mutation and his family history. This is the eleventh case of bilateral pheochromocytomas with VHL disease reported in Japanese literatures.

Effect of tamsulosin hydrochloride on lower urinary tract symptoms and quality of life in patients with benign prostatic hyperplasia. Evaluation using bother score.

Benign prostatic hyperplasia (BPH) affects quality of life (QOL), and the goal of treatment is to improve lower urinary tract symptoms (LUTS), thus improving patient QOL. However, the international prostate symptom score (IPSS) used for evaluating LUTS does not always reflect the level of patient bother, and improvement in the IPSS score does not always reflect patient QOL. Therefore, in the present study, we observed the therapeutic effects of alpha(1)-blockers on IPSS, QOL index, and the bother score for individual symptoms. Ninety-three men diagnosed with BPH who had not yet been treated were enrolled (mean age 70 years). The IPSS, QOL index, and bother score for each symptom of IPSS (maximum 42 points, 7 grades, from 0 to 6: not at all bothersome, not bothersome, not much bother, neutral, a little bothersome, somewhat bothersome, very bothersome) were assessed in order to examine the correlation between LUTS and QOL. After treatment with tamsulosin hydrochloride 0.2 mg/day for 4 weeks, the change in each IPSS and bother score and the correlation was reassessed. Furthermore, the contribution of improvements in each symptom score and bother score to improvement in QOL index was examined using a path analysis model. On the IPSS at initial evaluation, the score was highest for slow stream. The bother scores were high for slow stream, nocturia, and daytime frequency. For slow stream, patients with a high IPSS score also had a high bother score, but for nocturia, there was a large discrepancy between the IPSS and bother score. After treatment, total IPSS, QOL and total bother scores were significantly improved (p <0.01). Improvements in all individual symptom scores and bother scores were also observed (p <0.01). The most predictable symptom for improvement in QOL after treatment was the improvement in the bother score for nocturia (F test; p <0.01). Treatment with tamsulosin hydrochloride showed significant improvement of each IPSS and the bother score. For nocturia, there was a large discrepancy between the IPSS and bother score. After treatment, the improvement in bother score for nocturia showed the strongest contribution to improvement in QOL. The present study suggests that in addition to the IPSS, the evaluation of bother score for each symptom may be necessary in the management for patients with LUTS suggestive of BPH.

Prostaglandin E2 release from isolated bladder strips in rats with spinal cord injury.

AIM: Recent studies have shown that various factors contribute to the increased excitability into the bladder afferent nerves in spinal cord injury (SCI) rats. It has been reported that prostaglandins (PG) act as local modulators of reflex micturition in pathological conditions. In the present study, we measured the amount of PGE2 release from the bladder of chronic SCI rats. METHODS: Spinal cord was transected at the level of T8-9 in adult female Sprague-Dawley rats. After 10 weeks, specimens of the urinary bladder were obtained from SCI rats and sham-injured control rats, and bladder strips were dissected from the bladder. Using an muscle-bath technique and a microdialysis procedure, the dialysate, containing substance released from bladder strips, was collected. Then the amount of PGE2 in the dialysate was measured by radioimmunoassay. RESULTS: Excretion of urinary PGE2 was significantly higher in SCI rats than in control rats. PGE2 release from bladder strips was significantly higher in SCI rats than in control rats. Removal of urothelium caused significant decreases in PGE2 release in both control and SCI rats. Stretches of the bladder strips caused significant resting tension-dependent increases in PGE2 release from the strips with urothelium. CONCLUSIONS: The present data suggest that bladder urothelium partly contributes to the increase in PGE2 release from the bladder, and that bladder distension may cause increases in PGE2 release in SCI rats.

Non-neuronal cholinergic system in human bladder urothelium.

OBJECTIVES: To evaluate the non-neuronal cholinergic system in the isolated human bladder. Recently, several reports have demonstrated that non-nerve-evoked acetylcholine (ACh) release may contribute to various pathophysiologic conditions. METHODS: Human bladders were obtained from 15 patients. Bladder strips with or without urothelium were suspended in an organ bath filled with Krebs-Henseleit solution. ACh release was measured using microdialysis and high-performance liquid chromatography. The contribution of urothelium to non-nerve-evoked ACh release was tested, and the effects of age and elevation of the resting tension (0 to 40 mN) on ACh release were also evaluated. Furthermore, choline acetyltransferase (ChAT) immunohistochemical staining of bladder was performed. RESULTS: Immunohistochemistry showed marked ChAT-positive staining in the urothelium and suburothelial region. Tetrodotoxin-insensitive non-nerve-evoked ACh release occurred and was significantly greater in strips with than without urothelium. The non-nerve-evoked ACh release from strips with urothelium increased with age, and the positive correlation between age and release was significant. Stretching of the strips caused increases in non-nerve-evoked ACh release that was significantly greater in strips with than without urothelium. The stretch-induced release of non-nerve-evoked ACh increased with age. CONCLUSIONS: These data have demonstrated that a non-neuronal cholinergic system is present in the human bladder and that the urothelium/suburothelium partially releases ACh. We found a significant age-related and stretch-induced increase in non-nerve-evoked ACh release. This suggests that the non-neuronal cholinergic system may contribute to the physiology and pathophysiology of human bladder function.

Acetylcholine release from urinary bladder smooth muscles of non-insulin-dependent diabetic rats.

INTRODUCTION: To investigate the mechanism of voiding dysfunction in non-insulin-dependent diabetes mellitus, we attempted to measure the acetylcholine (ACh) release using an in vivo microdialysis technique and measuring the detrusor pressure after electrical field stimulation (EFS) of the pelvic nerve. MATERIALS AND METHODS: Eight- and 32-week-old female Goto-Kakizaki (GK) rats (non-insulin-dependent diabetes mellitus model) and age-matched female Wistar rats (controls) were used in this study. The pelvic nerve was exposed on a bipolar platinum electrode to EFS, and a cannula was inserted into the bladder to measure the detrusor pressure. The microdialysis probe was inserted into the bladder wall and was connected to a microinfusion syringe pump. Dialysate was constantly perfused, collected in a microtube, and then injected into the ACh assay system. Histological examinations were performed by staining with hematoxylin and eosin and S-100 immunohistochemical staining in bladder preparations of both GK and control rats. RESULTS: In 8-week-old rats, both detrusor pressures and amounts of ACh release of GK rats were not significantly different from those of control rats. In 32-week-old rats, both detrusor pressures and ACh releases were only significantly increased at 5 and 10 Hz of EFS. In the histological study, the number of nerve fibers or bundles of 32-week-old GK rats was significantly decreased as compared with control rats. CONCLUSION: The present data suggest that the decrease in EFS-induced ACh release in GK rats, which may be caused by the decreased number of nerve fibers, may contribute to the decrease in bladder contractions.