Morphological and biochemical investigation of nitric oxide synthase and related enzymes in the rat and pig urothelium.

We investigated the enzymes involved in the NADPH-diaphorase (d) reaction in the rat and pig bladder urothelium. The urothelial cell layer displayed intense and uniform NADPH-d activity. Preincubation with the flavoprotein inhibitor diphenyleneiodionium chloride (DPI) and the alkaline phosphatase inhibitor levamisole concentration-dependently decreased the urothelial NADPH-d activity. Immunoreactivities to neuronal (n), endothelial (e), or inducible (i) nitric oxide synthase (NOS) were not detected in rat or pig urothelial cells. In rats, the urothelium was uniformly immunoreactive for NADPH cytochrome P450 reductase, whereas the pig urothelium displayed inconsistent labeling. In lipopolysaccharide (LPS)-treated rats, the bladder urothelium showed positive iNOS immunoreactivity. The iNOS labeling was found predominantly in cells located in the basal layer of the urothelium. In the pig bladder mucosa, a Ca2+-dependent NOS activity was evident in cytosolic and particulate fractions that was quantitatively comparable to the NOS activity found in the smooth muscle. In ultrastructural studies of urothelial cells, NADPH-d reaction products were found predominantly on membranes of the nuclear envelope, endoplasmatic reticulum and mitochondria. In conclusion, NADPH-d staining of the urothelium cannot be taken as an indicator for the presence of constitutively expressed NOS. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. However, LPS treatment of rats caused expression of iNOS in urothelial cells.     

Immunohistochemical study of neuroendocrine differentiation in primary glandular lesions and tumors of the urinary bladder

OBJECTIVE: Neuroendocrine (NE) cells are uncommon in primary adenocarcinoma (AC) and other glandular lesions of the bladder, with no recent study series concerning its significance in differential diagnosis, prognosis or biologic significance. STUDY DESIGN: Sixteen primary bladder AC (enteric-type [n = 71, mucinous [n = 6] and not otherwise specified [NOS] [n = 31), 4 cases of urothelial carcinoma with glandular differentiation, 20 cases of glandular cystitis and 3 urachal remnants with intestinal metaplasia constituted the study series. In addition, 20 specimens of normal-looking urothelium, 15 conventional urothelial carcinomas and 5 small cell carcinoma (SCC) cases were included for comparison. NE differentiation included detection of chromogranin A, neuron-specific enolase (NSE) and synaptophysin by immunohistochemistry. The statistical analysis included the chi2 or Fisher exact test. RESULTS: Chromogranin A-positive cells were present in 60% (11 of 16) of primary AC, all of enteric or mucinous type, but not in any of the 3 NOS-type AC investigated. NE differentiation in bladder AC subtypes resulted in highly significant differences between enteric or mucinous vs. NOS type (p = 0.0023). NE differentiation was also different in urachal vs. nonurachal AC (p = 0.020) and primary bladder AC vs. conventional invasive urothelial carcinoma (p < 0.001). Synaptophysin-positive cells were seen in 2 (12.5%) of the 16 primary AC cases, and NSE was negative in the 16 primary bladder AC. All urachal remnants and 70% of glandular cystitis examples had chromogranin A-immunoreactive cells. One of 4 urothelial carcinomas with glandular differentiation had chromogranin A-immunoreactive cells, but this was not significant when compared with primary AC (p = 0.1). Normal-looking bladder urothelium and conventional urothelial carcinoma specimens had no chromogranin A-immunoreactive cells. The 5 SCC cases investigated were positive for chromogranin A. No correlation was found between NE differentiation and outcome of primary bladder AC or urothelial carcinoma with glandular differentiation. CONCLUSION: Primary bladder AC, cystitis glandularis and urachal remnants with intestinal metaplasia showed variable degrees of NE differentiation, with no apparent clinical correlation or prognostic significance. However, the absence of NE differentiation in NOS-type primary bladder AC may help in better defining this uncommon subtype of primary bladder AC.     

Oxidative Status and Lipofuscin Accumulation in Urothelial Cells of Bladder in Aging Mice

Age-related changes in various tissues have been associated with the onset of a number of age-related diseases, including inflammation and cancer. Bladder cancer, for instance, is a disease that mainly afflicts middle-aged or elderly people and is mostly of urothelial origin. Although research on age-related changes of long-lived post-mitotic cells such as neurons is rapidly progressing, nothing is known about age-related changes in the urothelium of the urinary bladder, despite all the evidence confirming the important role of oxidative stress in urinary bladder pathology. The purpose of this study was thus to investigate the oxidative status and age-related changes in urothelial cells of the urinary bladder of young (2 months) and aging (20 months) mice by means of various methods. Our results demonstrated that healthy young urothelium possesses a powerful antioxidant defence system that functions as a strong defence barrier against reactive species. In contrast, urothelial cells of aging bladder show significantly decreased total antioxidant capacity and significantly increased levels of lipid peroxides (MDA) and iNOS, markers of oxidative stress. Our study demonstrates for the first time that ultrastructural alterations in mitochondria and accumulation of lipofuscin, known to be one of the aging pigments, can clearly be found in superficial urothelial cells of the urinary bladder in aging mice. Since the presence of lipofuscin in the urothelium has not yet been reported, we applied various methods to confirm our finding. Our results reveal changes in the oxidative status and structural alterations to superficial urothelial cells similar to those of other long-lived post-mitotic cells.     

Regulation of IGF-1 but not TGF-β1 by NGF in the smooth muscle of the inflamed urinary bladder

Intraperitoneal injection of cyclophosphamide (CYP) causes hemorrhagic cystitis with excess growth of muscular layer leading to bladder hypertrophy; this could be attributable to changes in the expression profiles of growth factors in the inflamed urinary bladder. The growth factors characterized in the current study include nerve growth factor (NGF), insulin-like growth factor (IGF)-1, and transforming growth factor (TGF)-β1. We found that following CYP injection for 8 h and 48 h, the mRNA levels of all three factors were increased in the inflamed bladder when compared to control. The level of NGF mRNA was mainly increased in the urothelium layer while the levels of IGF-1 mRNA and TGF-β1 mRNA were increased in the smooth muscle layer. The level of NGF high affinity receptor TrkA mRNA was also increased in both the urothelium and the smooth muscle layers during bladder inflammation. When we blocked NGF action with NGF neutralizing antibody in vivo, we found that the up-regulation of IGF-1 in the inflamed bladder was reversed while the up-regulation of TGF-β1 was not affected by NGF neutralization. The effect of NGF on regulating IGF-1 expression was further confirmed in bladder smooth muscle culture showing that exogenous NGF increased the mRNA level of IGF-1 after 30 min to 1 h stimulation. These results suggested that bladder inflammation induced region-specific changes in the expression profiles of NGF, IGF-1 and TGF-β1. The up-regulation of NGF in the urothelium may have a role in affecting bladder smooth muscle cell physiology by regulating IGF-1 expression.     

Macrophage Migration Inhibitory Factor Mediates PAR-Induced Bladder Pain

IntroductionMacrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is constitutively expressed in urothelial cells that also express protease-activated receptors (PAR). Urothelial PAR1 receptors were shown to mediate bladder inflammation. We showed that PAR1 and PAR4 activator, thrombin, also mediates urothelial MIF release. We hypothesized that stimulation of urothelial PAR1 or PAR4 receptors elicits release of urothelial MIF that acts on MIF receptors in the urothelium to mediate bladder inflammation and pain. Thus, we examined the effect of activation of specific bladder PAR receptors on MIF release, bladder pain, micturition and histological changes.MethodsMIF release was measured in vitro after exposing immortalized human urothelial cells (UROtsa) to PAR1 or PAR4 activating peptides (AP). Female C57BL/6 mice received intravesical PAR1- or PAR4-AP for one hour to determine: 1) bladder MIF release in vivo within one hour; 2) abdominal hypersensitivity (allodynia) to von Frey filament stimulation 24 hours after treatment; 3) micturition parameters 24 hours after treatment; 4) histological changes in the bladder as a result of treatment; 5) changes in expression of bladder MIF and MIF receptors using real-time RT-PCR; 6) changes in urothelial MIF and MIF receptor, CXCR4, protein levels using quantitative immunofluorescence; 7) effect of MIF or CXCR4 antagonism.ResultsPAR1- or PAR4-AP triggered MIF release from both human urothelial cells in vitro and mouse urothelium in vivo. Twenty-four hours after intravesical PAR1- or PAR4-AP, we observed abdominal hypersensitivity in mice without changes in micturition or bladder histology. PAR4-AP was more effective and also increased expression of bladder MIF and urothelium MIF receptor, CXCR4. Bladder CXCR4 localized to the urothelium. Antagonizing MIF with ISO-1 eliminated PAR4- and reduced PAR1-induced hypersensitivity, while antagonizing CXCR4 with AMD3100 only partially prevented PAR4-induced hypersensitivity.ConclusionsBladder PAR activation elicits urothelial MIF release and urothelial MIF receptor signaling at least partly through CXCR4 to result in abdominal hypersensitivity without overt bladder inflammation. PAR-induced bladder pain may represent an interesting pre-clinical model of Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS) where pain occurs without apparent bladder injury or pathology. MIF is potentially a novel therapeutic target for bladder pain in IC/PBS patients.     

Temporal diabetes- and diuresis-induced remodeling of the urinary bladder in the rat

The natural history of diabetes mellitus-induced remodeling of the urinary bladder is poorly understood. In this study, we examined temporal remodeling of the bladder in diabetic and diuretic rats. Male Sprague-Dawley rats were divided into three groups: streptozotocin-induced diabetic, 5% sucrose-induced diuretic, and age-matched control. Micturition and morphometric characteristics were evaluated using metabolic cages and light-microscopic examination of the bladder 4 days and 1, 2, 3, and 9 wk after induction. Digital image analysis was used to quantify equatorial cross-sectional areas of bladder tissue and lumen, as well as relative content of the three primary tissue components: smooth muscle, urothelium, and collagen. Diabetes and diuresis caused significant increases in fluid intake, urine output, and bladder weight. In both groups, progressive increases were observed in lumen area from 4 days to 3 wk after induction and in wall area from 2 to 3 wk after induction. Wall thickness decreased within the first 2 wk in the diabetic and diuretic rats but returned to control at 3 and 9 wk. As a percentage of total cross-sectional area, smooth muscle area increased, urothelium area was unchanged, and collagen area decreased in diabetic and diuretic rats after 2-3 wk compared with control rats. In conclusion, diabetes and diuresis induced similar bladder remodeling. Diabetes-induced diuresis caused adaptive physical changes in rat bladder by 4 days after induction; remodeling was observed by 2-3 wk after induction and remained stable from 3 to 9 wk.     

Characterization of the Early Proliferative Response of the Rodent Bladder to Subtotal Cystectomy: A Unique Model of Mammalian Organ Regeneration

Subtotal cystectomy (STC; surgical removal of ∼75% of the rat urinary bladder) elicits a robust proliferative response resulting in complete structural and functional bladder regeneration within 8-weeks. The goal of these studies was to characterize the early cellular response that mediates this regenerative phenomenon, which is unique among mammalian organ systems. STC was performed on eighteen 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7-days post-STC, the bladder was harvested 2-hours after intraperitoneal injection of bromodeoxyuridine (BrdU). Fluorescent BrdU labeling was quantified in cells within the urothelium, lamina propria (LP), muscularis propria (MP) and serosa. Cell location was confirmed with fluorescently co-labeled cytokeratin, vimentin or smooth muscle actin (SMA), to identify urothelial, interstitial and smooth muscle cells, respectively. Expression of sonic hedgehog (Shh), Gli-1 and bone morphogenic factor-4 (BMP-4) were evaluated with immunochemistry. Three non-operated rats injected with BrdU served as controls. Less than 1% of cells in the bladder wall were labeled with BrdU in control bladders, but this percentage significantly increased by 5-8-fold at all time points post-STC. The spatiotemporal characteristics of the proliferative response were defined by a significantly higher percentage of BrdU-labeled cells within the urothelium at 1-day than in the MP and LP. A time-dependent shift at 3 and 5-days post-STC revealed significantly fewer BrdU-labeled cells in the MP than LP or urothelium. By 7-days the percentage of BrdU-labeled cells was similar among urothelium, LP and MP. STC also caused an increase in immunostaining for Shh, Gli-1 and BMP-4. In summary, the early stages of functional bladder regeneration are characterized by time-dependent changes in the location of the proliferating cell population, and expression of several evolutionarily conserved developmental signaling proteins. This report extends previous observations and further establishes the rodent bladder as an excellent model for studying novel aspects of mammalian organ regeneration.     

Developmental changes in actin and myosin heavy chain isoform expression in smooth muscle

Smooth muscle cells express isoforms of actin and myosin heavy chains (MHC). In early postnatal animals the nonmuscle (NM) actin and MHC isoforms in vascular (aorta) smooth muscle were present in relatively high percentages. More than 30% of the MHC and 40% of the actin isoforms were NM. The relative percentage of the NM isoforms decreased significantly as the animals reached maturity, with NM MHC less than 10% and NM actin less than 30% of the totals. Concurrent with this decrease in NM isoforms was an increase in the smooth muscle (SM) isoforms. The relative changes and time frame in which these changes occurred were very similar for the actin and MHC isoforms. In arterial tissue there were species differences for changes with development in the two SM MHC isoforms (SM1 and SM2). The ratio of SM1:SM2 in young rat aorta was approximately 0.5, while this same ratio was approximately 3 in young swine carotid. Both adult rats and swine had a SM1:SM2 MHC ratio of approximately 1.2. Rat bladder smooth muscle showed no significant change in NM vs SM ratio between young and old rats, while the SM1:SM2 ratio decreased from 2.7 to 1.7 between these age groups. The shifts in alpha and beta actin were similar to those in the vascular tissue, but of much smaller magnitude.     

Role for pituitary adenylate cyclase activating polypeptide in cystitis-induced plasticity of micturition reflexes

Pituitary adenylate cyclase activating polypeptide (PACAP) peptides are expressed and regulated in sensory afferents of the micturition pathway. Although these studies have implicated PACAP in bladder control, the physiological significance of these observations has not been firmly established. To clarify these issues, the roles of PACAP and PACAP signaling in micturition and cystitis were examined in receptor characterization and physiological assays. PACAP receptors were identified in various tissues of the micturition pathway, including bladder detrusor smooth muscle and urothelium. Bladder smooth muscle expressed heterogeneously PAC(1)null, PAC(1)HOP1, and VPAC(2) receptors; the urothelium was more restricted in expressing preferentially the PAC(1) receptor subtype only. Immunocytochemical studies for PAC(1) receptors were consistent with these tissue distributions. Furthermore, the addition of 50-100 nM PACAP27 or PACAP38 to isolated bladder strips elicited transient contractions and sustained increases in the amplitude of spontaneous phasic contractions. Treatment of the bladder strips with tetrodotoxin (1 muM) did not alter the spontaneous phasic contractions suggesting direct PACAP effects on bladder smooth muscle. PACAP also increased the amplitude of nerve-evoked contractions. By contrast, vasoactive intestinal polypeptide had no direct effects on bladder smooth muscle. In a rat cyclophosphamide (CYP)-induced cystitis paradigm, intrathecal or intravesical administration of PAC(1) receptor antagonist, PACAP6-38, reduced cystitis-induced bladder overactivity. In summary, these studies support roles for PACAP in micturition and suggest that inflammation-induced plasticity in PACAP expression in peripheral and central micturition pathways contribute to bladder dysfunction with cystitis.     

Enhanced ATP release from rat bladder urothelium during chronic bladder inflammation: effect of botulinum toxin A

The effects of mechanoreceptor stimulation and subsequent ATP release in cyclophosphamide evoked chronic bladder inflammation was examined to demonstrate: (1) whether inflammation modulates ATP release from bladder urothelium and (2) whether intravesical botulinum toxin A administration inhibits urothelial ATP release, a measure of sensory nerve activation. ATP release was measured from rat bladders in a Ussing chamber, an apparatus that allows one to separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Cystometry was utilized to correlate changes in ATP release with alterations in the frequency of voiding and non-voiding bladder contractions, in vivo measures of bladder afferent activity. The resting urothelial release of ATP was not significantly affected by either cyclophosphamide or botulinum toxin A treatment. However, evoked ATP release following hypoosmotic stimulation was significantly increased (i.e. 94%) in chronic cyclophosphamide treated bladder urothelium compared to control bladders. In addition, botulinum toxin A treatment significantly reduced hypoosmotic shock induced ATP release in cyclophosphamide treated animals by 69%. Cystometry revealed that cyclophosphamide and botulinum toxin A treatments altered non-voiding (i.e. cyclophosphamide increased, botulinum toxin A decreased) but not voiding contraction frequency suggesting that alterations in urothelial ATP release selectively diminished underlying bladder C-fiber nerve activity. Finally, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side implying that its effects were confined to the urothelial side of the bladder preparation.     

Induction of oxidative stress causes functional alterations in mouse urothelium via a TRPM8‐mediated mechanism: implications for aging

The incidence of bladder conditions such as overactive bladder syndrome and its associated urinary incontinence is highly prevalent in the elderly. However, the mechanisms underlying these disorders are unclear. Studies suggest that the urothelium forms a ‘sensory network’ with the underlying innervation, alterations in which, could compromise bladder function. As the accumulation of reactive oxygen species can cause functional alterations with age, the aim of this study was to investigate whether oxidative stress alters urothelial sensory signalling and whether the mechanism underlying the effect of oxidative stress on the urothelium plays a role in aging. Five‐month‐old(young) and 24‐month‐old (aged) mice were used. H₂O₂, used to induce oxidative stress, resulted in an increase in bladder afferent nerve activity and urothelial intracellular calcium in preparations from young mice. These functional changes were concurrent with upregulation of TRPM8 in the urothelium. Moreover, application of a TRPM8 antagonist significantly attenuated the H₂O₂‐induced calcium responses. Interestingly, an upregulation of TRPM8 was also found in the urothelium from aged mice, where high oxidative stress levels were observed, together with a greater calcium response to the TRPM8 agonist WS12. Furthermore, these calcium responses were attenuated by pretreatment with the antioxidant N‐acetyl‐cysteine. This study shows that oxidative stress affects urothelial function involving a TRPM8‐mediated mechanism and these effects may have important implications for aging. These data provide an insight into the possible mechanisms by which oxidative stress causes physiological alterations in the bladder, which may also occur in other organs susceptible to aging.     

Expression of nitric oxide synthase isoforms (NOS II and NOS III) in adult rat lung in hyperoxic pulmonary hypertension

Breathing air with a high oxygen tension induces an inflammatory response and injures the microvessels of the lung. The resulting development of smooth muscle cells in these segments contributes to changes in vasoreactivity and increased pulmonary artery pressure. This in vivo study determines the temporal and spatial expression of endogenous endothelial nitric oxide synthase (NOS III) and inducible NOS (NOS II), important enzymes regulating vasoreactivity and inflammation, in the adult rat lung during the development of experimental pulmonary hypertension induced by oxidant injury. We analyzed the cellular distribution of these NOS isoforms, using specific antibodies, and assessed enzyme activity at baseline and after 1-28 days of hyperoxia (FIO₂ 0.87). The number of NOS III-immuno-positive endothelial cells increased early in hyperoxia and then remained high. By day 28, the relative number of these cells had increased from 40% in proximal vessels and 13-16% in distal alveolar vessels of the normal lung to 73-86% and 40-59%, respectively, in hyperoxia. Pulmonary alveolar macrophages (PAMs), normally few in number and only weakly immunopositive for NOS II or III in the normal lung, increased in number in hyperoxia and were strongly immunopositive for each isoform. These morphological data were supported by a temporal increase in total and calcium-independent NOS activity. Thus NOS expression and activity significantly increased in hyperoxia as pulmonary hypertension developed, and NOS III expression increased selectively in vascular endothelial cells, while both NOS isoforms were expressed by the PAM population. We conclude that this increase in expression of a potent vasodilator, an antiproliferative agent for smooth muscle cells, and an antioxidant molecule represents an adaptive response to protect the lung from oxidant-induced vascular and epithelial injury.     

P2X2 and P2X3 receptor expression in postnatal and adult rat urinary bladder and lumbosacral spinal cord

P2X receptors mediate the effects of ATP in micturition and nociception. During postnatal maturation, a spinobulbospinal reflex and voluntary voiding replace primitive voiding reflexes. This may involve changes in neuroactive compounds and receptors in bladder reflex pathways. We examined P2X2 and P2X3 receptors in bladder and spinal cord from postnatal (P0-P36, indicating number of days) and adult Wistar rats. Western blot of whole bladders for P2X2 and P2X3 expression was performed. Immunostaining for P2X2 and P2X3 receptors in urothelium and detrusor smooth muscle whole mounts and spinal cord sections was examined. Western blot demonstrated an age-dependent decrease (R(2) = 0.96, P 相似文献   

The effect of intravesical Bacillus Calmette-Guerin instillations on the expression of inducible nitric oxide synthase in humans.

The activation of the inducible isoform of nitric oxide synthase (NOS) is associated with the production of large quantities of nitric oxide in response to cytokine stimulation. Bacillus Calmette-Guerin (BCG) mode of action against bladder carcinoma remains unclear, although a plethora of local and systemic events may follow its intravesical instillation. The present study was designed to investigate the expression of inducible NOS in normal and neoplastic urothelium and its alteration following tumor resection and subsequent intravesical immunotherapy. Bladder carcinoma and autologous normal bladder tissue specimens were procured from 36 patients undergoing transurethral resection. Tissue specimens were obtained from the same patients at first cystoscopy following six weekly intravesical instillations. Inducible NOS protein expression was assessed by immunohistochemistry in all tissue specimens. Immunostaining of normal urothelium for iNOS before treatment was negative in all but four cases. BCG treatment induced iNOS expression in tumor-free bladder tissue in 24 cases (66.6%). There were only four early tumor recurrences; interestingly, they corresponded to the cases with tumor cells expressing iNOS before BCG treatment, while novel tumors were also iNOS immunoreactive. BCG upregulated iNOS expression in normal human urothelial cells in vivo suggesting a role for nitric oxide in BCG mediated antitumor activity. Inducible NOS was detected in certain tumor specimens before and after BCG treatment implying a possible involvement in pro-tumor action.     

Pathophysiology of overactive bladder and urge urinary incontinence

Storage symptoms such as urgency, frequency, and nocturia, with or without urge incontinence, are characterized as overactive bladder (OAB). OAB can lead to urge incontinence. Disturbances in nerves, smooth muscle, and urothelium can cause this condition. In some respects the division between peripheral and central causes of OAB is artificial, but it remains a useful paradigm for appreciating the interactions between different tissues. Models have been developed to mimic the OAB associated with bladder instability, lower urinary tract obstruction, neuropathic disorders, diabetes, and interstitial cystitis. These models share the common features of increased connectivity and excitability of both detrusor smooth muscle and nerves. Increased excitability and connectivity of nerves involved in micturition rely on growth factors that orchestrate neural plasticity. Neurotransmitters, prostaglandins, and growth factors, such as nerve growth factor, provide mechanisms for bidirectional communication between muscle or urothelium and nerve, leading to OAB with or without urge incontinence.     

Overactive and underactive bladder dysfunction is reflected by alterations in urothelial ATP and NO release

ATP and NO are released from the urothelium in the bladder. Detrusor overactivity (DO) following spinal cord injury results in higher ATP and lower NO release from the bladder urothelium. Our aim was to study the relationship between ATP and NO release in (1) early diabetic bladders, an overactive bladder model; and (2) "diuretic" bladders, an underactive bladder model. To induce diabetes mellitus female rats received 65mg/kg streptozocin (i.v.). To induce chronic diuresis rats were fed with 5% sucrose. At 28 days, in vivo open cystometry was performed. Bladder wash was collected to analyze the amount of ATP and NO released into the bladder lumen. For in vitro analysis of ATP and NO release, a Ussing chamber was utilized and hypoosmotic Krebs was perfused on the urothelial side of the chamber. ATP was analyzed with luminometry or HPLC-fluorometry while NO was measured with a Sievers NO-analyzer. In vivo ATP release was increased in diabetic bladders and unchanged in diuretic bladders. In vitro release from the urothelium followed the same pattern. NO release was unchanged both in vitro and in vivo in overactive bladders whereas it was enhanced in underactive bladders. We found that the ratio of ATP/NO, representing sensory transmission in the bladder, was high in overactive and low in underactive bladder dysfunction. In summary, ATP release has a positive correlation while NO release has a negative correlation with the bladder contraction frequency. The urinary ATP/NO ratio may be a clinically relevant biomarker to characterize the extent of bladder dysfunction.