Is testosterone important in LUT function in men and women? ICI-RS 2015.

AIM: This review article is a collaborative report based upon the Authors' presentations and Group discussion on the role of testosterone (T) in the male and female lower urinary tract (LUT) which took place at the 6th International Consultation on Incontinence Research Society's (ICI-RS) annual meeting, in Bristol, UK (September 8-10, 2015). METHODS: It comprises overviews and opinions on both the current state of knowledge of the role of T in LUT function and dysfunction in both sexes. RESULTS: Results from animal studies suggest that T treatment may be beneficial for disorders of the LUT in women including urinary incontinence and pelvic organ prolapse. The need for clinical studies to evaluate the effect of T treatment in peri- and post-menopausal women, taking into account the type of applied androgen, the application form, timing and dosage, is especially emphasized. In males, findings on the impact of T on the male external urethral sphincter underscores that there is still much to learn about its role in male LUT physiology. The important topic of the use of T therapy in the treatment of enuresis in the young, both sexes, is also discussed. The importance of understanding the steroidogenic pathways linking T with estradiol is discussed as being of paramount importance in researching the unique actions of T in the LUT. CONCLUSION: The overall conclusion is that further research into the role of T in LUT function and dysfunction across genders and age groups (young to old) is extremely important. Neurourol. Urodynam. 36:859-862, 2017. © 2017 Wiley Periodicals, Inc.

Psychological and Physical Environmental Factors in the Development of Incontinence in Adults and Children: A Comprehensive Review.

The aim of this review was to identify etiological environmental factors related to incontinence in children and adults. A variety of etiological environmental factors for the development of incontinence were identified. In children, these encompass stressful life events and trauma, family dysfunction, parental psychopathology, school-related stressors, toilet or "potty" training, fluid consumption habits, housing conditions, and the availability of toilets. In adults, physical exercise, obesity, working conditions, fluid intake, and the availability of toilets play a role. Intervening variables such as hormonal variations due to work shifts have also been identified as influencing the likelihood of incontinence. Current research suggests that environmental factors influence the development of incontinence in children and adults. The interactions between biological factors, the immediate environment, and intervening variables need to be explored in greater detail. Practical solutions to reduce barriers to adequate fluid intake and healthy toileting habits should be implemented in school and work settings.

Path of translational discovery of urological complications of obesity and diabetes.

Diabetes mellitus (DM) is a prevalent chronic disease. Type 1 DM (T1DM) is a metabolic disorder that is characterized by hyperglycemia in the context of absolute lack of insulin, whereas type 2 DM (T2DM) is due to insulin resistance-related relative insulin deficiency. In comparison with T1DM, T2DM is more complex. The natural history of T2DM in most patients typically involves a course of obesity to impaired glucose tolerance, to insulin resistance, to hyperinsulinemia, to hyperglycemia, and finally to insulin deficiency. Obesity is a risk factor of T2DM. Diabetes causes some serious microvascular and macrovascular complications, such as retinopathy, nephropathy, neuropathy, angiopathy and stroke. Urological complications of obesity and diabetes (UCOD) affect quality of life, but are not well investigated. The urological complications in T1DM and T2DM are different. In addition, obesity itself affects the lower urinary tract. The aim of this perspective is to review the available data, combined with the experience of our research teams, who have spent a good part of last decade on studies of association between DM and lower urinary tract symptoms (LUTS) with the aim of bringing more focus to the future scientific exploration of UCOD. We focus on the most commonly seen urological complications, urinary incontinence, bladder dysfunction, and LUTS, in obesity and diabetes. Knowledge of these associations will lead to a better understanding of the pathophysiology underlying UCOD and hopefully assist urologists in the clinical management of obese or diabetic patients with LUTS.

Do we need to know more about the effects of hormones on lower urinary tract dysfunction? ICI-RS 2014.

This review article reflects the presentations and subsequent discussions during a think tank at the 5th International Consultation on Incontinence Research Society's annual meeting, held in Bristol, UK (September 22-24, 2014). It reviews the current state of knowledge on the role of hormones in lower urinary tract dysfunction (LUTD) and overactive bladder (OAB) and in particular: highlights some specific basic research findings from discussion participants; reviews future research topics; and discusses potential new therapeutic opportunities for LUTD and OAB. The role of the large conductance voltage- and Ca(2+) -activated K(+) (BK) channels, as novel therapeutic targets for OAB was discussed, in particular as recent studies on human detrusor smooth muscle suggest that estradiol exerts a direct non-genomic activation of the BK channels. Recent developments on the roles of sex hormones on diuresis, as well as the roles of melatonin and vitamin D on LUTD were also discussed. It was concluded that further basic science and translational studies are needed to better understand hormonal regulatory mechanisms of the lower urinary tract and the implications for novel treatment options for LUTD and OAB.

Effect of Polyuria on Bladder Function in Diabetics versus Non-Diabetics: An Article Review.

OBJECTIVES: To review studies that investigated the role of polyuria on bladder function. METHODS: We performed a search of the English literature through PubMed. We only included animal studies that assessed parameters of bladder function and had compared diabetic and non-diabetic polyuric animals. RESULTS: Fluid intake and urine output were increased in diabetic and polyuric animals; failure to appropriately gain weight was seen in diabetics only. All but 1 study reported increase in bladder weight in both groups. In cystometrograms, control mice showed reductions in basal bladder pressure over time, whereas diabetic and diuretic groups stabilized or increased. Both groups showed larger bladder capacity. Overall, many characteristic changes in cystometrographic studies in diabetic animals could be attributed to polyuria. In histological studies, bladder hypertrophy, increase in smooth muscle and urothelium, and increase in protein and DNA content per bladder were observed in diuretic and diabetic rats. Actual collagen cross-sectional area did not change during the progression of diabetes or diuresis but decreased over time in both groups as a percentage of total tissue area. Both groups expressed less type I collagen mRNA and TGF-beta-1 mRNA. CONCLUSIONS: Although lower urinary tract changes in anatomy and function in diabetic patients might arise from a number of causes, polyuria seems to play an important causative role.

Melatonin and Its Role in Lower Urinary Tract Function: An Article Review.

This article reviewed the results of studies done on animals that assessed effects of melatonin on bladder function. Melatonin does not change strip relaxation on its own. However, pre-treatment with melatonin decreases contractile responses induced by phenylephrine, acetylcholine, bethanechol and KCl in a dose-dependent manner. The contractile responses induced by the direct calcium channel openers are significantly decreased by melatonin pre-treatment. It also binds to Ca(2+)-activated calmodulin, and prevents it from activating myosin light-chain kinase. It may have direct effects on ion channels which are responsible for regulating bladder contraction. Its other mode of action on bladder occurs via the brain GABAA receptor. Melatonin is an antioxidant. In bladder, treatment with melatonin prevents elevations in malondialdehyde levels, reverses changes in glutathione levels, and decreases myeloperoxidase levels compared with oxidative injury. It can normalize age induced bladder dysfunction through its antioxidant effects, inhibiting smooth muscle contractility directly and restoring impaired contractility via normalization of Ca(2+) handling and sensitizations pathways. It attenuates the severity of cystitis and inflammation. Mast cell proliferation and activation are increased in cystitis, but decrease by melatonin treatment. Also, there is a decrease in expression levels of pro-inflammatory cytokines after melatonin treatment.

Effect of botulinum toxin A on urothelial-release of ATP and expression of SNARE targets within the urothelium.

AIMS: Botulinum neurotoxin serotype A (BoNT/A) has emerged as an effective treatment of urinary bladder overactivity. Intravesical lipotoxin (BoNT/A delivery using liposomes), which may target the urothelium, is effective in blocking acetic acid induced hyperactivity in animals. The objective of this study was to assess the possible site of toxin action within the urothelium. METHODS: We examined expression of the toxin receptor (SV2) and its cleavage targets (SNAP-25 and SNAP-23) within urothelium as well as effects of the toxin on mechanically evoked release of ATP from cultured rat urothelial cells. ATP release was measured using the luciferin-luciferase assay; we examined expression of SNAP-23 and -25 in urothelial cells and mucosa of rat and human bladders. RESULTS: BoNT/A (1.5 U; 1-3 hr) blocked hypotonic evoked release of urothelial ATP, without affecting morphology. The expression of protein targets for BoNT/A binding (SV2) was detected in human and rat bladder mucosa and catalytic action (SNAP-23, -25) in urothelial cells and mucosa (differed in intensity) from rat and human bladder. Incubation of cultured (rat) urothelial cells with BoNT/A decreased expression levels of both SNAP-23 (44%) and SNAP-25 (80%). CONCLUSIONS: Our findings reveal that the bladder urothelium expresses the intracellular targets and the binding protein for cellular uptake of BoNT/A; and that the toxin is able to suppress the levels of these targets as well as hypotonic-evoked ATP release. These data raise the possibility that intravesical treatment with BoNT/A suppresses bladder reflex and sensory mechanisms by affecting a number of urothelial functions including release of transmitters.

Corticotropin-releasing factor family peptide signaling in feline bladder urothelial cells.

Corticotropin-releasing factor (CRF) plays a central role in the orchestration of behavioral and neuroendocrine responses to stress. The family of CRF-related peptides (CRF and paralogs: urocortin (Ucn)-I, -II, and -III) and associated receptors (CRFR1 and CRFR2) are also expressed in peripheral tissues such as the skin and gastrointestinal tract. Local signaling may exert multiple effects of stress-induced exacerbation of many complex syndromes, including psoriasis and visceral hypersensitivity. Interstitial cystitis/painful bladder syndrome (IC/PBS), a chronic visceral pain syndrome characterized by urinary frequency, urgency, and pelvic pain, is reported to be exacerbated by stress. Functional changes in the epithelial lining of the bladder, a vital blood-urine barrier called the urothelium, may play a role in IC/PBS. This study investigated the expression and functional activity of CRF-related peptides in the urothelium of normal cats and cats with feline interstitial cystitis (FIC), a chronic idiopathic cystitis exhibiting similarities to humans diagnosed with IC/PBS. Western blots analysis showed urothelial (UT) expression of CRFR1 and CRFR2. Enzyme immunoassay revealed release of endogenous ligands (CRF and Ucn) by UT cells in culture. Evidence of functional activation of CRFR1 and CRFR2 by receptor-selective agonists (CRF and UCN3 respectively) was shown by i) the measurement of ATP release using the luciferin-luciferase assay and ii) the use of membrane-impermeant fluorescent dyes (FM dyes) for fluorescence microscopy to assess membrane exocytotic responses in real time. Our findings show evidence of CRF-related peptide signaling in the urothelium. Differences in functional responses between FIC and normal UT indicate that this system is altered in IC/PBS.

Pathophysiology of idiopathic overactive bladder and the success of treatment: a systematic review from ICI-RS 2013.

AIMS: To investigate the frequency of phenotype profiling of patients with idiopathic overactive bladder (OAB) syndrome, and to determine the effectiveness of treatment among individuals with different pathophysiologic profiles. METHODS: The electronic databases MEDLINE, EMBASE, Cochrane CENTRAL, Cochrane Database of Systematic Reviews, and CINAHL were searched from January 1, 1980 to August 12, 2013 for interventional randomized controlled treatment trials (RCTs) of idiopathic OAB. Phenotying for pathophysiologies originating in the urothelial/mucosal layer of the bladder, the detrusor muscle cell layer, and the central nervous system were sought. Articles that analyzed urgency outcomes based on pathophysiologic profiling were selected. Due to the heterogeneity of the included interventions and outcome assessment measures, meta-analysis was not appropriate and a qualitative synthesis was undertaken. RESULTS: Of 239 original RCTs of idiopathic OAB, 48 (20%) profiled participants on underlying pathophysiology. Less than half of these (n = 20) reported treatment efficacy for urgency symptoms by pathophysiological sub-type. One examined the effect of botulinum A toxin on interstitial cell protein expression. Four compared treatment efficacy in OAB patients with and without involuntary detrusor contractions. Fifteen compared the effect of treatment on urgency reduction in patients with detrusor overactivity. There were no consistent trends in treatment efficacy according to pathophysiologic sub-type. No studies examined urothelial dysfunction or abnormal central processing of bladder afferent signaling in response to treatment. CONCLUSIONS: In order to advance the field of idiopathic OAB, more trials are needed that profile and test urgency outcomes in participants according to suspected underlying pathophysiology. Neurourol. Urodynam. 33:611-617, 2014. © 2014 Wiley Periodicals, Inc.

Urothelial mucosal signaling and the overactive bladder-ICI-RS 2013.

There is abundant evidence that the lower urinary tract (LUT) mucosal layer is involved both in mechanosensory functions that regulate bladder contractile activity and in urethral sensation. Changes to the mucosa can be associated with a number of bladder pathologies. For example, alterations of the urothelium and underlying lamina propria at both the molecular and structural levels have been reported in both patients and animals associated with disorders such as bladder pain syndrome and diabetic cystopathy. In contrast to the urinary bladder, much less is known about the urothelium/lamina propria of the bladder neck/proximal urethra. There are important gender differences in the outflow region both anatomically and with respect to innervation, hormonal sensitivity, and location of the external urethral sphincter. There is reasonable evidence to support the view that the mucosal signaling pathway in the proximal urethra is important for normal voiding, but it has also been speculated that the proximal urethra can initiate bladder overactivity. When dysfunctional, the proximal urethra may be an interesting target, for example, botulinum toxin injections aiming at eliminating both urgency and incontinence due to detrusor overactivity.

Impact of diabetes mellitus on bladder uroepithelial cells.

Diabetic bladder dysfunction (DBD), a prevalent complication of diabetes mellitus (DM), is characterized by a broad spectrum of symptoms including urinary urgency, frequency, and incontinence. As DBD is commonly diagnosed late, it is important to understand the chronic impact of DM on bladder tissues. While changes in bladder smooth muscle and innervation have been reported in diabetic patients, the impact of DM on the specialized epithelial lining of the urinary bladder, the urothelium (UT), is largely unknown. Quantitative polymerase chain reaction analysis and electron microscopy were used to evaluate UT gene expression and cell morphology 3, 9, and 20 wk following streptozotocin (STZ) induction of DM in female Sprague-Dawley rats compared with age-matched control tissue. Desquamation of superficial (umbrella) cells was noted at 9 wk DM, indicating a possible breach in barrier function. One causative factor may be metabolic burden due to chronic hyperglycemia, suggested by upregulation of the polyol pathway and glucose transport genes in DM UT. While superficial UT repopulation occurred by 20 wk DM, the phenotype was different, with significant upregulation of receptors associated with UT mechanosensation (transient receptor potential vanilloid subfamily member 1; TRPV1) and UT autocrine/paracrine signaling (acetylcholine receptors AChR-M2 and -M3, purinergic receptors P2X(2) and P2X(3)). Compromised barrier function and alterations in UT mechanosensitivity and cell signaling could contribute to bladder instability, hyperactivity, and altered bladder sensation by modulating activity of afferent nerve endings, which appose the urothelium. Our results show that DM impacts urothelial homeostasis and may contribute to the underlying mechanisms of DBD.

Alterations in the non-neuronal acetylcholine synthesis and release machinery in esophageal epithelium.

AIMS: A non-neuronal cholinergic system has been described in epithelial cells including that of the urinary bladder (urothelium) and the upper gastrointestinal tract (esophagus). Epithelial dysfunction has been implicated in the pathophysiology of persistent pain conditions such as painful bladder syndrome as well as functional heartburn. For example, alterations in the ability to synthesize and release acetylcholine may contribute to changes in epithelial sensory and barrier function associated with a number of functional genitourinary and intestinal disorders. MAIN METHODS: We examined using immunoblot, acetylcholine (ACh)-synthesis and release components in cat esophageal mucosa and whether elements of these components are altered in a naturally occurring model of chronic idiopathic cystitis termed feline interstitial cystitis (FIC). KEY FINDINGS: We identified proteins involved in ACh synthesis and release (high affinity choline transporter, CHT1; ACh synthesizing enzyme choline acetyltransferase ChAT and carnitine acetyltransferase CarAT; vesicular ACh transporter VAChT and the organic cation transporter isoforms 1-3 or OCT-1-3) in cat esophageal mucosa. Significant alterations in CHT, ChAT, VAChT and OCT-1 were detected in the esophageal mucosa from FIC cats. Changes in the vesicular nucleotide transporter (VNUT) and the junctional protein pan-cadherin were also noted. SIGNIFICANCE: Taken together, these findings suggest that changes in the non-neuronal cholinergic system may contribute to alterations in cell-cell contacts and possibly communication with underlying cells that may contribute to changes in sensory function and visceral hyperalgesia in functional esophageal pain.

Botulinum neurotoxin serotype A suppresses neurotransmitter release from afferent as well as efferent nerves in the urinary bladder.

BACKGROUND: Botulinum neurotoxin A (BoNTA), which alleviates overactive bladder symptoms, is thought to act predominantly via the inhibition of transmitter release from parasympathetic nerves. However, actions at other sites such as afferent nerve terminals are possible. OBJECTIVE: To evaluate the effects of BoNTA on bladder afferent neuropeptide release and firing. DESIGN, SETTING, AND PARTICIPANTS: One side of the bladder of control and chronic (1-2 wk) spinal cord transected (SCT; T(8)-T(9)) adult female mice was injected with BoNTA (0.5 U/5 µl saline). After 48 h, bladders with L(6)-S(2) spinal nerves were prepared for in vitro recordings. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: In bladder preparations, tension and optical mapping of Ca(2+) transients were used to measure intrinsic contractions, those evoked by capsaicin or the electrical stimulation of spinal nerves. Afferent firing was evoked by stretch or intrinsic bladder contractions. The numbers of responding units and firing rates were measured. Animal numbers were used to detect moderate to large between-group differences based on Cohen's criteria. Two-way analysis of variance was used to test spatial/temporal differences in Ca(2+) signals as mean plus or minus standard deviation. Differences between data sets were tested with the student t test and skewed data sets with a Mann-Whitney U test (significant when p<0.05). RESULTS AND LIMITATIONS: In control and SCT bladders, BoNTA treatment decreased the contractions evoked by electrical stimulation of spinal nerves without altering intrinsic contractions. Afferent firing on untreated sides in response to stretch/intrinsic contractions was increased in SCTs versus controls. On BoNTA-treated sides, afferent firing rates were greatly attenuated in response to mechanical stimulation as were the capsaicin-evoked optical signals mediated by neuropeptide release. CONCLUSIONS: SCT caused an increased sensitivity of afferent nerves to mechanical stimulation that was reduced by BoNTA treatment. Increased intrinsic activity after SCT was unaffected by the toxin. Thus BoNTA suppresses neurogenic detrusor overactivity by targeting afferent as well as efferent pathways in the bladder.

Beyond neurons: Involvement of urothelial and glial cells in bladder function.

AIM: The urothelium, or epithelial lining of the lower urinary tract (LUT), is likely to play an important role in bladder function by actively communicating with bladder nerves, smooth muscle, and cells of the immune and inflammatory systems. Recent evidence supports the importance of non-neuronal cells that may extend to both the peripheral and central processes of the neurons that transmit normal and nociceptive signals from the urinary bladder. Using cats diagnosed with a naturally occurring syndrome termed feline interstitial cystitis (FIC), we investigated whether changes in physiologic parameters occur within 3 cell types associated with sensory transduction in the urinary bladder: 1) the urothelium, 2) identified bladder dorsal root ganglion (DRG) neurons and 3) grey matter astrocytes in the lumbosacral (S1) spinal cord. As estrogen fluctuations may modulate the severity of many chronic pelvic pain syndromes, we also examined whether 17beta-estradiol (E2) alters cell signaling in rat urothelial cells. RESULTS: We have identified an increase in nerve growth factor (NGF) and substance P (SP) in urothelium from FIC cats over that seen in urothelium from unaffected (control) bladders. The elevated NGF expression by FIC urothelium is a possible cause for the increased cell body size of DRG neurons from cats with FIC, reported in this study. At the level of the spinal cord, astrocytic GFAP immuno-intensity was significantly elevated and there was evidence for co-expression of the primitive intermediate filament, nestin (both indicative of a reactive state) in regions of the FIC S1 cord (superficial and deep dorsal horn, central canal and laminae V-VIl) that receive input from pelvic afferents. Finally, we find that E2 triggers an estrus-modifiable activation of p38 MAPK in rat urothelial cells. There were cyclic variations with E2-mediated elevation of p38 MAPK at both diestrus and estrus, and inhibition of p38 MAPK in proestrous urothelial cells. CONCLUSION: Though urothelial cells are often viewed as bystanders in the processing of visceral sensation, these and other findings support the view that these cells function as primary transducers of some physical and chemical stimuli. In addition, the pronounced activation of spinal cord astrocytes in an animal model for bladder pain syndrome (BPS) may play an important role in the pain syndrome and open up new potential approaches for drug intervention.

Diabetic bladder dysfunction: current translational knowledge.

PURPOSE: Diabetes mellitus, a metabolic disorder caused by an absolute or relative deficiency of insulin, is a debilitating and costly disease with multiple serious complications. Lower urinary tract complications are among the most common complications of diabetes mellitus. The most common, bothersome lower urinary tract complication of diabetes mellitus is diabetic cystopathy or diabetic bladder dysfunction. We reviewed the current translational knowledge of diabetic bladder dysfunction. MATERIALS AND METHODS: We performed a search of the English literature through PubMed. The key words used were diabetes and bladder dysfunction or cystopathy. Our data and perspective are provided for consideration of the future direction of research. RESULTS: Despite traditional recognition of diabetic bladder dysfunction as a voiding problem characterized by poor emptying and overflow incontinence, recent clinical and experimental evidence indicate storage problems such as urgency and urge incontinence in diabetes mellitus cases. Recent experimental evidence from studies of diabetic bladder dysfunction in small animal models of diabetes mellitus show a temporal effect on diabetic bladder dysfunction. Early phase diabetes mellitus causes compensated bladder function and the late phase causes decompensated bladder function. The temporal theory could plausibly provide the scientific road map to correlate clinical and experimental findings, and identify the role of mechanisms such as polyuria, hyperglycemia, oxidative stress, autonomic neuropathy and decompensation of the bladder contractile apparatus in the creation of clinical and experimental manifestations of diabetic bladder dysfunction. CONCLUSIONS: Diabetic bladder dysfunction includes time dependent manifestations of storage and emptying problems. Identifying mechanistic pathways would lead to the identification of therapeutic intervention.

New insights into the pharmacology of the bladder.

PURPOSE OF REVIEW: Pharmacotherapy of a number of bladder disorders has traditionally focused on targeting the 'sensory' component or bladder nerves and the smooth muscle. This review aims to provide an insight into recent (experimental and clinical) developments in mechanisms of existing therapies as well as novel targets. RECENT FINDINGS: Traditionally, sensory signaling in the urinary bladder has been attributed to activation of bladder afferents, but new findings have pointed to the urothelium and interstitial cells as key participants in the transduction of sensory events. Recent advances provide strong support for the development of subtype selective receptor agonists/antagonists, the modulation of signal transduction cascades and new and expanded uses for various neurotoxins. SUMMARY: The development of therapeutic options for the treatment of a number of bladder disorders is complicated, and most treatments are associated with an increased incidence of side effects or lack of specificity. Recent studies suggest that selective targeting of receptors/ion channels or a disease-specific (i.e. phosphorylated) form of the receptor may represent a viable therapeutic target. Though the mechanisms regulating ion channel expression under pathological conditions are not fully known, an increased understanding of these pathways has important implications for drug development.

Expression and function of rat urothelial P2Y receptors.

The control and regulation of the lower urinary tract are partly mediated by purinergic signaling. This study investigated the distribution and function of P2Y receptors in the rat urinary bladder. Application of P2Y agonists to rat urothelial cells evoked increases in intracellular calcium; the rank order of agonist potency (pEC(50) +/- SE) was ATP (5.10 +/- 0.07) > UTP (4.91 +/- 0.14) > UTPgammaS (4.61 +/- 0.16) = ATPgammaS (4.70 +/- 0.05) > 2-methylthio adenosine 5'-diphosphate = 5'-(N-ethylcarboxamido)adenosine = ADP (<3.5). The rank order potency for these agonists indicates that urothelial cells functionally express P2Y(2)/P2Y(4) receptors, with a relative lack of contribution from other P2Y or adenosine receptors. Real-time PCR, Western blotting, and immunocytochemistry confirmed the expression of P2Y(2) and to a lesser extent P2Y(4) in the urothelium. Immunocytochemical studies revealed expression of P2Y(2) staining in all layers of the urothelium, with relative absence of P2Y(4). P2Y(2) staining was also present in suburothelial nerve bundles and underlying detrusor smooth muscle. Addition of UTP and UTPgammaS was found to evoke ATP release from cultured rat urothelial cells. These findings indicate that cultured rat urothelial cells functionally express P2Y(2)/P2Y(4) receptors. Activation of these receptors could have a role in autocrine and paracrine signaling throughout the urothelium. This could lead to the release of bioactive mediators such as additional ATP, nitric oxide, and acetylcholine, which can modulate the micturition reflex by acting on suburothelial myofibroblasts and/or pelvic afferent fibers.

Non-neuronal acetylcholine and urinary bladder urothelium.

Non-neuronal release of acetylcholine (ACh) has been proposed to play a role in urinary bladder function. These studies investigated the expression and function of the non-neuronal cholinergic system in cultured urothelial cells isolated from the rat urinary bladder. Our findings have revealed that urothelial cells express the high-affinity choline transporter (CHT1) and acetylcholine-synthesizing enzymes, choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT). In contrast to neurons, urothelial cells do not express the vesicular acetylcholine transporter (VAChT) but do express OCT3, a subtype of polyspecific organic cation transporter (OCT) that is thought to be involved in the release of acetylcholine from non-neuronal cells. Following exposure of cultured urothelial cells to (3)H-choline, radioactivity was detected in the cells and increased release of radioactivity into the eternal media was evoked by mechanical stimulation (exposure of the cells to 50% hypotonic Krebs) or chemical stimulation of purinergic receptors by 100 muM ATP. The present experiments did not establish if the evoked release of radioactivity (termed (3)H-ACh release in this paper) was due to release of acetylcholine or choline. (3)H-ACh release was not evoked by application of acetylcholine alone, however pretreatment with the non-selective muscarinic receptor antagonist atropine prior to application of acetylcholine facilitated (3)H-ACh release, suggesting that the acetylcholine released from urothelial cells may participate in a negative feedback mechanism by acting on muscarinic receptors to inhibit its own release in the urothelium. Brefeldin, an agent which disrupts vesicular exocytosis, did not block hypotonic-evoked (3)H-ACh release. These observations indicate that acetylcholine release from urothelial cells is mediated by different mechanisms than those such as vesicular storage and exocytosis that underlie the release of neurotransmitters from nerves.

The hydroosmotic response of frog urinary bladder to serosal hypertonicity is dependent on adenylate cyclase for its maintenance and affected by [Cl-]o changes.

The role of adenylate cyclase (AC) in the maintenance of the hydroosmotic response to serosal hypertonicity (SH) in anuran urinary bladder is disputed. In this study, norepinephrine (NE) significantly reversed the hydroosmotic response of Rana temporaria bladders in hypertonic medium (330 mosmol/kgH2O). The reversal was inhibited by yohimbine but was unaffected by prazosin and propranolol, indicating that NE action was mediated via alpha2-adrenergic receptors. Preincubation of bladders with indomethacin did not interfere with the inhibitory action of NE, contraindicating a role for prostaglandins. The SH hydroosmotic response was abolished in the presence of 5-n-ethyl-N-isopropyl amiloride (EIPA), but the antidiuretic hormone (ADH) hydroosmotic response was not. EIPA inhibits Na+/H+, known to be activated by cell shrinkage. An investigation of the anionic requirement of the SH hydroosmotic response revealed that replacement of bath Cl- with the nonpermeable anion gluconate reversibly abolished this response. In contrast, the hydroosmotic response to ADH was unaffected by Cl- removal; however, when Cl- was absent, it was no longer augmented in hypertonic bath. The SH response was inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate but not by the Na/K/2Cl inhibitor bumetanide. Our results show that not only the onset but also the maintenance of the SH hydroosmotic response is dependent on AC activity and does not differ in this respect to the ADH hydroosmotic response. The effect of modifying extracellular Cl- concentration, suggests that this anion, possibly functionally linked with Na+/H+ activity, may be involved in invoking the SH hydroosmotic response in anuran urinary bladder.