Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions.     

Thrombin induces macrophage migration inhibitory factor release and upregulation in urothelium: a possible contribution to bladder inflammation

Purpose

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells.

Materials and Methods

MIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells (in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR.

Results

UROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both in vitro (UROtsa; 2.8-fold increase at 1 hr) and in vivo (rat; 4.5-fold) while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment.

Conclusions

Urothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression.     

Cyclophosphamide-Induced Cystitis Increases Bladder CXCR4 Expression and CXCR4-Macrophage Migration Inhibitory Factor Association

Background

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine involved in cystitis and a non-cognate ligand of the chemokine receptor CXCR4 in vitro. We studied whether CXCR4-MIF associations occur in rat bladder and the effect of experimental cystitis.

Methods and Findings

Twenty male rats received saline or cyclophosphamide (40 mg/kg; i.p.; every 3^rd day) to induce persistent cystitis. After eight days, urine was collected and bladders excised under anesthesia. Bladder CXCR4 and CXCR4-MIF co-localization were examined with immunhistochemistry. ELISA determined MIF and stromal derived factor-1 (SDF-1; cognate ligand for CXCR4) levels. Bladder CXCR4 expression (real-time RTC-PCR) and protein levels (Western blotting) were examined. Co-immunoprecipitations studied MIF-CXCR4 associations.Urothelial basal and intermediate (but not superficial) cells in saline-treated rats contained CXCR4, co-localized with MIF. Cyclophosphamide treatment caused: 1) significant redistribution of CXCR4 immunostaining to all urothelial layers (especially apical surface of superficial cells) and increased bladder CXCR4 expression; 2) increased urine MIF with decreased bladder MIF; 3) increased bladder SDF-1; 4) increased CXCR4-MIF associations.

Conclusions

These data demonstrate CXCR4-MIF associations occur in vivo in rat bladder and increase in experimental cystitis. Thus, CXCR4 represents an alternative pathway for MIF-mediated signal transduction during bladder inflammation. In the bladder, MIF may compete with SDF-1 (cognate ligand) to activate signal transduction mediated by CXCR4.     

Intraluminal Blockade of Cell-Surface CD74 and Glucose Regulated Protein 78 Prevents Substance P-Induced Bladder Inflammatory Changes in the Rat

Background

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine constitutively expressed by urothelial cells. During inflammatory stimuli, MIF is released into the lumen complexed to other proteins and these complexes can bind to urothelial cell-surface receptors to activate signaling pathways. Since MIF is complexed to α1-inhibitor III (A1-I3; a member of the α2-macroglubulin family) and glucose regulated protein 78 (GRP78) is a receptor for A1-I3 the goals of this study were to determine if substance P elicits urothelial cell-surface expression of GRP78 and to assess the functional role of CD74 (receptor for MIF) or GRP78 in substance P-induced bladder inflammatory changes.

Methodology/Principal Findings

Anesthetized male Sprague-Dawley rats received either saline or substance P (s.c.), bladders were collected 1 hour after treatment and processed for histology or protein/mRNA. The expression of GRP78 at urothelial cell-surface was determined by performing in vivo biotinylation of urothelial cell-surface proteins. Finally, in order to determine the effects of receptor blockade on substance P-induced MIF release and inflammatory changes, rats received either intraluminal antibodies to CD74, GRP78, both, or non-specific IgG (as a control).GRP78 and MIF immunostaining was simultaneously visualized in umbrella cells only after substance P treatment. Immunoprecipitation studies showed GRP78-MIF complexes increased after substance P while in vivo biotinylation confirmed substance P-induced GRP78 cell-surface expression in urothelial cells. Intraluminal blockade of CD74 and/or GRP78 prevented substance P-induced changes, including bladder edema, intraluminal MIF release by urothelial cells and production of inflammatory cytokines by urothelial cells.

Conclusions/Significance

GRP78 is expressed on the surface of urothelial cells after substance P treatment where it can bind MIF complexes. Blocking CD74 (receptor for MIF) and/or GRP78 prevented substance P-induced inflammatory changes in bladder and urothelium, indicating that these urothelial receptors are effective targets for disrupting MIF-mediated bladder inflammation.     

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.     

Chemoprevention of BBN-Induced Bladder Carcinogenesis by the Selective Estrogen Receptor Modulator Tamoxifen

Bladder cancer is the fifth most frequent tumor in men and ninth in women in the United States. Due to a high likelihood of recurrence, effective chemoprevention is a significant unmet need. Estrogen receptors (ERs), primarily ERβ, are expressed in normal urothelium and urothelial carcinoma, and blocking ER function with selective ER modulators such as tamoxifen inhibits bladder cancer cell proliferation in vitro. Herein, the chemoprotective potential of tamoxifen was evaluated in female mice exposed to the bladder-specific carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Carcinogen treatment resulted in a 76% tumor incidence and increased mean bladder weights in comparison to controls. In contrast, mice receiving tamoxifen concurrent (8–20 weeks) or concurrent and subsequent (8–32 weeks) to BBN administration had no change in bladder weight and only 10% to 14% incidence of tumors. Non-muscle-invasive disease was present in animals treated with tamoxifen before (5–8 weeks) or after (20–32 weeks) BBN exposure, while incidence of muscle-invasive bladder carcinoma was reduced. ERβ was present in all mice and thus is a potential mediator of the tamoxifen chemoprotective effect. Surprisingly, ERα expression, which was detected in 74% of the mice exposed to BBN alone but not in any controlmice, was correlated with tumor incidence, indicating a possible role for this receptor in carcinogen-induced urothelial tumorigenesis. Thus, these data argue that both ERα and ERβ play a role in modulating carcinogen-induced bladder tumorigenesis. Administration of tamoxifen should be tested as a chemopreventive strategy for patients at high risk for bladder cancer recurrence.     

MicroRNA expression signatures of bladder cancer revealed by deep sequencing

Background

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression. They are aberrantly expressed in many types of cancers. In this study, we determined the genome-wide miRNA profiles in bladder urothelial carcinoma by deep sequencing.

Methodology/Principal Findings

We detected 656 differentially expressed known human miRNAs and miRNA antisense sequences (miRNA*s) in nine bladder urothelial carcinoma patients by deep sequencing. Many miRNAs and miRNA*s were significantly upregulated or downregulated in bladder urothelial carcinoma compared to matched histologically normal urothelium. hsa-miR-96 was the most significantly upregulated miRNA and hsa-miR-490-5p was the most significantly downregulated one. Upregulated miRNAs were more common than downregulated ones. The hsa-miR-183, hsa-miR-200b∼429, hsa-miR-200c∼141 and hsa-miR-17∼92 clusters were significantly upregulated. The hsa-miR-143∼145 cluster was significantly downregulated. hsa-miR-182, hsa-miR-183, hsa-miR-200a, hsa-miR-143 and hsa-miR-195 were evaluated by Real-Time qPCR in a total of fifty-one bladder urothelial carcinoma patients. They were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium (p<0.001 for each miRNA).

Conclusions/Significance

To date, this is the first study to determine genome-wide miRNA expression patterns in human bladder urothelial carcinoma by deep sequencing. We found that a collection of miRNAs were aberrantly expressed in bladder urothelial carcinoma compared to matched histologically normal urothelium, suggesting that they might play roles as oncogenes or tumor suppressors in the development and/or progression of this cancer. Our data provide novel insights into cancer biology.     

Functional Role for Piezo1 in Stretch-evoked Ca2+ Influx and ATP Release in Urothelial Cell Cultures

The urothelium is a sensory structure that contributes to mechanosensation in the urinary bladder. Here, we provide evidence for a critical role for the Piezo1 channel, a newly identified mechanosensory molecule, in the mouse bladder urothelium. We performed a systematic analysis of the molecular and functional expression of Piezo1 channels in the urothelium. Immunofluorescence examination demonstrated abundant expression of Piezo1 in the mouse and human urothelium. Urothelial cells isolated from mice exhibited a Piezo1-dependent increase in cytosolic Ca²⁺ concentrations in response to mechanical stretch stimuli, leading to potent ATP release; this response was suppressed in Piezo1-knockdown cells. In addition, Piezo1 and TRPV4 distinguished different intensities of mechanical stimulus. Moreover, GsMTx4, an inhibitor of stretch-activated channels, attenuated the Ca²⁺ influx into urothelial cells and decreased ATP release from them upon stretch stimulation. These results suggest that Piezo1 senses extension of the bladder urothelium, leading to production of an ATP signal. Thus, inhibition of Piezo1 might provide a promising means of treating bladder dysfunction.     

The TRPV4 Cation Channel Mediates Stretch-evoked Ca2+ Influx and ATP Release in Primary Urothelial Cell Cultures

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD) evoked Ca²⁺ influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca²⁺ concentration and ATP release. Stretch stimulation evoked intracellular Ca²⁺ increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca²⁺ increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca²⁺ increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4α-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca²⁺. These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.Transient receptor potential vanilloid 4 (TRPV4),³ a member of the TRP superfamily of cation channels, is a Ca²⁺-permeable channel activated by a wide variety of physical and chemical stimuli (1, 2). TRPV4 was originally viewed as an osmo- or mechano-sensor, because the channel opens in response to hypotonicity-induced cell swelling (3–5) and shear stress (6). Alternatively, TRPV4 can be activated by diverse chemical stimuli such as synthetic phorbol ester 4α-phorbol 12,13-didecanoate (4α-PDD) (7), a botanical agent (bisandrographolide A), anandamide metabolites such as arachidonic acid and epoxyeicosatrienoic acids, as well as moderate warmth (>27 °C) (8–10). TRPV4 is widely expressed throughout the body, including renal epithelium, auditory hair cells, skin keratinocytes, hippocampus neurons, endothelial cells, and urinary bladder epithelium, thereby contributing to numerous physiological processes such as osmoregulation (11, 12), hearing (13), thermal and mechanical hyperalgesia (14, 15), neural activity in the brain (16), skin barrier recovery (17), and cell volume regulation (18). Therefore, the TRPV4 channel is now considered a multimodal transducer in various tissues and cells.Non-neuronal cells within the urinary bladder wall (notably the transitional epithelial cells (urothelial cells)) function as a barrier against ions, solutes, and infection and also participate in the detection of physical and chemical stimuli (19–21). The urothelium expresses various sensory receptors and channels (bradykinin receptors, adrenergic/cholinergic receptors, nerve growth factor receptors, purinergic receptors, amiloride-sensitive Na⁺ channels, and TRP channels), all of which are substantially implicated in modulating bladder functions (22).Recently, the potential roles of TRP channels have been explored in the bladder. Thus far, expression of TRPV1, TRPV2, TRPV4, TRPA1, and TRPM8 has been reported in different regions of urogenital tracts (21). TRPV1 is reportedly expressed in the epithelial cells lining the urothelium, in interstitial cells, and in sensory nerve terminals. TRPV1-deficient mice displayed a higher frequency of low amplitude non-voiding bladder contractions in comparison with wild-type (WT) mice (22), suggesting that TRPV1 is required for detection of bladder stretch, which involves stretch-evoked release of ATP and nitric oxide. The release of both mediators was reduced in the bladders of TRPV1-deficient mice. In a clinical setting, capsaicin or resiniferatoxin reduces bladder overactivity through desensitization of bladder afferents by acting on TRPV1 (23). Expression of other TRP channels, e.g. TRPM8 and TRPA1, was found in sensory C fibers in the bladder (24–27). The diagnostic ice water test is utilized to determine whether disturbance of bladder function involves neurogenic components, one of which could be related to TRPM8 function, in patients with spinal cord lesion (28). TRPA1 in sensory afferents is activated by several known ligands (allyl isothiocyanate and cinnamaldehyde), thereby inducing bladder overactivity (26). TRPV2 is expressed by several cell types in the rat bladder (29); however, its physiological function has not yet been investigated. TRPV4 is expressed in the urothelium and in smooth muscle cells of the urinary bladder (30, 31). Activation of the channel by specific ligands leads to augmentation of bladder contraction amplitude in cystometry and induction of bladder overactivity in vivo. In a separate cystometry analysis in conjunction with behavioral experiments, the intermicturitional interval was elongated and storage urine volume was increased in TRPV4-deficient mice compared with WT mice (32). Thus, TRPV4 may contribute to bladder function, especially to mediating bladder distention signals to primary afferent nerves during urine storage. However, whether urothelial TRPV4 is required for sensing mechanical stretch, or to what extent urothelial TRPV4 contributes to stretch-evoked ATP release, has not been precisely determined.In the present study, we examined the functional contribution of TRPV4 to stretch-dependent urothelial cell responses and stretch-evoked ATP release in vitro. We first established a primary cell culture for mouse urothelium and retention of TRPV4 expression was confirmed. Because urothelial cells are physically extended during urine storage in vivo, we reproduced this phenomenon in an in vitro experiment using the uni-axial cell stretch system. All the experiments were performed by comparing urothelial cells obtained from WT mice and TRPV4-deficient mice to evaluate the correlation between TRPV4 expression and stretch responses. We demonstrated that urothelial cells sense mechanical stretch stimuli via TRPV4 channels, which induces robust Ca²⁺ influx and contributes to ATP release upon extension.     

Hypoxia‐induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non‐cognate ligand of the CXC‐family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine‐like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC‐derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia‐induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non‐classical mechanism and originated from pre‐formed MIF stores. Early hypoxia‐triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia‐inducible factor (HIF)‐1α‐induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF‐1α‐induced de novo synthesis of MIF. To functionally investigate the role of hypoxia‐inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL⁺ KDR⁺ CD31⁺ EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose‐dependent bell‐shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia‐conditioned HUVECs, an effect that was completely abrogated by anti‐MIF‐ or anti‐CXCR4‐antibodies. Thus, hypoxia‐induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia‐induced myocardial damage.     

Store-operated Ca2+ entry suppresses distention-induced ATP release from the urothelium

Epithelial cells in the urinary bladder (urothelium) trigger sensory signals in micturition by releasing ATP in response to distention of the bladder wall. Our previous study revealed the distinct roles of extracellular Ca(2+) and the Ca(2+) stores in the endoplasmic reticulum (ER) in urothelial ATP release. In the present study, we investigated the regulation of urothelial ATP release by Ca(2+) influx from the extracellular space and Ca(2+) release from the ER using a distention assay of the mouse bladder wall in a small Ussing chamber. Stimulation of Ca(2+) release from the ER in the mucosal side of the bladder induced significant ATP release without distention. Blockade of the inositol 1,4,5-triphosphate receptor reduced distention-induced ATP release, suggesting that Ca(2+) release from the ER is essential for the induction of urothelial ATP release. On the other hand, blockade of store-operated Ca(2+) entry (SOCE) from the extracellular space significantly enhanced distention-induced ATP release. Thus Ca(2+) release from the ER causes urothelial ATP release and depletion of Ca(2+) stores in the ER, which in turn causes the depletion-inducing SOCE to suppress the amount of urothelial ATP released.     

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.     

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.     

Distinctive G Protein-Dependent Signaling by Protease-Activated Receptor 2 (PAR2) in Smooth Muscle: Feedback Inhibition of RhoA by cAMP-Independent PKA

We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated G_q, G₁₃, G_i1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Ga_q or Ga_i minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga₁₃ siRNA, and by Ga₁₃ minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075"}}U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC₂₀, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release catalytic subunit of PKA from its binding to IkBa and phosphorylation of RhoA at Ser¹⁸⁸.     

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.     

Conditional Expression of the Androgen Receptor Increases Susceptibility of Bladder Cancer in Mice

Bladder cancer represents a significant human tumor burden, accounting for about 7.7% and 2.4% of all cancer cases in males and females, respectively. While men have a higher risk of developing bladder cancer, women tend to present at a later stage of disease and with more aggressive tumors. Previous studies have suggested a promotional role of androgen signaling in enhancing bladder cancer development. To directly assess the role of androgens in bladder tumorigenesis, we have developed a novel transgenic mouse strain, R26hAR^LoxP/+:Upk3a^GCE/+, in which the human AR transgene is conditionally expressed in bladder urothelium. Intriguingly, both male and female R26hAR^LoxP/+:Upk3a^GCE/+ mice display a higher incidence of urothelial cell carcinoma (UCC) than the age and sex matched control littermates in response to the carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). We detect expression of the human AR transgene in CK5-positive and p63-positive basal cells in bladder urothelium. Further analyses of UCC tissues from R26hAR^LoxP/+:Upk3a^GCE/+ mice showed that the majority of tumor cells are of urothelial basal cell origin. Positive immunostaining of transgenic AR protein was observed in the majority of tumor cells of the transgenic mice, providing a link between transgenic AR expression and oncogenic transformation. We observed an increase in Ki67 positive cells within the UCC lesions of transgenic AR mice. Manipulating endogenous androgen levels by castration and androgen supplementation directly affected bladder tumor development in male and female R26hAR^LoxP/+:Upk3a^GCE/+ mice, respectively. Taken together, our data demonstrate for the first time that conditional activation of transgenic AR expression in bladder urothelium enhances carciongen-induced bladder tumor formation in mice. This new AR transgenic mouse line mimics certain features of human bladder cancer and can be used to study bladder tumorigenesis and for drug development.     

Prostaglandin D2 effects and DP1/DP2 receptor distribution in guinea pig urinary bladder out‐flow region

The proximal urethra and urinary bladder trigone play important roles in continence. We have previously shown that PGD₂ is released from guinea pig bladder urothelium/suburothelium and can inhibit detrusor contractile responses. We presently wished to investigate PGD₂ actions in guinea pig out‐flow region and the distribution of DP₁/DP₂ receptors. The effects of PGD₂ on urothelium‐intact trigone and proximal urethra contractility were studied in organ bath experiments. Expression of DP₁/DP₂ receptor proteins was analysed by western blot. Immunohistochemistry was used to identify distribution of DP₁/DP₂ receptors. PGD₂ in a dose‐dependent manner inhibited trigone contractions induced by electrical field stimulation (EFS) and inhibited spontaneous contractions of the proximal urethra. PGD₂ was equally (trigone) or slightly less potent (urethra) compared with PGE₂. Expression of DP₁ and DP₂ receptors was found in male guinea pig bladder trigone, neck and proximal urethra. In the trigone and proximal urethra, DP₁ receptors were found on the membrane of smooth muscle cells and weak immunoreactivty was observed in the urothelium. DP₂ receptors were distributed more widespread, weakly and evenly in the urothelium and smooth muscles. Inhibitory effects by PGD₂ on motor activity of guinea pig trigone and proximal urethra are consistent with finding DP₁ and DP₂ receptors located in the urothelium and smooth muscle cells of the trigone and proximal urethra, and PGD₂ may therefore be a modulator of the bladder out‐flow region, possibly having a function in regulation of micturition and a role in overactive bladder syndrome.