Protective effect of equol intake on bladder dysfunction in a rat model of bladder outlet obstruction.

OBJECTIVES: This study evaluates the impact of equol, a metabolite of soy isoflavone, on bladder dysfunction in rats with bladder outlet obstruction (BOO). In addition, we investigate its potential as a neuroprotective agent for the obstructed bladder and discuss its applicability in managing overactive bladder (OAB). METHODS: Eighteen male Sprague-Dawley rats were divided into three groups (six rats per group) during the rearing period. The Sham and C-BOO groups received an equol-free diet, while the E-BOO group received equol supplementation (0.25 g/kg). At 8 weeks old, rats underwent BOO surgery, followed by continuous cystometry after 4 weeks of rearing. The urinary oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde) were measured, and the bladder histology was analyzed using hematoxylin-eosin, Masson's trichrome, and immunohistochemical staining (neurofilament heavy chain for myelinated nerves, peripherin for unmyelinated nerves, and malondialdehyde). RESULTS: Equol reduced BOO-induced smooth muscle layer fibrosis, significantly prolonged the micturition interval (C-BOO: 193 s, E-BOO: 438 s) and increased the micturition volume (C-BOO: 0.54 mL, E-BOO: 1.02 mL) compared to the C-BOO group. Equol inhibited the increase in urinary and bladder tissue malondialdehyde levels. While the C-BOO group exhibited reduced peripherin alone positive nerve fibers within the smooth muscle layer, equol effectively attenuated this decline. CONCLUSIONS: Equol reduces lipid peroxidation and smooth muscle layer fibrosis in the bladder and exhibited neuroprotective effects on bladder nerves (peripheral nerves) and prevented the development of bladder dysfunction associated with BOO in rats. Consumption of equol is promising for the prevention of OAB associated with BOO.

Time-dependent progress of lower urinary tract dysfunction in streptozotocin-induced diabetic rats with or without low-dose insulin treatment.

Objectives: To examine time-dependent functional and structural changes of the lower urinary tract in streptozotocin-induced diabetic rats with or without low-dose insulin treatment and explore the pathophysiological characteristics of insulin therapy on lower urinary tract dysfunction (LUTD) caused by diabetes mellitus (DM). Methods: Female Sprague-Dawley rats were divided into five groups: normal control (NC) group, 4 weeks insulin-treated DM (4-DI) group, 4 weeks DM (4-DM) group, 8 weeks insulin-treated DM (8-DI) group and 8 weeks DM (8-DM) group. DM was initially induced by i.p. injection of streptozotocin (65 mg/kg), and then the DI groups received subcutaneous implantation of insulin pellets under the mid dorsal skin. Voiding behavior was evaluated in metabolic cages. The function of bladder and urethra in vivo were evaluated by simultaneous recordings of the cystometrogram and urethral perfusion pressure (UPP) under urethane anesthesia. The function of bladder and urethra in vitro were tested by organ bath techniques. The morphologic changes of the bladder and urethra were investigated using Hematoxylin-Eosin and Masson's staining. Results: Both 4-and 8-weeks diabetic rats have altered micturition patterns, including increased 12-h urine volume, urinary frequency/12 hours and voided volume. In-vivo urodynamics showed the EUS bursting activity duration is longer in 4-DM group and shorter in 8-DM group compared to NC group. UPP change in 8-DM were significantly lower than NC group. While none of these changes were found between DI and NC groups. Organ bath showed the response to Carbachol and EFS in bladder smooth muscle per tissue weights was decreased significantly in 4- and 8-weeks DM groups compared with insulin-treated DM or NC groups. In contrast, the contraction of urethral muscle and maximum urethral muscle contraction per gram of the tissue to EFS stimulation were significantly increased in 4- and 8-weeks DM groups. The thickness of bladder smooth muscle was time-dependently increased, but the thickness of the urethral muscle had no difference. Conclusions: DM-induced LUTD is characterized by time-dependent functional and structural remodeling in the bladder and urethra, which shows the hypertrophy of the bladder smooth muscle, reduced urethral smooth muscle relaxation and EUS dysfunction. Low-dose insulin can protect against diuresis-induced bladder over-distention, preserve urethral relaxation and protect EUS bursting activity, which would be helpful to study the slow-onset, time-dependent progress of DM-induced LUTD.

Novel pharmacotherapeutic avenues for bladder storage dysfunction in men.

INTRODUCTION: Bladder storage dysfunction is associated with low quality of life in men and remains a challenging field in pharmacotherapy because of low persistence followed by patient-perceived lack of efficacy and adverse effects. The persistent desire for the development of novel pharmacotherapy is evident, leading to numerous research efforts based on its pathophysiology. AREAS COVERED: This review describes the pathophysiology, current pharmacotherapeutic strategies, and emerging novel drugs for male bladder storage dysfunction. The section on emerging pharmacotherapy provides an overview of current research, focusing on high-potential target molecules, particularly those being evaluated in ongoing clinical trials. EXPERT OPINION: As pharmacotherapies targeting alpha-adrenergic, beta-adrenergic, and muscarinic receptors - the current primary targets for treating male bladder storage dysfunction - have demonstrated insufficient efficacy and side effects, researchers are exploring various alternative molecular targets. Numerous targets have been identified as central to regulating bladder afferent nerve activity, and their pharmacological effects and potential have been evaluated in animal-based experiments. However, there is a limited number of clinical trials for these new pharmacotherapies, and they have not demonstrated clear superiority over current treatments. Further research is needed to develop new effective pharmacotherapies for bladder storage dysfunction in men.

Characterizing the toxicological responses to inorganic arsenicals and their metabolites in immortalized human bladder epithelial cells.

Arsenic is highly toxic to the human bladder. In the present study, we established a human bladder epithelial cell line that closely mimics normal human bladder epithelial cells by immortalizing primary uroplakin 1B-positive human bladder epithelial cells with human telomerase reverse transcriptase (HBladEC-T). The uroplakin 1B-positive human bladder epithelial cell line was then used to evaluate the toxicity of seven arsenicals (iAsV, iAsIII, MMAV, MMAIII, DMAV, DMAIII, and DMMTAV). The cellular uptake and metabolism of each arsenical was different. Trivalent arsenicals and DMMTAV exhibited higher cellular uptake than pentavalent arsenicals. Except for MMAV, arsenicals were transported into cells by aquaglyceroporin 9 (AQP9). In addition to AQP9, DMAIII and DMMTAV were also taken up by glucose transporter 5. Microarray analysis demonstrated that arsenical treatment commonly activated the NRF2-mediated oxidative stress response pathway. ROS production increased with all arsenicals, except for MMAV. The activating transcription factor 3 (ATF3) was commonly upregulated in response to oxidative stress in HBladEC-T cells: ATF3 is an important regulator of necroptosis, which is crucial in arsenical-induced bladder carcinogenesis. Inorganic arsenics induced apoptosis while MMAV and DMAIII induced necroptosis. MMAIII, DMAV, and DMMTAV induced both cell death pathways. In summary, MMAIII exhibited the strongest cytotoxicity, followed by DMMTAV, iAsIII, DMAIII, iAsV, DMAV, and MMAV. The cytotoxicity of the tested arsenicals on HBladEC-T cells correlated with their cellular uptake and ROS generation. The ROS/NRF2/ATF3/CHOP signaling pathway emerged as a common mechanism mediating the cytotoxicity and carcinogenicity of arsenicals in HBladEC-T cells.

A pilot study exploring time- and dose-dependent DNA damage and chromosomal instability caused by benzo[a]pyrene in two urothelial cell types.

Environmental and occupational exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with adverse health effects in humans. Uncertainty exists regarding the causation of urinary bladder cancer by benzo[a]pyrene (B[a]P) due to a lack of sufficient data. In this work, we focused on in-vitro DNA damage and the formation of micronuclei and chromosomal aberrations as predictors of cancer risk, applying a wide range of dosages and time periods to quantify the onset, intensity, and duration of the response. We chose two urothelial cell types to compare susceptibility and the ability to increase the malignity of a pre-existing bladder cancer: a cancer cell line (T24) and a pooled sample of primary urinary bladder epithelia cells (PUBEC) from pigs. The highest level of DNA damage assessed by comet assay was observed following 24-h treatment in both cell types, whereas PUBEC cells were clearly more susceptible. Even 4-h treatment induced DNA damage in PUBEC cells with benchmark doses of 0.0027 µM B[a]P and 0.00023 µM after 4-h and 24-h exposure, respectively. Nearly no effect was observed for periods of 48 h. The frequency of micronucleus formation increased more markedly in T24 cells, particularly with 24-h treatment. In PUBEC cells, 48-h exposure notably induced the formation of nucleoplasmic bridges and nuclear buds. Even though only one biological replicate was studied due to the sophisticated study design, our results give a strong indication of the potential of B[a]P to induce and increase malignity in human-relevant cell types.

Improvement of lower urinary tract dysfunction by a monoacylglycerol lipase inhibitor in mice with spinal cord injury.

AIMS: Activation of the endocannabinoid system by monoacylglycerol lipase (MAGL) blockade may affect the lower urinary tract function. We investigated the effect of an MAGL inhibitor, MJN110, on neurogenic lower urinary tract dysfunction (LUTD) in the mouse model of spinal cord injury (SCI). METHODS: Female C57BL/6 mice that underwent spinal cord transection at T8-10 level were divided into three groups consisting of (1) vehicle-treated SCI mice, (2) 5 mg/kg, or (3) 10 mg/kg of MJN110-treated SCI mice. MJN110 and vehicle were administered intraperitoneally for 7 days from 4 weeks after spinal cord transection. We then conducted awake cystometrograms and compared urodynamic parameters between three groups. The expression of cannabinoid (CB) receptors, TRP receptors, and inflammatory cytokines in L6-S1 dorsal root ganglia (DRG) or the bladder mucosa were evaluated and compared among three groups. Changes in the level of serum 2-arachidonoylglycerol (2-AG) and bladder MAGL were also evaluated. RESULTS: In the cystometrogram, detrusor overactivity (DO) parameters, such as the number of nonvoiding contraction (NVC), a ratio of time to the 1st NVC to intercontraction interval (ICI), and NVC integrals were improved by MJN110 treatment, and some effects were dose dependent. Although MJN110 did not improve voiding efficiency, it decreased bladder capacity, ICI, and residual urine volume compared to vehicle injection. MJN110 treatment groups had lower CB2, TRPV1, TRPA1, and inflammatory cytokines mRNA levels in DRG and bladder mucosa. Serum 2-AG was increased, and bladder MAGL was decreased after MAGL inhibitor treatment. CONCLUSIONS: MAGL inhibition improved LUTD including attenuation of DO after SCI. Thus, MAGL can be a therapeutic target for neurogenic LUTD after SCI.

Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

Role of ERK signaling in bladder urothelium in response to cyclophosphamide injury.

Mice with inducible urothelial deletion of fibroblast growth factor receptor 2 (ShhCreERT2;Fgfr2Fl/Fl ) injured with cyclophosphamide had aberrant basal cell endoreplication and poor regeneration. The endoreplication correlated with an absence of phosphorylated (activated) ERK expression in urothelium. We assessed whether inhibiting ERK activity phenocopied the urothelial defects in injured Fgfr2 mutant mice. We co-administered cyclophosphamide and an ERK inhibitor (ERKi) systemically in mice and assessed general histology and immunofluorescence for various markers post injury. Since AKT also signals downstream of FGFR2, we assessed effects of an AKT inhibitor (AKTi) on cyclophosphamide injury. ERK knockdown did not affect urothelial injury or proliferation 24 h after cyclophosphamide. Conversely, ERK inhibition led to larger basal cell nuclei, more submucosal hemorrhage and attenuated uroplakin staining 3 days after injury versus vehicle-treated mice. Compared to vehicle-treated mice, ERKi-treated mice had a trend for more Ki67+ urothelial cells and had statistically fewer phospho-Histone H3+ cells normalized to Ki67 and higher basal cell DNA content, consistent with endoreplication 3 days after injury. Ten days after injury, ERKi-treated mice still had signs of poor urothelial regeneration with absent or aberrant expression of differentiation markers and ectopic lumenal expression of keratin 14 (basal progenitor marker). Co-administration of the AKTi led to no apparent urothelial defects 3 days after cyclophosphamide. Thus, ERK knockdown (but not AKT knockdown) leads to urothelial regenerative responses after cyclophosphamide reminiscent of Fgfr2 mutant mice. Together, it appears that FGFR2 acts through ERK to prevent aberrant urothelial basal cell endoreplication and ensure normal regeneration after cyclophosphamide.

Treatment with the soluble guanylate cyclase activator BAY 60-2770 normalizes bladder function in an in vivo rat model of chronic prostatitis.

BACKGROUND AND PURPOSE: Chronic pelvic pain syndrome (CPPS) is a common and bothersome condition for which no pharmacological treatment options with acceptable efficacy exist. The aim of this study was to investigate the effects of the soluble guanylate cyclase (sGC) activator BAY 60-2770 and the COX-2 inhibitor celecoxib on bladder function in a rat model of CPPS. EXPERIMENTAL APPROACH: Forty-eight male Sprague-Dawley rats were intraprostatically injected with either saline, serving as control, or zymosan, to induce prostatitis. On days 8-20, the rats were treated with either dimethylsulphoxide (DMSO; vehicle), celecoxib, BAY 60-2770 or a combination of celecoxib and BAY 60-2770. Thereafter, micturition parameters were assessed in a metabolic cage and urine samples were collected. The following day, cystometry was performed. Subsequently, the urinary bladder and prostate were removed and examined histopathologically. KEY RESULTS: Induction of prostatitis led to a significant increase of micturition frequency and corresponding decrease of volume per micturition. These alterations were ameliorated by celecoxib, and completely normalized by BAY 60-2770. Induction of prostatitis led to a significantly increased number of non-voiding contractions, decreased bladder compliance and increased voiding time. These parameters were normalized by treatment with BAY 60-2770, either alone or in combination with celecoxib. The immunohistochemical analysis showed signs of prostate inflammation, but not bladder inflammation. CONCLUSION AND IMPLICATIONS: Induction of prostatitis led to significant impairment in bladder function. These alterations could be prevented by BAY 60-2770, alone or in combination with celecoxib. This is the first study to show that sGC activators could be a promising option for the treatment of CPPS.

Vibegron inhibits enhanced spontaneous contractions induced by anoxia/reoxygenation in isolated whole bladder from rats.

It has been recently proposed that repeated bladder ischemia/reperfusion induced by chronic pelvic ischemia may lead to detrusor overactivity, followed by lower urinary tract symptoms. Vibegron is a selective ß3-adrenoceptor agonist approved for the treatment of overactive bladder. Several studies have tested ß3-adrenoceptor agonists using animal models with detrusor overactivity related to bladder ischemia/reperfusion. However, whether ß3-adrenoceptor agonists directly affect ischemia/reperfusion-evoked detrusor overactivity is unclear. Therefore, we examined whether bladder anoxia/reoxygenation could enhance spontaneous bladder contractions (SBCs) and investigated the effect of vibegron on enhanced SBCs. Isolated whole bladders from rats were incubated with Krebs solution aerated with 95% N2 + 5% CO2 for 5 h (anoxia). Subsequently, the bathing solution was replaced with an oxygen-saturated solution (reoxygenation). Anoxia/reoxygenation caused enhancement of the amplitude but not the frequency of SBC compared with that before reoxygenation. Vibegron (0.3-30 µM) inhibited this increase in SBC amplitude, but not the frequency, in a dose-dependent manner. The inhibitory effect of vibegron was not affected by pretreatment with the adenylyl cyclase inhibitor SQ22536 (100 µM) or protein kinase A inhibitor KT5720 (1 µM) and was not accompanied by considerable changes in cyclic adenosine monophosphate (cAMP) content in the bladder. In contrast, the large conductance potassium channel inhibitor iberiotoxin (100 nM) suppressed the inhibitory effect of vibegron. These results suggest that bladder ischemia/reperfusion induces SBC enhancement and vibegron directly inhibits detrusor overactivity via the large conductance potassium channel, which involves ß3-adrenoceptor, rather than the cAMP signaling pathway.

BoNT/A1 Secondary Failure for the Treatment of Neurogenic Detrusor Overactivity: An Ex Vivo Functional Study.

Management of neurogenic detrusor overactivity (NDO) remains a clinical priority to improve patients' quality of life and prevent dramatic urological complications. Intradetrusor injection of onabotulinumtoxinA (BoNT/A1, botulinum neurotoxin A1) is approved as second therapeutic line in these patients, demonstrating a good efficacy. However, a loss of its efficacy over time has been described, with no clear understanding of the underlying mechanisms. This paper aims at shedding new light on BoNT/A1 secondary failure in NDO through functional and structural analysis. Three groups of patients (either non-NDO, NDO with no toxin history or toxin secondary failure) were investigated using an ex vivo bladder strip assay. Detrusor strips were tensed in organ baths and submitted to electrical field stimulation to generate contractions. Recombinant BoNT/A1 was then added at various concentrations and contractions recorded for 4 h. Histology exploring BoNT/A1 targets, fibrosis and neuronal markers was also used. Detrusor strips from patients with BoNT/A1 secondary failure displayed a smaller sensitivity to toxin ex vivo at 3 nM compared to the other groups. Histological evaluation demonstrated the presence of cleaved Synaptosomal-Associated Protein, 25 kDa (c-SNAP25) in the detrusor from the toxin-secondary failure population, indicating some remaining in vivo sensitivity to BoNT/A1 despite the therapeutic escape. Moreover, residual c-SNAP25 did not affect parasympathetic-driven contractions observed ex vivo. This study confirms the slightly lower efficacy of BoNT/A1 in the BoNT/A1 secondary failure NDO group, suggesting that the escape from BoNT/A1 efficacy in NDO occurs at least at the parasympathetic level and could imply compensatory mechanisms for detrusor contraction.

Proteome changes in human bladder T24 cells induced by hydroquinone derived from Arctostaphylos uva-ursi herbal preparation.

ETHNOPHARMACOLOGICAL RELEVANCE: Arctostaphylos uva-ursi (L.) Spreng. (bearberry) is a well-known traditional herbal plant used as a urinary tract disinfectant. Its antiseptic and diuretic properties can be attributed to hydroquinone, obtained by hydrolysis of arbutin. AIM OF THE STUDY: This study aimed to determine the toxic profile of free hydroquinone on urinary bladder cells (T24) as a target of therapeutic action. MATERIALS AND METHODS: Quantitative and qualitative analysis of the extract and the digestive stability and bioavailability of arbutin and hydroquinone were performed by HPLC assay and simulated in vitro digestion, respectively. Cytotoxic effect, reactive oxygen species induction and proteome changes in T24 cells after hydroquinone treatment were determined using Neutral red assay, 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay and mass spectrometry, respectively. RESULTS: Through in vitro digestion, arbutin was stable, but hydroquinone increased after pepsin treatment (109.6%) and then decreased after the small intestine phase (65.38%). The recommended doses of Uva-ursi had a cytotoxic effect on T24 cells only when all hydroquinone conjugates were converted to free hydroquinone (320 and 900 µg/mL) and the toxic effect was enhanced by recovery. One cup of the therapeutic dose had a prooxidative effect after 4 h of incubation. Shorter time of cell exposure (2 h) to hydroquinone did not have any impact on reactive oxygen species induction. Proteomic analysis found 17 significantly up-regulated proteins compared to control. Hydroquinone activated proteins related to oxidative stress response, stress-adaptive signalling, heat shock response and initiation of translation. CONCLUSIONS: Despite the therapeutic properties of bearberry, up-regulated T24 cell proteins are evidence that plant compounds, although from a natural source, may exhibit negative properties.

BoNT/A in the Urinary Bladder-More to the Story than Silencing of Cholinergic Nerves.

Botulinum neurotoxin (BoNT/A) is an FDA and NICE approved second-line treatment for overactive bladder (OAB) in patients either not responsive or intolerant to anti-cholinergic drugs. BoNT/A acts to weaken muscle contraction by blocking release of the neurotransmitter acetyl choline (ACh) at neuromuscular junctions. However, this biological activity does not easily explain all the observed effects in clinical and non-clinical studies. There are also conflicting reports of expression of the BoNT/A protein receptor, SV2, and intracellular target protein, SNAP-25, in the urothelium and bladder. This review presents the current evidence of BoNT/A's effect on bladder sensation, potential mechanisms by which it might exert these effects and discusses recent advances in understanding the action of BoNT in bladder tissue.

Chidamide and mitomycin C exert synergistic cytotoxic effects against bladder cancer cells in vitro and suppress tumor growth in a rat bladder cancer model.

Intravesical instillation (IVI) of Bacillus Calmette-Guerin (BCG) can prevent bladder cancer recurrence, but this agent has been out of stock in recent years. IVI of other agents, like chidamide, a histone deacetylase (HDAC) inhibitor, may have the potential to exert a therapeutic effect against bladder cancer by modifying the gene expression profiles associated with histone modifications that occur during cancer tumorigenesis. Here, we investigated the in vitro therapeutic effect of chidamide and/or mitomycin C in bladder cancer cell lines and screened related molecular pathways using an antibody array. We also quantitatively analyzed the synergistic effect of IVI of chidamide and mitomycin C in vivo in an N-methyl-N-nitrosourea (MNU)-induced rat bladder cancer model. The synergistic cytotoxic effect of chidamide plus mitomycin C was confirmed in both T24 and UMUC3 cells, with significantly greater induction of apoptosis elicited with chidamide plus mitomycin C than with either drug alone. The antibody array identified the Axl signaling pathway as the key target of the synergistic effect. Expression of Axl and its related downstream molecules, including claspin and survivin, was significantly suppressed. In the rat bladder cancer model, IVI of chidamide plus mitomycin C reduced tumor burden (Ki67 index) to a greater extent than either drug alone. Our results suggest that chidamide and mitomycin act synergistically to reduce MNU-induced bladder cancer. These findings provide new insights into a new and potentially effective approach to treating bladder cancer.

Histamine receptors rapidly desensitize without altering nerve-evoked contractions in murine urinary bladder smooth muscle.

Histamine has been implicated in urinary bladder dysfunction as an inflammatory mediator driving sensory nerve hypersensitivity. However, the direct influence of histamine on smooth muscle has not been thoroughly investigated. We hypothesized that histamine directly contracts urinary bladder smooth muscle (UBSM) independent of effects on nerves. Single cell quantitative RT-PCR determined that only histamine H1 and H2 receptors were expressed on UBSM cells. In isolated tissue bath experiments, histamine (200 µM) caused a highly variable and rapidly desensitizing contraction that was completely abolished by the H1 receptor antagonist fexofenadine (5 µM) and the Gq/11 inhibitor YM254890 (1 µM). Neither the muscarinic receptor antagonist atropine (1 µM), the Na+ channel blocker tetrodotoxin (1 µM), nor the transient receptor potential vanilloid type 1 antagonist capsazepine (10 µM) altered responses to histamine, suggesting that nerve activation was not involved. UBSM desensitization to histamine was not due to receptor internalization, as neither the cholesterol-depleting agent methyl-ß-cyclodextrin (10 mM), the dynamin-mediated endocytosis inhibitor dynasore (100 µM), nor the clathrin-mediated endocytosis inhibitor pitstop2 (15 µM) augmented or prolonged histamine contractions. Buffer from desensitized tissues still contracted histamine-naïve tissues, revealing that histamine was not metabolized. Prolonged exposure to histamine also had no effect on contractions due to electrical field stimulation, suggesting that both efferent nerve and UBSM excitability were unchanged. Together, these data suggest that histamine, although able to transiently contract UBSM, does not have a lasting effect on UBSM excitability or responses to efferent nerve input. Thus, any acute effects of histamine directly on UBSM contractility are unlikely to alter urinary bladder function.NEW & NOTEWORTHY Histamine is commonly associated with inflammatory bladder pathologies. We sought to investigate the role of histamine on urinary bladder contractility. Histamine contracts the bladder, but this response is highly variable and desensitizes completely in minutes. This desensitization is not due to internalization of the receptor or metabolism of histamine. Because nerve-evoked contractions are also not increased in the presence of histamine, our findings suggest that histamine is not directly acting to change contractility.

Trypsin-induced elevated contractile responses in a rat model of interstitial cystitis/bladder pain syndrome: Involvement of PAR2 and intracellular Ca2+ release pathways.

AIMS: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disease with unclear etiology. Different receptors play a role in the pathophysiology including protease activated receptors (PARs). The present study aimed to investigate the subtypes and the effects of PARs on contractility using permeabilized detrusor smooth muscle strips in IC/BPS. MAIN METHODS: IC/BPS was induced by cyclophosphamide injection. Histopathological analysis, PCR for detecting PAR proteins, western blotting for indicating PAR2 protein expression levels and myograph recording for measuring contractile force were used. KEY FINDINGS: The present study reveals that in rat bladder PAR1 and PAR2 but not PAR4 were found to be expressed. The first evidence was revealed where trypsin-induced contractions in rat permeabilized detrusor were potentiated in CYP-induced cystitis. Moreover, the functional inhibition of trypsin-induced contractions by selective PAR2 antagonist (ENMD-1068) and the supporting immunoblotting results emphasized that the main PAR subtype involved in IC/BPS model in rat bladder is PAR2. Our data emphasize the prominent role of IP3 in cystitis pathology besides ryanodine channels. Trypsin-induced Ca2+sensitization contractions were also higher in cystitis. Both Rho kinase and protein kinase C played a role in this increased Ca2+sensitization situation. SIGNIFICANCE: The present paper highlights the intracellular pathways that are involved in trypsin-induced contractions mainly via PAR2 in permeabilized bladder detrusor smooth muscle in a rat model of IC/BPS.

The inhibitory effect of trimetazidine on detrusor contractility - a potential repositioning of trimetazidine for the treatment of overactive bladder.

OBJECTIVES: This study aimed to identify the effect of trimetazidine (TMZ), an antianginal drug, on detrusor smooth muscle (DSM) contractility and its possible mechanisms of action. METHODS: We performed in-vitro contractility studies on isolated mouse DSM strips and investigated the effect of TMZ on Ca2+ levels in fura-2-loaded A7r5 cells. KEY FINDINGS: TMZ (300 or 1000 µM) inhibited carbachol (CCh)- and KCl-induced contractions and produced a concentration-dependent (10-1000 µM) relaxation in KCl-precontracted DSM strips. TMZ-induced relaxation was markedly decreased by BaCl2, an inward-rectifying K+ channel blocker, but was not altered by preincubation with tetraethylammonium, glibenclamide, 4-aminopyridine, propranolol, L-NAME or methylene blue. TMZ (300 or 1000 µM) reduced both the CaCl2-induced contraction of depolarized DSM strips under Ca2+-free conditions and the CCh-induced contraction of DSM strips preincubated with nifedipine in Ca2+-containing Krebs solution. Furthermore, TMZ (1000 µM) significantly decreased the Ca2+ levels in fura-2-loaded A7r5 cells. CONCLUSIONS: TMZ decreased DSM contractility and caused a concentration-dependent relaxation of the tissue possibly through its actions on Ca2+ transients and K+ channels. Our results provide preclinical evidence that TMZ would be a potential candidate to treat disorders related to the overactivity of the bladder.

Validation of Fenoterol to Study ß2-Adrenoceptor Function in the Rat Urinary Bladder.

Fenoterol is a ß2-adrenoceptor (AR)-selective agonist that is commonly used to investigate relaxation responses mediated by ß2-AR in smooth muscle preparations. Some data have questioned this because fenoterol had low potency in the rat urinary bladder when a muscarinic agonist was used as a pre-contraction agent and because some investigators proposed that fenoterol may act in part via ß3-AR. We designed the present study to investigate whether fenoterol is a proper pharmacological tool to study ß2-AR-mediated relaxation responses in the rat urinary bladder. Firstly, we have compared the effect of pre-contraction agents on fenoterol potency and found that fenoterol potency was about 1.5 log units greater against KCl than carbachol (pEC50 7.19 ± 0.66 and 5.62 ± 1.09 of KCl and of carbachol, respectively). To test the selectivity of fenoterol, we have determined the effects of the ß2-AR antagonist ICI 118,551 and the ß3-AR antagonist L 748,337 on relaxation responses to fenoterol. While 300 nM L 748,337 had little effect on the potency of fenoterol (pEC50 6.56 ± 0.25 and 6.33 ± 0.61 in the absence and presence of L 748,337, respectively), the relaxation curve for fenoterol was right-shifted in the presence 300 nM ICI 118,551 (pEC50 5.03 ± 0.18). Thus, we conclude that fenoterol is a proper pharmacological tool to assess ß2-AR-mediated responses in the rat urinary bladder and most likely in other smooth-muscle preparations containing multiple subtypes of the ß-AR.