Ectonucleotidase Activity in Smooth Muscle Tissues of Rats with a Valproate Model of Autism.

Ectonucleotidases play an important role in regulating the level of extracellular nucleotides and nucleosides and are an important part of the regulation of the effects of adenosine and ATP on adenosine and P2 receptors, respectively. We have previously established the ambiguous effect of P2 receptor agonists on the contractile activity of smooth muscle tissue in rats with the valproate model of autism. In this work, HPLC was used to evaluate the activity of ectonucleotidases in the smooth muscle tissues of the internal organs of rats with a valproate model of autism. The activity of ectonucleotidases was significantly higher in the smooth muscle tissues of the duodenum, vas deferens, and bladder, but lower in the ileum and uterus. The results obtained make it possible to compare the activity of ectonucleotidases identified here with changes in P2 receptor-mediated contractility of smooth muscle tissues revealed in our previous experiments.

Divergent histopathological and molecular patterns in chemically induced interstitial cystitis/bladder pain syndrome rat models.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a complex chronic pain disorder with an elusive etiology and nonspecific symptoms. Although numerous animal models with phenotypes similar to human disease have been established, no available regimen can consistently alleviate clinical symptoms. This dilemma led us to question whether current animal models adequately represent IC/BPS. We compared four commonly used IC/BPS rat models to determine their diverse histopathological and molecular patterns. Female rats were given single treatments with hydrochloric acid (HCL), acetic acid (AA), protamine sulfate plus lipopolysaccharide (PS + LPS), or cyclophosphamide (CYP) to induce IC/BPS. Bladder sections were stained for histopathologic evaluation, and mRNA expression profiles were examined using next-generation sequencing and gene set analyses. Mast cell counts were significantly higher in the HCL and AA groups than in the PS + LPS, CYP, and control groups, but only the AA group showed significant collagen accumulation. The models differed substantially in terms of their gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our observations suggest that none of these rat models fully reflects the complexity of IC/BPS. We recommend that future studies apply and compare multiple models simultaneously to fully replicate the complicated features of IC/BPS.

Lower urinary dysfunction as a long-term effect of childhood vincristine treatment, with potential influences by sex and dose.

Vincristine (VCR) is one of the most widely used chemotherapy agents in treating pediatric cancer. Nonetheless, it is known to cause dose-dependent neurotoxicity which can impact virtually every organ system. Despite its widespread use, the precise impact of VCR on the lower urinary tract (LUT) remains inadequately elucidated. Our initial clinical and translational investigations suggest a sex-specific influence of childhood VCR exposure on LUT function. Thus, the current study aimed to investigate the late effects of systemic VCR exposure on LUT physiology and the underlying mechanisms, focusing on dosage and male-sex, employing juvenile CD-1 mice as a model. Male mice subjected to VCR exhibited augmented functional bladder capacity accompanied by frequent non-void contractions during awake cystometry, alongside mast cell accumulation within the bladder, compared to the saline-treated control group. Noteworthy functional changes were observed in bladder strips from the VCR group, including decreased nerve-mediated contraction, heightened contractile responses to cholinergic and purinergic agonists, enhanced responsiveness to histamine-primarily via histamine receptor 1 (Hrh1)-and an augmented relaxation effect with compound 48/80 (a mast cell degranulator), relative to the control group. Significant changes in gene expression levels associated with neuroinflammation and nociception were observed in both the bladder and lumbosacral dorsal root ganglia (Ls-DRG) of the VCR group. These findings suggest that VCR exposure during childhood, particularly in males, triggers neuroimmune responses in the bladder and Ls-DRG, amplifying responsiveness to neurotransmitters in the bladder, thereby contributing to LUT dysfunction characterized by a mixed bladder phenotype as a late effect during survivorship.

Specialized pro-resolution mediators in the bladder: effects of resolvin E1 on diabetic bladder dysfunction in the type 1 diabetic male Akita mouse model.

BACKGROUND: One of the most common, but least studied, diabetic complication is diabetic bladder dysfunction. Current therapies include glucose control and symptom-based interventions. However, efficacy of these therapies is mixed and often have undesirable side effects. Diabetes is now known to be a chronic inflammatory disease. Specialized pro-resolving mediators are a class of compounds that promote the resolution of inflammation and have been shown to be effective in treating chronic inflammatory conditions. In this study we examine the ability of resolvin E1 to improve signs of diabetic bladder dysfunction. METHODS: Male Akita mice (Type 1 diabetic) develop hyperglycemia at 4 weeks and signs of bladder underactivity by 15 weeks. Starting at 15 weeks, mice were given one or two weeks of daily resolvin E1 and compared to age-matched wild type and untreated Akita mice. RESULTS: Resolvin E1 did not affect diabetic blood glucose after one week, although there was a slight decrease after two weeks. Diabetes decreased body weight and increased bladder weights and this was not affected by resolvin E1. Evan's blue dye extravasation (an indirect index of inflammation) was dramatically suppressed after one week of resolvin E1 treatment, but, surprisingly, had returned to diabetic levels after two weeks of treatment. Using cystometry, untreated Akita mice showed signs of underactivity (increased void volumes and intercontraction intervals). One week of resolvin E1treatment restored these cystometric findings back to control levels. After two weeks of treatment, cystometric changes were changed from controls but still significantly different from untreated levels, indicating a durable treatment effect even in the presence of increased inflammation at 2 weeks. CONCLUSIONS: Resolvin E1 has a beneficial effect on diabetic bladder dysfunction in the type 1 diabetic male Akita mouse model.

Pirfenidone improves voiding function by suppressing bladder fibrosis in underactive bladder rats.

Underactive bladder (UAB), characterized by a complex set of symptoms with few treatment options, can significantly reduce the quality of life of affected people. UAB is characterized by hyperplasia and fibrosis of the bladder wall as well as decreased bladder compliance. Pirfenidone is a powerful anti-fibrotic agent that inhibits the progression of fibrosis in people with idiopathic pulmonary fibrosis. In the current study, we evaluated the efficacy of pirfenidone in the treatment of bladder fibrosis in a UAB rat model. UAB was induced by crushing damage to nerve bundles in the major pelvic ganglion. Forty-two days after surgery, 1 mL distilled water containing pirfenidone (100, 300, or 500 mg/kg) was orally administered once every 2 days for a total of 10 times for 20 days to the rats in the pirfenidone-treated groups. Crushing damage to the nerve bundles caused voiding dysfunction, resulting in increased bladder weight and the level of fibrous related factors in the bladder, leading to UAB symptoms. Pirfenidone treatment improved urinary function, increased bladder weight and suppressed the expression of fibrosis factors. The results of this experiment suggest that pirfenidone can be used to ameliorate difficult-to-treat urological conditions such as bladder fibrosis. Therefore, pirfenidone treatment can be considered an option to improve voiding function in patient with incurable UAB.

Effects of oral administration of nonselective Trk inhibitor on bladder overactivity in rodent models of prostatic inflammation.

BACKGROUND: Our research focused on the assessment of the impact of systemic inhibition of Trk receptors, which bind to nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), on bladder hypersensitivity in two distinct rodent models of prostatic inflammation (PI). METHODS: Male Sprague-Dawley rats were divided into three groups (n = 6 each): the control group (no PI, vehicle administration), the untreated group (PI, vehicle administration), and the treated group (PI, nonselective Trk inhibitor, GNF 5837, administration). PI in rats was induced by a intraprostatic injection of 5% formalin. Posttreatment, we carried out conscious cystometry and a range of histological and molecular analyses. Moreover, the study additionally evaluated the effects of a nonselective Trk inhibitor on bladder overactivity in a mouse model of PI, which was induced by prostate epithelium-specific conditional deletion of E-cadherin. RESULTS: The rat model of PI showed upregulations of NGF and BDNF in both bladder and prostate tissues in association with bladder overactivity and inflammation in the ventral lobes of the prostate. GNF 5837 treatment effectively mitigated these PI-induced changes, along with reductions in TrkA, TrkB, TrkC, and TRPV1 mRNA expressions in L6-S1 dorsal root ganglia. Also, in the mouse PI model, GNF 5837 treatment similarly improved bladder overactivity. CONCLUSIONS: The findings of our study suggest that Trk receptor inhibition, which reduced bladder hypersensitivity and inflammatory responses in the prostate, along with a decrease in overexpression of Trk and TRPV1 receptors in sensory pathways, could be an effective treatment strategy for male lower urinary tract symptoms associated with PI and bladder overactivity.

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.

Urothelium-derived prostanoids enhance contractility of urinary bladder smooth muscle and stimulate bladder afferent nerve activity in the mouse.

The transitional epithelial cells (urothelium) that line the lumen of the urinary bladder form a barrier between potentially harmful pathogens, toxins, and other bladder contents and the inner layers of the bladder wall. The urothelium, however, is not simply a passive barrier, as it can produce signaling factors, such as ATP, nitric oxide, prostaglandins, and other prostanoids, that can modulate bladder function. We investigated whether substances produced by the urothelium could directly modulate the contractility of the underlying urinary bladder smooth muscle. Force was measured in isolated strips of mouse urinary bladder with the urothelium intact or denuded. Bladder strips developed spontaneous tone and phasic contractions. In urothelium-intact strips, basal tone, as well as the frequency and amplitude of phasic contractions, were 25%, 32%, and 338% higher than in urothelium-denuded strips, respectively. Basal tone and phasic contractility in urothelium-intact bladder strips were abolished by the cyclooxygenase (COX) inhibitor indomethacin (10 µM) or the voltage-dependent Ca2+ channel blocker diltiazem (50 µM), whereas blocking neuronal sodium channels with tetrodotoxin (1 µM) had no effect. These results suggest that prostanoids produced in the urothelium enhance smooth muscle tone and phasic contractions by activating voltage-dependent Ca2+ channels in the underlying bladder smooth muscle. We went on to demonstrate that blocking COX inhibits the generation of transient pressure events in isolated pressurized bladders and greatly attenuates the afferent nerve activity during bladder filling, suggesting that urothelial prostanoids may also play a role in sensory nerve signaling.NEW & NOTEWORTHY This paper provides evidence for the role of urothelial-derived prostanoids in maintaining tone in the urinary bladder during bladder filling, not only underscoring the role of the urothelium as more than a barrier but also contributing to active regulation of the urinary bladder. Furthermore, cyclooxygenase products greatly augment sensory nerve activity generated by bladder afferents during bladder filling and thus may play a role in perception of bladder fullness.

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.

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.

Predicting muscarinic receptor occupancy in human bladder mucosa from urinary concentrations of antimuscarinic agents for overactive bladder.

To assess the pharmacologically relevant and selective muscarinic receptor occupancy in the bladder mucosa, we considered not only plasma drug concentrations but also urinary drug concentrations. The purpose of this study was to predict muscarinic receptor occupancy in the human bladder mucosa based on urinary concentrations in response to clinical dosages of antimuscarinic agents used to treat overactive bladder. The calculated mean plasma or serum unbound steady state concentrations were 0.06-11 nM in clinical dosages of five antimuscarinic agents. Urinary concentrations calculated from the mean plasma or serum and renal clearance ranged between 19 nM and 2 µM, which were >10-fold higher than the Ki values for bladder muscarinic receptors excluding propiverine. Bladder mucosal muscarinic receptor occupancy estimated from the urinary concentrations and the Ki values was >90 % at a steady state in clinical dosages of five antimuscarinic agents. The bladder muscarinic receptor occupancy was higher than that in the parotid gland calculated based on the mean plasma or serum unbound concentrations and Ki values for muscarinic receptors in the parotid gland. These results suggest that sufficient and selective muscarinic receptor occupancy by antimuscarinic agents, to exert pharmacological effects, in the bladder mucosa can be predicted using urinary concentrations.

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.

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.

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.