H2O2 enhances the spontaneous phasic contractions of isolated human-bladder strips via activation of TRPA1 channels on sensory nerves and the release of substance P and PGE2.

Several studies have indicated that reactive oxygen species (ROS) can lead to detrusor overactivity (DO), but the underlying mechanisms are not known. Hydrogen dioxide (H2O2) is used commonly to investigate the effects of ROS. In present study, we investigated the effects of H2O2 on phasic spontaneous bladder contractions (SBCs) of isolated human-bladder strips (iHBSs) and the underlying mechanisms. Samples of bladder tissue were obtained from 26 patients undergoing cystectomy owing to bladder cancer. SBCs of iHBSs were recorded in organ-bath experiments. H2O2 (1µM-10mM) concentration-dependently increased the SBCs of iHBSs. These enhancing effects could be mimicked by an agonist of transient receptor potential (TRP)A1 channels (allyl isothiocyanate) and blocked with an antagonist of TRPA1 channels (HC030031; 10 µM). H2O2 induced enhancing effects also could be attenuated by desensitizing sensory afferents with capsaicin (10 µM), blocking nerve firing with TTX (1 µM), blocking neurokinin effects with NK2 receptor antagonist (SR48968, 10 µM), and blocking PGE2 synthesis with indomethacin (10 µM), respectively. Our study: (i) suggests activation of TRPA1 channels on bladder sensory afferents, and then release of substance P or PGE2 from sensory nerve terminals, contribute to the H2O2-induced enhancing effects on SBCs of iHBSs; (ii) provides insights for the mechanisms underlying ROS leading to DO; (iii) indicates that targeting TRPA1 channels might be the promising strategy against overactive bladder in conditions associated with excessive production of ROS.

Activation of TRPA1 in Bladder Suburothelial Myofibroblasts Counteracts TGF-ß1-Induced Fibrotic Changes.

The activation of the transient receptor potential ankyrin 1 (TRPA1) channel has anti-fibrotic effects in the lung and intestine. Suburothelial myofibroblasts (subu-MyoFBs), a specialized subset of fibroblasts in the bladder, are known to express TRPA1. However, the role of the TRPA1 in the development of bladder fibrosis remains elusive. In this study, we use the transforming growth factor-ß1 (TGF-ß1) to induce fibrotic changes in subu-MyoFBs and assess the consequences of TRPA1 activation utilizing RT-qPCR, western blotting, and immunocytochemistry. TGF-ß1 stimulation increased α-SMA, collagen type I alpha 1 chain(col1A1), collagen type III (col III), and fibronectin expression, while simultaneously suppressing TRPA1 in cultured human subu-MyoFBs. The activation of TRPA1, with its specific agonist allylisothiocyanate (AITC), inhibited TGF-ß1-induced fibrotic changes, and part of these inhibition effects could be reversed by the TRPA1 antagonist, HC030031, or by reducing TRPA1 expression via RNA interference. Furthermore, AITC reduced spinal cord injury-induced fibrotic bladder changes in a rat model. The increased expression of TGF-ß1, α-SMA, col1A1 and col III, and fibronectin, and the downregulation of TRPA1, were also detected in the mucosa of fibrotic human bladders. These findings suggest that TRPA1 plays a pivotal role in bladder fibrosis, and the negative cross talk between TRPA1 and TGF-ß1 signaling may represent one of the mechanisms underlying fibrotic bladder lesions.

Piezo2 Channel Upregulation is Involved in Mechanical Allodynia in CYP-Induced Cystitis Rats.

Mechanical sensing Piezo2 channel in primary sensory neurons has been shown contribute to mechanical allodynia in somatic chronic pain conditions. Interstitial cystitis (IC)-associated pain is often triggered by bladder filling, a presentation that mimics the mechanical allodynia. In the present study, we aimed to examine the involvement of sensory Piezo2 channel in IC-associated mechanical allodynia using a commonly employed cyclophosphamide (CYP)-induced IC model rat. Piezo2 channels in dorsal root ganglia (DRGs) was knocked down by intrathecal injections of Piezo2 anti-sense oligodeoxynucleotides (ODNs) in CYP-induced cystitis rats, and mechanical stimulation-evoked referred bladder pain was measured in the lower abdomen overlying the bladder using von Frey filaments. Piezo2 expression at the mRNA, protein, and functional levels in DRG neurons innervating the bladder was detected by RNA-fluorescence in situ hybridization, western blotting, immunofluorescence, and Ca2+ imaging, respectively. We found that Piezo2 channels were expressed on most (> 90%) of the bladder primary afferents, including afferents that express CGRP, TRPV1 and stained with isolectin B4. CYP-induced cystitis was associated with Piezo2 upregulation in bladder afferent neurons at the mRNA, protein, and functional levels. Knockdown of Piezo2 expression in DRG neurons significantly suppressed mechanical stimulation-evoked referred bladder pain as well as bladder hyperactivity in CYP rats compared to CYP rats treated with mismatched ODNs. Our results suggest upregulation of Piezo2 channels is involved in the development of bladder mechanical allodynia and bladder hyperactivity in CYP-induced cystitis. Targeting Piezo2 might be an attractive therapeutic approach for IC-related bladder pain.

The enhancing effect of 5-HT on phasic contractions of human isolated distal ureter and the mechanisms mediating these effects.

5-Hydroxytryptamine (5-HT) can enhance human ureteral contractions. However, the mediating receptors have not been clarified. This study sought to further characterize the mediating receptors using several selective antagonists and agonists. Human distal ureters were obtained from 96 patients undergoing cystectomy. The mRNA expression levels of 5-HT receptors were examined using RT-qPCR experiments. The phasic contractions of ureter strips, either spontaneous or evoked with neurokinin, were recorded in an organ bath. Among the 13 5-HT receptors, 5-HT2A and 5-HT2C receptors showed the highest mRNA expression levels. 5-HT (10-7-10-4 M) increased the frequency and baseline tension of phasic contractions in a concentration-dependent manner. However, a desensitization effect was observed. The 5-HT2C receptor selective antagonist, SB242084 (10,30,100 nM), shifted the 5-HT concentration-response curves (frequency and baseline tension) rightward with a pA2 value of 8.05 and 7.75, respectively. 5-HT2C receptor selective agonist, vabicaserin, increased contraction frequency with an Emax of 35% of 5-HT. 5-HT2A receptor selective antagonist, volinanserin (1,10,100 nM), only reduced baseline tension with a pA2 of 8.18. The selective antagonists of 5-HT1A, 1B, 1D, 2B, 3, 4, 5, 6, and 7 had no antagonism. Blockade of voltage-gated sodium channels, α1-adrenergic receptors, adrenergic neurotransmission, and neurokinin-2 receptors using tetrodotoxin, tamsulosin, guanethidine, and Men10376, respectively, and desensitization of sensory afferents using capsaicin (100 µM), significantly reduced 5-HT effects. We conclude that 5-HT enhanced ureteral phasic contractions mainly by activating 5-HT2C and 5-HT2A receptors. Sympathetic nerve and sensory afferents partly contributed to 5-HT effects. 5-HT2C and 5-HT2A receptors could be promising targets for ureteral stone expulsion.

Upregulation of transient receptor potential cation channel subfamily M member-3 in bladder afferents is involved in chronic pain in cyclophosphamide-induced cystitis.

ABSTRACT: The transient receptor potential cation channel subfamily M member-3 (TRPM3) channel is a recently recognized noxious heat sensor that is involved in inflammatory thermal hyperalgesia. To examine its involvement in the development of hyperalgesia in interstitial cystitis/painful bladder syndrome (IC/PBS), rats with cyclophosphamide (CYP)-induced chronic cystitis were used as a model of IC/PBS. Mechanical and thermal hyperalgesia in lower abdominal region overlying the bladder in CYP rats were measured using von Frey filaments and radiant heat, respectively. Transient receptor potential cation channel subfamily M member-3 expression at the mRNA, protein, and functional levels in dorsal root ganglion neurons innervating the bladder was detected using RNA in situ hybridization (RNAscope), Western blotting, immunohistochemistry, and Ca 2+ imaging, respectively. Transient receptor potential cation channel subfamily M member-3 channels were expressed on most of the bladder primary afferent nerve terminals containing calcitonin gene-related peptide and their cell bodies in L6-S1 dorsal root ganglion. Activation of TRPM3 in the bladder wall by its specific agonist pregnenolone sulphate or CIM0216 induced spontaneous bladder pain, calcitonin gene-related peptide release, and neurogenic inflammation that was evidenced by edema, plasma extravasation, inflammatory cell accumulation, and mast cell infiltration. In CYP rats, pretreatment with the TRPM3 antagonist primidone (2 mg/kg, i.p.) significantly alleviated the mechanical and thermal hyperalgesia, bladder submucosal edema, mast cell infiltration, and bladder hyperactivity. Cyclophosphamide-induced cystitis was associated with TRPM3 upregulation at the mRNA, protein, and functional levels in bladder afferent neurons. Our results suggest that upregulation of TRPM3 channels is involved in the development of chronic pain in CYP-induced cystitis, and targeting TRPM3 may be a pharmacological strategy for treating bladder pain in IC/PBS.

Cell Deformation at the Air-Liquid Interface Evokes Intracellular Ca2+ Increase and ATP Release in Cultured Rat Urothelial Cells.

Urothelial cells have been implicated in bladder mechanosensory transduction, and thus, initiation of the micturition reflex. Cell deformation caused by tension forces at an air-liquid interface (ALI) can induce an increase in intracellular Ca2+ concentration ([Ca2+]i) and ATP release in some epithelial cells. In this study, we aimed to examine the cellular mechanisms underlying ALI-induced [Ca2+]i increase in cultured urothelial cells. The ALI was created by stopping the influx of the perfusion but maintaining efflux. The [Ca2+]i increase was measured using the Ca2+ imaging method. The ALI evoked a reversible [Ca2+]i increase and ATP release in urothelial cells, which was almost abolished by GdCl3. The specific antagonist of the transient receptor potential vanilloid (TRPV4) channel (HC0674) and the antagonist of the pannexin 1 channel (10panx) both diminished the [Ca2+]i increase. The blocker of Ca2+-ATPase pumps on the endoplasmic reticulum (thapsigargin), the IP3 receptor antagonist (Xest-C), and the ryanodine receptor antagonist (ryanodine) all attenuated the [Ca2+]i increase. Degrading extracellular ATP with apyrase or blocking ATP receptors (P2X or P2Y) with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) significantly attenuated the [Ca2+]i increase. Our results suggest that both Ca2+ influx via TRPV4 or pannexin 1 and Ca2+ release from intracellular Ca2+ stores via IP3 or ryanodine receptors contribute to the mechanical responses of urothelial cells. The release of ATP further enhances the [Ca2+]i increase by activating P2X and P2Y receptors via autocrine or paracrine mechanisms.

Age-related reductions in the excitability of phasic dorsal root ganglion neurons innervating the urinary bladder in female rats.

Previous studies have revealed an impairment in bladder sensory transduction in aged animals. To examine the contributions of electrical property changes of bladder primary afferents to this impairment, we compared the electrical properties of dorsal root ganglion (DRG) neurons innervating the bladder among young (3 months), middle-aged (12 months), and old (24 months) female rats. The DRG neurons were labeled using axonal tracing techniques. Whole-cell current-clamp recordings of small and medium-sized neurons were performed to assess their passive and active properties. Two patterns of firing were identified based on responses to super-threshold stimuli (1.5, 2.0, 2.5, and 3.0 × rheobase): tonic neurons fired more action potentials (APs), whereas phasic neurons fired only one AP at the onset of stimulus. Tonic neurons were smaller and had a slower rate of AP rise, longer AP duration, more depolarized voltage threshold, and greater rheobase than phasic neurons. In phasic neurons, there was an age-associated increase in voltage threshold and an increase of rheobase (P < 0.05), suggesting an age-related decrease in excitability. In addition, both middle-aged and old rats had longer AP durations and slower rates of AP rise than young rats (P < 0.05). In tonic neurons, old rats had a greater AP overshoot and greater rate of AP rise, but no age-associated changes were identified in any other electrical properties. Our results suggest that the electrical properties of tonic and phasic bladder afferents are differentially altered with aging. A decrease in excitability may contribute to age-related reductions in bladder sensory function.

Functional Expression of Transient Receptor Potential and Piezo1 Channels in Cultured Interstitial Cells of Human-Bladder Lamina Propria.

The interstitial cells in bladder lamina propria (LP-ICs) are believed to be involved in sensing/afferent signaling in bladder mucosa. Transient receptor potential (TRP) cation channels act as mechano- or chemo-sensors and may underlie some of the sensing function of bladder LP-ICs. We aimed to investigate the molecular and functional expression of TRP channels implicated in bladder sensory function and Piezo1/Piezo2 channels in cultured LP-ICs of the human bladder. Bladder tissues were obtained from patients undergoing cystectomy. LP-ICs were isolated and cultured, and used for real-time reverse transcription-quantitative polymerase chain reaction, immunocytochemistry, and calcium-imaging experiments. At the mRNA level, TRPA1, TRPV2, and Piezo1 were expressed most abundantly. Immunocytochemical staining showed protein expression of TRPA1, TRPV1, TRPV2, TRPV4, TRPM8, as well as Piezo1 and Piezo2. Calcium imaging using channel agonists/antagonists provided evidence for functional expression of TRPA1, TRPV2, TRPV4, Piezo1, but not of TRPV1 or TRPM8. Activation of these channels with their agonist resulted in release of adenosine triphosphate (ATP) from LP-ICs. Inhibition of TRPV2, TRPV4 and Piezo1 blocked the stretch induced intracellular Ca2+ increase. Whereas inhibition of TRPA1 blocked H2O2 evoked response in LP-ICs. Our results suggest LP-ICs of the bladder can perceive stretch or chemical stimuli via activation of TRPV2, TRPV4, Piezo1 and TRPA1 channels. LP-ICs may work together with urothelial cells for perception and transduction of mechanical or chemical signals in human-bladder mucosa.

Significance of piezo-type mechanosensitive ion channel component 2 in premature ejaculation: An animal study.

BACKGROUND: Penile hypersensitivity is one of the main pathological mechanisms of premature ejaculation. However, little is known about the neurophysiological mechanism of penile peripheral nerve sensitization. Piezo Type Mechanosensitive Ion Channel Component 2 (PIEZO2), was recently identified as a mechanically sensitive channel. OBJECTIVES: This study explored the possible neural mechanisms of PIEZO2 action in the mechanisms of premature ejaculation using molecular biology and electrophysiology approaches. MATERIALS AND METHODS: One hundred seventy male rats and 85 female rats were recruited. The females were induced estrus by injection of estradiol benzoate and progesterone followed by surgically castrated. Subsequently, the copulatory behaviors were record by a video camera six times, once a week. The last three mating processes of 134 male rats were successfully recorded. The males were divided into three groups according to ejaculation frequency value. Immunocytochemical and molecular methods as well as whole-cell patch clamp recording were used to show the difference between premature ejaculation rats and control rats. To further clarify the involvement of PIEZO2 in premature ejaculation, we constructed a PIEZO2 knockdown model in rats by intrathecal injection of PIEZO2 antisense oligodeoxynucleotides. RESULTS: We showed that PIEZO2 in the penis head and in the dorsal root ganglia(DRG) were significantly increased in premature ejaculation rats. Whole-cell patch clamp recording demonstrated that mechanical stimulation evoked a higher inward current density in premature ejaculation rats compared with control rats, which could be inhibited by the PIEZO2-specific antagonist, FM1-43. PIEZO2 knockdown experiments revealed that the inward current density induced by mechanical stimulation was significantly decreased in PIEZO2 knockdown rats, and that the mount frequency and ejaculation latency and frequency were significantly improved in PIEZO2 knockdown rats. DISCUSSION AND CONCLUSION: Our data demonstrate PIEZO2 involvement in peripheral nerve sensitization, indicating that pharmacological antagonism of PIEZO2 may be a useful strategy for treating premature ejaculation.

Reduced bladder responses to capsaicin and GSK-1016790A in retired-breeder female rats with diminished volume sensitivity.

Literature documents an age-related reduction of bladder sensory function. Transient receptor potential vanilloid (TRPV)1 or TRPV4 channels have been implicated in bladder mechanotransduction. To investigate contributions of TRPV1 or TRPV4 to the age-related reduction of bladder sensory function, bladder responses to capsaicin (CAP; TRPV1 agonist) and GSK-1016790A (GSK; TRPV4 agonist) in retired breeder (RB; 12-15 mo) and young adult (2-3 mo) female rats were compared using multiple methods. Metabolic cage and continuous infusion cystometry [cystometrogram (CMG)] recordings revealed that RB rats exhibit larger bladder capacity and lower voiding frequency. RB rats also have a greater intravesical pressure threshold for micturition; however, the voiding contraction strength was equivalent to that in young rats. CAP (1 µM) or GSK (20 nM) administered intravesically evoked smaller changes in all CMG parameters in RB rats. In vitro, CAP (1 µM) or GSK (20 nM) evoked smaller enhancement of bladder strip contractions, while the muscarinic receptor agonist carbachol (at 100, 300, and 1,000 nM) elicited greater amplitude contractions in RB rats. Patch-clamp recording revealed smaller CAP (100 nM) induced inward currents in bladder primary sensory neurons, and Ca2+ imaging revealed smaller GSK (20 nM) evoked increases in intracellular Ca2+ concentration in urothelial cells in RB rats. These results suggest that RB rats have a decreased bladder sensory function commonly observed in elderly women, and could be used as an animal model to study the underling mechanisms. Reduced functional expression of TRPV1 in bladder afferents or reduced functional expression of urothelial TRPV4 may be associated with the diminished sensory function.

Heavy metals contamination and accumulation in submerged macrophytes in an urban river in China.

Deteriorating urban water quality has attracted considerable attention in China. We investigated the contamination levels and distribution of heavy metals (As, Cd, Cu, Ni, Pb, and Zn) in Yuxi River water and sediments, and assessed the heavy metal accumulation capability of five species of submerged macrophytes: Vallisneria natans (Lour.) Hara, Potamogeton pectinatus L., Hydrilla verticillata (L. f.) Royle, Myriophyllum spicatum L., and Potamogeton crispus L. Samples were collected from upstream and downstream locations in different season. The results showed that the levels of heavy metals in the downstream areas were higher than in the upstream areas. Heavy metal concentrations in the river water during the dry seasons were higher than those during the rainy seasons, and the opposite results appeared in sediments and submerged macrophytes. In general, the river was slightly contaminated by heavy metals, and the concentrations of Pb and Ni in this river should serve as a warning, while Cd and Zn pollution in the sediments desperately needs to be removed. Furthermore, Potamogeton pectinatus L. showed a higher accumulation capacity for these metals among the five native submerged macrophytes and could be defined as a hyperaccumulator for Cd. Therefore, the potential use of native aquatic plants in contaminated rivers is worth further exploration.

Paris Polyphylla-Derived Saponins Inhibit Growth of Bladder Cancer Cells by Inducing Mutant P53 Degradation While Up-Regulating CDKN1A Expression.

OBJECTIVES: Paris polyphylla var. yunnanensis (PPVY), a Chinese herb, has long been used for cancer treatment, and its steroidal saponins are suggested to exert an anti-tumor activity, however, the underlying mechanism is incompletely understood and their effect on bladder cancer (BC) remains unknown. The present study is thus designed to address these issues. MATERIAL AND METHODS: Total steroidal saponins were extracted with ethanol from PPVY and used to treat BC cells (HT1197 and J82 carrying mutant p53). Gene expression was determined using qPCR and immunoblotting and cell cycle analyzed using flow cytometry. DNA damage response activation was assessed using immunofluorescence staining. RESULTS: PPVY saponins treatment led to dose-dependent declines in the number of both HT1197 and J82 cells with IC50 approximately 1.2 µg/ml, which was coupled with strong growth arrest at G2/M phase and the activation of DNA damage response pathway. Moreover, the clonogenic potential of these cells was severely impaired even in the presence of low concentrations of PPVY saponins. Mechanistically, PPVY saponins induced the degradation of mutant p53 while stimulated CDKN1A gene transcription. Phosphorylated AKT was diminished in PPVY saponin-treated cells, but its specific inhibitor LY294002 exhibited significantly weaker efficacy in inducing CDKN1A expression than did PPVY saponins. CONCLUSION: PPVY saponins activate DNA damage response pathway, degrade mutant p53 and stimulate CDKN1A expression, thereby inhibiting BC cell growth. Given their poor absorption via oral administration, PPVY saponins may be applicable for intravesical instillations in BC treatment.

Anti-Cancer Effects of Paris Polyphylla Ethanol Extract by Inducing Cancer Cell Apoptosis and Cycle Arrest in Prostate Cancer Cells.

OBJECTIVE: To evaluate the potential anti-prostate cancer effects of Paris polyphylla ethanol extract (PPEE) and its underlying mechanisms. MATERIALS AND METHODS: The anti-proliferation activity of PPEE was tested on PC3 and DU145 cells using Cell Counting Kit-8 assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by flow cytometry. Apoptosis of prostate cancer cells was induced by PPEE through endogenous and exogenous pathways. A mouse xenograft model was used to examine its anti-prostate cancer effects in vivo. RESULTS: We found that the IC50 of PPEE on PC3 cells was 3.98 µg/ml and the IC50 of PPEE on DU145 cells was 8 µg/ml. PPEE induced prostate cancer cell apoptosis in a concentration dependent manner, through endogenous and exogenous pathways. PPEE induced PC3 cell cycle arrest in G0/G1 and G2/M phases, while in DU145cell it induced cell arrest in the G0/G1 phase. PPEE inhibited the growth of prostate cancer cells in vivo. CONCLUSION: PPEE could inhibit prostate cancer growth in vitro and in vivo, induce apoptosis of prostate cancer cells, and cause cell cycle arrest, which laid the foundation for further research on the anti-tumor mechanism of PPEE.

Kidney Injury Molecule-1 Level is Associated with the Severity of Renal Interstitial Injury and Prognosis in Adult Henoch-Schönlein Purpura Nephritis.

BACKGROUND AND AIMS: Kidney injury molecule-1 (KIM-1) was identified the most highly upregulated protein in chronic kidney diseases and prolonged KIM-1 expression may be maladaptive. The present study was aimed to investigate urinary, renal and plasma KIM-1 levels and to analyze association between KIM-1 levels with clinical and pathological indexes in adult Henoch-Schönlein purpura (HSP) patients. METHODS: Twenty healthy individuals, 20 HSP patients without nephritis and 35 HSP patients with nephritis were recruited. Urinary and plasma KIM-1 levels were determined by ELISA and Luminex, respectively. Renal KIM-1 expression was evaluated by immunohistochemistry. RESULTS: HSP patients with nephritis were characterized as elevated levels of urinary, renal and plasma KIM-1. Those with more severe tubular injury of renal biopsy tissues presented significantly higher urinary and renal KIM-1 levels compared to control and patients without nephritis. Urinary and renal levels of KIM-1 were positively correlated with blood urea nitrogen and proteinuria, while they were negatively correlated with eGFR at both baseline and after two years follow-up. Moreover, plasma KIM-1 levels were associated with blood urea nitrogen and proteinuria as well. Further univariate correlation analysis indicated urinary and renal KIM-1 levels were positively correlated with interstitial inflammation index and tubulointerstitial chronicity index. Only urinary KIM-1 levels were associated with interstitial inflammation index, tubulointerstitial chronicity index and extracapillary glomerular activity index, after logistic regression analysis. The area under the curve (AUC) for urinary KIM-1/Cr predicting progression of renal damage was significantly greater than the AUC for proteinuria. CONCLUSIONS: This finding suggests that measurement of urinary and renal KIM-1 level may be helpful to evaluate severity of renal pathological damage and prognosis in adult HSP patients with nephritis.