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

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

The urothelium is a sensory structure that contributes to mechanosensation in the urinary bladder. Here, we provide evidence for a critical role for the Piezo1 channel, a newly identified mechanosensory molecule, in the mouse bladder urothelium. We performed a systematic analysis of the molecular and functional expression of Piezo1 channels in the urothelium. Immunofluorescence examination demonstrated abundant expression of Piezo1 in the mouse and human urothelium. Urothelial cells isolated from mice exhibited a Piezo1-dependent increase in cytosolic Ca 2؉ concentrations in response to mechanical stretch stimuli, leading to potent ATP release; this response was suppressed in Piezo1-knockdown cells. In addition, Piezo1 and TRPV4 distinguished different intensities of mechanical stimulus. Moreover, GsMTx4, an inhibitor of stretch-activated channels, attenuated the Ca 2؉ influx into urothelial cells and decreased ATP release from them upon stretch stimulation. These results suggest that Piezo1 senses extension of the bladder urothelium, leading to production of an ATP signal. Thus, inhibition of Piezo1 might provide a promising means of treating bladder dysfunction.
Bladder urothelium has classically been considered a passive barrier against water, ions, solutes, and infections. However, it is currently thought that urothelial cells are involved in sensory mechanisms in response to physical and chemical stimuli (1)(2)(3)(4) and that bladder urothelium can sense bladder extension and respond to stimuli by releasing neurotransmitters, including ATP (5,6). ATP released from the urothelium has an important role in acting on P2X receptors in afferent nerves to transmit signals of bladder filling (7). In this mechanosensory transduction system, the opening of stretch-activated channels (SACs) 3 is the first step, leading to various intracellular events. Most SACs are nonselective cation channels with high Ca 2ϩ permeability (8), indicating that SACs convert mechanical force into Ca 2ϩ influx. We hypothesized that SACs sense the extension of the bladder urothelium. The molecular candidates for SACs in mammals include two-pore domain K ϩ channels, acid-sensing ion channels (ASICs), and the transient receptor potential (TRP) family of channels (4, 9 -16).
Gating of the two-pore domain K ϩ channels (TREK1, TREK2, and TRAAK) is regulated by mechanical perturbation of the cell membrane as well as polyunsaturated fatty acids, other lipids, and temperature (17)(18)(19). ASICs were initially implicated in mechanotransduction because they are essential for perception of touch (20,21). On the other hand, TRPs sense thermal stimulation, and TRPV4 is involved in mechanosensation (22)(23)(24)(25). In the mouse bladder urothelium, urothelial cells sense mechanical stretch stimuli via TRPV4 channels, which induce Ca 2ϩ influx and contribute to ATP release upon extension (26). Although TRPs and ASICs are candidate mechanosensors of stretch stimulation, there is little unambiguous evidence indicating that these channels are directly mechanically gated. Moreover, these proteins do not fulfill all of the criteria for SACs. In other words, when expressed heterologously, none of these channels recapitulates the electrical signature of sensory mechanosensitive currents observed in their native environment (10). Thus, other mechanically activated channels in addition to TRPs and ASICs could also sense stretch stimuli in the bladder urothelium.
True SACs are conserved and present in bacteria, yeast, and plants (27). Although large conductance mechanosensitive channels function as SACs in bacteria (28), mammalian SACs have not been well described. Whereas TRPV4 and two-pore domain K ϩ channels are reportedly gated by mechanical stimuli (17,25,29,30), the mechanism through which the cell senses mechanical stimulation remains poorly understood. Recently, Coste et al. (31) reported a novel family of mechanically activated cation channels, consisting of Piezo1 and Piezo2 (also called Fam 38A and Fam 38B, respectively) in mammals. These channels have most of the properties of real SACs as described above. Piezo ion channels, first identified in the Neuro2A mouse cell line, are members of a new family of mechanosensitive ion channels found in higher eukaryotic cells. Moreover, they are associated with the physiological response to touch, pressure, and stretch. These channels are ϳ2500 amino acids long and contain 24 -32 transmembrane regions. It appears that they do not require any additional proteins for their opening, and therefore they could directly sense lipid membrane extension (32,33). Piezo1 currents are similar to those of Piezo2 but have quantitatively different kinetics and conductance. Piezo2 is inactivated more rapidly than Piezo1 and is present in somatosensory neurons. Piezo proteins are also expressed in the mouse lung, colon, and bladder (31). Therefore, we studied whether Piezo1 mediated stretch-evoked Ca 2ϩ influx and ATP release in mouse primary urothelial culture cells. We found that Piezo1 is present in the mouse and human bladder urothelium and has a functional role in stretch-evoked Ca 2ϩ influx and ATP release in mouse urothelial cells in vitro. Additionally, we examined differences between Piezo1 and TRPV4 channels, the latter of which is involved in mechanosensation in the urothelium (26). Finally, we examined the effects of GsMTx4, a peptide isolated from the venom of the tarantula spider. GsMTx4 is widely used to study mechanically activated channels (32,34,35) upon stretch stimulation of the urothelium because it inhibits Piezo1 (36).

EXPERIMENTAL PROCEDURES
Animals-Wild-type (C57BL/6Cr) mice and TRPV4-knockout mice (37) backcrossed on a C57BL/6Cr background were used. All experiments were performed with 8 -12-week-old male mice. All procedures were conducted in accordance with the Guiding Principles in the Care and Use of Animals in the Field of the Physiologic Society of Japan and the policies of the Institutional Animal Care and Use Committee of the University of Yamanashi (Chuo, Yamanashi, Japan).
Human Subjects-Bladder specimens were harvested from 29 patients with prostate cancer or benign prostatic hyperplasia who had undergone surgery at a single medical hospital (Kawahara Kidney-Urology Clinic, Kagoshima, Japan). This study was approved by the institutional review board of the Kawahara Kidney-Urology Clinic and was conducted according to the principles of the Declaration of Helsinki. All samples were collected with informed consent from the patients.
Preparation of Primary Urothelial Cell Cultures-Whole bladders were removed from anesthetized mice, and urothelial cells were prepared by methods described previously (26), with slight modifications. The bladder was incubated with papain (9.2 units/ml) for 25 min in a water bath at 37°C. The dissociated and isolated urothelial cells were seeded on elastic silicone chambers (STB-CH-04, STREX, Osaka, Japan) or 8-well chambers (Fisher) coated with fibronectin (AGC Techno Glass Co., Ltd., Shizuoka, Japan) and cultivated in urothelium-dedicated culture medium (CNT-16, CELLnTEC, Bern, Switzerland). All experiments with primary urothelial cell cultures were performed after the cells had formed clusters, which occurred after 72-84 h of cultivation.
Quantitative Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay-Quantitative real-time RT-PCR assays were performed as reported previously (38). The primer sequences used are shown in Table 1.
In Situ Hybridization Analysis-Mice were anesthetized, and the bladder was removed and frozen with OCT compound (Sakura Finetek, Tokyo, Japan). The frozen tissue was cut on a cryostat and mounted on glass slides. Slides were incubated in PBS with 4% paraformaldehyde. The mouse bladder sections were treated with anti-digoxigenin-AP Fab fragments (1:1000; Roche Applied Science) with blocking reagent. Sense and antisense RNA probes were created from the plasmid constructs of mouse Piezo1 cDNA inserted into pCR2.1 (Invitrogen) and were synthesized by using the digoxigenin RNA labeling kit (Roche Applied Science). Mouse Piezo1 plasmids were linearized with BamHI, and cRNA probes were transcribed from a fragment containing 499 bp (nucleotides 7409 -7908, NM_ 001037298). Digoxigenin labeling was confirmed by the dot blot method.
Immunofluorescence Staining-For immunohistochemistry, whole bladder samples excised from mouse and human specimens were embedded with tissue-TEK OCT compound and frozen in liquid nitrogen. Cryostat sections (10 m) mounted on glass slides were fixed with 4% paraformaldehyde. The samples were incubated with the primary antibody (rabbit anti-Piezo1 antibodies (Proteintech, Chicago, IL), 1:400) and then covered with the secondary antibody (Alexa Rb488 and Ms546, Molecular Probes, Inc.). For immunocytochemistry, primary urothelial cell cultures were seeded on fibronectin-coated 8-well chambers at a density of 5.0 ϫ 10 4 cells/well. Primary urothelial cell cultures were incubated with the primary antibodies (rabbit anti-Piezo1 antibodies, 1:100; mouse anti-cytokeratin-7 antibodies, 1:25) and subsequently incubated with the secondary antibodies (Alexa Rb488 and Ms546 (Molecular Probes, Inc.) and DAPI). Immunofluorescence images were obtained with a confocal laser microscope (Fluoview 1000, Olympus, Tokyo, Japan), as described previously (39). For the comparison of double-stained patterns, images were processed with Photoshop version 5 (Adobe Systems, Mountain View, CA). Piezo1 siRNA Treatment-siRNA against mouse Piezo1 was used as reported previously (target sequence, TCGGCGCTT-GCTAGAACTTCA) (31). Primary urothelial cells were prepared as described above; after 3 h of culture, urothelial cells were transfected with 10 nM siRNAs by using Lipofectamine RNAi MAX (Invitrogen). The culture medium was exchanged 48 h after transfection. The expression of mRNA was measured by quantitative real-time RT-PCR after 72 h of cultivation, and Western blotting was performed after 84 h of cultivation. Mechanical stretch experiments were performed after 72-84 h of cultivation.
Immunoblot Analysis-After transfection with control or Piezo1 siRNA, primary urothelial cells were lysed in radioimmunoprecipitation assay buffer (Takara, Ootsu, Japan), and lysates were subjected to SDS-PAGE on 7.5% gels by using a Power Station 1000VC system at 20 mA for 120 min. The membranes were incubated with mouse anti-Piezo1 antibodies (1:1000; Proteintech) and mouse anti-␤ actin antibodies (1:5000) diluted with Can Get Signal solution 1 (TOYOBO, Osaka, Japan). The proteins were visualized as bands by chemiluminescence (ECL Advance Western blotting Detection Kit, GE Life Sciences).
Direct Mechanical Cell Stretch Experiment and Hypotonicity Cell Swelling Examination-The mechanical stretch experiments were conducted as described previously (26). An elastic silicone chamber (STB-CH-04, STREX) was attached to two pieces of coverglass by an adhesive agent, in which a 1,000-mwide slit (from glass edge to edge) was formed in the center of the observation area. This customized design enabled only part of the chamber to be extended upon stretching. Chambers were attached to an extension device (modified version of STB-150, STREX) on the microscope stage. Stretch stimulation was applied using preset stretch speed and distance. A stretch distance of 100 -300 m theoretically induces 10 -30% elongation (strain) of the 1,000-m-wide slit in the silicone chamber, but the actual extents of cell elongation in the chamber were 9.2 Ϯ 0.7% at 100 m, 17.5 Ϯ 1.8% at 200 m, and 25.5 Ϯ 2.1% at 300 m. Upon comparing multiple speeds, we found that significant differences in the changes of intracellular Ca 2ϩ concentrations, [Ca 2ϩ ] i , were most clearly seen at a speed of 100 m/s.
In the hypotonicity-induced cell swelling examination, hypotonic swelling with 60% of normal osmolarity (340 mosM) was achieved by adding distilled water in equal volume to the bath solution. The isotonic solution was prepared by adding mannitol.
Measurement of Intracellular Ca 2ϩ Concentrations-Intracellular Ca 2ϩ concentrations were measured as reported previously (26). Primary urothelial culture cells were loaded with the fluorescent Ca 2ϩ indicator fura-2 AM (Invitrogen). The fura-2 fluorescence was measured in a standard bath solution containing 150 mM NaCl, 5 mM KCl, 1.8 mM CaCl 2 , 1.2 mM MgCl 2 , 25 mM HEPES, and 10 mM glucose, pH 7.4, adjusted with NaOH. Chemical stimulants were applied directly to the chamber. Ionomycin (5 M; Sigma-Aldrich) was applied at the final step in each experiment for normalization and to check cell viability.
[Ca 2ϩ ] i values were measured by ratiometric imaging with fura-2 at 340 and 380 nm, and the emitted light signal was read at 510 nm. F 340 /F 380 was acquired and calculated with an imaging processing system (Aquacosmos version 2.6, Hamamatsu Photonics, Hamamatsu, Japan). Changes in ratios (⌬) were calculated by subtracting basal values from peak values.
Photon Imaging of ATP Release-Photon imaging of ATP release was performed as reported previously (40). Stretch-induced ATP release from primary urothelial cell cultures was detected with a luciferin-luciferase bioluminescence assay (ATP bioluminescence assay kit CLS II, Roche Applied Science) and visualized with a VIM camera (C2400-35; Hamamatsu Photonics) by using an integration time of 30 s. The absolute ATP concentration was estimated by using a standard ATP solution (Roche Applied Science). The standard calibration curve yielded a correlation coefficient for bioluminescence versus ATP concentration of 0.9917 over a concentration range of 0 nM to 10.0 M. Data were imaged with Aquacosmos software (Hamamatsu Photonics) and analyzed with ImageJ 1.41 software (National Institutes of Health).
Whole-cell Patch Clamp Recording for HEK293 Cells Overexpressing TRPV4-Human embryonic kidney-derived 293 (HEK293) T cells were maintained in Dulbecco's modified Eagle's medium (WAKO Pure Chemical Industries, Ltd., Osaka, Japan), and cells were transfected with 1.0 g of mouse TRPV4 plasmid by using Lipofectamine Plus reagent (Invitrogen). Whole-cell patch clamp recordings were performed 24 h after transfection. HEK293 cells on coverslips were mounted in a chamber and superfused with the standard bath solution that was used in the Ca 2ϩ imaging experiments. The pipette solution contained 140 mM KCl, 5 mM EGTA, and 10 mM HEPES, pH 7.4. Data were sampled at 10 kHz and filtered at 4 kHz for analysis (Axon 700B amplifier with pCLAMP software, Axon Instruments, Molecular Devices, Tokyo, Japan). Membrane potential was clamped at Ϫ60 mV, and voltage ramp pulses from Ϫ100 to ϩ100 mV (500 ms) were applied every 5 s. GsMTx4 (100 l/min) was applied via another pipette close to the recorded cell. All experiments were performed at room temperature.
Statistical Analyses-The experimental results were expressed as means Ϯ S.E. The statistical significance of differences between two groups was determined by Student's t test. The statistical significance for multiple comparisons was determined by using the Tukey-Kramer analysis method. p values of Ͻ0.05 were considered statistically significant.

RESULTS
Expression of Piezo1 in the Mouse Urothelium-Piezo1 is highly expressed in the mouse lung tissue and the urinary bladder (31). To confirm the distribution of Piezo1 and Piezo2, we examined Piezo1 and Piezo2 mRNA expression levels in various organs, including the urinary tract tissues. Real-time RT-PCR analyses revealed abundant Piezo1 mRNA expression in the mouse urinary tract, especially in the bladder (Fig. 1A). To examine the localization of Piezo1 in the mouse bladder, we performed in situ hybridization. The Piezo1 signals were pre-dominantly found in the urothelial layer rather than the smooth muscle layer (Fig. 1B). In contrast, little Piezo2 expression was detected in the urinary tract tissues (Fig. 1A). Thus, we focused on Piezo1 expressed in the mouse urothelium.
Next, we performed immunofluorescence assays for Piezo1 in the mouse whole bladder sections and in primary cultured urothelial cells. Immunohistochemistry revealed that Piezo1like immunoreactivity was mainly observed in the urothelium, with less immunoreactivity in the stromal tissues, including the smooth muscle (Fig. 1C). Immunocytochemistry showed that Piezo1-like immunoreactivity occurred at the plasma membrane and cytoplasm near the nucleus, and the expression of cytokeratin-7 (CK-7), an intermediate filament protein present in all urothelial layers and considered to be an urothelial marker (41), overlapped well (Fig. 1D). The cytoplasmic Piezo1 signals were no longer observed after preincubation of the antibody with a blocking peptide, indicating the specificity of the Piezo1 signals detected by the antibody (Fig. 1D, far right).

Effects of siRNA-mediated Knockdown of Piezo1 on Primary
Urothelial Cultured Cells-To determine the involvement of Piezo1 in the responses to cell swelling or cell stretch stimulation, we treated primary urothelial cultured cells with a Piezo1specific siRNA. This intervention reduced Piezo1 mRNA levels by ϳ60%, whereas treatment with control siRNA did not ( Fig.  2A). Western blotting analyses revealed that Piezo1 siRNA treatment also suppressed Piezo1 protein expression (Fig. 2B).
Electrophysiological investigations have shown that mechanical stretch (application of positive or negative pressure to a patch membrane through a patch pipette) activates cationic currents in HEK293 cells overexpressing Piezo1 (31,42). Therefore, to determine whether cell swelling stimulation activates Piezo1 channels, we evaluated changes in cytosolic Ca 2ϩ concentrations ([Ca 2ϩ ] i ) upon exposure to hypotonic osmolarity in urothelial cells by using a Ca 2ϩ -imaging system. [Ca 2ϩ ] i increase upon hypotonic stimulus was significantly suppressed in Piezo1-knockdown (KD) cells with siRNA compared with that of cells treated with control siRNA (Fig. 2, C and D). These results clearly showed that mouse primary urothelial cell cultures retained functional Piezo1 expression.
Mechanical Stretch Stimulation Evokes Piezo1 Activation in Urothelial Cells-We previously reported that urothelial cells sense mechanical stretch stimuli by using a uniaxial cell extension system. Mechanical stretch stimulation caused Ca 2ϩ influx, leading to ATP release in mouse primary urothelial cell cultures (26). Therefore, we investigated whether Piezo1 is involved in [Ca 2ϩ ] i changes by direct mechanical stretch stimulation by using a cell extension device and a Ca 2ϩ -imaging system. Urothelial cells were cultivated on a stretch silicon chamber, and [Ca 2ϩ ] i changes in response to stretch stimulation were examined. Fig. 3A demonstrates representative [Ca 2ϩ ] i changes with pseudocolor images before and after stretch stimulation. Stretch-evoked [Ca 2ϩ ] i increases were significantly suppressed in Piezo1 siRNA-treated cells (Piezo1-KD cells) compared with control siRNA-treated cells (Fig. 3, A and  B). Stretch stimulation evoked [Ca 2ϩ ] i increases in a stretch distance-dependent manner in urothelial cells (Fig. 3C). Because stretch-evoked [Ca 2ϩ ] i increases started at a stretch distance of 100 m (actual extent of cell elongation rate, 9.2 Ϯ 0.7%) and were saturated at 400 m (cell extension rate, 30.5 Ϯ 2.1%), a stretch distance of 200 m (cell extension rate, 17.5% Ϯ 1.8%) was chosen for the cell extension system in this experiment. The stretch speed of the cell extension system was fixed at 100 m/s as explained under "Experimental Procedures." The average peak of [Ca 2ϩ ] i increase in response to stretch stimulation was significantly reduced in Piezo1-KD cells in the presence of extracellular Ca 2ϩ , and Ca 2ϩ responses were small in both cell types in the absence of extracellular Ca 2ϩ (Fig. 3D), suggesting that Ca 2ϩ influx through Piezo1 channels caused [Ca 2ϩ ] i increases upon mechanical stretch stimulation in primary urothelial cell cultures.
Stretch-induced ATP Release from Urothelial Cells via Piezo1-Bladder urothelium releases ATP upon stretch stimulation, which mediates signaling to primary afferent neurons via purinergic receptors (43,44). To determine whether activation of Piezo1 is required for the stretch-induced ATP release from urothelial cells, we measured the amount of extracellular ATP following mechanical cell stretch stimulation by using an ATP photon imaging system. Urothelial cells on a stretch chamber were extended by using the same conditions as in the Ca 2ϩimaging experiment (stretch distance, 200 m; stretch speed, 100 m/s). Upon stretch stimulation, prominent ATP release occurred in control cells, whereas ATP release from Piezo1-KD cells was significantly reduced (Fig. 4, A and B), which was consistent with the results of the Ca 2ϩ -imaging experiment (Fig.  3D). The amount of released ATP was suppressed by chelation of extracellular Ca 2ϩ , indicating that activation of Piezo1 and the resulting [Ca 2ϩ ] i increase were essential for ATP release upon stretch stimulation in primary urothelial cell cultures.
Differences in the Properties of the Piezo1 and TRPV4 Channels-The TRPV4 channel functions as a mechanosensor in mouse primary urothelial cell cultures (26,43). Therefore, we compared the functions of Piezo1 and TRPV4 channels by measuring stretch-evoked changes in [Ca 2ϩ ] i . To explore the functional role of these channels, we compared the stretch-  [Ca 2ϩ ] i increase was significantly suppressed in Piezo1-KD cells, TRPV4-KO cells, and TRPV4-KO/Piezo1-KD cells compared with control cells (Fig. 5A). Interestingly, when TRPV4-KO urothelial cells were treated with Piezo1 siRNA, the stretch-evoked increase in [Ca 2ϩ ] i was further suppressed to a level near that observed in the absence of extracellular Ca 2ϩ (Fig. 3D), suggesting that Piezo1 and TRPV4 contributed to the stretch-induced [Ca 2ϩ ] i increases in primary mouse urothelial cells. At a 100-m stretch length, the stretch-evoked [Ca 2ϩ ] i increases in Piezo1-KD cells and TRPV4-KO/Piezo1-KD cells were significantly lower than that in control cells, but the [Ca 2ϩ ] i increase in TRPV4-KO cells was not significantly different from that in control cells. In contrast, at a 300-m stretch length, stretch-evoked [Ca 2ϩ ] i increases were not significantly different among the four cell types, probably due to saturation of the mechanisms causing [Ca 2ϩ ] i increases. These results indicate that Piezo1 senses stretch stimulation over a wider range than the TRPV4 channel and that the sensitivity of Piezo1 to stretch stimulation is higher than that of TRPV4.
Next, we compared the amount of ATP release from each cell type upon stretch stimulation with the same conditions as in the Ca 2ϩ -imaging experiments. At a 200-m stretch length, stretch-induced ATP release from Piezo1-KD and TRPV4-KO/ Piezo1-KD cells was significantly reduced compared with control cells. At a 100-m stretch length, small amounts of ATP  were released from all cell types without significant differences, probably because [Ca 2ϩ ] i increases under these conditions were not sufficient to cause ATP releases. At a 300-m stretch length, large amounts of ATP were released from all the four cell types with no significant differences (Fig. 5B) similar to the [Ca 2ϩ ] i increase (Fig. 5A). Collectively, these results suggested that activation of both Piezo1 and TRPV4 and the resultant [Ca 2ϩ ] i increase critically contributed to ATP release upon stretch stimulation (at least under the 200-m stretch length) in primary urothelial cell cultures.
GsMTx4 Inhibited Piezo1 in Mouse Urothelial Cells-Bae et al. (36) previously reported that extracellular GsMTx4 (3-5 M) inhibited mechanical stimulus-evoked currents by ϳ80% in outside-out patches using HEK293 cells overexpressing Piezo1. Therefore, we evaluated the effects of GsMTx4 in stretch experiments, examining the dose-dependent effects of GsMTx4 in our urothelial cell stretch study. In control urothelial cells, GsMTx4 significantly attenuated the stretch-evoked [Ca 2ϩ ] i increase to a level observed in Piezo1-KD cells at concentrations greater than 10 M. In contrast, GsMTx4 failed to affect the stretch-evoked [Ca 2ϩ ] i increase in Piezo1-KD cells (Fig. 6A), suggesting that the GsMTx4-dependent component could be attributed to Piezo1. Stretch-induced ATP release was also inhibited by application of GsMTx4 in control urothelial cells (Fig. 6B). Taken together, these data suggested that inhibition of Piezo1 by GsMTx4 suppressed the resulting increase in [Ca 2ϩ ] i , and this suppression was essential to the reduction in ATP release upon stretch stimulation in primary urothelial  cell cultures. To rule out the possibility that GsMTx4 inhibits TRPV4 channel activity, we examined the [Ca 2ϩ ] i response to GSK1016790A (GSK), a TRPV4-selective agonist (45), in urothelial cells. GSK evoked a prominent increase in [Ca 2ϩ ] i in a dose-dependent manner, but the Ca 2ϩ response to GSK was not affected by GsMTx4 treatment at any of the tested GSK concentrations (Fig. 6C). Moreover, we performed a whole-cell patch clamp recording to examine whether GsMTx4 inhibits the GSK-evoked current in HEK293T cells expressing mouse TRPV4. In this experiment, we also confirmed that GsMTx4 did not inhibit TRPV4 activity, whereas HC067047, a specific TRPV4 inhibitor, almost completely blocked the TRPV4 activity (Fig. 6D).
Next, to determine whether the inhibitory effect of GsMTx4 was reversible, we performed a GsMTx4 wash-out study upon stretch stimulation. In the mechanical stretch experiment, the stretch-evoked [Ca 2ϩ ] i increase was relatively small in the presence of GsMTx4, and it became larger after wash-out of GsMTx4 (Fig. 6, E and F), approaching the level in the control cells (Fig. 5A). These results indicated that inhibition of Piezo1 by GsMTx4 was reversible upon mechanical stretch stimulation in urothelial cells.
Human Piezo1 Is Highly Expressed in the Human Bladder Urothelium-Finally, to confirm the expression of Piezo1 in the human bladder, we performed immunohistochemical analysis with human bladder specimens. We found that Piezo1-like immunoreactivity was mainly present in the urothelium layer (Fig. 7A), which is consistent with the findings in the mouse bladder (Fig. 1C). Moreover, we examined the expression of human PIEZO1 and human TRPV4 mRNAs in the human bladder urothelium by quantitative RT-PCR. Fig. 7B shows that PIEZO1 mRNA was much more abundant than TRPV4 mRNA in the human urothelium. These results suggested that Piezo1 might be more important than TRPV4 for detecting urothelial extension during bladder distention in humans.

DISCUSSION
The present study provides evidence that the Piezo1 channel is involved in mechanosensory transduction in the urinary bladder. First, we demonstrated that Piezo1 was predominantly present in the mouse urothelium, and Piezo1 expression was also confirmed in mouse primary urothelial cell cultures (Fig.  1). Second, we demonstrated that mechanical stretch stimulation of urothelial cells activated Piezo1, leading to [Ca 2ϩ ] i increases and ATP release in primary urothelial cell cultures . Third, we showed that GsMTx4 attenuated the stretch-evoked [Ca 2ϩ ] i increase and ATP release by urothelial cells (Fig. 6). Finally, we showed that Piezo1 was highly expressed in the human bladder urothelium (Fig. 7). These findings indicate that Piezo1 plays a crucial role in sensing mechanical force in the urothelium and participates in the mechanosensory-transduction pathway in the urinary bladder. To the best of our knowledge, this is the first study to reveal the expression of Piezo1 in the bladder urothelium and to describe the function of Piezo1 upon mechanical stretch stimulation in primary urothelial cell cultures.
In our immunocytochemical study, Piezo1 expression was detected in the plasma membrane and cytoplasm near the nucleus in mouse urothelial cell cultures (Fig. 1D), data that are consistent with previous reports (31,46). Piezo1 expression in the plasma membrane is consistent with its function as a SAC that mediates Ca 2ϩ influx.
We previously found that TRPV4 is responsible for sensing mechanical stretch and causing Ca 2ϩ entry into urothelial cells (26). Therefore, in the present study, we compared the functional roles of Piezo1 and TRPV4 channels between urothelial cells obtained from TRPV4-KO mice with and without Piezo1 siRNA treatment. At a 200-m stretch length, the stretchevoked [Ca 2ϩ ] i increase was strongly suppressed almost to control cell levels in the absence of extracellular Ca 2ϩ when Piezo1 mRNA was knocked down in TRPV4-KO cells (Figs. 3D and 5A). These findings indicate that Piezo1 and TRPV4 are activated independently and that the stretch-evoked Ca 2ϩ influx pathway is mediated mainly through Piezo1 and TRPV4 channels. In contrast, at a 100-m stretch length, stretch-evoked Ca 2ϩ influx in Piezo1-KD cells was significantly lower than that in control cells, whereas that in TRPV4-KO cells was not significantly different from that in control cells (Fig. 5A). These findings imply that urothelial cells might recognize and distinguish the different intensity of mechanical force by using different mechanosensors. The sensitivity of Piezo1 for stretch stimulation was higher than that of TRPV4, and Piezo1 might be a more sensitive mechanosensor than TRPV4. This heightened sensitivity might be related to the fact that Piezo1 does not require any other proteins for its activation and could be directly activated by membrane stretch (32,33,42). In contrast, TRPV4 interacts with the actin cytoskeleton (47) to form a mechanosensory complex (48) and is activated by an endogenous ligand, epoxyeicosatrienoic acid, a metabolite of arachidonic acid, although TRPV4 expressed in Xenopus oocytes has been reported to be directly activated by membrane stretch (25).
ATP released from urothelial cells depends on the increase in intracellular Ca 2ϩ concentrations (26,49) and plays an important role for signal transduction to afferent nerve endings; ATP regulates bladder functions, including the micturition reflex and nonvoiding contractions (44). In the present study, the amount of ATP released from the urothelium paralleled the changes in the cytosolic Ca 2ϩ concentrations at a 200-m stretch length. However, Ca 2ϩ influx in Piezo1-KD cells was significantly reduced compared with that in control cells and TRPV4-KO cells at a 100-m stretch length (Fig. 5A), whereas ATP released from urothelial cells was not significantly different among control cells, Piezo1-KD cells, and TRPV4-KO cells (Fig. 5B). These results could imply that the small increase in cytosolic Ca 2ϩ concentrations at a 100-m stretch length is not sufficient to cause ATP release. Two mechanisms for ATP release by epithelia have been proposed: a conductive release through ion channels (50,51) and an exocytotic release with ATP-enriched vesicles (52,53), which is regulated by intracellular Ca 2ϩ concentrations. Ca 2ϩ influx via Piezo1 might activate anion channels or connexin hemichannels (54) through which ATP is released. Alternatively, ATP could also be released through vesicular exocytosis by a PI3K-mediated pathway (55)(56)(57). Given that Piezo1 constitutes one of the Ca 2ϩ influx pathways, its activation could be critical for ATP release upon stretch stimulation, although the mechanism for Piezo1mediated ATP release remains unclear.
Piezo1 is inhibited by ruthenium red, a nonspecific inhibitor of many cation channels (31); this lack of specificity makes it difficult to use ruthenium red to identify the targets of Piezo1 (33). We used GsMTx4, a peptide isolated from tarantula venom that is a much more specific inhibitor of endogenous cationic SACs (35), to determine the involvement of Piezo1 activity in the stretch-evoked responses. It is likely that GsMTx4 inhibits Piezo1 by acting as a gating modifier that is inserted at the channel-lipid interface and prestresses the channel toward the closed conformation (36). We showed that the stretch-evoked [Ca 2ϩ ] i increase in control urothelial cells was significantly attenuated by GsMTx4 to the level of Piezo1-KD cells, suggesting that GsMTx4 acts on the Piezo1 channel more selectively than other SACs. We found that GsMTx4 inhibited Piezo1 channel activity and attenuated the stretch-evoked Ca 2ϩ influx and ATP release in urothelial cells, although GsMTx4 might have other targets.
We demonstrated that the effect of GsMTx4 on the stretchevoked [Ca 2ϩ ] i increase was reversible (Fig. 6, E and F) and that Piezo1 was expressed in both mouse and human urothelium (Fig. 7A). Those results suggest that GsMTx4 could be useful for the development of agents to treat diseases involving Piezo1, such as urine storage disorders. In the clinical setting, pathological conditions, such as overactive bladder and interstitial cystitis are considered to be correlated with urothelial pathogenesis (58 -60). It has recently been speculated that distension during urine storage stimulates urothelial cells to release various chemical mediators like ATP, prostaglandins, nitric oxide, and acetylcholine, which then transfer signals to afferent nerves or muscle cells. Some abnormalities in this pathway could cause overactive bladder and/or interstitial cystitis. We currently have no evidence showing the involvement of mechanical stretch stimulation in the release of other neurotransmitters (besides ATP), which needs to be explored in the future.
In summary, Piezo1 functions as a mechanosensor and plays an important role in the stretch-evoked Ca 2ϩ influx and ATP release in the mouse bladder urothelium. Moreover, GsMTx4 inhibits Piezo1 activity in the urothelium. Based on the findings of our current study, we suggest that Piezo1 is an important regulator of bladder function and that pharmacological inhibition of Piezo1 might improve urine storage disorders. It is possible that the specific inhibition of urothelial Piezo1 with compounds such as GsMTx4 might become a novel therapy for overactive bladder and/or interstitial cystitis.