Functional role for Piezo1 in stretch-evoked Ca²⁺ influx and ATP release in urothelial cell cultures

Abstract

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.

Keywords: ATP; Calcium; Ion Channel; Mechanotransduction; Transient Receptor Potential Channels (TRP Channels).

FIGURE 1.

Expression of Piezo1 mRNA and Piezo1 protein in mouse urothelium. A, quantitative real-time RT-PCR analysis of Piezo1 and Piezo2 mRNA expression in tissues and cells, including urinary tract organs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference gene. White bars and black bars, Piezo1 and Piezo2, respectively. BSM, bladder smooth muscle; DRG, dorsal root ganglion. Data are presented as means ± S.E. B, in situ hybridization (ISH) analysis of Piezo1 expression in mouse bladder. An arrowhead indicates the urothelial layer. SM, smooth muscle; L, lumen; scale bar, 500 μm. C, Piezo1-like immunoreactivity in the mouse bladder. Scale bars, 100 μm. D, expression of Piezo1 (green) and cytokeratin-7 (CK-7) (red), a urothelium marker, in cultured primary mouse urothelial cells. The far right panel indicates that no Piezo1-like immunoreactivity was detected in cells by an anti-Piezo1 antibody preincubated with antigenic peptide. Blue, DAPI. Scale bars, 20 μm.

FIGURE 2.

Effects of siRNA-mediated knockdown of Piezo1 on primary urothelial cultured cells. A, quantitative analysis of Piezo1 mRNA levels by real-time RT-PCR in control mouse primary-cultured urothelial cells (Naïve) and cells treated with control siRNA or Piezo1 siRNA. Data are presented as percentage of naive cells (n = 6). B, detection of Piezo1 protein by immunoblotting in lysates from mouse primary-cultured urothelial cells. Representative gel images (left) and analyzed data (right) are shown (Piezo1, 233 kDa; β-actin, 42 kDa). Data are presented as percentage of naive cells (n = 3). C, changes in [Ca²⁺]_i from control siRNA-treated urothelial cells and Piezo1-KD urothelial cells upon exposure from isotonic (340 mosm) to hypotonic (200 mosm) solution, as indicated by the fura-2 ratio with pseudocolor expression. Scale bars, 100 μm. D, average changes in [Ca²⁺]_i (difference between base and peak (Δ ratio)) by hypotonic stimulation (n = 12 experiments (233 cells) for control and 13 experiments (254 cells) for Piezo1-KD). All data are presented as means ± S.E. (error bars). *, p < 0.05; **, p < 0.01, Student's t test, as compared with the control.

FIGURE 3.

Ca²⁺ responses to mechanical stretch stimulation in mouse primary cultured urothelial cells. A, representative pseudocolor images of [Ca²⁺]_i increase upon stretch stimulation in control siRNA-treated urothelial cells (top) and Piezo1-KD cells (bottom). Stretch speed was fixed at 100 μm/s, and the distance was 200 μm. Cells were extended transversely (indicated by arrows) in both cell types. Ionomycin (5 μm) was applied after stretching. Scale bars, 100 μm. B, quantification of [Ca²⁺]_i changes (fura-2 ratio changes) in individual cells. All traces were obtained from the cells shown in A. Black arrowheads denote the onset of stretch. Gray bars, 5 μm ionomycin application. C, stretch distance-dependent [Ca²⁺]_i responses in mouse primary-cultured urothelial cells. n = 8 (151 cells), n = 8 (168 cells), n = 8 (166 cells), n = 7 (138 cells), and n = 7 experiments (124 cells) for 100-, 200-, 300-, 400-, and 500-μm extension, respectively. The stretch speed was fixed at 100 μm/s. Data are presented as means ± S.E. (error bars). D, stretch-evoked changes in fura-2 ratio normalized to the ratio changes by 5 μm ionomycin (percentage) in control siRNA-treated urothelial cells (Control) and Piezo1-KD cells (KD) in the presence (+) or absence (−) of extracellular Ca²⁺. Data are from eight experiments for each group with 168, 183, 173, and 150 cells for control Ca²⁺(+), Piezo1-KD Ca²⁺(+), Control Ca²⁺(−), and Piezo1-KD Ca²⁺(−), respectively. Data are presented as means ± S.E. **, p < 0.01, Tukey-Kramer method.

FIGURE 4.

Visualization of stretch-induced ATP release from urothelial cells. A, left panels show urothelial cells in phase-contrast images, and the middle and right panels show photon count images of control cells (Control) and Piezo1-KD cells, respectively. The top and bottom panels show the images before and after mechanical stretch, respectively. Stretch speed was 100 μm/s, and stretch distance was 200 μm. Cells were extended to the vertical axis (indicated by arrows). Scale bars, 100 μm. B, average amount of ATP released from control siRNA-treated urothelial cells (Control) and Piezo1-KD cells in the presence (+) or absence (−) of extracellular Ca²⁺. Data are from eight experiments for each group. Data are presented as means ± S.E. (error bars). *, p < 0.01, Tukey-Kramer method.

FIGURE 5.

Different functional properties between Piezo1 and TRPV4 channels. A, [Ca²⁺]_i responses (normalized to ionomycin responses) in mouse primary-cultured urothelial cells at different stretch distances (100, 200, and 300 μm) in control siRNA-treated urothelial cells obtained from wild type mice (Control), Piezo1 siRNA-treated cells from wild type mice (Piezo1-KD), control siRNA-treated urothelial cells from TRPV4 knock-out mice (TRPV4-KO) and Piezo1 siRNA-treated cells from TRPV4 knock-out mice (TRPV4-KO/Piezo1-KD). B, comparison of stretch-induced ATP release from mouse primary-cultured urothelial cells at different stretch distances (100, 200, and 300 μm) in control, Piezo1-KD, TRPV4-KO, and TRPV4-KO/Piezo1-KD cells. Data are from eight experiments for each group for both [Ca²⁺]_i responses and ATP release. Cell numbers examined for [Ca²⁺]_i responses were 151 cells (100 μm), 168 cells (200 μm), and 166 cells (300 μm) for control; 201 cells (100 μm), 183 cells (200 μm), and 187 cells (300 μm) for Piezo1-KD; 149 cells (100 μm), 122 cells (200 μm), and 202 cells (300 μm) for TRPV4-KO; and 160 cells (100 μm), 131 cells (200 μm), and 212 cells (300 μm) for TRPV4-KO/Piezo1-KD. All data are presented as means ± S.E. (error bars). *, p < 0.05; **, p < 0.01, Tukey-Kramer method.

FIGURE 6.

Inhibitory effects of GsMTx4 for Piezo1 in the urothelial cells. A, concentration-dependent inhibition of stretch-evoked [Ca²⁺]_i responses by GsMTx4 in control siRNA-treated urothelial cells (Control) and Piezo1-KD cells. The stretch speed and stretch distance were 100 μm/s and 200 μm. Data were from 7 (control/Piezo1-KD), 10/10, 12/10, and 10/10 experiments for 0, 1, 10, and 30 μm GsMTx4, respectively. Cell numbers were 140/152 (control/Piezo1-KD), 199/202, 233/201, and 178/188 for 0, 1, 10, and 30 μm, respectively. Data are presented as means ± S.E. (error bars). *, p < 0.05; **, p < 0.01, Student's t test. n.s., not significant. B, effects of GsMTx4 (10 μm) on the stretch-evoked ATP release from urothelial naive cells. The stretch distance was 200 μm. Data were from 13 and 14 experiments for GsMTx4(−) and GsMTx4(+), respectively, and are presented as percentage of control and means ± S.E. *, p < 0.05, Student's t test. C, GSK-evoked [Ca²⁺]_i increases (normalized to ionomycin responses) at different concentrations in urothelial cell cultures with (+) or without (−) GsMTx4. Data were from five experiments for all groups. Cell numbers were 92/81 (without/with GsMTx4 treatment), 132/89, and 130/145 for 30, 100, and 500 nm GsMTx4, respectively. D, representative traces of GSK (100 nm)-evoked TRPV4 currents in the presence of GsMTx4 (10 μm) (top) or HC067047 (a TRPV4-selective antagonist; 10 μm) (bottom). Yellow, red, and blue bars indicate application of GsMTx4, GSK, and HC067047, respectively. E, reversible effects of GsMTx4 (10 μm) on the stretch-evoked [Ca²⁺]_i increases in mouse primary-cultured urothelial cells. The first stretch stimulus was applied (black arrowhead) in the presence of GsMTx4 (yellow bar), and GsMTx4 was washed out, and the stretch was halted (a white arrowhead). The cells were then restretched (a red arrowhead) after a 20-min interval in the absence of GsMTx4. The first stretch and restretch stimulations were applied under the same conditions (stretch speed and distance are 100 μm/s and 200 μm, respectively). 5 μm ionomycin was applied to confirm cell viability (gray bar). Data were from 20 experiments (366 cells). F, comparison of the stretch-evoked [Ca²⁺]_i increases (normalized to ionomycin responses) in the GsMTx4 treatment and after its wash out. **, p < 0.01, Student's t test.

FIGURE 7.

Piezo1 expression in human bladder. A, Piezo1-like and cytokeratin-7 (CK 7) immunoreactivity in the urothelial cell layer of human bladder. White arrowheads, urothelium. SM, smooth muscle; L, lumen. Scale bar, 100 μm. B, expression levels of PIEZO1 and TRPV4 mRNAs (normalized to β-ACTIN) in the human bladder urothelium determined by quantitative RT-PCR. Data are presented as means ± S.E. (error bars).