The TRPV4 cation channel mediates stretch-evoked Ca2+ influx and ATP release in primary urothelial cell cultures

Abstract

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) evoked Ca(2+) influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca(2+) concentration and ATP release. Stretch stimulation evoked intracellular Ca(2+) increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca(2+) increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca(2+) increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4alpha-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca(2+). These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.

FIGURE 1.

Gene expression of TRP channels in mouse primary urothelial cell cultures. A, RT-PCR shows expression of trpv4 and trpa1 in wild-type (WT, upper panel) and expression of trpa1 in TRPV4-deficient (TRPV4KO, lower panel) primary urothelial cell cultures from mice. The cytokeratin 7 (ck7) gene is detected in both WT and TRPV4KO cells. In RT (−) lanes, reverse transcriptase enzyme was omitted (negative control). B, real-time PCR analysis shows that trpv4 is uniquely abundant in primary urothelial cell cultures. Copy numbers were calculated by standard curves of specific primer sets and normalized by the copy numbers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 × 10⁶ copies).

FIGURE 2.

Immunofluorescent analyses with mouse bladder and cultured urothelial cells. A, TRPV4 staining in mouse whole bladder tissue merged with a phase-contrast image. TRPV4-like immunoreactivity is mainly detected in basal and intermediate urothelial cell layers in wild-type (WT), but not in TRPV4-deficient (TRPV4KO) cells. A muscle layer, a suburothelial layer, and an urothelial apical cell layer (umbrella cells) appear to express much less TRPV4 in WT. An inset in the WT image was expanded from the square box to show TRPV4 localization in urothelial cell layers. M, muscle; L, lumen. Scale bars: 100 μm. B, immunocytochemical analysis of primary urothelial cell cultures. Cytokeratin 7 (CK7)-like immunoreactivity indicates that the cells are urothelial origin both in WT and TRPV4KO cell clusters, and TRPV4 signals are present in WT cells, but not in TRPV4KO cells. Green, TRPV4; red, CK7; and blue, 4′,6-diamidino-2-phenylindole. Scale bars: 100 μm.

FIGURE 3.

Ca²⁺ imaging in response to 4α-PDD in primary urothelial cell cultures. A, representative pseudo-color images show that 4α-PDD stimulation causes robust [Ca²⁺]_i increases in wild-type (WT) urothelial cells (upper left and middle panels), whereas TRPV4-deficient (TRPV4KO) cells fail to respond to 4α-PDD (lower left and middle panels). The right panels show the TRPV4-positive cells (shown in “green”) merged with a phase-contrast image after a Ca²⁺-imaging experiment. Scale bars: 100 μm. B, quantification of [Ca²⁺]_i changes in individual cells displays 4α-PDD-evoked [Ca²⁺]_i increases in WT cells, but not in TRPV4KO cells. All traces were obtained from the cells shown in A both in WT and TRPV4KO cells. Black bars: 10 μm 4α-PDD application; gray bars: 5 μm ionomycin application. C, the average peak [Ca²⁺]_i increases in response to 10 μm 4α-PDD are significantly reduced in TRPV4KO cells in the presence of extracellular Ca²⁺ (Ca²⁺ (+)), but Ca²⁺ responses are negligible in both cell types in the absence of extracellular Ca²⁺ (Ca²⁺ (−)). All data are normalized to the responses to ionomycin as 100% and presented as means ± S.E. (n > 6). **, p < 0.005 (Student's t test).

FIGURE 4.

Ca²⁺ responses to stretch stimulation in primary urothelial cell cultures. A, [Ca²⁺]_i increases upon stretch stimulation by STREX in wild-type (WT) urothelial cells (upper panels), but the response is poor in TRPV4-deficient (TRPV4KO) cells (lower panels). All images were taken from cells seeded on the 1-mm slit area in the stretch chamber (refer to supplemental Fig. S1). The stretch speed was 400 μm/s, and the distance was 500 μm. Cells were extended to a vertical axis (indicated by arrows) in both cell types. Scale bars: 100 μm. B, quantification of [Ca²⁺]_i changes in individual cells. Stretch evokes transient [Ca²⁺]_i increases in WT cells, whereas only small and sustained [Ca²⁺]_i increases are observed in TRPV4KO cells. All traces were obtained from the cells shown in A both in WT and TRPV4KO cells. Black arrowheads denote the onset of stretch. Gray bars: 5 μm ionomycin application. C, the average peak of [Ca²⁺]_i increases is significantly reduced in TRPV4KO (KO) urothelium upon stretching. Ruthenium red (RR, 10 μm) attenuates the stretch-evoked [Ca²⁺]_i increase in WT cells to the level achieved in TRPV4KO cells without RR treatment, but shows no effect on TRPV4KO cells. [Ca²⁺]_i increases are similar between WT and TRPV4KO cells in the absence of extracellular Ca²⁺ (Ca²⁺ (−)). All the data are normalized to the values induced by 5 μm ionomycin application as 100% and presented as means ± S.E. (n > 7). An asterisk indicates significant difference between WT and the other five groups. *, p < 0.05 (Fisher's protected least significant difference).

FIGURE 5.

Visualization of stretch-evoked ATP release from primary urothelial cell cultures. A, ATP is robustly released from wild-type (WT) urothelial cells, but poorly released from TRPV4-deficient (TRPV4KO) cells upon stretching. The upper panels show urothelial cells in phase-contrast images, and the lower panels show photon count images (white dots) in the same field. The stretch speed was 400 μm/s, and the distance was 500 μm. Cells were extended transversely (indicated by arrows). Scale bars: 100 μm. B, the average amount of ATP released from TRPV4KO cells in response to mechanical stretch stimulation is significantly smaller than that in WT cells. ATP release is diminished by RR treatment or by the absence of extracellular Ca²⁺ (Ca²⁺ (−)) in WT cells. Data presented as means ± S.E. (n > 6). An asterisk indicates a significant difference between WT and the other five groups. *, p < 0.05 (Fisher's protected least significant difference). C, ATP release is minimal at a stretch distance of 300 μm and saturated at 1000 μm in both cell types (also refer to supplemental Fig. S4). Data are presented as means ± S.E. (n = 6). D, 4α-PDD (10 μm) induces robust ATP release by WT cells, but not by TRPV4KO cells. Data are presented as means ± S.E. (n = 6). Significant difference: **, p < 0.005 (Student's t test).