Pannexin 1 channels contribute to mechanically-induced ATP release from bladder mucosa.

<p>Whole isolated rat bladders were bathed and instilled with PBS+glucose (1 g/L) in the absence and presence of the Panx1 channel blocker mefloquine (MFQ, 100 nM). A filling-voiding cycle was simulated by bladder instillation for 6 min at 10 mL/h flow rate followed by 5 min no flow, after which the bladder was emptied and ATP release in the bladder lumen was quantified. (A) Representative recording of intravesical pressure before, during instillation and at the moment of sample collection (“voiding”). (B) Bladder wall distension induced release of significant amounts of ATP that was blunted in the presence of 100 nM MFQ. Data represent mean±SEM (N = 3 bladders per experimental condition. **<i>P<</i>0.01 by Student’s <i>t</i>-test).</p

Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

<div><p>Urothelial cells respond to bladder distension with ATP release, and ATP signaling within the bladder and from the bladder to the CNS is essential for proper bladder function. In other cell types, pannexin 1 (Panx1) channels provide a pathway for mechanically-induced ATP efflux and for ATP-induced ATP release through interaction with P2X₇ receptors (P2X₇Rs). We report that Panx1 and P2X₇R are functionally expressed in the bladder mucosa and in immortalized human urothelial cells (TRT-HU1), and participate in urothelial ATP release and signaling. ATP release from isolated rat bladders induced by distention was reduced by the Panx1 channel blocker mefloquine (MFQ) and was blunted in mice lacking Panx1 or P2X₇R expression. Hypoosmotic shock induced YoPro dye uptake was inhibited by MFQ and the P2X₇R blocker A438079 in TRT-HU1 cells, and was also blunted in primary urothelial cells derived from mice lacking Panx1 or P2X₇R expression. Rinsing-induced mechanical stimulation of TRT-HU1 cells triggered ATP release, which was reduced by MFQ and potentiated in low divalent cation solution (LDPBS), a condition known to enhance P2X₇R activation. ATP signaling evaluated as intercellular Ca²⁺ wave radius was significantly larger in LDPBS, reduced by MFQ and by apyrase (ATP scavenger). These findings indicate that Panx1 participates in urothelial mechanotransduction and signaling by providing a direct pathway for mechanically-induced ATP release and by functionally interacting with P2X₇Rs.</p></div

Pannexin 1 channels and P2X₇Rs participate in ATP-mediated paracrine signaling among urothelial cells.

<p>Intercellular communication in TRT-HU1 cultures was induced by focal mechanical stimulation of single cells (white asterisks) and measured as radius of the intercellular Ca²⁺ wave (ICW) spread. (A) Representative pseudocolor images illustrating the radius (indicated by a red line circle) of ICW spread in PBS, in low divalent cation solution (LDPBS) and in LDPBS+mefloquine (MFQ, 100 nM). (B) Amplification of ICW spread in LDPBS is prevented by treatment with the Panx1 channel blocker MFQ (N = 6, 6 and 9, respectively. *<i>P</i><0.05 and **<i>P</i><0.01 by Kruskal-Wallis test). (C) Apyrase, an ATP catalase, greatly reduced the radius of ICW spread in TRT-HU1 cultures bathed in both PBS (N = 3 and 5, ^$<i>P</i><0.05 by Mann-Whitney U test) and LDPBS (N = 5 and 6, ^$$<i>P</i><0.01 by Mann-Whitney U test). Bar: 100 µm.</p

Immortalized human bladder urothelial (TRT-HU1) cells express Panx1 and P2X₇R.

<p>Detection of Panx1 and P2X₇R mRNA by PCR in (A) and protein by immunoblotting in (B). Total RNA from human bladder and HeLa cells were used as reference for the PCR analyses, and whole HeLa cell lysates was used as reference for immunoblotting.</p

Stretch-induced ATP release is reduced in bladders of pannexin 1 and P2X₇ receptor deficient mice.

<p>Whole bladders isolated from wildtype (WT), Panx1 deficient (Panx1^−/−) and P2X₇R deficient (P2X₇R^−/−) mice were bathed and instilled with PBS+glucose (1 g/L). A filling-voiding cycle was simulated by bladder instillation for 8 min at 1.5 mL/h flow rate followed by 5 min no flow, after which the bladder was emptied and ATP release in the bladder lumen was quantified. Data represent mean ± SEM (N = 9 WT, 4 Panx1^−/− and 7 P2X₇R^−/− bladders. Compared to WT: *<i>P<</i>0.05 and **<i>P<</i>0.01 by Student’s <i>t</i>-test).</p

Rat bladder mucosa is immuno-positive for Pannexin 1 (Panx1) channels and P2X₇ receptors.

<p>Confocal fluorescence Z stack images of flat mount bladder mucosa taken from the urothelial towards the serosal surface. (A) Granular staining for Panx1 channels (red) is observed throughout the mucosa. Staining for the intermediate filament vimentin (green) is observed on the apical urothelial region and particularly on a few cells in the lamina propria, which likely correspond to suburothelial myofibroblasts. Note partial colocalization of Pannexin 1 with vimentin-positive cells. (B) Positive staining for P2X₇R is observed in the urothelium, blood vessels (white arrows) and lamina propria, while staining for cytokeratin 7/17 is restricted to urothelial cells. Note intense P2X₇R immunoreactivity on the basal region of the mucosa, which is likely localized to the lamina propria myofibroblasts. DAPI nuclear staining in blue. Scale bar = 20 µm.</p

YoPro-1 uptake by urothelial cells in response to hypoosmotic shock involves Panx1 and P2X₇R activation.

<p>(A) TRT-HU1 cells: YoPro-1 uptake induced by cell swelling (hypoosmotic shock, black line) was significantly reduced in the presence of the Panx1 channel blocker mefloquine (MFQ 100 nM; yellow line), the P2X₇R blocker A438079 (10 µM; red line) and when both Panx1 and P2X₇R were blocked (blue line). Except for hypoosmotic with MFQ vs. A438079 and hypoosmotic with A438079+MFQ vs. isoosmotic, all other comparisons were significantly different at time 2800 sec (<i>P</i><0.01, N≥6) by two-way repeated measures ANOVA, followed by Tukey’s multiple comparison. (B) Primary mouse urothelial cells: YoPro-1 uptake induced by hypoosmotic shock was significantly lower in P2X₇R^−/− (red line) compared to wildtype (WT) urothelial cells (black line). Dye uptake by P2X₇R^−/− cells was abolished in the presence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; green line) and was absent in Panx1^−/− urothelial cells (blue line). WT vs P2X₇R^−/−, P2X₇R^−/−+MFQ and Panx1^−/− (<i>P<</i>0.001, N = 4) by ANOVA followed by Tukey’s multiple comparison.</p

GJA1 expression and gap junction formation in the bladders of mice with cystitis.

<p><i>A</i>: qPCR revealed significantly elevated expression of <i>Gja1</i> mRNA in the whole bladder at 6 hours after treatment (n = 4). Dunnett's multiple comparison test was performed. <i>P</i><0.05 was regarded as statistically significant; *<i>P</i><0.05. <i>B</i>: Immunoblotting data revealed up-regulation of GJA1 protein in the whole bladder at 24 hours after treatment (n = 3). The representative data shown were consistently replicated in other experiments. <i>C</i>: Immunoblotting data revealed that GJA1 expression was stronger in the urothelium with suburothelium (U) than in the smooth muscle layer (S) in the CYP-induced cystitis group (n = 3). The representative data shown were consistently replicated in other experiments. W: the whole bladder. <i>D</i>: TEM study revealed that urothelial cells were attached with each other in the sham-treated control group (CTRL; n = 3) but disconnected in the CYP-induced cystitis group (CYP; n = 3). Gap junctions were not observed in either group. In contrast, gap junctions were not found in the smooth muscle layer of the CTRL group but appeared in that of the CYP group. The representative data shown were consistently replicated in other experiments. Red arrow heads indicate gap junction. Scale bars indicate 2 µm.</p

Voiding behavior in mice with cystitis treated with 18α-glycyrrhetinic acid (GA).

<p>Micturition patterns were analyzed in the CYP-induced cystitis group in the presence (CYP+GA; n = 5) or absence (as control, CYP; n = 6) of 18α-GA with the aVSOP method. <i>A</i>: Maximum voided volume per void. The CYP+GA group demonstrated a significantly increased volume at 48 and 60 hours when compared to the control group. <i>B</i>: Mean voided volume per void. The CYP+GA group revealed a significant increase in volume at 48 hours. <i>C</i>: Urinary frequency per 12 hours. The CYP+GA group revealed lower frequency at each period examined. <i>D</i>: Total urine volume per 12 hour. The CYP+GA group revealed greater volume at each period. The white and black triangles show the time of 18α-GA and CYP administration, respectively. On the X-axis, the black squares indicate dark periods (9∶00 pm to 9∶00 am) and the white squares indicate light periods (9∶00 am to 9∶00 pm). Two way repeated measure ANOVA and Bonferroni post-test were performed. <i>P</i><0.05 was regarded as statistically significant; *<i>P</i><0.05, ***<i>P</i><0.001.</p

The sequences of primers used for real-time quantitative PCR.

<p>The sequences of primers used for real-time quantitative PCR.</p