Figure legends
Figure 1. H2O2concentration-dependently enhanced the spontaneous bladder contractions (SBCs) of isolated human-bladder strips, which could be inhibited by an antagonist of TRPA1: HC-030031 . (A ) Representative traces showing H2O2 (1μM–10 mM) enhanced SBCs. Enhancement manifested mainly as an increase in the baseline tone and amplitude, but not the frequency, of phasic contractions. Each strip was treated with only one concentration of H2O2. (B ) Representative traces showing H2O2-induced increase in SBCs was attenuated in the presence of an antagonist of TRPA1: HC-030031 (HC03; 30μM). HC03 was applied 5-10 min before and during application of H2O2. (C ) Noncumulative concentration–response curves of H2O2in the absence and presence of HC03. AUC and baseline tension were measured at 15-min intervals immediately before and after H2O2 treatment. H2O2-induced effects were evaluated by the percent increase in AUC and net increase in baseline tension (Δg = after minus before). Δg was normalized further with the wet weight of the strip (Δg/g). Solid lines represent the fitted curves using the Hill equation, from which were obtained an EC50 of 175.8μM (H2O2alone) and 1.29mM (HC03 + H2O2) for AUC; 40.3μM (H2O2 alone) and 743.9μM (HC03 + H2O2) for Δg. *p < 0.05,**p < 0.01, ***p < 0.001 by one-sample t-test. Data are the mean ± SEM. Data from n = 4–6 strips were averaged for each concentration.
Figure 2. Activation of TRPA1 produced similar enhancing effects to H2O2 in isolated human-bladder strips. (A and B ) Representative traces (A) and summary data (B) showing that AITC (10–1000μM) increased SBCs in a concentration-dependent manner. The concentration–response curves of AITC-induced increase in AUC (top) gave an EC50 of 195.3μM for AUC, and a reliable EC50 for Δg (below) could not be obtained because saturation was not reached even at 1 mM of AITC. (C and D ) Representative traces (C) and summary data (D) showing AITC (300μM)-induced increase in SBCs was blocked partially by the TRPA1 antagonist HC-030031 (HC03; 30μM). HC03 was applied 5-10 min before and during AITC application. n: number of the bladder strips; N: number of human samples. ***p < 0.001.
Figure 3. Desensitization of bladder sensory afferents with capsaicin or blockade of sodium channels with TTX attenuated H2O2-induced enhancing effects.(A and B ) Representative traces (A) and summary data (B) showing pretreatment with capsaicin (10μM) or TTX (1μM) reduced H2O2-induced increases in SBCs of human-bladder strips significantly. Capsaicin or TTX was applied 15 min before and during H2O2(100μM) administration. (C and D ) Representative traces (C) and summary data (D) showing pretreatment with capsaicin (10μM) or TTX (1μM) reduced AITC (100μM)-induced increases in SBCs of human-bladder strips significantly. n: number of the bladder strips; N: number of human samples. ***p < 0.001.
Figure 4. TRPA1 channels are expressed on the sensory afferents located in sub-urothelial and detrusor layer of the human bladder . Immunofluorescence for SP (red, 400× magnification) and TRPA1 (green, 400× magnification) in sub-urothelial (A) and intermuscular (B) nerve terminals and their co-localization (yellow, 400× magnification).
Figure 5. A selective antagonist of NK2 receptors attenuated H2O2-induced enhancing effects.(A and B ) Representative traces (A) and summary data (B) showing SP (500nM) administration induced a prominent increase in SBCs of human-bladder strips, which were attenuated by pretreatment with a selective antagonist of NK2 receptors: SR 48968 (10μM). SR 48968 was applied 5 min before and during H2O2 (100μM) administration. (C and D ) Representative traces (C) and summary data (D) showing pretreatment with SR 48968 attenuated H2O2-induced increase in SBCs. n: number of bladder strips, N: number of human samples. ***p < 0.001.
Figure 6. Blockade of PGE2 synthesis with indomethacin attenuated H2O2-induced enhancing effects. (A-B ) Representative traces showing PGE2 (1µM) administration induced enhancement in SBCs (A), and that pretreatment with indomethacin attenuated H2O2-induced increases in SBCs (B). Indomethacin was applied 10–15 min before and during H2O2 (100μM) administration. (C ) Summary data showing pretreatment with indomethacin reduced H2O2 (100μM)-induced increases in AUC and Δg significantly. *p < 0.05.
Supplementary Figure 1 . Desensitization effect of repeat application of H2O2. (A)Typical traces showing that, on the same bladder strip, second-time application of H2O2 evoked a lower response compared with first-time application. (B) Summary data from five strips. **p < 0.01.