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.