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\begin{document}

\title{H2O2 enhances spontaneous contractions of human-bladder strips via
activation of TRPA1 channels on sensory nerves and release of substance
P and PGE2}



\author[1]{Haoyu Wang}%
\author[2]{Mengmeng Zhao}%
\author[1]{jiaxin liu}%
\author[1]{lei liu}%
\author[1]{hanwen liu}%
\author[1]{ning ding}%
\author[3]{Shaoyong Wang}%
\author[1]{Nan Ge}%
\author[1]{Xiulin Zhang}%
\affil[1]{The Second Hospital of Shandong University}%
\affil[2]{Shandong Provincial Hospital Affiliated to Shandong First Medical University}%
\affil[3]{Second Hospital of Shandong University}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Abstract Background and purpose: Several studies have indicated that
reactive oxygen species (ROS) can lead to detrusor overactivity (DO),
but the underlying mechanisms are not known. Hydrogen dioxide (H2O2) is
used commonly to investigate the effects of ROS. In present study, we
aimed to investigate the effects of H2O2 on phasic spontaneous bladder
contractions (SBCs) of isolated human-bladder strips (iHBSs) and the
underlying mechanisms. Experimental approach: Samples of bladder tissue
were obtained from 26 patients undergoing cystectomy owing to bladder
cancer. SBCs of iHBSs were recorded in organ-bath experiments.
Immunofluorescence staining was conducted to confirm the TRPA1
expression on sensory nerves. Key results: H2O2 (1\selectlanguage{greek}μ\selectlanguage{english}M--10mM)
concentration-dependently increased the SBCs of iHBSs. These enhancing
effects could be mimicked by an agonist of transient receptor potential
(TRP)A1 channels (allyl isothiocyanate) and blocked with an antagonist
of TRPA1 channels (HC030031; 10\selectlanguage{greek}μ\selectlanguage{english}M). H2O2 induced enhancing effects also
could be attenuated by desensitizing sensory afferents with capsaicin
(10\selectlanguage{greek}μ\selectlanguage{english}M), blocking nerve firing with TTX (1\selectlanguage{greek}μ\selectlanguage{english}M), blocking neurokinin
effects with NK2 receptor antagonist (SR48968, 10\selectlanguage{greek}μ\selectlanguage{english}M), and blocking PGE2
synthesis with indomethacin (10\selectlanguage{greek}μ\selectlanguage{english}M), respectively. Conclusions and
implications: Our study: (i) suggests activation of TRPA1 channels on
bladder sensory afferents, and then release of substance P or PGE2 from
sensory nerve terminals, contribute to the H2O2-induced enhancing
effects on SCs of iHBSs; (ii) provides insights for the mechanisms
underlying ROS leading to DO; (iii) indicates that targeting TRPA1
channels might be the promising strategy against overactive bladder in
conditions associated with excessive production of ROS.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

Overactive bladder (OAB) is a common condition affecting millions of
people worldwide. It has been defined by the International Continence
Society as a syndrome characterized by urgency (with or without urge
incontinence), usually with frequency, or nocturia {[}1, 2{]}. It has
been proposed that OAB symptoms are closely associated with exaggerated
spontaneous bladder contractions (SBCs), referred to as ``detrusor
overactivity'' (DO){[}3, 4{]} or ``no-voiding contraction.''
Consequently, DO is the target for medical treatment of the storage
symptoms of OAB{[}5{]}. Thus, unraveling the underlying mechanisms of DO
is valuable to identify novel drug targets for OAB treatments{[}3, 5{]}.

OAB is prevalent in patients or animal models with bladder outlet
obstruction (BOO){[}6{]}, injury to the spinal cord {[}7{]}, diabetes
mellitus{[}8{]}, chronic ischemia{[}9{]}, or aging{[}10{]}. These
pathologic conditions are characterized by enhanced oxidative stress and
excessive accumulation of reactive oxygen species (ROS) due to ischemia,
ischemia--reperfusion, or inflammation{[}5, 11-13{]}. Thus, oxidative
stress and the resulting increased levels of ROS has been proposed to be
an important mechanism for DO development{[}14-16{]}.

Hydrogen dioxide (H\textsubscript{2}O\textsubscript{2}) is the most
likely ROS involved in signal transduction, and has been used commonly
to investigate the effects of ROS{[}15, 17{]}. In support of the role of
ROS in DO, an\emph{in vivo} study in rats showed that intravesical
instillation of H\textsubscript{2}O\textsubscript{2} (0.003--0.3\%)
could induce OAB{[}15{]}. Intravesical injection of
H\textsubscript{2}O\textsubscript{2} has been utilized to establish an
animal model of OAB{[}18{]}. \emph{In vitro} studies in rats showed that
H\textsubscript{2}O\textsubscript{2}increased the spontaneous
contractions (SBCs) of isolated bladder strips in a
concentration-dependent manner{[}17, 19{]}. However, the exact
mechanisms underlying how excessive ROS leads to DO are not known.
Furthermore, most of these types of studies have been from animal
bladders, and studies on the human bladder are lacking.

Pathologic DO has been considered to be an amalgamation of small and
localized asynchronous SBCs, which could be measured \emph{in
vivo}during normal filling of the bladder and could also be recorded
\emph{in vitro} in isolated whole bladder or bladder strips from animal
and human sources{[}20{]}. Studies on the origin or modulation of SBCs
may help to unravel the underlying mechanisms of pathologic DO{[}20{]}.
\selectlanguage{greek}β\selectlanguage{english}3-adrenoceptor agonists which have been used to treat OAB were shown to
suppress SBCs in human-bladder strips{[}21{]}. \emph{In vitro} recording
of SBCs has indicated that SBCs could be increased by nerve stimulation,
muscarinic agonists{[}22{]}, nicotinic ligands, adenosine triphosphate
(ATP), and substance P (SP){[}23{]}, and decreased by noradrenaline and
calcitonin gene-related peptide (CGRP){[}24, 25{]}. The modulatory
effects of SP and CGRP (the two neuropeptide transmitters released from
bladder sensory afferents) implicate a role of the axon reflex or the
``efferent'' role of sensory afferents in SBCs regulation{[}25, 26{]}.

Transient receptor potential (TRP)A1 channels are expressed on C-fiber
bladder afferents {[}27, 28{]}. TRPA1 activation by intravesical
instillation of TRPA1 activators such as allyl isothiocyanate (AITC) can
initiate DO{[}27{]}. \emph{In vitro} recordings have indicated that AITC
enhanced the SBCs of isolated bladder strips from rats or guinea pigs by
activating TRPA1 on bladder sensory afferents{[}29-31{]}. Those studies
suggest a close correlation between activation of TRPA1 on sensory
afferents and DO. Furthermore, Andrade et al{[}31{]}found that the
release of SP and prostaglandin (PG)E2 contributed to AITC-mediated
enhancement of SBCs, which suggests that TRPA1 activation-induced DO may
result from the efferent role of bladder sensory afferents.

TRPA1 channels expressed on sensory afferents have been shown to be the
main molecular target for H\textsubscript{2}O\textsubscript{2}{[}28,
32{]}. In rats or mice, H\textsubscript{2}O\textsubscript{2}
administration has been demonstrated to evoke increases in inward
current or the intracellular calcium (Ca\textsuperscript{2+})
concentration in a subset of dorsal-root ganglia neurons that were also
responsive to TRPA1 agonists{[}32-34{]}.
H\textsubscript{2}O\textsubscript{2} application evoked a long-lasting
firing of most capsaicin-sensitive high-threshold afferents \emph{via}
TRPA1 activation in the guinea-pig bladder{[}28{]}.

Given the excitatory role of H\textsubscript{2}O\textsubscript{2} on
bladder sensory afferents and the modulatory role of bladder sensory
afferents on SBCs, we hypothesized that TRPA1 activation by ROS induced
the release of neurotransmitters from peripheral sensory nerve terminals
of the bladder contributed to DO. There are pronounced differences in
SBCs patterns between humans and animals{[}35{]}. Also, studies
examining the effects of ROS on the contractile activity of the bladder
have been conducted almost exclusively on laboratory animals. Hence, in
the present study we investigated
H\textsubscript{2}O\textsubscript{2}-induced effects on bladder
contractile activity and the mediating mechanisms using the human
bladder tissue.

\textbf{2. Materials and Methods}

\textbf{2.1 Ethical approval of the study protocol}

The experimental protocol was approved (KYLL-2023LW038) by the Ethics
Committee of the Second Hospital, Cheeloo College of Medicine in
Shandong University (Jinan, China). Our study was conducted in
accordance with the Declaration of Helsinki 1964 and its later
amendments. All patients provided written informed consent.

\textbf{2.1 Samples of human bladder}

Samples of human bladder were obtained from 26 patients (11 women, 15
men; patients mean age = 49 \selectlanguage{ngerman}± 10 years; age range = 39--59 years)
undergoing cystectomy owing to bladder cancer in the Second Hospital or
Qilu Hospital of Shandong University. Tissues were examined by a
uropathologist. No histological signs of inflammation or carcinoma were
observed. After removal, each specimen was transported immediately to
the laboratory for organ-bath experiments. A portion of each tissue was
placed in formalin for immunofluorescence experiments.

\textbf{2.3 Organ-bath experiments}

All samples were taken from the bladder body. From each patient, 5--10
longitudinal bladder strips (length = 20 ± 1.5 mm; width = 8--9 mm) were
studied. Bladder strips were placed in warm Krebs solution (composition
in mM: NaCl, 118; KCl, 4.7; CaCl\textsubscript{2}, 1.9;
MgSO\textsubscript{4}, 1.2; NaHCO\textsubscript{3}, 24.9;
KH\textsubscript{2}PO\textsubscript{4}, 1.2; glucose, 11.7; pH = 7.4)
and bubbled with 95\% O\textsubscript{2} and 5\% CO\textsubscript{2}.
Strips were tied at each end using a fine thread, mounted in a vertical
organ bath in oxygenated Krebs solution (volume = 20 mL), and heated to
37 °C in a circulating warm water-bath. The longitudinal tension of the
isolated preparations was recorded continuously with an isometric
transducer and then processed with Lab Chart 7 (AD Instruments, New
South Wales, Australia). Tissues were stretched to a baseline tension of
15mN (1.5 g). Contractions were allowed to equilibrate for 30--40 min,
until the appearance of stable SBCs. In strips with no SBCs, carbachol
(1--10nM) was applied to initiate SBCs. Strips that had a spontaneous or
carbachol-evoked SBCs were considered as having ``good'' viability. All
the strips from the 26 patients had good viability.

In a pilot study, we found that repeated application of high
concentrations (\selectlanguage{english}[?]100\selectlanguage{greek}μ\selectlanguage{english}M) of H\textsubscript{2}O\textsubscript{2} in the
same strip induced desensitization (supplementary figure 1). Thus, the
noncumulative concentration--response relationship of
H\textsubscript{2}O\textsubscript{2}was determined. On completion of
experiments, threads were removed and strips were weighed after water
absorption with tissue paper.

\textbf{2.4 Immunofluorescence staining}

Immunofluorescence staining was conducted to confirm the TRPA1
expression on sensory nerves. Bladder tissues were fixed with 4\%
formalin for \textgreater{}24 h. Bladder tissues were embedded in
paraffin and then cut in the transverse direction into sections of
thickness 8\selectlanguage{greek}μ\selectlanguage{english}m. Sections were deparaffinized, rehydrated, and followed by
antigen retrieval for 10 min. Then, sections were blocked with 5\%
albumin from goat serum in a 37 \selectlanguage{ngerman}°C oven for 1 h, followed by incubation
with mixed primary antibodies (TRPA1, 1:100, Alomone labs; Substance P,
SANTA CRUZ, 1:100) overnight at 4 °C. Primary antibodies were visualized
with Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L; diluted 1:200
in phosphate-buffered saline, Elabscience Biotechnology Co., Ltd.,
Wuhan, China) or fluorescein-conjugated goat anti-rabbit IgG (H+L;
diluted 1:50). Sections were analyzed by Confocal laser scanning
microscope ZEISS Observer.Z1 (Carl Zeiss Microscopy GmbH, Baden
Wurttemberg, Germany). Images were acquired with the ZEN 2.1 (blue
version) (Carl Zeiss Microscopy GmbH, Baden-Wurttemberg, Germany).
Control staining was performed with PBS rather than primary antibodies.

\textbf{2.5 Drugs}

H\textsubscript{2}O\textsubscript{2}, HC-03003, AITC, capsaicin,
tetrodotoxin (TTX) and Carbachol were purchased from Sigma-Aldrich
(Darmstadt, USA). While PGE2, SP and a selective antagonist of
neurokin-2 receptors (NK2Rs), SR 48968, were sourced from MCE (New
Jersey, USA).

Stock solution of capsaicin were prepared in 100\% ethanol and stored at
\selectlanguage{english}-20 \selectlanguage{english} until use. AITC, SR 48968, PGE2, and SP solutions were made in
dimethyl sulfoxide and diluted immediately before use. TTX was dissolved
in deionized water. The final concentrations of ethanol or dimethyl
sulfoxide were \textless{}0.1\%. In all experimental groups, at least
one parallel control experiment was carried out in vehicle. The vehicles
we used had no pharmacological effects on the tone of preparations or
agonist-induced contractions. Chemicals were used at concentrations
shown to be effective in preliminary experiments, or at concentrations
used in studies published previously{[}29, 30{]}.

\textbf{2.6 Data analyses}

Data are the mean +- SEM. SBCs was measured at 15-min intervals
immediately before (control) and after drug application. The response to
H\textsubscript{2}O\textsubscript{2} was evaluated as the percent
increase in the area under the curve (AUC, an integrative of the
amplitude, baseline tension, and frequency of phasic contractions) as
well as the net increase in baseline tension (\selectlanguage{greek}Δ\selectlanguage{english}g) from the control. \selectlanguage{greek}Δ\selectlanguage{english}g
was normalized with the wet weight of strips (\selectlanguage{greek}Δ\selectlanguage{english}g/g).
Concentration--response curves were fitted with the Hill equation:
Percent enhancement = (MAX\textsubscript{enhancement}/{[}drug
concentration + EC\textsubscript{50}{]})\textsubscript{n} where
MAX\textsubscript{enhancement} is maximal \% enhancement, drug
concentration is the agonist concentration, EC\textsubscript{50} is the
half-maximal effective concentration, and n is the Hill coefficient.
Statistical significance was tested with the one-sample \emph{t} -test
or paired \emph{t} -test (two-tailed). Statistical analyses were
undertaken with Excel (Microsoft, Redmond, WA, USA) and Prism 8
(GraphPad, La Jolla, CA, USA). \emph{p} \textless{} 0.05 was considered
significant.

\textbf{3. Results}

\textbf{3.1 H\textsubscript{2}O\textsubscript{2} enhanced the
spontaneous phasic contractions of isolated human-bladder strips in a
concentration-dependent manner}

We found that 112 of 186 (60\%) of human-bladder strips displayed phasic
SBCs during an equilibration period of 30--40 min. Nevertheless, SBCs
were evoked with carbachol (1nM). Carbachol (1nM) treatment increased
the baseline tension by 1.2 \selectlanguage{ngerman}± 0.3 g (n = 62 strips).

Application of H\textsubscript{2}O\textsubscript{2}(from 1\selectlanguage{greek}μ\selectlanguage{english}M to 10mM)
produced a concentration-dependent increase in the SBCs of human-bladder
strips (Fig. 1A). This enhancement manifested mainly as an increase in
the baseline tone and amplitude of phasic contractions, and there was no
change in the frequency of phasic contractions (Fig.1A). The enhancement
could last \textgreater{}20 min. Therefore, the response to
H\textsubscript{2}O\textsubscript{2} was evaluated with the percent
increase in AUC as well as the net change of \selectlanguage{greek}Δ\selectlanguage{english}g from the control. A
significant desensitization effect was observed at higher doses ([?]100\selectlanguage{greek}μ\selectlanguage{english}M)
of H\textsubscript{2}O\textsubscript{2}(Supplementary Fig. 1). Thus,
noncumulative concentration--response curves of
H\textsubscript{2}O\textsubscript{2} were established (Fig. 1C) and
revealed an EC\textsubscript{50} of 175.8\selectlanguage{greek}μ\selectlanguage{english}M and 40.3\selectlanguage{greek}μ\selectlanguage{english}M for AUC and \selectlanguage{greek}Δ\selectlanguage{english}g,
respectively (Fig. 1C). We did not see the inhibitory effects of
H\textsubscript{2}O\textsubscript{2} during a 30-min observation period
for any concentration. There was no significant difference in the
amplitude of the H\textsubscript{2}O\textsubscript{2} (100\selectlanguage{greek}μ\selectlanguage{english}M)-induced
increase in \selectlanguage{greek}Δ\selectlanguage{english}g between carbachol-treated (\selectlanguage{greek}Δ\selectlanguage{english}g = 0.85 \selectlanguage{ngerman}± 0.12g/g, n = 12)
and untreated strips (\selectlanguage{greek}Δ\selectlanguage{english}g = 0.78 \selectlanguage{ngerman}± 0.23g/g, n = 7).

\textbf{3.2 Activation of TRPA1 channels is involved in
H\textsubscript{2}O\textsubscript{2}-induced enhancement effects}

Studies have shown that H\textsubscript{2}O\textsubscript{2} can target
TRPA1 channels{[}28, 32, 36{]}. To determine the involvement of TRPA1
activation in H\textsubscript{2}O\textsubscript{2}-induced effects,
HC-030031 (30\selectlanguage{greek}μ\selectlanguage{english}M), a potent and selective inhibitor of TRPA1{[}37{]}, was
applied 5--10 min before and during
H\textsubscript{2}O\textsubscript{2}application. HC-030031 applied alone
had no effects on contractile activity.
H\textsubscript{2}O\textsubscript{2}-induced enhancement was attenuated
significantly in the presence of HC-030031 (Fig. 1B), and the
concentration--response curve of AUC and \selectlanguage{greek}Δ\selectlanguage{english}g was shifted to the right
(Fig. 1C). In the presence of HC-030031, EC\textsubscript{50} was 1.29
mM and 743.9\selectlanguage{greek}μ\selectlanguage{english}M for AUC and \selectlanguage{greek}Δ\selectlanguage{english}g, respectively, which was much larger than
that of the control\textsubscript{.} The maximum response
(E\textsubscript{max}) was reduced by 37.8\% and 15.3\% for AUC and \selectlanguage{greek}Δ\selectlanguage{english}g,
respectively, in the presence of HC-030031. These observations suggested
that TRPA1 activation was involved in
H\textsubscript{2}O\textsubscript{2}-induced effects.

Similar to H\textsubscript{2}O\textsubscript{2,} AITC (10\selectlanguage{greek}μ\selectlanguage{english}M, 30\selectlanguage{greek}μ\selectlanguage{english}M,
100\selectlanguage{greek}μ\selectlanguage{english}M, 300\selectlanguage{greek}μ\selectlanguage{english}M, 1mM), a specific agonist of TRPA1, also produced a
concentration-dependent increase in the SBCs of human-bladder strips
(Fig. 2A). The concentration--response curve of AITC revealed an
EC\textsubscript{50} of 195.3\selectlanguage{greek}μ\selectlanguage{english}M for AUC (Fig. 2B). However, a reliable
EC\textsubscript{50} for \selectlanguage{greek}Δ\selectlanguage{english}g could not be obtained because saturation was
not reached even at 1 mM of AITC. The enhancement effect of AITC (300\selectlanguage{greek}μ\selectlanguage{english}M)
could be blocked by HC-030031 (30\selectlanguage{greek}μ\selectlanguage{english}M) (Fig. 2C). The AUC and \selectlanguage{greek}Δ\selectlanguage{english}g were
reduced by 73\% and 62.4\%, respectively, in the presence of HC-030031
(Fig. 2D). Because H\textsubscript{2}O\textsubscript{2} at 100\selectlanguage{greek}μ\selectlanguage{english}M and
AITC at 300\selectlanguage{greek}μ\selectlanguage{english}M produced a prominent increase in the SBCs of human-bladder
strips, they were used as the test concentrations in subsequent
experiments.

\textbf{3.3 TRPA1 expressed on sensory nerves is involved in
H\textsubscript{2}O\textsubscript{2}-inducedenhancement effects}

Nicholas et al showed that H\textsubscript{2}O\textsubscript{2}increased
the firing activity of bladder sensory afferents mainly by activating
TRPA1 channels{[}28{]}. It is accepted that sensory afferents regulate
SBCs by releasing neurotransmitter CGRP or SP, a process termed
``efferent actions of sensory afferents''{[}23, 24, 26, 38, 39{]}. To
examine the involvement of TRPA1 on bladder sensory nerves in
H\textsubscript{2}O\textsubscript{2}-induced effects, bladder strips
were treated with a high dose of capsaicin (10\selectlanguage{greek}μ\selectlanguage{english}M) 10--15 min before and
during H\textsubscript{2}O\textsubscript{2} to desensitize bladder
sensory nerves. Capsaicin applied alone did not impact the contractile
activity. Desensitizing sensory afferents with capsaicin reduced the
H\textsubscript{2}O\textsubscript{2} (100\selectlanguage{greek}μ\selectlanguage{english}M)-induced increase in AUC and
\selectlanguage{greek}Δ\selectlanguage{english}g significantly by 68.1\% and 71.5\%, respectively (\emph{p}\textless{}
0.05) (Fig. 3A and B). After desensitization of sensory nerves, the AITC
(300\selectlanguage{greek}μ\selectlanguage{english}M)-induced increase in AUC and \selectlanguage{greek}Δ\selectlanguage{english}g was reduced by 71.1\% and 73.7\%,
respectively (\emph{p} \textless{} 0.05) (Fig. 3C and D).

To further confirm the involvement of activation of sensory afferents,
TTX (1\selectlanguage{greek}μ\selectlanguage{english}M) was applied to block nerve firing. TTX applied alone did not
impact the contractile activity. However, application of TTX (1\selectlanguage{greek}μ\selectlanguage{english}M)
reduced the H\textsubscript{2}O\textsubscript{2} (100\selectlanguage{greek}μ\selectlanguage{english}M)-induced
increase in the AUC and \selectlanguage{greek}Δ\selectlanguage{english}g significantly by 61.3\% and 72.9\%,
respectively (Fig. 3A and B). Application of TTX (1\selectlanguage{greek}μ\selectlanguage{english}M) reduced AITC
(300\selectlanguage{greek}μ\selectlanguage{english}M)-induced increase in AUC and \selectlanguage{greek}Δ\selectlanguage{english}g significantly by 73.5\% and
70.9\%, respectively (Fig. 3C and D).

Immunofluorescence experiments revealed TRPA1 channels on sensory
afferents located in sub-urothelial and intramuscular layers. These
sensory afferents also expressed SP (Fig. 4A and B).

\textbf{3.4 Release of SP and PGE2 contribute to
H\textsubscript{2}O\textsubscript{2}-induced enhancement effects}

Tachykinins are neuropeptides present in the capsaicin-sensitive primary
afferents of the urinary bladder of various species (including humans).
Release of these peptides in the periphery produced prominent effects on
the SBCs of isolated bladder strips{[}3{]}. In agreement with this
report, application of SP (500nM) increased the SBCs of human-bladder
strips significantly (Fig. 5A), and the enhancing effect of SP could be
blocked by a specific antagonist of NK2Rs: SR 48968 (10\selectlanguage{greek}μ\selectlanguage{english}M). AUC and \selectlanguage{greek}Δ\selectlanguage{english}g
were reduced by 84.5\% and 64.3\%, respectively (Fig. 5A, B). NK2R is
the main subtype of neurokinin receptor that mediates the effects of
neurokinins in the human bladder{[}40-42{]}. SR 48968 applied alone did
not impact the contractile activity. As expected, the NK2R antagonist SR
48968 (10\selectlanguage{greek}μ\selectlanguage{english}M) reduced the
H\textsubscript{2}O\textsubscript{2}(100\selectlanguage{greek}μ\selectlanguage{english}M)-induced increase in SBCs
significantly (Fig. 5C). AUC and \selectlanguage{greek}Δ\selectlanguage{english}g were reduced by 74.1\% and 49.1\%,
respectively (Fig. 5D), which suggested that SP release contributed to
the enhancement effects of H\textsubscript{2}O\textsubscript{2}.

PGE2 released from sensory afferents, urothelial cells, or the detrusor
has been shown have important modulatory effects on SBCs \emph{in vitro}
{[}43{]} and voiding behavior \emph{in vivo} {[}44{]}. In agreement with
those reports, application of PGE2 (1\selectlanguage{greek}μ\selectlanguage{english}M) evoked a significant increase
in the SBCs of human-bladder strips (Fig. 6A). Pretreatment (15 min
before H\textsubscript{2}O\textsubscript{2}) with indomethacin (10\selectlanguage{greek}μ\selectlanguage{english}M) to
block the synthesis and release of PGE2 led to significant attenuation
of H\textsubscript{2}O\textsubscript{2} (100\selectlanguage{greek}μ\selectlanguage{english}M)-induced effects, with
AUC and \selectlanguage{greek}Δ\selectlanguage{english}g being reduced by 65.9\% and 35.9\%, respectively (Fig. 6B and
C). Indomethacin (10\selectlanguage{greek}μ\selectlanguage{english}M) applied alone did not affect contractile
activity. This observation indicated that PGE2 release contributed to
the enhancing effects of H\textsubscript{2}O\textsubscript{2}.

\textbf{4. Discussion}

Excessive ROS can lead to DO. To investigate the underlying mechanisms,
the effects of H\textsubscript{2}O\textsubscript{2} on SBCs of isolated
human-bladder strips were investigated in the present study. To our
knowledge, this is the first study to examine the mechanisms in human
bladder. We found that H\textsubscript{2}O\textsubscript{2}(1\selectlanguage{greek}μ\selectlanguage{english}M to 10mM)
concentration-dependently increased the SBCs of human-bladder strips.
These enhancing effects could be mimicked by an agonist of TRPA1
channels, and could be blocked with an antagonist of TRPA1 channels.
H\textsubscript{2}O\textsubscript{2} induced enhancing effects could be
attenuated by desensitizing sensory afferents with capsaicin, blocking
nerve firing with TTX, blocking neurokinin effects with NK2 receptor
antagonist and blocking PGE2 synthesis with indomethacin, respectively.
Our results suggested activation of TRPA1 channels on bladder sensory
afferents, then causing the release of SP or PGE2 from nerve terminals
is one of the underling mechanisms for ROS leading to DO.

In the present study, low concentration of
H\textsubscript{2}O\textsubscript{2} produced similar stimulatory
effects on SBCs in isolated strips of human bladder and rat bladder
{[}17, 19{]}. H\textsubscript{2}O\textsubscript{2} can act on several
cell targets or pathways to induce enhanced contraction of the bladder.
H\textsubscript{2}O\textsubscript{2} could target the detrusor muscle
directly to enhance SBCs \emph{via} activation of cyclooxygenase or
rho-kinase pathways which, in turn, increase extracellular
Ca\textsuperscript{2+} influx into the detrusor{[}17, 19{]}.
H\textsubscript{2}O\textsubscript{2} may target urothelium cells to
evoke DO \emph{via} ATP release, as suggested by Stephany et
al.{[}45{]}. Activation of bladder sensory afferents has been shown to
be another important mechanism for
H\textsubscript{2}O\textsubscript{2}-induced DO {[}15, 46{]}.

Activation of sensory afferents may work through two mechanisms to
enhance bladder contractile activity: (i) by increasing the spinal
reflex or (ii) by increasing neurotransmitter release in the
periphery\emph{via} a local axon reflex (as suggested by Maggie and
Gillespie){[}25, 26{]}. Our most important finding was that peripheral
activation of bladder sensory afferents and SP release had crucial roles
in H\textsubscript{2}O\textsubscript{2} induced effects in the human
bladder, which support (ii). This conclusion was based on our two
results. First, desensitization of bladder sensory afferents with
capsaicin or blockade of nerve firing with TTX attenuated the effects of
H\textsubscript{2}O\textsubscript{2} significantly. Second, the
H\textsubscript{2}O\textsubscript{2}-induced enhancement of SBCs was
mimicked with SP and reduced significantly by antagonists of NK2Rs,
which suggested SP release. However, our results did not deny the
importance of the spinal reflex pathway evoked by sensory-afferent
activation \emph{in vivo} {[}15{]} and did not exclude the other
possible underlying mechanisms mentioned above.

TRPA1 channels are expressed predominantly in the sensory-afferent nerve
endings of the bladder{[}27{]}. One study in guinea pigs showed that
H\textsubscript{2}O\textsubscript{2} evoked long-lasting firing of
capsaicin-sensitive high-threshold bladder afferents by activating
TRPA1{[}28{]}. We also found that activation of TRPA1 channels on
bladder sensory afferents mediated the enhancing effects of
H\textsubscript{2}O\textsubscript{2}in the human bladder. The supporting
evidence was: (1) a specific antagonist of TRPA1, HC-030031, attenuated
the H\textsubscript{2}O\textsubscript{2}-induced increase in SBCs
significantly (Fig. 1); (2) the
H\textsubscript{2}O\textsubscript{2}-induced increase in SBCs was
mimicked by TRPA1 agonists (Fig. 2); (3) TRPA1 channels were expressed
on bladder sensory afferents (Fig. 4); (4) studies have shown that
H\textsubscript{2}O\textsubscript{2} mainly targets TRPA1 in sensory
afferents{[}28, 32{]}. In addition to sensory afferents, TRPA1 has been
shown to be localized in the urothelial cells of the urinary
bladder{[}47{]}, but urothelial expression of TRPA1 is
species-specific{[}28{]}. TRPA1 in the bladder urothelium might be
involved in sensory transduction in the bladder and OAB induction by
BOO{[}47, 48{]}. Our immunohistology study also revealed TRPA1
expression in human urothelial cells (Fig. 4A) but
Ca\textsuperscript{2+} imaging studies did not reveal functional
expression of TRPA1 in disassociated human urothelial cells (data not
shown), which indicated a low possibility for urothelial TRPA1
contribution to the effects of H\textsubscript{2}O\textsubscript{2}.
However, we could not exclude the involvement of TRPA1 on sub-urothelial
interstitial cells (ICs) given the important modulatory role of these
ICs in SBCs{[}20{]} and functional expression of TRPA1 in human
sub-urothelial ICs revealed by our previous study{[}49{]}. However, the
significant reduction of H\textsubscript{2}O\textsubscript{2}-induced
effects by desensitization of sensory afferents with capsaicin and
blockade of nerve firing with TTX may suggest that the effect of TRPA1
on sensory afferents has a dominant role.

SP is a neuropeptide present in the capsaicin-sensitive primary afferent
nerves of the urinary bladder. SP release in the periphery produced
enhancing effects on detrusor contractions{[}3{]}. In accordance with
studies in the human bladder{[}40, 41{]}, SP produced prominent
enhancement in SBCs (Fig. 5A). Furthermore, an antagonist of NK2Rs (the
dominant receptor subtypes mediating the effects of neurokinins in the
human bladder){[}40-42{]} attenuated the
H\textsubscript{2}O\textsubscript{2}-induced increase in SBCs
significantly (Fig. 5C and D), which suggested the involvement of SP and
NK2Rs in the effects of H\textsubscript{2}O\textsubscript{2}. This
result is in accordance with studies showing SP release contribute to
increased SBCs induced by a TRPA1 agonist in isolated rat
bladder{[}31{]}.

PGE2 is an important modulator of bladder function and micturition.
Studies have shown PGE2 release contributed to TRPA1 activation-induced
increase in SBCs in the bladder of rats and guinea pigs{[}29, 31, 50{]}.
Our data also indicated the involvement of PGE2 in the
H\textsubscript{2}O\textsubscript{2}-induced enhancement of SBCs in
human-bladder strips. First, PGE2 reproduced similar enhancing effects
to those elicited by H\textsubscript{2}O\textsubscript{2}. Second,
blockade of the synthesis and release of PGE2 with indomethacin
attenuated H\textsubscript{2}O\textsubscript{2} (100\selectlanguage{greek}μ\selectlanguage{english}M)-enhancing
effects significantly (Fig. 6A-B). The role of PGE2 in
H\textsubscript{2}O\textsubscript{2}-induced effects may further prove
sensory-afferent activation by H\textsubscript{2}O\textsubscript{2}.
However, unlike SP (which is released mainly from sensory nerve
terminals), PGE2 can be synthetized and released by cells in the
urothelium or detrusor in addition to sensory nerves. An interaction
between SP and PGE2 has been proposed that release of SP in response to
TRPA1 activation on sensory afferents could stimulate PGE2 production in
nerve endings via an autocrine process{[}50{]}, this idea may support
our proposal.

Our study had three main limitations. First, we did not provide direct
evidence for the release of SP and PGE2: only pharmacological blockade
experiments were undertaken. Second, we did not test the involvement of
other TRP channels (e.g., TRPV1), because literature indicates that
TRPA1 is the dominant channel responsive to
H\textsubscript{2}O\textsubscript{2}{[}28{]}. Third, we did not test the
role of TRPA1 on ICs in H\textsubscript{2}O\textsubscript{2}-induced
effects.

\textbf{5. Conclusions}

Our study provides evidence that H\textsubscript{2}O\textsubscript{2},
at a concentration range associated with inflammation and
ischemia--reperfusion{[}28{]}, enhanced SBCs of human-bladder.
Activation of TRPA1 channels on peripheral sensory nerves and then the
release of SP or PGE2 were important mechanism for
H\textsubscript{2}O\textsubscript{2} induced enhancing effects. Our
study provides new insights for the mechanisms underlying ROS leading to
OAB. Targeting TRPA1 channels might be the promising strategy for
clinical treatment of OAB in conditions associated with excessive
production of ROS.

\textbf{Acknowledgments}

This work was supported by: The National Natural Science Funds of China
(82070783, Zhang) and Natural Science Funds of Shandong Province
(ZR2020MH083, Ge;ZR2022QH097, Liu).

\textbf{Data availability}

Data will be made available on request.

\textbf{Conflict of interest}

None of the contributing authors have any conflicts of interest to
disclose.

\textbf{Figure legends}

\textbf{Figure 1.
H\textsubscript{2}O\textsubscript{2}concentration-dependently enhanced
the spontaneous bladder contractions (SBCs) of isolated human-bladder
strips, which could be inhibited by an antagonist of TRPA1: HC-030031} .
(\textbf{A} ) Representative traces showing
H\textsubscript{2}O\textsubscript{2} (1\selectlanguage{greek}μ\selectlanguage{english}M--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
H\textsubscript{2}O\textsubscript{2}. (\textbf{B} ) Representative
traces showing H\textsubscript{2}O\textsubscript{2}-induced increase in
SBCs was attenuated in the presence of an antagonist of TRPA1: HC-030031
(HC03; 30\selectlanguage{greek}μ\selectlanguage{english}M). HC03 was applied 5-10 min before and during application of
H\textsubscript{2}O\textsubscript{2}. (\textbf{C} ) Noncumulative
concentration--response curves of H\textsubscript{2}O\textsubscript{2}in
the absence and presence of HC03. AUC and baseline tension were measured
at 15-min intervals immediately before and after
H\textsubscript{2}O\textsubscript{2} treatment.
H\textsubscript{2}O\textsubscript{2}-induced effects were evaluated by
the percent increase in AUC and net increase in baseline tension (\selectlanguage{greek}Δ\selectlanguage{english}g =
after minus before). \selectlanguage{greek}Δ\selectlanguage{english}g was normalized further with the wet weight of
the strip (\selectlanguage{greek}Δ\selectlanguage{english}g/g). Solid lines represent the fitted curves using the Hill
equation, from which were obtained an EC\textsubscript{50} of 175.8\selectlanguage{greek}μ\selectlanguage{english}M
(H\textsubscript{2}O\textsubscript{2}alone) and 1.29mM (HC03 +
H\textsubscript{2}O\textsubscript{2}) for AUC; 40.3\selectlanguage{greek}μ\selectlanguage{english}M
(H\textsubscript{2}O\textsubscript{2} alone) and 743.9\selectlanguage{greek}μ\selectlanguage{english}M (HC03 +
H\textsubscript{2}O\textsubscript{2}) for \selectlanguage{greek}Δ\selectlanguage{english}g. *\emph{p} \textless{}
0.05,**\emph{p} \textless{} 0.01, ***\emph{p} \textless{} 0.001 by
one-sample t-test. Data are the mean \selectlanguage{ngerman}± SEM. Data from n = 4--6 strips
were averaged for each concentration.

\textbf{Figure 2. Activation of TRPA1 produced similar enhancing effects
to H\textsubscript{2}O\textsubscript{2} in isolated human-bladder
strips.} (\textbf{A} and \textbf{B} ) Representative traces (A) and
summary data (B) showing that AITC (10--1000\selectlanguage{greek}μ\selectlanguage{english}M) increased SBCs in a
concentration-dependent manner. The concentration--response curves of
AITC-induced increase in AUC (top) gave an EC\textsubscript{50} of
195.3\selectlanguage{greek}μ\selectlanguage{english}M for AUC, and a reliable EC\textsubscript{50} for \selectlanguage{greek}Δ\selectlanguage{english}g (below)
could not be obtained because saturation was not reached even at 1 mM of
AITC. (\textbf{C} and \textbf{D} ) Representative traces (C) and summary
data (D) showing AITC (300\selectlanguage{greek}μ\selectlanguage{english}M)-induced increase in SBCs was blocked
partially by the TRPA1 antagonist HC-030031 (HC03; 30\selectlanguage{greek}μ\selectlanguage{english}M). HC03 was
applied 5-10 min before and during AITC application. n: number of the
bladder strips; N: number of human samples. ***\emph{p} \textless{}
0.001.

\textbf{Figure 3. Desensitization of bladder sensory afferents with
capsaicin or blockade of sodium channels with TTX attenuated
H\textsubscript{2}O\textsubscript{2}-induced enhancing
effects.}(\textbf{A} and \textbf{B} ) Representative traces (A) and
summary data (B) showing pretreatment with capsaicin (10\selectlanguage{greek}μ\selectlanguage{english}M) or TTX (1\selectlanguage{greek}μ\selectlanguage{english}M)
reduced H\textsubscript{2}O\textsubscript{2}-induced increases in SBCs
of human-bladder strips significantly. Capsaicin or TTX was applied 15
min before and during H\textsubscript{2}O\textsubscript{2}(100\selectlanguage{greek}μ\selectlanguage{english}M)
administration. (\textbf{C} and \textbf{D} ) Representative traces (C)
and summary data (D) showing pretreatment with capsaicin (10\selectlanguage{greek}μ\selectlanguage{english}M) or TTX
(1\selectlanguage{greek}μ\selectlanguage{english}M) reduced AITC (100\selectlanguage{greek}μ\selectlanguage{english}M)-induced increases in SBCs of human-bladder
strips significantly. n: number of the bladder strips; N: number of
human samples. ***\emph{p} \textless{} 0.001.

\textbf{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\selectlanguage{ngerman}× magnification) and TRPA1 (green,
400× magnification) in sub-urothelial (A) and intermuscular (B) nerve
terminals and their co-localization (yellow, 400× magnification).

\textbf{Figure 5. A selective antagonist of NK2 receptors attenuated
H\textsubscript{2}O\textsubscript{2}-induced enhancing
effects.}(\textbf{A} and \textbf{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
(10\selectlanguage{greek}μ\selectlanguage{english}M). SR 48968 was applied 5 min before and during
H\textsubscript{2}O\textsubscript{2} (100\selectlanguage{greek}μ\selectlanguage{english}M) administration. (\textbf{C}
and \textbf{D} ) Representative traces (C) and summary data (D) showing
pretreatment with SR 48968 attenuated
H\textsubscript{2}O\textsubscript{2}-induced increase in SBCs. n: number
of bladder strips, N: number of human samples. ***\emph{p} \textless{}
0.001.

\textbf{Figure 6. Blockade of PGE2 synthesis with indomethacin
attenuated H\textsubscript{2}O\textsubscript{2}-induced enhancing
effects.} (\textbf{A-B} ) Representative traces showing PGE2 (1\selectlanguage{greek}µ\selectlanguage{english}M)
administration induced enhancement in SBCs (A), and that pretreatment
with indomethacin attenuated
H\textsubscript{2}O\textsubscript{2}-induced increases in SBCs (B).
Indomethacin was applied 10--15 min before and during
H\textsubscript{2}O\textsubscript{2} (100\selectlanguage{greek}μ\selectlanguage{english}M) administration. (\textbf{C}
) Summary data showing pretreatment with indomethacin reduced
H\textsubscript{2}O\textsubscript{2} (100\selectlanguage{greek}μ\selectlanguage{english}M)-induced increases in AUC
and \selectlanguage{greek}Δ\selectlanguage{english}g significantly. *\emph{p} \textless{} 0.05.

\textbf{Supplementary Figure 1} . Desensitization effect of repeat
application of H\textsubscript{2}O\textsubscript{2}. \textbf{(A)}Typical
traces showing that, on the same bladder strip, second-time application
of H\textsubscript{2}O\textsubscript{2} evoked a lower response compared
with first-time application. \textbf{(B)} Summary data from five strips.
**\emph{p} \textless{} 0.01.

\textbf{References:}

{[}1{]}. Abrams, P., et al., Fourth International Consultation on
Incontinence Recommendations of the International Scientific Committee:
Evaluation and treatment of urinary incontinence, pelvic organ prolapse,
and fecal incontinence. Neurourol Urodyn, 2010. 29(1): p. 213-40.

{[}2{]}. Haylen, B.T., et al., An International Urogynecological
Association (IUGA)/International Continence Society (ICS) joint report
on the terminology for female pelvic floor dysfunction. Int Urogynecol
J, 2010. 21(1): p. 5-26.

{[}3{]}. Andersson, K.E. and A. Arner, Urinary bladder contraction and
relaxation: physiology and pathophysiology. Physiol Rev, 2004. 84(3): p.
935-86.

{[}4{]}. Fry, C.H., et al., Spontaneous activity and electrical coupling
in human detrusor smooth muscle: implications for detrusor overactivity?
Urology, 2004. 63(3 Suppl 1): p. 3-10.

{[}5{]}. Saitoh, S., et al., Hydrogen peroxide: a feed-forward dilator
that couples myocardial metabolism to coronary blood flow. Arterioscler
Thromb Vasc Biol, 2006. 26(12): p. 2614-21.

{[}6{]}. Grivas, N., et al., The effectiveness of otis urethrotomy
combined with six weeks urethral dilations until 40 Fr in the treatment
of bladder outlet obstruction in women: a prospective study. Urol J,
2014. 10(4): p. 1063-6.

{[}7{]}. Kennelly, M., et al., Efficacy and Safety of AbobotulinumtoxinA
in Patients with Neurogenic Detrusor Overactivity Incontinence
Performing Regular Clean Intermittent Catheterization: Pooled Results
from Two Phase 3 Randomized Studies (CONTENT1 and CONTENT2). Eur Urol,
2022. 82(2): p. 223-232.

{[}8{]}. Xiao, N., et al., Roles of polyuria and hyperglycemia in
bladder dysfunction in diabetes. J Urol, 2013. 189(3): p. 1130-6.

{[}9{]}. Zhang, Q., et al., Effects of ischemia and oxidative stress on
bladder purinoceptors expression. Urology, 2014. 84(5): p. 1249.e1-7.

{[}10{]}. Gomez-Pinilla, P.J., et al., Melatonin restores impaired
contractility in aged guinea pig urinary bladder. J Pineal Res, 2008.
44(4): p. 416-25.

{[}11{]}. Swindle, E.J., J.A. Hunt and J.W. Coleman, A comparison of
reactive oxygen species generation by rat peritoneal macrophages and
mast cells using the highly sensitive real-time chemiluminescent probe
pholasin: inhibition of antigen-induced mast cell degranulation by
macrophage-derived hydrogen peroxide. J Immunol, 2002. 169(10): p.
5866-73.

{[}12{]}. Yu, H.J., et al., Hypoxia preconditioning attenuates bladder
overdistension-induced oxidative injury by up-regulation of Bcl-2 in the
rat. J Physiol, 2004. 554(Pt 3): p. 815-28.

{[}13{]}. Allen, C.L. and U. Bayraktutan, Oxidative stress and its role
in the pathogenesis of ischaemic stroke. Int J Stroke, 2009. 4(6): p.
461-70.

{[}14{]}. Huang, Y.B., et al., Anti-oxidant activity and attenuation of
bladder hyperactivity by the flavonoid compound kaempferol. Int J Urol,
2014. 21(1): p. 94-8.

{[}15{]}. Masuda, H., et al., BJU Int, 2008. 101(6): p. 775-80.

{[}16{]}. Azadzoi, K.M., S.V. Yalla and M.B. Siroky, Oxidative stress
and neurodegeneration in the ischemic overactive bladder. J Urol, 2007.
178(2): p. 710-5.

{[}17{]}. Wang, M., et al., Regulation of Spontaneous Contractions in
Intact Rat Bladder Strips and the Effects of Hydrogen Peroxide. Biomed
Res Int, 2018. 2018: p. 2925985.

{[}18{]}. Homan, T., et al., Novel mouse Reactive oxygen species mediate
detrusor overactivity via sensitization of afferent pathway in the
bladder of anaesthetized rats. model of chronic inflammatory and
overactive bladder by a single intravesical injection of hydrogen
peroxide. J Pharmacol Sci, 2013. 121(4): p. 327-37.

{[}19{]}. Han, J.H., et al., Effect of low concentrations of hydrogen
peroxide on the contractile responses of rat detrusor smooth muscle
strips. Eur J Pharmacol, 2010. 638(1-3): p. 115-20.

{[}20{]}. Fry, C.H. and K.D. McCloskey, Spontaneous Activity and the
Urinary Bladder. Adv Exp Med Biol, 2019. 1124: p. 121-147.

{[}21{]}. Biers, S.M., J.M. Reynard and A.F. Brading, The effects of a
new selective beta3-adrenoceptor agonist (GW427353) on spontaneous
activity and detrusor relaxation in human bladder. BJU Int, 2006. 98(6):
p. 1310-4.

{[}22{]}. Finney, S.M., L.H. Stewart and J.I. Gillespie, Cholinergic
activation of phasic activity in the isolated bladder: possible evidence
for M3- and M2-dependent components of a motor/sensory system. BJU Int,
2007. 100(3): p. 668-78.

{[}23{]}. Maggi, C.A., et al., Regional differences in the motor
response to capsaicin in the guinea-pig urinary bladder: relative role
of pre- and postjunctional factors related to neuropeptide-containing
sensory nerves. Neuroscience, 1988. 27(2): p. 675-88.

{[}24{]}. Lee, K., et al., Role of PTHrP and Sensory Nerve Peptides in
Regulating Contractility of Muscularis Mucosae and Detrusor Smooth
Muscle in the Guinea Pig Bladder. J Urol, 2016. 196(4): p. 1287-94.

{[}25{]}. Gillespie, J.I., Inhibitory actions of calcitonin gene-related
peptide and capsaicin: evidence for local axonal reflexes in the bladder
wall. BJU Int, 2005. 95(1): p. 149-56.

{[}26{]}. Maggi, C.A., et al., Further studies on the motor response of
the human isolated urinary bladder to tachykinins, capsaicin and
electrical field stimulation. Gen Pharmacol, 1989. 20(5): p. 663-9.

{[}27{]}. Streng, T., et al., Distribution and function of the hydrogen
sulfide-sensitive TRPA1 ion channel in rat urinary bladder. Eur Urol,
2008. 53(2): p. 391-9.

{[}28{]}. Nicholas, S., et al., Hydrogen peroxide preferentially
activates capsaicin-sensitive high threshold afferents via TRPA1
channels in the guinea pig bladder. Br J Pharmacol, 2017. 174(2): p.
126-138.

{[}29{]}. Daugherty, S.L., et al., TRP Channel Agonists Activate
Different Afferent Neuromodulatory Mechanisms in Guinea Pig Urinary
Bladder. Front Physiol, 2021. 12: p. 692719.

{[}30{]}. Artim, D.E., et al., Nitro-oleic acid targets transient
receptor potential (TRP) channels in capsaicin sensitive afferent nerves
of rat urinary bladder. Exp Neurol, 2011. 232(1): p. 90-9.

{[}31{]}. Andrade, E.L., et al., Contractile mechanisms coupled to TRPA1
receptor activation in rat urinary bladder. Biochem Pharmacol, 2006.
72(1): p. 104-14.

{[}32{]}. Sawada, Y., et al., Activation of transient receptor potential
ankyrin 1 by hydrogen peroxide. Eur J Neurosci, 2008. 27(5): p. 1131-42.

{[}33{]}. Andersson, D.A., et al., Transient receptor potential A1 is a
sensory receptor for multiple products of oxidative stress. J Neurosci,
2008. 28(10): p. 2485-94.

{[}34{]}. Sugiyama, D., et al., Hydrogen Peroxide Induces Muscle
Nociception via Transient Receptor Potential Ankyrin 1 Receptors.
Anesthesiology, 2017. 127(4): p. 695-708.

{[}35{]}. Sibley, G.N., A comparison of spontaneous and nerve-mediated
activity in bladder muscle from man, pig and rabbit. J Physiol, 1984.
354: p. 431-43.

{[}36{]}. Taylor-Clark, T.E., Role of reactive oxygen species and TRP
channels in the cough reflex. Cell Calcium, 2016. 60(3): p. 155-62.

{[}37{]}. McNamara, C.R., et al., TRPA1 mediates formalin-induced pain.
Proc Natl Acad Sci U S A, 2007. 104(33): p. 13525-30.

{[}38{]}. Saitoh, C., et al., The differential contractile responses to
capsaicin and anandamide in muscle strips isolated from the rat urinary
bladder. Eur J Pharmacol, 2007. 570(1-3): p. 182-7.

{[}39{]}. Gillespie, J.I., Inhibitory actions of calcitonin gene-related
peptide and capsaicin: evidence for local axonal reflexes in the bladder
wall. BJU Int, 2005. 95(1): p. 149-56.

{[}40{]}. Maggi, C.A., et al., Contractile response of the human
isolated urinary bladder to neurokinins: involvement of NK-2 receptors.
Eur J Pharmacol, 1988. 145(3): p. 335-40.

{[}41{]}. Dion, S., et al., Substance P and neurokinins as stimulants of
the human isolated urinary bladder. Neuropeptides, 1988. 11(2): p. 83-7.

{[}42{]}. Dion, S., et al., Receptors for neurokinins in human bronchus
and urinary bladder are of the NK-2 type. Eur J Pharmacol, 1990. 178(2):
p. 215-9.

{[}43{]}. Kobayter, S., J.S. Young and K.L. Brain, Prostaglandin E2
induces spontaneous rhythmic activity in mouse urinary bladder
independently of efferent nerves. Br J Pharmacol, 2012. 165(2): p.
401-13.

{[}44{]}. Quinn, T., C. Collins and A.W. Baird, Mechanisms of neurokinin
A- and substance P-induced contractions in rat detrusor smooth muscle in
vitro. BJU Int, 2004. 94(4): p. 651-7.

{[}45{]}. Stephany, H.A., et al., Chronic cyclic bladder over distention
up-regulates hypoxia dependent pathways. J Urol, 2013. 190(4 Suppl): p.
1603-9.

{[}46{]}. Nomiya, M., K.E. Andersson and O. Yamaguchi, Chronic bladder
ischemia and oxidative stress: new pharmacotherapeutic targets for lower
urinary tract symptoms. Int J Urol, 2015. 22(1): p. 40-6.

{[}47{]}. Du S, et al., Differential expression profile of cold (TRPA1)
and cool (TRPM8) receptors in human urogenital organs. Urology, 2008.
72(2): p. 450-5.

{[}48{]}. Andersson, K.E., C. Gratzke and P. Hedlund, The role of the
transient receptor potential (TRP) superfamily of cation-selective
channels in the management of the overactive bladder. BJU Int, 2010.
106(8): p. 1114-27.

{[}49{]}. Zhao, M., et al., Functional Expression of Transient Receptor
Potential and Piezo1 Channels in Cultured Interstitial Cells of
Human-Bladder Lamina Propria. Front Physiol, 2021. 12: p. 762847.

{[}50{]}. Philyppov, I.B., et al., TRPA1-dependent regulation of bladder
detrusor smooth muscle contractility in normal and type I diabetic rats.
J Smooth Muscle Res, 2016. 52: p. 1-17.\selectlanguage{english}
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