4. Discussion
Excessive ROS can lead to DO. To investigate the underlying mechanisms,
the effects of H2O2 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 H2O2(1μM 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.
H2O2 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 H2O2 produced similar
stimulatory effects on SBCs in isolated strips of human bladder and rat
bladder [17, 19]. H2O2 can act on
several cell targets or pathways to induce enhanced contraction of the
bladder. H2O2 could target the detrusor
muscle directly to enhance SBCs via activation of cyclooxygenase
or rho-kinase pathways which, in turn, increase extracellular
Ca2+ influx into the detrusor[17, 19].
H2O2 may target urothelium cells to
evoke DO via ATP release, as suggested by Stephany et
al.[45]. Activation of bladder sensory afferents has been shown to
be another important mechanism for
H2O2-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 peripheryvia 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 H2O2 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
H2O2 significantly. Second,
the
H2O2-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 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
H2O2 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
H2O2in the human bladder. The supporting evidence was: (1) a specific
antagonist of TRPA1, HC-030031, attenuated the
H2O2-induced increase in SBCs
significantly (Fig. 1); (2) the
H2O2-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
H2O2 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
Ca2+ 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
H2O2. 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
H2O2-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
H2O2-induced increase in SBCs
significantly (Fig. 5C and D), which suggested the involvement of SP and
NK2Rs in the effects of H2O2. 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
H2O2-induced enhancement of SBCs in
human-bladder strips. First, PGE2 reproduced similar enhancing effects
to those elicited by H2O2. Second,
blockade of the synthesis and release of PGE2 with
indomethacin attenuated
H2O2 (100μM)-enhancing effects
significantly (Fig. 6A-B). The role of PGE2 in
H2O2-induced effects may further prove
sensory-afferent activation by
H2O2.
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
H2O2[28]. Third, we did not test the
role of TRPA1 on ICs in H2O2-induced
effects.