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.