TRP Channel Agonists Activate Different Afferent Neuromodulatory Mechanisms in Guinea Pig Urinary Bladder

Activation of TRP channels expressed in urinary bladder afferent nerves and urothelium releases neurotransmitters that influence bladder function. Experiments were undertaken to examine the mechanisms underlying effects of TRPA1 (allyl isothiocyanate, AITC), TRPV1 (capsaicin, CAPS), and TRPC (oleoyl-2-acetyl-sn-glycerol, OAG) agonists on guinea pig bladder activity. Effects of these agonists were compared with effects of nitro-oleic acid (OA-NO2), an electrophilic nitro-fatty acid, known to activate TRPV1, TRPA1 or TRPC channels in sensory neurons. AITC (100 μM) increased (231%) area of spontaneous bladder contractions (SBCs) an effect reduced by a TRPA1 antagonist (HC3-03001, HC3, 10 μM) and reversed to inhibition by indomethacin (INDO, 500 nM) a cyclooxygenase inhibitor. The post-INDO inhibitory effect of AITC was mimicked (39% depression) by calcitonin gene-related peptide (CGRP, 100 nM) and blocked by a CGRP antagonist (BIBN, 25 μM). CAPS (1 μM) suppressed SBCs by 30% in 81% of strips, an effect blocked by a TRPV1 antagonist (diarylpiperazine, 1 μM) or BIBN. SBCs were suppressed by OA-NO2 (30 μM, 21% in 77% of strips) or by OAG (50 μM, 30%) an effect blocked by BIBN. OA-NO2 effects were not altered by HC3 or diarylpiperazine. OA-NO2 also induced excitation in 23% of bladder strips. These observations raise the possibility that guinea pig bladder is innervated by at least two types of afferent nerves: [1] Type A express TRPA1 receptors that induce the release of prostaglandins and excite the detrusor, [2] Type B express TRPV1, TRPA1 and TRPC receptors and release CGRP that inhibits the detrusor.


INTRODUCTION
The urinary bladder is innervated by small myelinated (Aδ) and unmyelinated (C-fiber) afferent nerves that respond to mechanical and chemical stimuli and then transmit information to the central nervous system about bladder filling and the chemical environment in the bladder (de Groat et al., 2015). Activation of C-fiber bladder afferents also releases neurotransmitters that act on the smooth muscle to directly regulate bladder contractility (Maggi, 1993). This "efferent function" of bladder afferent nerves is mediated in part by neuropeptides (Maggi and Meli, 1986;Maggi, 1991Maggi, , 1993. Thus, bladder afferents are classified, based on receptors and transmitters that they express as well as by axonal size and myelination. A common type of bladder afferent has an unmyelinated axon, expresses multiple types of transient receptor potential (TRP) channels and synthesizes and releases neuropeptides such as neurokinins or calcitonin gene-related peptide (CGRP) (de Groat and Yoshimura, 2009). In many species these afferents respond to agonists for TRPV1 (capsaicin, CAPS) (Maggi and Meli, 1986;de Groat and Yoshimura, 2009) or TRPA1 receptors (allyl isothiocyanate, AITC) by releasing neurotransmitters that enhance (Maggi, 1993;Andrade et al., 2006;Artim et al., 2011) or inhibit contractions (Maggi, 1993;Gillespie, 2005) of the bladder smooth muscle. In rats the excitatory effect is also mimicked by application of OA-NO 2 , a thiol-reactive electrophilic nitrofatty acid (Artim et al., 2011) that mediates the post-translational modification of susceptible protein thiols, thus activating TRPV1 and TRPA1 receptors in rat dorsal root ganglion (DRG) cells (Sculptoreanu et al., 2010;Zhang et al., 2014b). OA-NO2 or other nitro-fatty acids which have exhibited anti-inflammatory properties in both animal and clinical studies (Rudolph et al., 2010;Freeman et al., 2018;Schopfer et al., 2018) may find use in the future in the treatment of inflammatory or painful bladder conditions. However, the effect of OA-NO 2 on sensory nerves is complicated because this thiol-reactive electrophile will target different receptors in different afferent nerves and in different species. While OA-NO 2 activates TRPV1 and TRPA1 both in rat DRG neurons (Sculptoreanu et al., 2010) and bladder afferent nerves (Artim et al., 2011), OA-NO 2 selectively targets TRPA1 in mouse vagal afferent neurons (Taylor-Clark et al., 2009) and targets TRPV1, TRPA1, or TRPC in guinea pig DRG neurons (Zhang et al., 2014a).
The present experiments were undertaken to determine the mechanisms underlying effects of OA-NO 2 on the guinea pig bladder and to compare its effects with those of more selective activators of TRPA1, TRPV1, and TRPC (oleoyl-2-acetyl-snglycerol, OAG). The effects of four agonists (CAPS, AITC, OAG, and OA-NO 2 ) were examined on spontaneous activity of bladder strips with intact mucosa from adult guinea pigs.

Data Analysis
All data are presented as mean ± S.E.M. To study the onset and duration of agonist-evoked responses, majority of the bladder strip activity was measured in 5 min intervals immediately before (basal activity) and at varying intervals after application of the agonists, during the time period when they exerted their maximal or prolonged effects: [a] 0-30 s for CAPS early transient response (compared to a 30 s interval immediately before application of CAPS), [b] 0-5 min for OA-NO 2 , CAPS, SP, and CGRP, [c] 10-15 min for AITC and prostaglandin E 2 , [d] 0-65 min for OA-NO 2 , AITC, CAPS, and OAG. The effects were measured as a change in area under the curve (AUC) which reflects changes in amplitude and frequency of SBCs and as a change in baseline tension. Data are reported as percent change from basal activity. Eighty percent of the experiments were conducted on tissue from female animals; and the data from both sexes were combined for data analysis because strips from male and female guinea pig did not exhibit obvious differences in response to agonists and antagonists. Statistical significance was tested on raw data using a paired two tailed t-test, * P < 0.05 and * * P < 0.001, a one-way ANOVA with Dunnett's multiple comparison posthoc test ( + P < 0.05) or a two-way ANOVA and Bonferroni multiple comparisons post-hoc test (brackets, # P < 0.05) when appropriate. Excel and Graph Pad Prism 7 software were used for analysis. Data were considered statistically significant when P < 0.05.

AITC, a TRPA1 Agonist, Enhances SBCs
When stretched with 1 g tension, guinea pig bladder strips displayed spontaneous bladder contractions (SBCs) of variable amplitude and frequency (Figures 1A-F) that persisted over many hours. Consistent with its excitatory effect in rat bladder strips (Andrade et al., 2006;Artim et al., 2011) the TRPA1 agonist AITC (100 µM) increased AUC of SBCs by 231 ± 47% and baseline tension by 37 ± 6% in all strips (n = 16 strips from nine female and two male guinea pigs; P < 0.001; Figure 1A). The excitatory effect reached a maximum at 4-5 min after application and persisted for over 30 min.

CAPS, a TRPV1 Agonist, Has Bimodal Facilitatory and Inhibitory Effects on SBCs
CAPS at a concentration (1 µM) that enhances rat SBCs (Artim et al., 2011), elicited a bimodal response consisting of a transient excitation lasting on average 35 s that increased the AUC of a few contractions by 128 ± 50% (P < 0.002) followed by a more prominent inhibition in 81% of 26 strips from 1 male and 16 female guinea pigs ( Figure 1B). The inhibition decreased the AUC by 30 ± 5% and baseline tension by 11 ± 4% (P < 0.01, Figure 1B). Inhibition reached a maximum at an average of 2 min after application and persisted for 25-30 min. In the remaining strips CAPS increased the SBCs by 29 ± 16% for a period of several minutes ( Figure 1E). However, the magnitude of the prolonged excitatory effect was variable between experiments and the average increase in the AUC was not statistically significant (P > 0.05).

OANO 2 , a Pleiotropic-TRP Agonist, Has Mixed Inhibitory, and Facilitatory Effects on Guinea Pig SBCs
In contrast to its excitatory effect in rat bladder strips (Artim et al., 2011), OA-NO 2 at 30 µM elicited either inhibitory or excitatory effects on SBCs (n = 48 strips from 5 male and 24 female guinea pigs). Inhibition was present in 77% of strips ( Figure 1C), where it decreased the area under the curve (AUC) by 21 ± 3%, (P < 0.001) measured 0-5 min after application, while excitation occurred in 23% of strips ( Figure 1F), where it significantly increased the AUC by 28 ± 9% (P < 0.01). Neither response was accompanied by a significant change in the baseline tension (P > 0.05; Figures 1C,F). The inhibitory and excitatory effects reached a maximum at 2-3 min, after application and the effects persisted for 25-30 min and 5 min, respectively (P < 0.05). Oleic acid (30 µM), a non-electrophilic fatty acid control, did not alter AUC or baseline tension (P > 0.05, n = 5 strips from three animals; data not shown).

Effect of Tetrodotoxin (TTX, 1 µM) on Agonist Evoked Responses
To examine the role of efferent neurons or an intramural neural network, in the responses of TRP channel agonists the agonists were administered in the presence of TTX. TTX did not significantly alter: [1] control SBCs (five strips from two guinea pigs; Figures 2A, 3E), [2] the excitatory effect of AITC (100 µM, three strips from three guinea pigs, 124 ± 30% increase in AUC, data not shown), [3] the inhibitory effect of OA-NO 2 (three strips from three guinea pigs, 15 ± 11% decrease of AUC, Figure 2A) or [4] the inhibitory effect of CAPS (1 µM, six strips from three guinea pigs, 19 ± 7% decrease in AUC, data not shown). However, the initial transient excitatory effect of CAPS which preceded the inhibition was blocked by TTX (P < 0.05, n = 6 strips from three guinea pigs, 19.5 ± 18% decrease of AUC, measured 30 s after application of CAPS, data not shown).

Desensitization of Agonist Responses
In a separate group of experiments, agonists were applied multiple times to the same bladder strip after washout and long recovery periods (60-100 min). Neither OA-NO 2 or CAPS elicited responses during the second application (n = 5 and 9 strips from three and four guinea pigs respectively, P < 0.05, data not shown). On the other hand, the second application of AITC elicited a response (203 ± 62% increase in the SBCs AUC, n = 5 strips), that was not significantly different (P > 0.05) from that elicited by the first application (data not shown).
In another group of experiments where multiple agents were administered sequentially to the same bladder strip (Figure 2) application of CAPS blocked the responses to the subsequent application of OA-NO 2, even at a time point when the SBCs had recovered from the initial inhibitory effect of CAPS ( Figure 2B, n = 14 strips from 11 guinea pigs, P < 0.01). On the other hand, OA-NO 2 did not alter the inhibitory response to the subsequent application of CAPS ( Figure 2C, n = 17 strips from 13 guinea pigs). Administration of AITC during the inhibition induced by the combined application of OA-NO 2 and CAPS rapidly reversed Drugs were applied at the arrows and maintained in the tissue bath. Graphs summarize the major responses to the three agonists as % change in SBC area under the curve (AUC) compared to basal activity, baseline tension (BLT) and the percentage of strips exhibiting the responses. (D-F) Upper traces show the slow onset suppression of SBCs by OAG and the less common excitatory effects of CAPS and OA-NO 2 . Graphs summarize the responses of the three agonists as % change in SBC area under the curve (AUC) compared to basal activity, baseline tension (BLT) and the percentage of strips exhibiting the responses. Calibration bars refer to time (min) and tension (gram, g). Statistics were performed on raw numbers: 2-tailed t-test, *P < 0.05 and **P < 0.00, compared with basal activity; n, number of strips.

Effects of TRPA1, TRPV1, and TRPC Antagonists
After application of a combination of diarylpiperazine (DP, 1 µM), a TRPV1 antagonist and HC3-03001 (HC3, 10 µM), a selective TRPA1 receptor antagonist, at concentrations used in previous studies (Sculptoreanu et al., 2010;Artim et al., 2011;Zhang et al., 2014a) the bimodal facilitatory-inhibitory effect of CAPS was eliminated ( Figure 3B, n = 12 strips from five guinea pigs) and the facilitatory response of AITC was significantly reduced (Figure 3C, n = 16 strips from four guinea pigs). Application of DP or HC3 alone or in combination did not change the SBCs (n = 10 strips from four guinea pigs; Figures 2D, 3E).
Release of CGRP Mediates OA-NO 2 , OAG, and Capsaicin (CAPS) Induced Inhibition CGRP released from bladder C-fiber afferents has been reported to suppress spontaneous contractions of the guinea pig bladder (Maggi, 1993;Gillespie et al., 2006;Lee et al., 2016). In our experiments, CGRP (100 nM) significantly reduced the AUC and baseline tension by 39 ± 6% and 11 ± 6% respectively (n = 9 strips from nine guinea pigs, P < 0.001) and this inhibition was reversed (Figure 4A) or blocked in the presence of a CGRP antagonist (BIBN, 25 µM; n = 7 strips from three guinea pigs, 3 ± 6% decrease; Figure 4G) (Doods et al., 2000). To test the possibility that the inhibitory effects of other agonists on SBCs were mediated by release of CGRP, bladder strips were treated with BIBN (25 µM) before ( Figure 4G) and after OA-NO 2 , OAG, or CAPS application (Figures 4B-D). BIBN blocked the inhibitory effects of OA-NO 2 (n = 6 strips from four guinea pigs, 4 ± 4%), CAPS (n = 9 strips from five guinea pigs, 3 ± 5%) and OAG (n = 8 strips from four guinea pigs, 11 ± 7% decrease; Figure 4G). However, the transient facilitatory effect of CAPS which was detected in the majority of untreated preparations was similar in magnitude (172% increase in SBCs AUC) and duration in preparations treated with BIBN (n = 9 strips, data not shown). Thus, the block of inhibition by BIBN did not unmask prolonged facilitatory effects of CAPS or OANO 2 that occurred in the minority of untreated preparations (Figures 1E,F). BIBN (25 µM) administered alone increased SBC AUC by 20 ± 5% (P < 0.001, n = 13 strips from five guinea pigs, Figures 3E, 4F) suggesting that in the distended bladder there is tonic release of CGRP which inhibits bladder contractility and that OA-NO 2 , CAPS, and OAG inhibit SBCs by increasing the release of CGRP.

Release of Substance P Is Not the Primary Mechanism of AITC Induced Excitation
Substance P (SP) released from bladder afferents contributes to the excitatory effects of CAPS, OA-NO 2 , and AITC on SBC in rat bladder strips (Andrade et al., 2006;Artim et al., 2011). In the present experiments, application of SP (1 µM) mimicked the excitatory effect of AITC on spontaneous contractile activity (Figures 5A,D) significantly increasing SBC AUC by 128 ± 48% (P < 0.01) but only slightly increased baseline tension (5 ± 2%, P < 0.05; Figure 5D, n = 8 strips from five guinea pigs). To determine the role of SP release in AITC induced excitation, bladder strips were pretreated with a combination of neurokinin receptor 1, 2, and 3 antagonists (SR 140333, SR 48968, and SR 142801; 1 µM each) which blocks the excitatory effect of substance P in the guinea pig ileum (Holzer et al., 1998;Takahashi et al., 2002). The neurokinin receptor antagonists alone (n = 8 strips from eight guinea pigs) decreased AUC by 33 ± 9% (P < 0.05), without changing the baseline tension and significantly reduced the SP induced enhancement of SBCs (n = 8 strips from seven guinea pigs, Figures 3E,  5F,G). However, pretreatment with the neurokinin receptor antagonists did not significantly change the AITC induced enhancement of SBCs (n = 5 strips from five guinea pigs, Figures 5F,G, p > 0.05) indicating that SP released from bladder afferents is not the primary mechanism for AITC induced excitatory responses.  Figure 2. Data was obtained from single experiments with either a pristine agonist or an antagonist followed by an agonist and graphs are presented as % change in SBC area under the curve (AUC) compared to basal activity. Statistics were performed on raw numbers: (A-C) 2-tailed t-test, *P < 0.05 and **P < 0.001, and a two-way ANOVA with Bonferroni multiple comparisons post-hoc tests (brackets), # P < 0.05; (D) one-way ANOVA with Dunnett's multiple comparison post-hoc test (OAG; + P < 0.05) and two-way ANOVA (bracket) with Bonferroni multiple comparisons post-hoc test (La 3+ ; # P < 0.05); (E) 2-tailed t-test, *P < 0.05, compared with basal activity; n, number of strips].

DISCUSSION
This study revealed that activation of TRP channels in guinea pig urinary bladder can either enhance or suppress spontaneous contractile activity by releasing prostaglandins or CGRP, respectively. The main findings are: [1] a TRPA1 agonist (AITC) enhances SBCs by releasing prostaglandins. This effect is blocked by indomethacin (INDO) a COX inhibitor. AITC also inhibits SBCs by releasing CGRP an effect blocked by BIBN, a CGRP receptor antagonist.
[2] A TRPV1 agonist (CAPS), and TRPC agonists (OA-NO 2 or OAG) suppress the contractile activity in the majority of bladder strips by releasing CGRP [3]. The inhibitory effect of CAPS is preceded by a transient facilitation that is blocked by TTX and therefore likely mediated by activation of neurons in the bladder wall. The mechanisms underlying the effects of the TRP agonists on bladder activity are commonly ascribed to activation of transmitter release from intramural afferent nerves because: [1] TRP channels are expressed in bladder afferent neurons (Maggi, 1993;Franken et al., 2014;Shimizu et al., 2017Shimizu et al., , 2018, [2] the TRP agonists are known to activate primary afferent neurons in the guinea pig (Zhang et al., 2014a), [3] CGRP, a peptide neurotransmitter synthesized and released by a large percentage of visceral afferent nerves (Maggi et al., 1988b;Keast and de Groat, 1992;Hwang et al., 2005;Shimizu et al., 2017), is involved in the CAPS (Maggi et al., 1988b) and OA-NO 2 induced inhibition of bladder activity (Figures 1A,B), [4] CGRP release in the guinea pig bladder by CAPS or bradykinin is reduced by desensitization of afferent nerves following local or systemic administration of CAPS (Maggi et al., 1988a(Maggi et al., , 1989, [5] neuropeptides released from bladder afferent nerves modulate smooth muscle activity (Maggi and Meli, 1986;Artim et al., 2011;Franken et al., 2014), and [6] synthesis of prostaglandin can be increased in the urinary bladder by distension or noxious stimuli that activate afferent nerves (Bjorling et al., 1994). However, TRP channels are also expressed in other cells in the bladder, e.g., smooth muscle cells and cells in the urothelial and lamina propria layers of the mucosa (Birder et al., 2001;Kullmann et al., 2009;Everaerts et al., 2010;Yu et al., 2011), which can also release neurotransmitters that influence smooth muscle activity. Because prostaglandins enhance tonic and phasic contractions of isolated mucosal tissue (Stromberga et al., 2020), it is possible that AITC release of prostaglandins in full thickness bladder strips facilitates smooth muscle activity in part via an indirect route involving the mucosa. Thus additional experiments showing: [1] that effects of the agonists are abolished by elimination of the afferent innervation of the bladder or [2] that the responses persist in bladder strips after removal of the mucosa are needed to establish that the agonists act solely via a neurogenic mechanism or if they act on multiple targets in the bladder.
Assuming that afferent nerves are the principal target for the agonists (Figure 6), it is likely that TRPV1, TRPA1, and TRPC channels are co-expressed by one population of afferent nerves  Figure 4E. Statistics were performed on raw numbers: (G,H), 2-tailed t-test, *P < 0.05 and **P < 0.001, and a two-way ANOVA with Bonferroni multiple comparisons post-hoc tests were performed to compare the antagonist effects (brackets), # P < 0.05; n, number of strips. that produces bladder inhibition by releasing CGRP which is known to suppress spontaneous contractions of the guinea pig bladder (Maggi et al., 1988a;Giuliani et al., 1992;Gillespie, 2005;Lee et al., 2016). CAPS releases CGRP in the guinea pig bladder by a calcium dependent mechanism (Maggi, 1991(Maggi, , 1993 and calcium imaging experiments revealed that a percentage of OA-NO 2 responsive guinea pig DRG neurons also respond to CAPS indicating co-expression of TRPV1 and TRPC channels in these neurons (Zhang et al., 2014a). The present experiments showed that pretreatment with CAPS suppresses the inhibitory effect of a subsequent administration of OA-NO 2 ( Figure 2B) further suggesting the co-expression of TRPV1 and TRPC.
CGRP could act at multiple sites to modulate the contractile activity of the bladder strips. CGRP has excitatory or facilitatory effects on peripheral (Cornelissen et al., 2001) and central neurons (Fisher et al., 1983;Han et al., 2010) and therefore it could excite intrinsic neural or neural-interstitial cell networks in the guinea pig bladder (Gillespie et al., 2006) to release inhibitory transmitters. However, a neural mechanism seems unlikely because tetrodotoxin (TTX), which blocks nerve activity, did not alter the spontaneous activity of the bladder strips or the inhibitory responses to OA-NO 2 or CAPS (Figures 2A, 3E). Thus, CGRP released by TRP agonists is likely to act directly on bladder smooth muscle by stimulating the synthesis of cyclic AMP (Maggi et al., 1988b;Russell et al., 2014).
AITC has facilitatory and inhibitory effects on bladder activity (Figure 5). The facilitatory effect (Figure 1A) was blocked by pretreatment with the COX inhibitor, INDO (Figures 4E, 5H) and reduced by a TRPA1 receptor antagonist (HC3; Figures 2D,  3C) indicating that TRPA1 channels are expressed on a second population of afferents and/or on unidentified non-neuronal cells that release prostaglandins (Figure 6) that in turn excite the bladder smooth muscle (Figure 5C). However, in contrast to the action of AITC in the rat bladder where the excitatory effects are suppressed by neurokinin receptor antagonists (Andrade et al., 2006;Artim et al., 2011) the effects in the guinea pig bladder were unaffected by a combination of antagonists targeting neurokinin receptor subtypes 1, 2, and 3. In the presence of FIGURE 6 | Pharmacological experiments suggest that at least two types of sensory nerves expressing TRP channels innervate guinea pig urinary bladder. TRPA1 channels are located on one population of nerves (Type A). Activation of TRPA1 in these nerves and/or unidentified non-neuronal cells by AITC excites bladder smooth muscle; an effect blocked by indomethacin indicating that the excitation is mediated by the synthesis and release of prostaglandins (PGs). Another population of sensory nerves (Type B) expresses TRPV1, TRPA1, and TRPC channels. Activation of these channels by capsaicin (CAPS), AITC, or OA-NO 2 releases CGRP causing inhibition of the bladder smooth muscle. However, in comparison to the prominent effects of CAPS and OA-NO 2 the inhibitory effect of AITC mediated by CGRP release from Type B nerves is relatively weak and only detectable when prostaglandin (PG) synthesis is blocked by indomethacin indicating that it is normally masked by the PG excitation. (+): excitatory; (-): inhibitory.
INDO, AITC decreased bladder contractions, an effect blocked by BIBN (Figures 4E, 5H) indicating that AITC activates TRPA1 receptors that may be co-localized with TRPV1 and TRPC receptors on afferent nerves releasing CGRP (Figure 6). This inhibitory effect of AITC mediated via CGRP release was clearly masked by the more prominent excitatory effect mediated by prostaglandins.
The distribution of TRPA1 channels in two populations of bladder afferents (Figure 6) is consistent with electrophysiological studies of afferents in the guinea pig bladder (Zagorodnyuk et al., 2007(Zagorodnyuk et al., , 2009Nicholas et al., 2017) which revealed five populations of muscle and mucosal mechanosensitive afferents based on the threshold to stretch and the sensitivity to AITC and CAPS. In these studies of urothelium free bladder preparations, low threshold stretch-sensitive afferents respond to AITC but only a small proportion of these afferents respond to CAPS (Nicholas et al., 2017), corresponding to the type A afferent population we show in Figure 6. However, a large percentage (72-86%) of high threshold stretch-sensitive afferents respond to AITC or CAPS, corresponding to the type B afferent population we show in Figure 6.
In the majority of strips CAPS produced a small initial transient enhancement followed by the CGRP mediated inhibition. A similar bimodal effect of CAPS in guinea pig bladder preparations was reported in earlier studies (Maggi et al., 1987;Gillespie, 2005). The transient facilitation but not the inhibition was blocked by pretreatment with TTX suggesting that the facilitatory effect of CAPS is mediated by activation of efferent neurons or an intramural neural network that is known to be present in the guinea pig bladder (Gillespie et al., 2006;Eastham and Gillespie, 2013). The transmitters involved in this facilitatory response were not examined. The TTX resistant release of CGRP is presumably elicited by the ligand gated increase in intracellular Ca 2+ in the sensory nerve terminals or non-neural cells.
TRPV1 and TRPA1 antagonists did not change spontaneous activity (Figures 2D, 3E); however, the CGRP antagonist, BIBN, significantly increased the contractions (Figures 3E,  4F) indicating the existence of tonic CGRP inhibition. This inhibition might be induced by CGRP released from mechanosensitive afferent nerves in response to stretch of the bladder strips and/or the ongoing intrinsic contractions. Spontaneous contractile activity was also significantly reduced by application of a combination of three neurokinin receptor subtype antagonists (Figures 3E, 5F) indicating that neurokinins released from afferent nerves generate a tonic facilitation of spontaneous contractile activity. The tonic release of neurokinins may occur in a third type of afferent other than the two shown in Figure 6.

Limitations of This Study
The main limitation of our study is the use of a single technique, i.e., the measurement of spontaneous contractions of bladder strips and the use of only pharmacological approaches to examine the functions of TRP channels in the bladder. More detailed information about the distribution of TRP channels in the bladder will be obtained in future experiments using immunohistochemical techniques. Mucosa free bladder strips will also be used to limit the possible sites of drug action.

Perspectives and Significance
In summary, activation of TRP channels in guinea pig bladder strips enhances or suppresses SBCs. It is likely that one mechanism underlying these effects is release of signaling molecules from bladder afferent nerves which express multiple TRP channels. Activation of TRPV1, TRPA1 and TRPC channels expressed by one type of afferent nerve releases CGRP that elicits inhibition; while activation of TRPA1 channels in another type of afferent nerve and/or in non-neuronal cells releases prostaglandins that elicit excitation (Figure 6). CGRP and neurokinins which are tonically released are involved in an ongoing inhibition and facilitation, respectively, of SBCs that occurs in the absence of TRP stimulation. This suggests that a third type of mechanosensitive afferent nerve releases neurokinins in response to stretch. These findings further indicate that bladder afferent nerves have complex inhibitory and facilitatory efferent functions in addition to their primary role in bladder sensation and that they release multiple transmitters that modulate bladder smooth activity to influence the storage and elimination of urine. The type B nociceptive afferent population is unusual because it presumably co-releases CGRP with glutamate as excitatory transmitters in the spinal cord (Bird et al., 2006) that can potentially activate central micturition pathways and induce bladder overactivity; while simultaneously releasing CGRP which acts as an inhibitory transmitter in the periphery to suppress spontaneous bladder contractions. It is not known if these two functions can be turned on independently. The ability of OANO 2 , to selectively target the type B afferent population and induce CGRP mediated inhibition combined with its known anti-inflammatory actions might generate a synergistic interaction that could result in an effective treatment for some types of overactive bladder dysfunction.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Ethics Committee of Pittsburg University.

AUTHOR CONTRIBUTIONS
WG, BF, and XZ designed experiments. SD, KK, and JL carried out experiments and analyzed data. JB, SW, and XZ wrote the manuscript. WG critically edited the manuscript. All authors contributed to the article and approved the submitted version.