To determine if activation of the TGR5 receptor could influence bladder sensation we determined the impact of peripheral TGR5 activation in an ex vivo bladder afferent preparation. Intravesical application of Pg5α, a progesterone sulphate previously identified as an endogenous TGR5 agonist 28, resulted in a significant increase in bladder afferent firing in response to graded bladder distension compared to saline (Fig. 1A), indicating mechanical hypersensitivity following TGR5 activation. No change was observed in bladder compliance (pressure/volume relationship) between saline and Pg5α experiments (Fig. 1B), indicating that afferent hypersensitivity was not caused by changes in detrusor muscle tone.
Single-unit analysis of the bladder afferent nerve fibres revealed that 44.4% (20/45) of the mechanosensitive bladder afferents demonstrated mechanical hypersensitivity following instillation of Pg5α (‘responders’), whereas 55.6% (25/45) of mechanosensitive afferents were unresponsive to Pg5α (‘non-responders’) (Fig. 1C). Pg5α-responsive afferents showed a significantly increased afferent firing rate at pressures of 20 mmHg and above (Fig. 1Di) and a significant increase in the maximum peak of afferent firing in response to distension following Pg5α instillation (Fig. 1Dii). In comparison, no change in afferent firing was observed between saline and Pg5α experiments for non-responding afferents, neither at any particular distension pressure (Fig. 1Ei) nor in the maximum peak response (Fig. 1Eii). These data demonstrate that the progesterone metabolite Pg5α causes mechanical hypersensitivity of a sub-population of bladder afferents in response to graded bladder distension (Fig. 1F).
The synthetic TGR5 agonist CCDC replicates Pg5α-induced increases in bladder afferent mechanosensitivity
We next aimed to further define the specific involvement of the TGR5 receptor in mediating bladder afferent mechanosensitivity. To this end, we used a potent, synthetic TGR5 agonist, 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N,5-dimethyl-4-isoxazolecarboxamide (CCDC), reported to be TGR5-specific 46. Similar to Pg5α, on applying CCDC to the ex vivo bladder afferent preparation we observed an increase in afferent mechanosensitivity to bladder distension compared to saline (Fig. 2A). No change to muscle compliance was observed between saline and CCDC intravesical instillation (Fig. 2B), demonstrating that the effect of CCDC on mechanosensitivity is a direct afferent effect and not secondary to muscular changes. Single unit analysis of the mechanosensitive bladder afferents showed 50% (20/40) of afferents responded to CCDC with mechanical hypersensitivity to bladder distension compared to saline (‘responders’), while 50% (20/40) showed no change in mechanosensitivity following CCDC instillation (‘non-responders’) compared to saline (Fig. 2C). CCDC-responsiveness was indicated by an mechanical hypersensitivity compared to saline at pressures of 10 mmHg and above (Fig. 2Di) and by a significant increase in the maximum peak of afferent firing (Fig. 2Dii) following CCDC instillation compared to saline. No changes in afferent firing at any individual pressures (Fig. 2Ei) or at maximum peak (Fig. 2Eii) were observed in the non-responding afferents. These data demonstrate that CCDC causes mechanical hypersensitivity of a sub-population of bladder afferents (Fig. 2F).
CCDC-induced bladder afferent mechanical hypersensitivity is reduced in Gpbar1-/- mice
To confirm that the CCDC-evoked mechanical hypersensitivity in ex vivo bladder afferents we observed were mediated by TGR5, we determined the effect of CCDC on bladder afferents in a Gpbar1−/− mouse model. In Gpbar1−/− mice, the mechanical hypersensitivity to distension following CCDC compared to saline was completely abolished (Fig. 3A). As in wildtype mice there was no change in muscle compliance between CCDC and saline experiments (Fig. 3B), confirming that the absence of altered sensitivity was not caused by changes in bladder muscle function. Interestingly, single unit analysis of the afferents showed that while the majority of afferents (82.9%; 29/35) reflected the overall lack of sensitivity to CCDC, mechanical hypersensitivity was observed in 17.1% (6/35) of mechanosensitive afferent fibres (Fig. 3C). These CCDC-responsive neurons demonstrated mechanical hypersensitivity specifically at 10 mmHg (Fig. 3Di) and an increase in the maximum peak of afferent firing (Fig. 3Dii). In line with the overall bladder afferent data, the vast majority of mechanosensitive single units (82.9%; 29/35) showed no difference in mechanosensitivity following CCDC instillation compared to saline (Fig. 3Ei-ii).
In vivo intravesical CCDC in wild-type mice increases the number of activated dorsal horn neurons to bladder distension, an effect not apparent in Gpbar1-/- mice
Our results thus far have established a role for TGR5 agonists in peripheral bladder afferent hypersensitivity. To determine whether this mechanical hypersensitivity is conveyed to the central nervous system, we assessed dorsal horn neuronal activation in response to in vivo bladder distension at 40 mmHg induced pERK-immunoreactivity in lumbosacral (LS) dorsal horn neurons (Fig. 4A). We observed LS dorsal horn pERK-immunoreactive (-IR) neurons in response to saline bladder distension at 40 mmHg in wild-type mice (Fig. 4Ai). The pERK-IR neurons were identified predominantly in the regions of the dorsal grey commissure and the superficial dorsal horn, with some pERK-IR neurons in the lateral spinal nuclei and the sacral parasympathetic nucleus in sacral spinal segments (Fig. 4Aii). Following intra-bladder CCDC application we observed a significant increase in the number of pERK-IR neurons within LS dorsal horn which were distributed throughout the same regions of the dorsal horn as seen in the saline experiments (Fig. 4Ai, Aiii). As these regions are known to play a role in bladder afferent input, integration, and projection of autonomic and nociceptive visceral signalling 36,47, an increase in pERK-IR neurons indicates an increase in central sensitivity to bladder distension and hence projection of the peripheral mechanosensitivity observed ex vivo in wild-type mice.
LS spinal dorsal horn neuronal pERK-IR induced by in vivo bladder distensions following intravesical incubation with CCDC or saline was also determined in Gpbar1−/− mice. The CCDC-induced enhanced spinal dorsal horn neuronal response to bladder distension was abolished in the Gpbar1−/− model, with no difference in the number of LS dorsal horn pERK-IR neurons between CCDC and saline in these mice (Fig. 4Bi). The pERK-IR neurons activated by bladder distension following intravesical instillation of both saline and CCDC were observed in the same dorsal horn regions as seen in the wildtype, suggesting no differences in spinal bladder afferent signalling pathways in the Gpbar1−/− mice (Fig. 4Bii, Biii). Taken together, these results demonstrate that activation of the TGR5 receptor is able to mediate an increase in peripheral and spinal sensitivity to bladder distension indicative of sensory neuron activation.
Gpbar1 (TGR5) mRNA is expressed in the bladder and bladder innervating DRG neurons
To begin unravelling the mechanisms through which TGR5 agonists CCDC and Pg5a exert mechanical hypersensitivity of bladder afferents we determined the relative mRNA expression of the TGR5 gene Gpbar1 within the bladder and bladder-innervating sensory neurons. Gpbar1 mRNA expression was identified throughout the bladder wall; in isolated urothelial cells, the mucosa, and the detrusor smooth muscle (Fig. 5A), as well as in isolated LS (L5-S1) DRG (Fig. 5B). Gpbar1 expression was absent in all tissues from Gpbar1−/− mice (Fig. 5A, 5B), whilst the relative abundance of either Transient receptor potential (TRP) vanilloid 1 (trpv1) or ankyrin 1 (trpa1) observed in LS DRG from Gpbar1−/− mice was unchanged compared to wildtype (Fig. 5B). TRP channels are common downstream targets for GPCR signalling cascades, with previous studies identifying downstream coupling of irritant-sensing receptors with TRPV1 and TRPA1 16,24,26,48,49. To assess potential TRP channel coupling with TGR5 in bladder neurons, we used single cell RT-PCR to determine the co-expression of Gpbar1 with trpv1 and trpa1. Single cell RT-PCR identified Gpbar1 expression in 13.0% (10/77) of retrogradely traced bladder afferent neurons (Fig. 5C). 84.4% (65/77) and 18.5% (12/77) of bladder-innervating DRG expressed trpv1 and trpa1 mRNA respectively (Fig. 5C). Of the bladder DRG neurons expressing Gpbar1 mRNA, 100% co-expressed trpv1, and only 30% co-expressed trpa1 (Fig. 5Cii, 5Ciii), despite previous functional data in other DRG neurons showing distinct coupling of TGR5 with TRPA1 24.
In vitro application of CCDC to bladder-innervating DRG neurons increases intracellular Ca2+
To gain insights into the mechanism of action of neuronal TGR5 in mediating bladder afferent sensitivity, we performed live cell calcium imaging in LS DRG neurons isolated from retrogradely bladder-traced mice. Following direct application of CCDC to the neurons, a significant increase in intracellular Ca2+, indicated by an increase in the Fura-2 fluorescence ratio (Fig. 6Ai, Aii), was observed in 58.82% (40/68) of bladder-traced DRG neurons (Fig. 6Bi). Interestingly, the percentage of bladder-traced neurons responding to CCDC was substantially higher than the percentage on non-traced neurons, of which only 24.65% (126/511) responded (Fig. 6Bi). The average magnitude of CCDC-induced increases in intracellular Ca2+ was also greater in bladder-traced compared to non-traced neurons, as indicated by a significantly higher maximum peak value of Fura-2 fluorescence and area under the curve of response (Fig. 6Bii, 6Biii). These results demonstrate the TGR5 agonist CCDC is able to cause direct activation of LS DRG neurons and indicate the possibility that bladder-innervating DRG neurons are tuned to evoke greater responses to TGR5 agonists.
In vitro CCDC-induced increases in intracellular Ca2+ in bladder-innervating DRG neurons are partially mediated by TRPV1
Coupling between TGR5 and TRPA1 has been previously demonstrated in cutaneous 24 and colon 26 innervating DRG neurons. Here our single-cell mRNA expression data from bladder-innervating DRG neurons demonstrated 100% co-expression of trpv1 with Gpbar1 compared to only 30% co-expression of trpa1 with Gpbar1 (Fig. 5C), suggesting a distinct intracellular signalling pathway in bladder-innervating DRG neurons. To functionally explore potential coupling between TGR5 and TRP channels in bladder afferent DRG neurons in vitro, we determined co-activation of TGR5, TRPA1, and TRPV1 in individual neurons with successive administration of CCDC, AITC (TRPA1 agonist), and capsaicin (TRPV1 agonist) using live cell calcium imaging (Fig. 7A, 7B). In line with our single-cell co-expression data, 87.5% (21/24) of bladder traced neurons that responded to CCDC also responded to capsaicin, whilst 33.3% (8/24) of CCDC-responsive bladder-traced neurons also responded to AITC (Fig. 7B). Only 16.7% (4/24) of CCDC-responsive bladder-traced neurons were unresponsive to both AITC and capsaicin (Fig. 7B).
To further explore the potential functional relationships between TGR5 and TRP channels TRPV1 and TRPA1, we measured bladder-traced DRG neuronal responses to CCDC in vitro using trpv1−/− and trpa1−/− mice. The percentage of bladder-traced DRG neurons responding to CCDC was reduced by approximately 50% in trpv1−/− mice (27.3%) compared to wildtype mice (58.8%), with no obvious change observed in the trpa1−/− mice (60.9%) compared to wildtype mice (Fig. 7C). The profile and magnitude of CCDC responses between wildtype and trpv1−/− and trpa1−/− mice was also examined (Fig. 7Di-iii). Whilst the few trpv1−/− neurons that responded to CCDC show a trend towards reduced response amplitudes, there was no statistical differences in the average maximum peak of Fura-2 fluorescence (Fig. 7Dii) and area under the curve (Fig. 7Diii) of the CCDC responses. Overall, these data implicate an interaction between TRPV1 and TGR5 in bladder-innervating DRG neurons.
In vitro CCDC-induced increases in intracellular Ca2+ in bladder-innervating DRG neurons remain in Gpbar1-/- mice
To confirm the in-vitro effects of CCDC on isolated bladder-innervating DRG neurons were due to activation of the TGR5 receptor we examined CCDC responses firstly in cells isolated from Gpbar1−/− mice and subsequently in NaV1.8Cre-Gpbar1−/− mice. To our surprise, responses to CCDC remained in neurons from these mice (Fig. 8). The percentage of cells responding to CCDC from Gpbar1−/− and NaV1.8Cre-Gpbar1−/− mice was roughly equivalent to that from wild-type mice (Fig. 8Ai-iii, 8Bi-ii). The total fluorescent response to CCDC, depicted as the area under the response curve (AUC) was significantly reduced in cells from NaV1.8Cre-Gpbar1−/− (neuronal knockout of Gpbar1) but not Gpbar1−/− (global knockout of Gpbar1) mice (Fig. 8Aiii, 8C), however, a robust response still remained. No significant differences in the maximum peak of Fura-2 fluorescence in response to CCDC were observed in cells from wildtype, Gpbar1−/− or NaV1.8Cre-Gpbar1−/− mice (Fig. 8D).