Abdominal pain is the leading cause of morbidity for people living with gastrointestinal diseases, however pain management remains an unmet clinical challenge. TRPV4 is a member of the vanilloid subtype of transient receptor potential ion channels and possesses pronociceptive functions in the bowel. Our recent single-cell RNA sequencing study revealed a relative paucity of TRPV4 mRNA within colonic sensory neurons in contrast to the expression of other algogenic receptors and the considerable stimulatory effect of TRPV4 on colonic afferent activity, indicating that non-neuronal cells such as those of the gut mucosa may also contribute to TRPV4-mediated nociceptor stimulation. Several gut hormone receptors are also expressed including cholecystokinin (CCK) which is associated with a worsening of postprandial abdominal pain and visceral hypersensitivity in IBS. Consequently, this thesis sought to understand the contribution of signalling mediators and hormones of the gut mucosa to colonic sensory transduction.

Selective TRPV4 agonist GSK1016790A was found to evoke a rapid and robust increase in colonic sensory afferent firing which was abolished upon the removal of the gut mucosa, identifying a site of action for TRPV4 in sensory afferent activation in the gut. These effects were also eliminated following purinoreceptor and glutamate receptor blockade demonstrating that ATP and glutamate drive TRPV4-mediated afferent firing. This was confirmed by the enhancement of the afferent response to GSK1016790A in the presence of ectonucleotidase inhibition and the release of ATP and glutamate in the supernatant of colonic tissue treated with GSK1016790A. Furthermore, the co-culture of DRG sensory neurons with mucosal cells derived from the colon enhanced the proportion of neurons responding to GSK1016790A with an influx of intracellular Ca²⁺, validating the need for mucosal cells in the neuronal response to TRPV4. This co-culture was enriched with epithelial cells and mesenchymal cells such as fibroblasts which both expressed TRPV4 in primary culture.

CCK elicited a dose-dependent increase in both ileal mesenteric afferents, which was abolished by a CCK₁ antagonist, and colonic spinal afferents. CCK also evoked Ca²⁺ influxes in DRG sensory neurons via CCK₁ in non-peptidergic and peptidergic DRG neurons and a small population of capsaicin-responding neurons, indicative of nociceptor activation.

Overall, the findings outlined in this thesis establish that the gut mucosa drives colonic sensory afferent firing through TRPV4-driven release of ATP and glutamate, and gut hormone CCK. Together, this highlights how targeting mucosal signalling mediators and hormones may be effective for the gut restricted treatment of pain in GI diseases.