TRPV4 is expressed by enteric glia and muscularis macrophages of the colon but does not play a prominent role in colonic motility

Abstract

AbstractBackgroundMechanosensation is an important trigger of physiological processes in the gastrointestinal tract. Aberrant responses to mechanical input are associated with digestive disorders, including visceral hypersensitivity. Transient Receptor Potential Vanilloid 4 (TRPV4) is a mechanosensory ion channel with proposed roles in visceral afferent signaling, intestinal inflammation, and gut motility. While TRPV4 is a potential therapeutic target for digestive disease, current mechanistic understanding of how TRPV4 may influence gut function is limited by inconsistent reports of TRPV4 expression and distribution.MethodsIn this study we profiled functional expression of TRPV4 using Ca2+imaging of wholemount preparations of the mouse, monkey, and human intestine in combination with immunofluorescent labeling for established cellular markers. The involvement of TRPV4 in colonic motility was assessedin vitrousing videomapping and contraction assays.ResultsThe TRPV4 agonist GSK1016790A evoked Ca2+signaling in muscularis macrophages, enteric glia, and endothelial cells. TRPV4 specificity was confirmed using TRPV4 KO mouse tissue or antagonist pre-treatment. Calcium responses were not detected in other cell types required for neuromuscular signaling including enteric neurons, interstitial cells of Cajal, PDGFRα+ cells, and intestinal smooth muscle. TRPV4 activation led to rapid Ca2+responses by a subpopulation of glial cells, followed by sustained Ca2+signaling throughout the enteric glial network. Propagation of these waves was suppressed by inhibition of gap junctions or Ca2+release from intracellular stores. Coordinated glial signaling in response to GSK1016790A was also disrupted in acute TNBS colitis. The involvement of TRPV4 in the initiation and propagation of colonic motility patterns was examinedin vitro.ConclusionsWe reveal a previously unappreciated role for TRPV4 in the initiation of distension-evoked colonic motility. These observations provide new insights into the functional role of TRPV4 activation in the gut, with important implications for how TRPV4 may influence critical processes including inflammatory signaling and motility.SummaryTRPV4 is expressed by equivalent cell types in the rodent and primate (monkey and human) colon. This mechanosensitive ion channel has proposed roles in inflammation, visceral afferent signaling, and colonic motility.New analysis methods were developed to examine cellular communication in the enteric glial network. This approach revealed new insights into inflammation-associated changes in glial connectivity.New roles for TRPV4 in transduction of distension-evoked responses in the colon and colonic motility were identified.Key findingsWe have defined the cell types that functionally express TRPV4 in the gut wall. These include enteric glia, endothelia of blood and lymphatic vessels, mMac, and extrinsic afferent nerves. TRPV4- dependent Ca2+signaling was not detected in enteric neurons, PDGFRα cells, interstitial cells of Cajal and smooth muscle cells, which are important drivers of gut motility. These observations align with our experimental evidence for limited involvement of TRPV4 in neuromuscular transmission and propagating colonic motility.New and NoteworthyNovel cellular sites of functional TRPV4 expression in the GI tract were identified and compared across multiple vertebrate species. New analytical approaches to characterize enteric glial communication in a spatiotemporal manner were developed.A supporting role for TRPV4 in the initiation of propagating colonic contractions in response to distension was demonstrated. Potential mechanisms that contribute to TRPV4-mediated effects on GI function were identified.TRPV4-dependent activity in enteric glia is enhanced in inflammation, consistent with current evidence for inflammation-associated sensitization of TRPV4 on visceral afferents and a major role in mechanically evoked nociceptive signaling.Pair correlation analysis was used to examine spatial connectivity of Ca2+signaling, enabling demonstration of dysregulated glial communication in acute inflammation.