Expression of the relaxin family peptide 4 receptor by enterochromaffin cells of the mouse large intestine

Abstract

The gastrointestinal hormone, insulin-like peptide 5 (INSL5), is found in large intestinal enteroendocrine cells (EEC). One of its functions is to stimulate nerve circuits that increase propulsive activity of the colon through its receptor, the relaxin family peptide 4 receptor (RXFP4). To investigate the mechanisms that link INSL5 to stimulation of propulsion, we have determined the localisation of cells expressing Rxfp4 in the mouse colon, using a reporter mouse to locate cells expressing the gene. The fluorescent signal indicating the location of Rxfp4 expression was in EEC, the greatest overlap of Rxfp4-dependent labelling being with cells containing 5-HT. In fact, > 90% of 5-HT cells were positive for Rxfp4 labelling. A small proportion of cells with Rxfp4-dependent labelling was 5-HT-negative, 11–15% in the distal colon and rectum, and 35% in the proximal colon. Of these, some were identified as L-cells by immunoreactivity for oxyntomodulin. Rxfp4-dependent fluorescence was also found in a sparse population of nerve endings, where it was colocalised with CGRP. We used the RXFP4 agonist, INSL5-A13, to activate the receptor and probe the role of the 5-HT cells in which it is expressed. INSL5-A13 administered by i.p. injection to conscious mice caused an increase in colorectal propulsion that was antagonised by the 5-HT3 receptor blocker, alosetron, also given i.p. We conclude that stimuli that excite INSL5-containing colonic L-cells release INSL5 that, through RXFP4, excites 5-HT release from neighbouring endocrine cells, which in turn acts on 5-HT3 receptors of enteric sensory neurons to elicit propulsive reflexes.

Evidence that RXFP4 is located in enterochromaffin cells and can regulate production and release of serotonin

“…However, IGFBP3 has been implicated in regulation of gut stem cell proliferation and differentiation [ 29 ]. Interestingly, we also saw a small amount of GCG co-expression with RXFP4 which agrees with a recent immunohistological analysis of mouse gut that found a small amount of RXFP4 co-expression with Oxyntamodulin [ 15 ]. We looked at TPH2 and TAC1 to indicate the presence of enteric neuronal cell types but found little evidence for co-expression of RXFP4, although these markers were only expressed in a few cells suggesting these cell types were not sufficiently surveyed in these datasets.…”

“…Most 5-HT positive cells have some Rxfp4 speckles, however there is Rxfp4 signal in areas not positive for 5-HT. This aligns with immunohistological analysis of a Rxfp4-reporter mouse where full quantification showed co-expression to vary across the lower GI tract, over 90% of 5-HT-positive cells were also immunoreactive for GFP [ 15 ]. These findings make it clear that INSL5-RXFP4 signalling has great potential to affect serotonin synthesis and/or secretion from EC cells.…”

“…This seems paradoxical as there is strong evidence that serotonin promotes anterograde gut motility [ 38 ] and agonists of RXFP4 also increase gut motility, at least as shown by colonic bead expulsion assays [ 9 ]. Further, a 5-HT3 receptor antagonist Alosteron was recently shown to block INSL5-A13 action on gut motility in the bead expulsion assay [ 15 ] However, given that INSL5 is produced in cells in the distal colon [ 5 ] and that we find RXFP4 is only found in a subset of EC cells we postulate that the INSL5-RXFP4 system may be having a specific effect on the retrograde propulsion waves that are generated in this part of the colon [ 39 ]. Interestingly these are shown to increase after a meal, as does production of INSL5.…”

“…A recent publication commented that Rxfp4 was expressed in colonic Tph1 positive cells [ 11 ] in their mouse single cell RNASeq dataset [ 14 ] although details were not provided. In addition, recently published histological analysis of thick sections from the colon, distal colon and rectum of Rfxp4-reporter mice have revealed a large proportion (60–90%) of co-expression of Rxfp4 and 5-HT, and using super-resolution microscopy, 5-HT expressing cells were also found to be in proximity ‘intertwined’ with oxyntamodulin/PYY expressing L-cells [ 15 , 16 ]. Here we use scRNAseq datasets to show, for the first time, that RXFP4 colocalises with enterochromaffin cells (EC) in humans.…”

History of key regulatory peptide systems and perspectives for future research

“…The expression of Insl5 and Rxfp4 mRNA has been detected in the pancreas, thymus and in the gastrointestinal tract, 202,291 particularly in the L‐cells of distal gut 292–294 ; INSL5 has been shown to function as an orexigenic hormone, 291 and to influence colonic propulsion, 232,295 immune cell function, 296 and even human sperm motility 297 . Finally, a recent study identified a potential role for INSL5 (or relaxin‐3) signaling via ventral hypothalamic RXFP4 in the control of high‐fat and high‐protein diet consumption in mice 298 .…”