PROJECT SUMMARY Regulation of the peristaltic reflex and propulsion depends on the ability of the enteric nervous system to respond to signals derived from luminal contents. While activation by distension and by mucosal mechanical stimulation are well studied, much less is known about the activation of sensory pathways involved in the motility response elicited by chemical (i.e. dietary-derived) components of luminal contents. The process likely involves activation of specific chemoreceptors on enteroendocrine cells (EEC), release of specific paracrine agents, and activation of enteric CGRP-containing sensory neurons. Two major components of colonic luminal contents are free fatty acids (short chain (SCFA) and branched chain (BCFA)) which are detected by free fatty acid receptors (especially FFAR2 & FFAR3), and amino acids (AA) which are detected by the umami (T1R1/T1R3) taste receptor identical to that of taste buds. The expression and co-localization of these receptors on specific EECs, the specific paracrine agent(s) released by EECs following activation of these receptors, and the coupling of chemoreceptor-paracrine agent pair(s) to generation of motility patterns in the proximal and distal colon are not known. The Central Hypothesis is that SCFAs generated in the proximal colon from microbial fermentation of carbohydrates, and AAs/BCFAs generated in more distal colon from digestion/fermentation of proteins activate specific receptors on subtypes of EECs to release specific paracrine agents that determine the types of motility pattern and propulsion which are characteristic of the two regions of the colon. We propose three aims. Specific Aim 1: To identify the mechanism by which amino acids, acting via umami (T1R1/T1R3) receptors, mediate release of paracrine agents in the proximal and distal colon. Specific Aim 2: To identify the mechanism by which free fatty acids (SCFA & BCFA), acting via free fatty acid receptors (FFAR2 & FFAR3), mediate the release of paracrine agents in the proximal and distal colon. In these two aims, we will identify the co-localization of T1R1/T1R3, and FFAR2 and FFAR3 with each other and with peptide YY (PYY), neurotensin (NT), serotonin (5-HT), and brain-derived neurotrophic factor (BDNF) in colonic EECs. We will also measure the release of these paracrine agents following activation of T1R1/T1R3, and FFAR2 and FFAR3 in EEC cells. We have focused on these four paracrine agents present in EECs of the colon based on published and our preliminary data suggesting their key role in motility. Specific Aim 3: To identify the effect of SCFA, BCFA and AAs on motility patterns in proximal and distal colon and to determine the interaction between these luminal stimuli on motility patterns and propulsion. We will identify the coupling of these chemoreceptors and paracrine agents to activation of the peristaltic reflex and specific motility patterns, and to propulsion of fluids in proximal colon and solids (fecal pellets) in distal colon. These results will provide new mechanistic understanding of the regulation of colonic motility and propulsion by bioactive components derived from processing of luminal contents by microbiota resident in different regions of the colon.