PROJECT SUMMARY/ABSTRACT The gastrointestinal (GI) epithelium directly senses components of foods we eat, which contain a variety of nutritive and non-nutritive stimuli. These stimuli come from foods with a range of osmolality from zero (water) to >1,500 mmol/kg-H2O for some of the saltiest foods. GI epithelial cells are not only first to sense luminal osmoles, but these cells, along with the kidney tubular cells, encounter the largest osmole swings of all cells in the body. In functional and motility GI disorders, such as irritable bowel syndrome (IBS), which affect 10 - 20% of the US population, non-nutritive aspects of diets such as osmolality are often first line therapies. For example, FODMAPs (Fermentable Oligo-, Di-, Monosaccharides and Polyols) are targeted for elimination in IBS diets. FODMAPs are also highly osmotically active – they are known to drive luminal volume shifts which may lead to symptoms. Both hypo- and hyper-osmotic stimuli invoke downstream signaling that regulates GI motility and systemic physiologic responses. The enteroendocrine cells (EECs) are specialized sensory epithelial cells that interact with luminal stimuli, both nutritive and non-nutritive, and these cells are capable of regulating both local and systemic physiology including GI motility. While EECs are traditionally considered to be nutrient sensors, our lab discovered an EEC subpopulation that transduces mechanical signals, thereby opening the door to EECs being sensors of non-nutritive stimuli. Literature has shown that EEC receptor activation drives two main signal transduction pathways: calcium (Ca2+) and cAMP which lead to release of a range of signaling molecules, including serotonin (5HT). To investigate how EECs sense hyper and hypoosmotic stimuli respectively, our lab is manipulating EEC receptors: V1aR, VRAC, and Piezo2. In VRAC proteins, Ca2+ signaling drives Cl- currents. Understanding osmo-transduction will provide mechanistic insights into commonly used clinical therapies. The overall goal of this proposal is to uncover the mechanisms by which osmolality is sensed by EECs and how osmotic stimuli may engage EEC signal transduction to alter release of signaling molecules and systemic GI physiology. The hypothesis is that EECs transduce osmotic stimuli in location and subtype-specific ways - via cytoplasmic Ca2+, Cl- and cAMP, through osmoregulatory proteins, and secrete signaling molecules to modulate GI motility. Aim 1 investigates the cellular pathways by which EECs transduce osmotic stimuli. Aim 2 investigates the osmotically induced extracellular release of signaling molecules by EECs and subsequent changes in GI motility. The results of this work are poised to bridge knowledge gaps in EEC osmo-transduction, as well as inform broader osmosensing mechanisms in sensory epithelia. The proposed work will be carried out in an environment that provides expert knowledge towards achieving the specified goal, including collaborations with experts in v...