Control of motility in the lower esophageal sphincter

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Program Director/Principal Investigator (Last, First, Middle): Cobine, Caroline, A Project Summary The lower esophageal sphincter (LES) is a thickened region of muscle between the distal esophagus and proximal stomach. The LES develops contractile tone that forms a protective barrier and restricts acidic contents of the stomach from being refluxed into and damaging the esophagus. LES tone is thought to be ‘myogenic’, but LES muscles are not just composed of smooth muscle cells (SMCs) and neurons. Rather, interstitial cells of Cajal (ICC) are electrically coupled to SMCs and regulate the excitability of SMCs. For example, we found that responses to nitrergic input, an important regulator of relaxation after swallowing, are reduced in animals with loss of ICC (W/WV mice). This concept was criticized because manometric measurements in W/WV mice displayed evidence of nitrergic relaxation. An important observation in these studies, but not investigated or discussed in terms of mechanism, was that the LES in W/WV mice is hypotensive, suggesting that ICC provide important functions in the LES, such as development and maintenance of tone. This project seeks to better understand the mechanism of LES tone. Preliminary findings suggest that activation of Ca2+-dependent Cl- channels (CaCC encoded by Ano1), most highly expressed by ICC in the LES, are responsible for a significant portion of tone in these muscles. Ano1 channels are activated by spontaneous Ca2+ transients in ICC. The inward current activated by Ca2+ causes depolarization of the syncytium and increases the open probability of voltage-dependent Ca2+ channels in SMCs. The degree of contraction by this mechanism appears also to be regulated by a mechanism known as Ca2+ sensitization, a process suggested in previous papers, but previous findings are subject to other explanations. Here we propose to investigate the following: 1. Determine the molecular and functional apparatus responsible for the electromechanical and pharmacomechanical regulation of tone in the LES, 2. Examine how inputs from intrinsic neurons modulate the electromechanical and pharmacomechanical mechanisms underlying LES tone. This study will utilize a variety of cutting-edge techniques, such as transgenic mice, optogenetic monitoring of Ca2+ transients and biochemical analysis of phosphorylation steps that either increase or inhibit Ca2+ sensitivity in SMCs. Many of these pre-clinical studies will be performed on mice, but we will also perform parallel studies on Cynomolgus monkey LES, a primate with significant genetic homology to humans. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page