Bile Acid on Esophageal Nociceptor

Summary Esophageal nociceptive sensations, such as heartburn and chest pain, are the major complaints that present in many esophageal disorders, including GERD. More than 30% of these symptoms are refractory to acid inhibition by PPIs, making it a significant treatment challenge. Bile acid is a potent substance that can trigger esophageal nociceptive sensations in addition to acid, but the underlying mechanism of its neuronal action is still unknown. Our recent study demonstrated that bile acid not only directly activates esophageal nociceptive C-fibers but also sensitizes their responses to TRPV1 agonist capsaicin. Whether MrgprX4, a newly discovered bile acid receptor, mediates bile acid-elicited activation and sensitization of esophageal C-fibers and enhances their responses to acid are important yet unanswered questions. In the present proposal, we address the hypothesis that MrgprX4 contributes to PPI-refractory esophageal nociception by mediating bile acid-induced activation (independent of acid) and sensitization (increasing acid sensitivity) of esophageal nociceptive C-fibers. Aim 1 will characterize MrgprX4 expression in esophageal afferent C-fiber neuron subtypes. Using MrgprX4-expressing mice, we will first clarify MrgprX4 mRNA expression and co-expression with transcripts of nociceptive neurons by single-cell RT-PCR in esophageal-labeled afferent neurons. We will then characterize MrgprX4 protein expression in esophageal-labeled afferent neurons regarding neuron diameters and co-expression of C-fiber markers (i.e. CGRP/IB4). We hypothesize that MrgprX4-positive neurons are small-size unmyelinated C-fiber neurons (but not NF-200-positive Ad-fiber neurons). Next, we address the hypothesis that MrgprX4 is selectively expressed in subsets of TRPV1-positive (acid-sensitive) nociceptive C-fiber neurons with their nerve fibers richly distributed in esophageal mucosa. Aim 2 will determine MrgprX4-mediated activation of esophageal nociceptive C-fiber subtypes. Our previous study demonstrated that bile acid DCA directly activates esophageal vagal nodose and jugular C-fibers. Using our newly established MrgprX4-expressing and MrgprX4-/- mouse lines, we will address the hypothesis that MrgprX4 specific agonists (SML3371 and nateglinide), mimicking bile acid-induced effect, directly activate esophageal nociceptive C-fiber subtypes. We will then address the hypothesis that bile acid- induced activation effects on the neuron soma and the nerve terminals of esophageal vagal and DRG C-fiber subtypes are absent in the MrgprX4-/- mouse line. Aim 3 will determine MrgprX4-mediated sensitization of esophageal C-fiber subtypes. We will address the hypothesis that MrgprX4 mediates bile acid-induced enhancement of acid response in esophageal TRPV1-positive C-fibers. Using MrgprX4-expressing mouse line, we will first determine bile acid-induced sensitization of acid response in esophageal C-fiber subtypes. We will then determine whether MrgprX4 selective agonists mimic bile acid-induced excessive acid responses in esophageal C-fiber subtypes. Lastly, we will determine, in MrgprX4-/- mouse line, bile acid and MrgprX4 agonist- induced effects on acid responses in esophageal C-fiber subtypes.