This application represents a continuation of the principal investigator's FIRST Award. The long-term goal of this project is to evaluate, at the cellular level, the mechanisms that protect gastric mucosa from the damaging effects of luminal acid. Until recently, it was accepted that a Cl-/HCO3- exchanger, located in the basolateral cell membrane, is responsible for regulation of intracellular Cl- levels (aiCl) and intracellular pH (pHi) in surface cells and oxyntic cells. We recently found evidence to suggest that a basolateral Na-K-Cl cotransport process plays a dominant and previously unsuspected role in preserving intracellular Cl- levels of the surface cells and in regulating HCl secretion by oxyntic cells. These findings have altered the previous model and led to the following hypotheses: 1) a basolateral, HCO3--independent, Na-K-Cl cotransporter is the dominant mechanism for regulating aiCl of oxyntic cells and surface cells in Necturus gastric fundus during stimulation of acid secretion; 2) a complementary, Cl--independent mechanism of basolateral HCO3-extrusion is also present in the oxyntic cell and contributes significantly to the """"""""alkaline tide"""""""" generated by high rates of acid secretion; 3) expression of basolateral transporters that are dominant in preserving aiCl and pHi in the oxyntic cells is regulated by the gastric neurohumoral milieu; 4) secretory activity of the oxyntic glands plays a major role in regulating cell pH and ion composition of the neighboring surface epithelial cells during high rates of acid secretion and during exposure to ulcerogenic conditions; and 5) the ammonium (NH4+) ion that is produced by the pathogen H. pylori may gain access to the cell via the Na-K-Cl cotransporter, leading to disturbances in both aiCl and pHi. Studies proposed in this application will address these hypotheses using intracellular microelectrode and fluorescence techniques for measuring pH and ion composition, and molecular methods for evaluating expression of membrane transporters.
The specific aims of this application are organized into four parts. First, we will evaluate the relationship of the basolateral Cl--transporting processes and their implications for regulation of HCO3- transport and other protective functions in both surface cells and oxyntic cells in the mucosa of the gastric fundus of Necturus. Second, we will examine, in both amphibian and mammalian models, the effects of secretory agonists such as gastrin, histamine, carbachol in stimulating synthesis and expression of the basolateral HCO3- and Cl- transport systems in surface cells and oxyntic cells of gastric mucosa. Third, we will evaluate the effects of ulcerogens such as aspirin or indomethacin on activity and expression of basolateral Cl- transport systems in both surface cells and oxyntic cells. Finally, we will explore the interaction between ammonium (NH4+), which is produced by the bacterium Helicobacter pylori, and cell pH and Cl- homeostasis. Using these techniques, we hope to provide detailed information regarding activity and expression of transport processes that preserve ion composition and pH in gastric mucosal cells during acid secretion or exposure to ulcerogenic conditions.
