John G. Forte, Ph.D. - US grants

Secretion of HCl by the gastric oxyntic cell is a highly specialized process involving coordinated 1) secretagogue-receptor interaction, 2) cytoplasmic activation, 3) recruitment of cytoplasmic membranes into the apical cell surface, and 4) operation of highly energetic transport pathways. The developing system offers a unique opportunity to study the molecular features and limitations in the series of events that underlie HCl secretion. Morphological differentiation of the oxyntic cell occurs rather rapidly, indicating defined periods of high synthesis for particular elements of the secretory apparatus. We propose to evaluate the ontogeny of individual components of the secretory apparatus along with the ultrasturctural development of the oxyntic cell and the ability to transport HCl. The development of the H+ pump enzyme, H,K-ATPase, will be studied and correlated with the pattern of tubulovesicle synthesis within the cell. Specific enzyme activity, total enzyme quantity and relative rates of synthesis of the pump enzyme will be determined. H+ transport activity of isolated membrane fractins deriving from cytoplasmic tubulovesicles will be studied and compared with H+, K+ and C1- transport with membranes that have been transformed (by fusion) into the apical cell surface. The ontogeny of oxyntic cell secretagogue-receptor activity will be established and compared with the appearance of intracellular second messenger activating systems as well as the capacity of the various transport pathways. Pathways for membrane synthesis during cytodifferentiation and membrane flow during the secretory cycle will be established. Analysis of mRNA durig oxyntic cell differentiation will seek to identify stages where specifically coded messages, such as that for H,K-ATPase synthesis, are in high abundance. These studies will provide a comprehensive picture of the chronology for expression of surface receptors. intracellular activators, transport enxymes, and ion channels that constitute the integrated secretory machinery of the oxyntic cell. The ontogenic appearance of specific proteins and transport pathways will also serve as identifying markers and help to establish their relative importance in the process of HCl secretion.

adenosinetriphosphatase; hydrochloric acid; gastric acid; hydrogen transport; gastric mucosa; hydrogen potassium exchanging ATPase; protein isoforms; phosphoproteins; cytoskeletal proteins; gastrointestinal epithelium; ion transport; membrane permeability; secretion; cytoskeleton; microfilaments; cell differentiation; membrane structure; phosphorylation; protein structure function; actins; charge coupled device camera; laboratory mouse; laboratory rabbit; X ray crystallography; spectrometry; immunocytochemistry;

Funds are requested for a high-resolution low-temperature scanning electron microscope. The instrument isan Hitachi S5000 scanning electron microscope with cryosystem, backscattered electron detector, digital image system, and is matrixed for remote operation. With low-temperature microscopy the sample is held at liquid nitrogen temperature during examination, allowing water to be preserved for imaging in the frozen hydrated state and the surface and interior of cells to be analyzed without the need for chemical fixation, dehydration or drying.

Gastric ezrin is a 80kDa protein, which is highly enriched in apical microvilli of gastric parietal cells and with sequence homology to talin and erythrocyte band 4.1. Ezrin has beensuggested to play a key role in mediating the apical surface rearrangements involved in acid secretion by parietal cells. The mediation of stimulation of acid secretion by the cAMP-dependent protein kinase A pathway has been correlated with the phosphorylation of ezrin. These studies suggest that phosphorylation is at serine/threonine residues and not at tyrosine residues. On the other hand, ezrin in A431 cells which overexpresses EGF receptors was characterized as a substrate for an EGF-stimulated tyrosine kinase and was phosphorylated at bothserine and tyrosine residues (Brestcher et al., 1989). EGF inhibited acid secretion in parietal cells and it has been suggested that EGF-stimulated tyrosine phosphorylation of ezrin might itself inhibit stimulation. In this project we propose to examine the site-directed phosphorylation of ezrin by histamine and EGF which stimulate and inhibit acid secretion respectively and identify the in vivo phosphorylation sites in each case. Our experimental strategy would involve proteolysis of ezrin in gel slices, followed by purification of phosphopeptides on iron-loaded (FeIII) nitriloacetic (NTA) sepaharose columns, the separation of the phosphopeptides by HPLC and the identification of the specific Ser, Thr or Tyr phosphorylated, by MALDI mass spectrometry and post-source decay analysis.

This proposal requests funds in partial support of the 9th International Proton Transport Conference, "Mechanisms and Consequences of Gastric Proton Transport ", to be held August 19-21, 2001, in Leura, Australia. This is the 9th in a series that has met in association with the Congress of the International Union of Physiological Sciences every 3 to 4 years since 1971. Previous meetings have been distinguished by success in achieving objectives to promote (1) the exceptional progression of discoveries in gastric secretory physiology and pharmacology over the past 30 years, and (2) effective cross fertilization of research through interaction with outstanding scientists working outside the gastric field. Published proceedings of previous meetings attest to these successes as does reported enthusiasm of participants at all levels of training and experience. The Organizing Committee for the Conference is composed of notable contributors to gastric secretory physiology as well as three former Conference chairs, including: Drs. Catherine S. Chew, Gunnar Flemstrom, John G. Forte, James R. Goldenring, Barry Hirst, George Sachs, Tetsuro Urushidani (Secretariat). The Committee developed the program and will act as referees in selecting contributed abstracts for presentation and travel awards to young investigators. There have been major advances in the molecular biology of the gastric proton pump, the regulation of cellular secretory function, and the modulation of gastric pathophysiology by a variety of drugs. The principal objectives of the Conference are to discuss the implications of these findings, to enrich their efforts with presentations by scholars in closely related fields, and to develop strategies for future research and targets for pharmacological intervention. We now know that Helicobacter pylori plays a central role in the pathogenesis of peptic ulcer disease. This has not minimized the role of gastric acid but rather has directed focus on the synergism of injurious factors for the disease. Toward these goals the slate of topics will attract a broad audience of participation and focus current issues in the field, including (1) structure and function of H,K-ATPase - molecular basis as a drug target; (2) Helicobacter pylori - molecular microbiology and strategy for eradication; (3) gene targeting studies in the mouse - mechanisms of acid secretion and cell proliferation; (4) signal transduction in the parietal cell - pathway to activation; (5) cytoskeleton & membrane trafficking - the gastric parietal cell as a model for membrane recruitment.

DESCRIPTION (provided by applicant): The overall goal of this research is to provide, for basic scientists and clinicians, a complete understanding of the mechanism of HCl secretion by the stomach. The proposed studies specifically examine interactions between plasma membranes and the cytoskeleton elements that support and promote the dynamic apical surface structure associated with secretory activity. The gastric parietal cell is the principal experimental model, with special emphasis on mechanisms regulating and effecting the enormous membrane recruitment and recycling processes associated with acid secretion, and which are now recognized as the means for cycling specific transport proteins in all cells. Though the parietal cell is the principal model (because its extensive surface remodeling), comparative studies will examine other epithelial systems (e.g., renal tubules, intestinal villi). The stability and turnover of actin microfilaments, and accessory bundling and capping proteins in parietal cells, will be characterized throughout the course of apical membrane expansion associated with the onset of secretion, and the reverse of this process as the cells return to rest. The functional activity of an essential membrane-cytoskeleton linker protein, ezrin, potentially provides the most important factor in these surface interactions, and proposed experiments will test the nature of its bivalent linking activity. Specific questions address: i) The role of phosphorylation, the number of phosphorylation sites, and the turnover of phosphorylation within the concept of actin binding specificity and flexibility. ii) Relative functional differences between membrane binding and actin binding domains during the secretory cycle in the parietal cell. iii) The basis for differences in actin/ezrin interactions in highly flexible, functionally variable, parietal cell microvilli, and the highly regular brush borders of intestinal and proximal tubular cells. PUBLIC HEALTH RELEVANCE: A complete understanding of gastric secretory mechanisms, and the application of these principles to all secretory and epithelia, has been and continues to be an important objective for NIDDK. Past discoveries led to the development and application of drugs that control acidity by receptor intervention or by directly targeting essential acid pump proteins. Aggravated conditions such as dyspepsia, ulcers, and GERD can be alleviated, and cured, by the efficacious use of these drugs. While there is a certain level of comprehension, more complete understanding will offer further insight for pathological analysis and new targets for pharmacological intervention.