The major objective of this project is to define the cellular mechanisms by which hormones, neurotransmitters, paracrines and growth factors interact to control hydrochloric acid secretion by parietal cells in the gastric mucosa. Peptic ulcer disease is a widespread clinical problem that results from or is exacerbated by gastric acid secretion. A better understanding of the mechanisms by which the major acid secretory agonists, histamine, acetylcholine and gastrin, and other factors control parietal cell acid secretion will allow for development of improved treatment and prevention of this disease. This research may also generate useful information on stimulus-secretion coupling mechanisms that are applicable to other secretory cell types including, for example, those that are affected in diseases such as cystic fibrosis. There are three specific aims in this proposal. First, to develop and characterize newly developed cultured parietal cell models that can be used to define how agonists and antagonists interact to modulate parietal cell acid secretion. Second, to use acutely isolated and cultured cell models to continue to define the second messenger pathways by which cAMP and calcium-dependent agonists initiate acid secretion and the mechanisms by which inhibitors of secretion such as prostaglandins and somatostatin modulate the stimulatory effects of these agonists. Third, to continue to characterize, isolate and sequence parietal cell proteins that are phosphorylated in response to agonist stimulation with the ultimate goal of cloning the genes for these proteins. Little is presently known about intracellular events that occur between initial agonist-receptor binding to the basolateral cell membrane and ultimate activation of the H,K-ATPase or proton pump at the apical cell membrane. Results to date suggest, however, that in all cell types second messengers activate several different protein kinases and these kinases phosphorylate a variety of cellular proteins on serine, threonine and, rarely, on tyrosine residues. The identification and characterization of these agonist-responsive proteins is essential if progress is to be made in our understanding of secretory control mechanisms in parietal cells and other cells as well. Methodology to be used to address the specific aims of the proposal include parietal cell isolation and purification, cell culture, in vitro measurement of acid secretory responses, digitized video image analysis to characterize acid secretory and calcium signalling mechanisms in single parietal cells, and utilization of a variety of biochemical techniques including high resolution two dimensional gel electrophoresis and computer-based gel and autoradiographic analyses to identify, isolate and obtain partial sequence information on relevant phosphoproteins. Molecular biological techniques will then be employed to obtain DNA sequence information that will be used for structure-function predictions, antibody generation and gene cloning when necessary. High resolution digitized video image analysis and three dimensional, computer- based image reconstruction methodologies will be employed to localize cellular proteins involved in intracellular activation events. Ultimately, the tissue distribution of relevant proteins can be defined using RNA isolation/Northern blot methodology and a protein database that will complement the genomic database developed. Another long-term goal is to develop an immortalized parietal cell line that retains appropriate in vivo characteristics.
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