GASTROINTESTINAL SIGNIFICANCE OF GASTRIN REGULATION

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Although the physiological importance of gastrin in regulating gastric acid secretion has been established, and thus the relationship between gastrin and the pathogenesis of peptic ulcer disease implied, little is known about the mechanisms by which the synthesis of this gastrointestinal hormone is regulated. Recent development of molecular biological and cell culture techniques has made it possible to examine the control of hormone synthesis directly. I will use these techniques to study the biosynthesis, post-translational processing and secretion of gastrin. To determine the structure of the human gastrin precursor, gastrin cDNA will be synthesized from human antral and Zollinger-Ellison tumor mRNA with reverse transcriptase using the gastrin-specific synthetic oligodeoxynucleotide primer, d(G-A-A-G-T-C-C-A-T-C-C-A). The cDNA will be sequenced and used as a probe for selection of full-length gastrin cDNA clones from a cDNA library constructed using antral and Zollinger-Ellison tumor mRNA. The biosynthesis of gastrin will be examined with a newly developed antibody (GL-9), which recognized gastrin precursor, and with antibody 5135, which is specific for the carboxyl terminus of gastrin. Three systems will be used for biosynthetic studies: 1) cell-free translation of mRNA with wheat germ extract, 2) primary cultures of isolated canine antral gastrin cells, and 3) gastrin secreting single cell clones formed by hybridization of Zollinger-Ellison tumor cells with thymidine kinase deficient mouse fibroblasts. The critical step in post-translational processing of gastrin precursor to its active form is the amidation of the carboxyl terminal Phe. The enzyme responsible for this amidation will be identified and purified using a synthetic probe (Tyr-Gly-Trp-Met-Asp-Phe-Gly-Arg-Arg), an antibody specific for this probe (GL-9), and an antibody (5135) specific for amidated gastrin. Intermediates formed during amidation will be identified by high pressure liquid chromatography. The pathophysiological importance of alterations in gastrin synthesis and processing will be examined in vivo in rats treated chronically with methyl prednisolone, or in vitro in chronically stimulated somatic cell hybrids. In the vivo studies, induced alterations in gastrin mRNA content, biosynthesis, processing, and release will be examined and related to the development of gastric ulcers.