Zymogen secretion is of critical relevance in the digestive process of the gastrointestinal tract and is considered to contribute to pathological processes such as reflux esophagitis, peptic ulcer disease and pancreatitis.
The aim of the present studies is to delineate and characterize aspects of the molecular mechanisms of zymogen secretion utilizing an isolated zymogen granule model. The secretory granule is the key organelle of exocytosis, therefore, studies on characterization of zymogen granules are crucial for the understanding of exocrine secretion in general. Specifically, the aim of the present proposal is to delineate the physiological role of a novel zymogen granule membrane- associated protein kinase activity in gastric chief cells, pancreatic and parotid acinar cells. First, to identifying an important signature of this novel kinase, the endogenous substrate(s) for the kinase will be identified in a gastric chief cell model, and the specific peptide sequence of the substrate will be determined by phospho-peptide sequence analysis of phosphorylated substrates. This sequence will then be utilized to construct pseudo-substrate peptide inhibitors against the kinase. Second, purification of the kinase from peptic granule membranes will be undertaken by a combination of affinity and conventional chromatography strategies in order to obtain the microsequence. Third, monoclonal antibody against the partial peptide sequence of the kinase will be produced to determine the specific cellular localization and redistribution of the kinase utilizing immunocytochemisty at the light and confocal microscopic as well as the electron microscopic level. Fourth, the effect of the kinase on enzyme secretion will be evaluated in the permeabilized gastric gland and pancreatic acinar cell system by introduction of peptide inhibitors and specific antibodies, and finally, the determination of the complete sequence of the kinase will be undertaken utilizing the molecular cloning techniques of Polymerase Chain Reaction and screening of a gastric chief cell cDNA library. The recombinant protein will be produced for raising monoclonal antibody for further functional studies. These studies will allow the characterization of a novel putative regulator of zymogen exocytosis. The elucidation of the mechanism of zymogen secretion will facilitate the identification of its relevance to human disease processes. The ability to regulate zymogen secretion may become an important therapeutic strategy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29DK048820-01
Application #
2149293
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1995-01-01
Project End
1999-11-30
Budget Start
1995-01-01
Budget End
1995-11-30
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Yale University
Department
Surgery
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Kidd, M; Tang, L H; Modlin, I M et al. (2000) Gastrin-mediated alterations in gastric epithelial apoptosis and proliferation in a mastomys rodent model of gastric neoplasia. Digestion 62:143-51
Lauffer, J M; Modlin, I M; Hinoue, T et al. (1999) Pituitary adenylate cyclase-activating polypeptide modulates gastric enterochromaffin-like cell proliferation in rats. Gastroenterology 116:623-35
Schmid, S W; Modlin, I M; Tang, L H et al. (1998) Telenzepine-sensitive muscarinic receptors on rat pancreatic acinar cells. Am J Physiol 274:G734-41
Kidd, M; Tang, L H; Schmid, S W et al. (1998) A polyamine pathway-mediated mitogenic mechanism in enterochromaffin-like cells of Mastomys. Am J Physiol 275:G370-6
Luque, E A; Tang, L H; Bortecen, K H et al. (1998) Gastrin-regulated expression of p53 in transformed enterochromaffin-like cells in the African rodent mastomys. J Clin Gastroenterol 27 Suppl 1:S116-24
Tang, L H; Luque, E A; Efstathiou, J A et al. (1997) Gastrin receptor expression and function during rapid transformation of the enterochromaffin-like cells in an African rodent. Regul Pept 72:9-18
Tang, L H; Modlin, I M; Lawton, G P et al. (1996) The role of transforming growth factor alpha in the enterochromaffin-like cell tumor autonomy in an African rodent mastomys. Gastroenterology 111:1212-23
Borin, J F; Tang, L H; Kidd, M et al. (1996) Somatostatin receptor regulation of gastric enterochromaffin-like cell transformation to gastric carcinoid. Surgery 120:1026-32