The continuing objectives of this research are to elucidate factors involved in regulating intestinal growth and differentiation and to delineate the regulatory mechanisms by which regulators exert their effects. To determine hormonal regulation of intestinal growth and maturation, infant rats will be hypophysectomized or adrenalectomized and artificially reared by isocaloric infusion of a hormone-free diet to serve as an in vivo model system. Using this model, the effects of hormones, including growth hormone, corticosterone, thyroxine and epidermal growth factor, alone or in various combinations on epithelial cell proliferation, migration and maturation will be determined. The criteria of cell proliferation, including cell cycle time (G-1, S, G-2 and M phases), crypt cell production rates and crypt cell population, will be determined by autoradiography after H-thymidine injection and by metaphase accumulation rates after vincristine administration. Decrease of lactase activity and appearance or increase of sucrase activity will be used as maturational markers. The differential effects of corticosterone and thyroxine on the de novo synthesis of sucrase-isomaltase (S-I) will be examined by the techniques of immunofluorescent staining, immunohistochemistry, rocket immunoelectrophoresis, isoelectric focusing, immunoblotting and in vitro mRNA translation. The defect in post-translational modification of S-I which is believed to lead to sucrose intolerance in human hormonal regulation on glycosylation of S-I will be studied by pulse-chase experiments with 35S-methionine, 14C-mannose and fucose. Catalytic activity and structural changes will be analyzed. The hormonal effect on intestinal development will also be examined in an in vitro system to determine: 1) the role of mesenchymal cells in epithelial growth and differentiation, 2) hormonal effect on the organization of contractile proteins and cell migration, and 3) the mechanism by which epithelial cell sorting, migration and organogenesis occur.
