The goal of the proposed research is to define the physiological regulation and functional significance of vitamin K-dependent delta- carboxylation of protein substrates by rat alveolar type II cells. Surfactant homeostasis is a major metabolic function of these cells, and the surfactant-associated protein (SP-A) was recently found to be a predominant substrate for the microsomal carboxylase enzyme complex. Addition of Delta-carboxyglutamic acid (Gla) to SP-A likely explains the calcium dependence of its known properties by analogy to the coagulation system. This research will define microsomal carboxylase activity and substrate specificity in freshly isolated type II epithelial cells from normal, hormonally and nutritionally manipulated adult rats. The role of type II cell differentiation in modulation Delta-carboxylase activity and substrate specificity will be determined during primary culture where differentiated morphology and function can be modified by both laminin- and fibronectin-rich extracellular matrices. The physiological significance of SP-A Delta-carboxylation and glycosylation will be determined in functional studies of type II cell surfactant secretion and reuptake and from physiochemical studies of lipid-protein interactions and calcium binding stability. These experiments will utilize both naturally produced SP-A molecules and proteins with chemically modified oligosaccharide and Gla content. The importance of cell-cell interactions on adult and fetal type II cell carboxylase activity and substrate specificity will also be defined. Physiologically significant regulatory factors which influence the maturation of fetal type Ii cells will be studied using cocultures of fetal fibroblasts and type II cells and in mixed cell alveolar-like structures. The long term goals of this research are to understand the physiological regulation and function of vitamin K- dependent Delta-carboxylation in lung tissue. These studies will contribute to a better understand of surfactant homeostasis during development and in the adult and may provide new insights into the treatment of respiratory distress syndromes, the pulmonary consequences of anticoagulant therapies, and the involvement of these pathways in the response of the lung to injury.
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