Alveolar type II cells are critical to the function of normal lung. These cells synthesize and secrete pulmonary surface active material and are the stem cells for the alveolar epithelium. In this proposal we will use rat type II cells to investigate the synthesis and secretion of surface active material and the proliferation of type II cells in vitro. Pulmonary surface active material is an heterogeneous mixture of phospholipids and the surfactant associated proteins (SP-A, SP-B, and SP-C). Surfactant protein A inhibits the secretion of phosphatidylcholine in vitro and stimulates the uptake of liposomal lipid into type II cells. We have recently shown that SP-A binds to type II cells with high affinity and specificity. To date, the receptor has not been isolated. However, digestion of type II cell plasma membranes with endoglycoceramidase indicates that the high affinity receptor is a glycosphingolipid. Our first specific aim is to isolate and characterize the type II cell receptor for SP-A. We will determine if the receptor required for inhibition of secretion is the same as the receptor required for the stimulation of uptake of liposomal lipid by SP-A.
The second aim i s to determine how phospholipids and SP-A inhibit secretion in vitro. We will elucidate the structural basis for phospholipid mediated regulation of secretion and determine at which point in the secretory cascade phospholipids act. Since SP-A inhibits multiple agonists, we believe that its effects are distal to the initial receptor activation and generation of second messengers. Our hypothesis is that small molecular weight guanine nucleotide binding proteins or the subplasma membrane cytoskeleton are altered. We recently developed a primary culture system for maintaining differentiation of type II cells in vitro. In our third specific aim we will use this culture system to study longer term regulation of surfactant synthesis and secretion in vitro. We will determine whether or not the protein and the lipids of surfactant are secreted together and the regulation of secretion in the presence of extracellular surfactant. Type II cells proliferate to maintain the alveolar epithelium and can be stimulated to proliferate in vitro. Two sources of growth factors that stimulate proliferation in vitro and are potentially important in vivo are bronchoalveolar lavage fluid and media conditioned by macrophages. Our fourth specific aim is to identify and purify growth factors from these sources and establish whether or not they are known or novel growth factors. Our fifth specific aim is to demonstrate that the type II cells which proliferate in vitro can re- express the type II cell phenotype under appropriate culture conditions in vitro.
Showing the most recent 10 out of 96 publications
