Secretion of lung surfactant occurs by exocytosis of lamellar body contents into the alveolar lumen. Regulation of fusion between the lamellar body membrane and the plasma membrane that occurs during exocytosis, has not been investigated. Our characterization of lamellar bodies shows that they are surrounded by a limiting membrane and maintain an ATP-dependent pH gradient with an acid pH inside. We have prepared lamellar body membranes that shows enrichment of ATPase activity. We have also purified an approximately 47 kDa protein, synexin, from the cytosolic fraction of bovine lung, which promotes in vitro fusion between the lamellar bodies and the plasma membrane. In this proposal, we plan to understand the regulation of fusion of lamellar bodies with the plasma membranes. In doing so, we will determine the role of lipid and protein components of lamellar body membranes. We will also determine the role of pH, ATPases, and calcium in the fusion process. The pH of the lamellar bodies, or their membrane vesicles, will be altered with ATP in the absence or presence of inhibitors of H+-ATPase, and weakly basic compounds. The lamellar body and plasma membranes will be investigated for the synexin binding characteristics. Since surfactant apoproteins, SP-A and SP-B, increase aggregation of liposomes, a prerequisite for fusion, we will investigate their role, and the mechanism of their interaction with synexin, in the regulation of membrane fusion. To establish a correlation of synexin activity and lung surfactant secretion, we will determine the effect of inhibitors of synexin activity on the secretion of lung surfactant. We will purify synexin from rat lungs and raise monospecific polyclonal antibodies to rat synexin. Surfactant secretion will be followed after introduction of synexin antibodies into type II cells by incubation with liposome encapsulated antibodies. Using an oligonucleotide probe we have determined that the size of synexin mRNA is approximately 1.8 Kb. We will use this probe, and probes generated by reverse transcription polymerase chain reaction using primer based on human synexin cDNA and synexin antibodies to measure the synexin mRNA and synexin protein levels in cells cultured for varying periods of time, and correlate these with the secretion of surfactant from type II cells. Finally, we will measure changes in synexin and its mRNA in fetal rat lung which might increase with fetal lung development with gestational age, and in response to agents that promote lung maturation.
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