IDENTIFICATION AND CHARACTERIZATION OF MATERIALS SECRETED BY CLARA CELLS

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The functions of the bronchiolar Clara cell are not known although numerous morphological investigations in many species have led to the hypothesis that the cell is secretory. The objectives of this research are to elucidate the secretory nature of the Clara cell, identify and characterize those secretions and determine their extracellular functions. We have developed a model system for the study of Clara cell functions and metabolism using cells isolated from the lungs of rabbits. Clara cells are dispersed from the lungs with pancreatic proteases and purified to over 90% by using, sequentially, a variety of techniques involving centrifugal elutriation, density gradient centrifugation, differential adherence and in vitro cultivation. Incubation of the isolated Clara cells with 35S-methionine for 4 hrs results in the radiolabeling of many intracellular proteins some of which were released into the incubation medium. Biosynthesis and secretion of proteins by Clara cell have been compared with those synthesized and secreted by other isolated and purified pulmonary cells including alveolar macrophages, Type II cells, and basal cells. The labeled cellular proteins and proteins released from the cells were identified by using sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions followed by fluorography and quantified by microdensitometry. Four major newly synthesized proteins with molecular weights of 9 kd, 30 kd, 90 kd and 180 kd were released from the Clara cells. The 9 kd protein accounted for 42% of the released and 10% of the cellular 35S-protein after 4 hrs of incubation. The 9 kd and 180 kd proteins were not synthesized by any of the other cell types. In addition, antisera prepared against Clara cell antigens were used to identify the presence of the 9 kd protein in lavage effluents from the lungs of rabbits. These data demonstrate that Clara cells make and secrete proteins different from those made and secreted by several other major pulmonary cells and that some of those proteins are present in the pulmonary extracellular lining.