Monoclonal antibodies to surfactant proteins SP28-36 label canine type II and nonciliated bronchiolar cells by immunofluorescence.

The major 28,000- to 36,000-dalton proteins of pulmonary surfactant (SP28-36) have been shown by various techniques to be synthetic and secretory products of alveolar type II cells. Surfactant lipids are also secreted by these cells. Immunocytochemical studies of human and rodent lungs have indicated that nonciliated epithelial cells of small bronchioles appear to contain SP28-36 in their synthetic organelles and secretory granules. Because these observations were obtained with polyclonal antibodies against SP28-36, it was possible that bronchiolar cell staining was due to contaminant antibodies not detected by biochemical analyses. To clarify the role of bronchiolar cells in the metabolism of SP28-36, we have prepared 5 monoclonal antibodies against canine SP28-36. Electrophoresis and immunoblots of surfactant showed that each antibody reacted with SP32 and 36, as well as SP28, the nonglycosylated species. This indicates that the antibodies are directed against the protein rather than carbohydrate moieties of SP28-36. Immunoblot analysis of collagenase-treated SP28-36 showed that the antibodies DS-3 and DS-1 were directed against the noncollagen region of the protein. Immunoblot analysis of whole canine lung homogenates showed that a single protein species was recognized by the antibodies. Immunofluorescence studies of cryostat sections of canine lung showed that both type II and nonciliated bronchiolar cells were specifically labeled with each antibody. These and previous data are consistent with and support the idea that bronchiolar cells synthesize and secrete SP28-36.

Purification and subunit composition of atrial natriuretic peptide receptor.

A receptor for atrial natriuretic peptide (ANP) was purified 2700-fold, to apparent homogeneity, from cultured bovine aortic smooth muscle cells by affinity chromatography. The native ANP receptor has a molecular weight of 125,000 as determined by both metrizamide gradient centrifugation and nonreducing NaDodSO4/polyacrylamide gel electrophoresis. With 125I-labeled ANP as ligand, the purified receptor bound a maximum of 5.70 nmol of ligand per mg of protein and the dissociation constant was 4.0 X 10(-10)M. Upon treatment with 10 mM dithiothreitol, the purified receptor migrated as a single band at Mr 60,500 in NaDodSO4/polyacrylamide gel electrophoresis. These findings show that the holoreceptor for ANP in vascular tissue is composed of two subunits of identical apparent molecular weight, presumably linked by a disulfide bridge(s).

Identification of the receptor for atrial natriuretic factor on cultured vascular cells.

Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration-dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor.