[' ', '—', 'EXPERIMENTAL', 'CELL', 'RESEARCH', '226,', '90--97', '(1996)', 'ARTICLE', 'NO.', '0206', '60', 'Yo', 'Receptor-Mediated', 'Regulation', 'of', 'Pulmonary', 'Surfactant', 'Secretion', 'Davip', 'S.', 'STRAYER,*”', 'R.', 'PINDER,*', 'AND', 'AVINASH', 'CHANDERt', 'Nr', 'neg', '*Department', 'of', 'Pathology,', 'Anatomy,', 'and', 'Cell', 'Biology', 'and', '{Department', 'of', 'Pediatrics—', 'Division', 'of', 'Neonatology,', 'Thomas', 'Jefferson', 'University,', 'Philadelphia,', 'Pennsylvania', '19107', ' ', 'Surfactant', 'protein', 'A', '(SP-A)', 'regulates', 'surfactant', 'se-', 'cretion', 'via', 'an', 'SP-A', 'specific', 'type', 'II', 'cell', 'membrane', 're-', 'ceptor', '(SPAR).', 'We', 'report', 'here', 'that', 'two', 'anti-SPAR', 'monoclonal]', 'antibodies', 'can', 'modulate', 'the', 'secretory', 'in-', 'hibition', 'caused', 'by', 'SP-A.', 'A2C', 'and', 'A2R', 'are', 'rat', 'mono-', 'clonal', 'antibodies', 'raised', 'independently', 'and', 'recognize', 'a', '32-kDa', 'protein', 'on', 'rat', 'alveolar', 'type', 'I', 'cell', 'mem-', 'branes.', 'Immunocytochemical', 'studies', 'show', 'that', 'these', 'antibodies', 'bind', 'to', 'isolated', 'type', 'II', 'cells.', 'Scatchard', 'analysis', 'confirms', 'that', 'SP-A', 'binds', 'alveolar', 'type', 'II', 'cells', 'through', 'a', 'single', 'affinity', 'receptor', 'and', 'shows', 'that', 'A2C', 'and', 'A2R', 'recognize', 'that', 'same', 'receptor.', 'Both', 'antibod-', 'ies', 'inhibit', 'the', 'binding', 'of', "'*I-SP-A", 'to', 'isolated', 'type', 'II', 'cells.', 'The', 'functional', 'activity', 'of', 'this', '32-kDa', 'protein', 'was', 'studied', 'by', 'examining', 'surfactant', 'secretion', 'in', 'isolated', 'type', 'Il', 'cells.', 'Surfactant', 'phospholipid', 'secretion', 'was', 'measured', 'in', 'cells', 'that', 'were', 'exposed', 'to', 'various', 'surfac-', 'tant', 'phospholipid', 'secretagogues', '(ATP,', 'dibutyryl', 'cAMP,', 'terbutaline,', 'or', 'ionomycin),', '+SP-A', '(100', 'ng/m),', '+A2C', 'or', 'A2R.', 'Both', 'antibodies', 'block', 'the', 'negative', 'feed-', 'back', 'loop', 'by', 'which', 'SP-A', 'inhibits', 'surfactant', 'secretion.', 'This', 'activity', 'of', 'A2C', 'and', 'A2R', 'is', 'dose-dependent', 'and', 'is', 'independent', 'of', 'the', 'secretagogue', 'used.', 'Thus,', 'the', '32-', 'kDa', 'type', 'II', 'cell', 'membrane', 'protein', 'bound', 'by', 'A2C', 'and', 'A2R', 'is', 'the', 'functional', 'receptor', 'on', 'alveolar', 'type', 'II', 'cell', 'membranes', 'and', 'regulates', 'type', 'II', 'cell', 'surfactant', 'secre-', 'tion.', '©', '1996', 'Academic', 'Press,', 'Inc.', ' ', 'INTRODUCTION', 'Pulmonary', 'surfactant', 'is', 'produced', 'by', 'type', 'II', 'alveolar', 'cells', 'and', 'lowers', 'the', 'surface', 'tension', 'of', 'the', 'air—liquid', 'in-', 'terface', 'in', 'the', 'alveolar', 'lumen,', 'thus', 'facilitating', 'lung', 'expansion', 'and', 'preventing', 'atelectasis', '[1].', 'Chemically,', 'pulmonary', 'surfactant', 'is', 'a', 'complex', 'mixture', 'of', '~80%', 'glyc-', 'erophospholipids,', '~10%', 'cholesterol,', 'and', '~10%', 'protein.', 'The', 'dipalmitoylated-phosphatidylcholine', '(DPPC)', 'com-', 'prises', 'the', 'largest', 'proportion', '(~75%)', 'of', 'the', 'lipids', 'found', 'in', 'pulmonary', 'surfactant', '[2].', 'To', 'whom', 'correspondence', 'and', 'reprint', 'requests', 'should', 'be', 'ad-', 'dressed', 'at', 'Department', 'of', 'Pathology,', 'Anatomy', 'and', 'Cell', 'Biology,', 'Jef-', 'ferson', 'Medical', 'College,', '1020', 'Locust', 'St.,', 'Philadelphia,', 'PA', '19107.', 'Fax:', '(215)-923-2218.', 'Internet:', 'david.strayer@mail.tju.edu.', '0014-4827/96', '$18.00', 'Copyright', '©', '1996', 'by', 'Academic', 'Press,', 'Inc.', 'All', 'rights', 'of', 'reproduction', 'in', 'any', 'form', 'reserved.', 'PUBLICATIONS', ' ', 'At', 'least', 'four', 'unique', 'proteins', '(SP-A,', '-B,', '-C,', 'and', '-D)', 'have', 'been', 'isolated', 'from', 'pulmonary', 'surfactant', '[3].', 'The', 'most', 'abundant', 'of', 'these', 'is', 'surfactant', 'protein', 'A', '(SP-A),', 'a', 'heavily', 'glycosylated,', 'apoprotein', 'of', '~34', 'kDa', 'under', 'reduced', 'conditions', '(4,', '5].', 'SP-A', 'serves', 'multiple', 'func-', 'tions,', 'including', 'stabilizing', 'the', 'structure', 'of', 'intraal-', 'veolar', 'tubular', 'myelin', '[3].', 'It', 'may', 'also', 'participate', 'in', 'antimicrobial', 'defenses', '[5].', 'In', 'addition,', 'SP-A', 'regulates', '‘surfactant', 'secretion', '[6].', 'Intracellular', 'and', 'extracellular', 'reserves', 'of', 'surfactant', 'are', 'not', 'extensive;', 'so', 'secretion', 'is', 'tightly', 'regulated.', 'It', 'has', 'been', 'shown', 'that', 'SP-A', 'regu-', 'lates', 'surfactant', 'secretion', 'via', 'a', 'specific', 'type', 'II', 'cell', 'membrane', 'receptor', '[7,', 'SPAR}.', 'Rice', 'et', 'al.', '[8]', 'showed', 'that', 'SP-A', 'inhibits', 'surfactant', 'phospholipid', 'secretion.', 'This', 'inhibition', 'is', 'mediated', 'by', 'a', 'specific,', 'high-affinity', 'cell', 'membrane', 'receptor', 'for', 'SP-A', '[7,', '9].', 'We', 'have', 'identified', 'that', 'receptor', '[10]', 'and', 'report', 'here', 'on', 'its', 'functional', 'characteristics.', 'Using', 'rat', 'monoclonal', 'anti-idiotype', 'antibodies', '[11,', 'aiMAbs]', 'raised', 'against', 'monoclonal', 'antibodies', 'that', 'bind', 'surfactant', 'proteins,', 'we', 'identified,', 'structurally', 'characterized,', 'and', 'cloned', 'a', '~~32-kDa', 'protein', 'that', 'binds', 'SP-A', 'and', 'is', 'present', 'on', 'alveolar', 'type', 'II', 'cells.', 'However,', 'the', 'relationship', 'of', 'this', 'protein', 'to', 'surfactant', 'secretion', 'was', 'not', 'clear.', 'We', 'de-', 'scribe', 'here', 'the', 'role', 'of', 'that', '32-kDa', 'protein', 'in', 'regulating', 'of', 'surfactant', 'secretion', 'and', 'the', 'ability', 'of', 'the', 'aiMAbs', 'that', 'recognize', 'that', 'protein', 'to', 'prevent', 'the', 'inhibition', 'of', 'surfactant', 'secretion', 'by', 'SP-A.', 'MATERIALS', 'AND', 'METHODS', 'Animals.', 'Specific', 'pathogen-free,', 'male', 'Sprague-Dawley', 'rats', '(180-200', 'g)', 'were', 'obtained', 'from', 'Charles', 'River', 'Laboratories.', 'The', 'animals', 'were', 'used', 'as', 'sources', 'of', 'alveolar', 'type', 'II', 'cells', 'within', '1', 'week', 'of', 'receipt.', 'Chemicals', 'and', 'reagents.', 'SP-A', 'from', 'bovine', 'lung', 'was', 'purified', 'and', 'analyzed', 'as', 'described', '[12,', '13].', 'When', 'labeled', 'SP-A', 'was', 'used,', 'it', 'was', 'labeled', 'with', "'5I", '(Dupont-NEN,', 'Wilmington,', 'DE)', 'using', 'chloramine-', 'T', 'as', 'described', '[11].', '**I-SP-A', '(average', 'specific', 'activity', '2', 'pCi/ug)', 'was', 'purified', 'from', 'unbound', 'iodine', 'using', 'gel', 'filtration', 'chromatography.', 'Isolation', 'of', 'type', 'II', 'cells.', 'Alveolar', 'type', 'II', 'cells', 'were', 'isolated', 'from', 'rat', 'lungs', 'as', 'described', 'by', 'Dobbs', '—', '4', 'co-workers', '[14]', 'and', 'modified', 'by', 'Sen', 'and', 'Chander', '[15].', 'Essentially,', 'lungs', 'of', 'anesthetized', 'rats', 'were', 'cleared', 'of', 'blood', 'and', 'endotracheally', 'treated', 'with', 'elastase', '(Wor-', 'thington', 'Biochemical,', 'Freehold,', 'NJ)', 'to', 'obtain', 'free', 'cells.', 'Once', 'the', 'cells', 'were', 'separated', 'from', 'lung', 'debris,', 'they', 'were', 'plated', 'on', 'bacterio-', 'logical', 'plates', 'coated', 'with', 'normal', 'rat', 'IgG', '(Sigma', 'Chemical', 'Co.,', 'St', '0054743', ' ']