Assembly of high-areal-density deuterium-tritium fuel from indirectly driven cryogenic implosions.

The National Ignition Facility has been used to compress deuterium-tritium to an average areal density of ~1.0±0.1 g cm(-2), which is 67% of the ignition requirement. These conditions were obtained using 192 laser beams with total energy of 1-1.6 MJ and peak power up to 420 TW to create a hohlraum drive with a shaped power profile, peaking at a soft x-ray radiation temperature of 275-300 eV. This pulse delivered a series of shocks that compressed a capsule containing cryogenic deuterium-tritium to a radius of 25-35 µm. Neutron images of the implosion were used to estimate a fuel density of 500-800 g cm(-3).

Altered stability of pulmonary surfactant in SP-C-deficient mice.

The surfactant protein C (SP-C) gene encodes an extremely hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the lung. To discern the role of SP-C in lung function, SP-C-deficient (-/-) mice were produced. The SP-C (-/-) mice were viable at birth and grew normally to adulthood without apparent pulmonary abnormalities. SP-C mRNA was not detected in the lungs of SP-C (-/-) mice, nor was mature SP-C protein detected by Western blot of alveolar lavage from SP-C (-/-) mice. The levels of the other surfactant proteins (A, B, D) in alveolar lavage were comparable to those in wild-type mice. Surfactant pool sizes, surfactant synthesis, and lung morphology were similar in SP-C (-/-) and SP-C (+/+) mice. Lamellar bodies were present in SP-C (-/-) type II cells, and tubular myelin was present in the alveolar lumen. Lung mechanics studies demonstrated abnormalities in lung hysteresivity (a term used to reflect the mechanical coupling between energy dissipative forces and tissue-elastic properties) at low, positive-end, expiratory pressures. The stability of captive bubbles with surfactant from the SP-C (-/-) mice was decreased significantly, indicating that SP-C plays a role in the stabilization of surfactant at low lung volumes, a condition that may accompany respiratory distress syndrome in infants and adults.

Intra-amniotic endotoxin increases pulmonary surfactant proteins and induces SP-B processing in fetal sheep.

Intra-amniotic (IA) endotoxin induces lung maturation within 6 days in fetal sheep of 125 days gestational age. To determine the early fetal lung response to IA endotoxin, the timing and characteristics of changes in surfactant components were evaluated. Fetal sheep were exposed to 20 mg of Escherichia coli 055:B5 endotoxin by IA injection from 1 to 15 days before preterm delivery at 125 days gestational age. Surfactant protein (SP) A, SP-B, and SP-C mRNAs were maximally induced at 2 days. SP-D mRNA was increased fourfold at 1 day and remained at peak levels for up to 7 days. Bronchoalveolar lavage fluid from control animals contained very little SP-B protein, 75% of which was a partially processed intermediate. The alveolar pool of SP-B was significantly increased between 4 and 7 days in conjunction with conversion to the fully processed active airway peptide. All SPs were significantly elevated in the bronchoalveolar lavage fluid by 7 days. IA endotoxin caused rapid and sustained increases in SP mRNAs that preceded the increase in alveolar saturated phosphatidylcholine processing of SP-B and improved lung compliance in prematurely delivered lambs.

Adenovirus infection increases iNOS and peroxynitrite production in the lung.

Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 x 10(9) plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.

Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice.

Targeted ablation of the surfactant protein D (SP-D) gene caused chronic inflammation, emphysema, and fibrosis in the lungs of SP-D (-/-) mice. Although lung morphology was unperturbed during the first 2 weeks of life, airspace enlargement was observed by 3 weeks and progressed with advancing age. Inflammation consisted of hypertrophic alveolar macrophages and peribronchiolar-perivascular monocytic infiltrates. These abnormalities were associated with increased activity of the matrix metalloproteinases, MMP2 and MMP9, and immunostaining for MMP9 and MMP12 in alveolar macrophages. Hydrogen peroxide production by isolated alveolar macrophages also was increased significantly (10-fold). SP-D plays a critical role in the suppression of alveolar macrophage activation, which may contribute to the pathogenesis of chronic inflammation and emphysema.

IL-4 increases surfactant and regulates metabolism in vivo.

Mice that express interleukin (IL)-4 in Clara cells (CCSP-IL-4) develop chronic airway inflammation and an alveolar proteinosis-like syndrome. To identify the role of IL-4 in surfactant homeostasis, we measured lipid and protein metabolism in the lungs of CCSP-IL-4 mice in vivo. Alveolar saturated phosphatidylcholine (Sat PC) pools were increased 6.5-fold and lung tissue Sat PC pools were increased 4. 8-fold in the IL-4 transgenic mice. Whereas surfactant protein (SP) A was increased proportionately to Sat PC, SP-D was increased approximately 90-fold in the IL-4 mice compared with wild-type mice and was associated with 2.8-fold increase in SP-D mRNA. The incorporation of palmitate and choline into Sat PC was increased about twofold in CCSP-IL-4 mice. Although trace doses of radiolabeled Sat PC were cleared from the air spaces and lungs of CCSP-IL-4 mice more slowly than in wild-type mice, net clearance of Sat PC from the lungs of CCSP-IL-4 mice was sixfold higher in the IL-4 mice than in wild-type mice because of the larger Sat PC pool sizes. Expression of IL-4 in Clara cells increased surfactant lipid synthesis and clearance, establishing a new equilibrium with increased surfactant pools and an alveolar proteinosis associated with a selective increase in SP-D protein, demonstrating a previously unexpected effect of IL-4 in pulmonary surfactant homeostasis.

Surfactant protein C in fetal and ventilated preterm rabbit lungs.

The developing lung contains surfactant protein (SP) C mRNA levels comparable to term values before mature type II cells and alveolar surfactant lipids are detectable. Estimates of the amount of mature SP-C in the alveolar lavages of preterm lungs are not available. We used an antibody to a recombinant human SP-C to measure the amount of SP-C in alveolar lavages of preterm fetal rabbits, ventilated preterm rabbits, and term rabbits. The amounts of SP-C were compared with the amounts of saturated phosphatidylcholine (Sat PC). Median Sat PC amounts increased about 680-fold, and median SP-C values increased by over 5,000-fold in alveolar washes from 27 days gestation to term. There was no increase in Sat PC or SP-C with ventilation at 27 and 28 days gestation, but ventilation increased both Sat PC and SP-C at 29 days gestation. The molar percent of SP-C relative to Sat PC also increased with gestational age and with ventilation at 29 days gestation. proSP-C was abundant in a membrane fraction from lung tissue at 27 and 28 days gestation when minimal mature SP-C was detected in alveolar washes. At 29 days and at term, proSP-C decreased in membrane fractions. The preterm lung that is surfactant lipid deficient is also severely deficient in mature SP-C.

Transcriptional regulation of the murine surfactant protein-A gene by B-Myb.

Surfactant protein A (SP-A) is selectively synthesized in subsets of cells lining the respiratory epithelium, where its expression is regulated by various transcription factors including thyroid transcription factor-1 (TTF-1). Cell-specific transcription of the mouse SP-A promoter is mediated by binding of TTF-1 at four distinct cis-active sites located in the 5'-flanking region of the gene. Mutation of TTF-1-binding sites (TBE) 1, 3, and 4 in combination markedly decreased transcriptional activity of SP-A promoter-chloramphenicol acetyltransferase constructs containing SP-A gene sequences from -256 to +45. In contrast, the same mutations enhanced transcriptional activity in constructs containing additional 5' SP-A sequences from -399 to +45 suggesting that cis-acting elements within the region -399 to -256 influence effects of TTF-1 on SP-A promoter activity. A consensus Myb-binding site was identified within the region, located at positions -380 to -371 in the mouse gene. Mutation of the Myb-binding site decreased activity of SP-A promoter constructs in MLE-15 cells. MLE-15 cells, a cell line expressing SP-A mRNA, also expressed B-Myb. B-Myb bound to the MBS in the SP-A gene as assessed by electrophoretic mobility shift assay. While co-transfection of HeLa cells with a B-Myb expression plasmid activated the transfected SP-A promoter about 3-fold, co-transfection of B-myb with cyclin A and cdk-2, to enhance phosphorylation of B-Myb, increased transcriptional activity of SP-A constructs approximately 20-fold. Taken together, the data support activation of SP-A gene promoter activity by B-Myb which acts at a cis-acting element in the SP-A gene.

Surfactant protein-A binds group B streptococcus enhancing phagocytosis and clearance from lungs of surfactant protein-A-deficient mice.

Surfactant protein-A (SP-A) gene-targeted mice clear group B streptococcus (GBS) from the lungs at a slower rate than wild-type mice. To determine mechanisms by which SP-A enhances pulmonary clearance of GBS, the role of SP-A in binding and phagocytosis of GBS was assessed in SP-A (-/-) mice infected with GBS in the presence and absence of exogenous SP-A. Coadministration of GBS with exogenous SP-A decreased GBS colony counts in lung homogenates of SP-A (-/-) mice. SP-A bound to GBS in a calcium-dependent manner. Although pulmonary infiltration with macrophages was not altered in SP-A (-/-) versus wild-type mice after GBS infection, the number of alveolar macrophages with phagocytosed bacteria was lower in the SP-A (-/-) mice than in the wild-type mice. When SP-A was coadministered with GBS, phagocytosis was significantly increased. Oxygen radical production by alveolar macrophages from SP-A (-/-) mice infected with GBS was decreased compared with wild-type controls and was increased when SP-A (-/-) mice were infected in the presence of exogenous SP-A. Superoxide (SO) radical generation was deficient in macrophages from SP-A (-/-) mice. SP-A plays an important role in GBS clearance in vivo, mediated in part by binding to and enhancing GBS phagocytosis and by increasing SO production by alveolar macrophages.

Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo.

Surfactant protein D (SP-D) is a 43-kDa member of the collectin family of collagenous lectin domain-containing proteins that is expressed in epithelial cells of the lung. The SP-D gene was targeted by homologous recombination in embryonic stem cells that were used to produce SP-D (+/-) and SP-D (-/-) mice. Both SP-D (-/-) and SP-D (+/-) mice survived normally in the perinatal and postnatal periods. Whereas no abnormalities were observed in SP-D (+/-) mice, alveolar and tissue phosphatidylcholine pool sizes were markedly increased in SP-D (-/-) mice. Increased numbers of large foamy alveolar macrophages and enlarged alveoli were also observed in SP-D (-/-) mice. Phospholipid composition was unaltered in SP-D (-/-) mice, but surfactant morphology was abnormal, consisting of dense phospholipid membranous arrays with decreased tubular myelin. The pulmonary lipoidosis in the SP-D (-/-) mice was not associated with accumulation of surfactant proteins B or C, or their mRNAs, distinguishing the disorder from alveolar proteinosis syndromes. Surfactant protein A mRNA was reduced and, SP-A protein appeared to be reduced in SP-D (-/-) compared with wild type mice. Targeting of the mouse SP-D gene caused accumulation of surfactant lipid and altered phospholipid structures, demonstrating a previously unsuspected role for SP-D in surfactant lipid homeostasis in vivo.

Interleukin-4 enhances pulmonary clearance of Pseudomonas aeruginosa.

To determine the effects of interleukin-4 (IL-4) on bacterial clearance from the mouse lung, transgenic mice expressing IL-4 in respiratory epithelial cells under the control of the Clara cell secretory protein promoter (CCSP-IL-4 mice) were infected intratracheally with Pseudomonas aeruginosa. Survival of CCSP-IL-4 mice following bacterial administration was markedly improved compared with that of control mice. While bacteria proliferated in lungs of wild-type mice, a rapid reduction in the number of bacteria was observed in the IL-4 mice as early as 6 h postinfection. Similarly, intranasal administration of IL-4 enhanced bacterial clearance from the lungs of wild-type mice. While acute and chronic IL-4 increased the numbers of neutrophils in bronchoalveolar lavage fluid, bacterial infection was associated with acute neutrophilic pulmonary infiltration, and this response was similar in the presence or absence of IL-4. Local administration or expression of IL-4 in the mouse lung enhanced pulmonary clearance of P. aeruginosa in vivo and decreased mortality following infection.

Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor.

Cellular nuclease activity is a potential barrier to the successful delivery of foreign genes to mammalian cells. We tested the hypothesis that transfection in the presence of a specific DNase inhibitor can enhance the expression of foreign gene products. We have used DMI-2, a polyketide metabolite of Streptomyces sp. strain 560 to enhance the expression of bacterial chloramphenicol acetyltransferase (CAT) in the human lung adenocarcinoma cell line H441. DMI-2 has been shown previously to inhibit porcine DNase II, an acid pH nuclease contained in the endosomal/lysosomal compartment. Transfection of H441 cells in the presence of 0.1-1 microgram/ml DMI-2 caused: (1) 10-fold enhancement of CAT activity when the bacterial plasmid was complexed with either surfactant protein A-poly-lysine or transferrin-poly-lysine; (2) 1.5- to two-fold enhancement of CAT activity in cells exposed to lipofectin-DNA complexes: (3) no effect on transfection via calcium phosphate co-precipitation. DMI-2 alone showed no inherent transfection activity. In experiments using SP-A-poly-lysine and plasmid containing the beta-galactosidase reporter gene, DMI-2 increased the number of transfected cells. Methanolysis products of DMI-2 did not inhibit DNase II and did not enhance transfection efficiency. Taken together, the data support the hypothesis that nuclease action is a significant barrier to expression of foreign genes and inhibition of specific nucleases may facilitate transfection.

Surfactant protein A-deficient mice are susceptible to group B streptococcal infection.

To determine the role of surfactant protein A (SP-A) in host defense, the murine SP-A locus was targeted by homologous recombination to produce mice lacking SP-A. SP-A -/- and control mice were infected with group B streptococcus (GBS) by intratracheal instillation. Pulmonary infiltration 6 and 24 h following infection was more severe in SP-A -/- than in control mice, and was associated with increased numbers of GBS in lung homogenates. Dissemination of GBS to the spleen was observed more frequently in SP-A -/- mice. Pulmonary infiltration with macrophages was similar in both groups; however, the number of bacteria associated with alveolar macrophages was decreased in the SP-A-deficient mice. There was no detectable compensatory increase in surfactant protein D, the other known pulmonary collectin, in response to GBS instillation. SP-A plays an important role in vivo, enhancing clearance of GBS from the lung and inhibiting systemic dissemination of the organism.

Altered surfactant function and structure in SP-A gene targeted mice.

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.

Surfactant protein A-polylysine conjugates for delivery of DNA to airway cells in culture.

Surfactant protein A (SP-A) was modified by covalent linkage with polylysine of average M(r) 21 kD ([Lys]21kD-SP-A) and utilized to transfect human airway epithelial cells (H441) in vitro. Transfection of H441 cells was more efficient with [Lys]21kD-SP-A than with polylysine-DNA or unmodified SP-A-DNA complexes. Transfection with [Lys]21kD-SP-A was effective at a protein-to-DNA molar ratio of 400:1 and in the presence of an exogenous surfactant preparation, Survanta. Transfection with [Lys]21kD-SP-A was reduced in the presence of unmodified SP-A consistent with the concept of a receptor mediated uptake of protein-DNA complexes. Increased transfection efficiency correlated with decreasing diameter of the [Lys]21kD-SP-A-DNA complexes, and these complexes bound to the cell surface and pseudopodia of H441 cells. Transfection was enhanced by co-incubation with replication-deficient adenovirus. Cotransfection by [Lys]21kD-SP-A-DNA and [Lys]10kD-SP-B resulted in an additive level of reporter gene (CAT) expression. [Lys]21kD-SP-A-DNA is likely to be useful as a component of a surfactant-based DNA delivery system for transfection of airway cells.

Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids.

Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem. 262, 13877-13880). Chromatogram overlays in conjunction with in situ liquid secondary ion mass spectrometry (TLC-LSIMS) of several purified glycosphingolipids and neoglycolipids as well as binding assays with glycolipids immobilized on plastic wells, demonstrate recognition of galactose (human and dog SP-A), glucose, and lactose (human SP-A) in association with specific lipids. In addition, the occurrence of several neutral and acidic glycosphingolipids in human and rat extracellular surfactants and rat alveolar type II cells is described. Selected components among the neutral glycolipids are bound by radiolabeled human SP-A; these are identified by TLC-LSIMS as predominantly ceramide mono- and disaccharides (human surfactant) and ceramide tri- and tetrasaccharides (rat surfactant and type II cells). A recombinant carbohydrate recognition domain (CRD) of human SP-A inhibits the binding of human SP-A to galactosyl ceramide and to galactose- and mannose-bovine serum albumin, indicating that the CRD is directly involved in the binding of SP-A to these ligands. These results provide evidence for a novel type of binding specificity for proteins that have Ca(2+)-dependent CRDs and raise the possibility that glycosphingolipids are endogenous ligands for SP-A.

Lectin activity of the major surfactant protein (SP-A) may participate in, but is not required for, binding to rat type II cells.

Pulmonary surfactant is a complex mixture of lipids and proteins, of which surfactant protein A (SP-A) is the most abundant glycoprotein. The SP-A molecule has several distinct structural features that include a lectin-like domain, sharing structural features with other mammalian lectins. We have tested the hypothesis that lectin activity of the SP-A molecule is required for the binding to its receptor on the surface of alveolar Type II cells. By using colloidal gold immunocytochemistry in conjunction with electron microscopy, we evaluated the ability of mannosylated proteins to inhibit canine SP-A binding to rat Type II cells in vitro. After preincubation of SP-A with the mannosylated protein horse-radish peroxidase (HRP), SP-A was incubated with isolated filter-grown Type II cells. HRP did not alter the binding of SP-A to the Type II cell surface. Evidence that SP-A did bind to HRP was shown by coincident observation of gold-labeled SP-A and HRP precipitates. These results provide visual evidence that the lectin activity associated with SP-A is not required for its binding to receptor on rat alveolar Type II epithelial cells.

Human surfactant protein-A contains blood group A antigenic determinants.

A major blood group antigenic epitope was identified on human pulmonary surfactant protein A (SP-A). MAb and polyclonal antibodies generated against purified human SP-A aggregated blood group A human erythrocytes and immunostained epithelial cells in a variety of human tissues, consistent with the tissue distribution of major blood group antigens. SP-A MAb (MAb-8) agglutinated red cells and immunostained tissues from A or AB blood groups, but did not react with cells or tissues from O or B individuals. MAb-8 immunostaining of tissue from blood group A individuals was ablated by incubation with blood group A red cells. MAb and polyclonal antibodies directed against A blood group antigens reacted strongly with purified SP-A obtained from a blood group A individual with alveolar proteinosis. MAb and polyclonal antibodies specific for B blood group antigen failed to react with SP-A from this patient or from patients who were in blood group B. Reactivity of anti-blood group MAb was lost after treatment of SP-A with endoglycosidase-F, demonstrating its reactivity with an epitope dependent on the asparagine-linked oligosaccharide at asparagine 187. Reactivity of MAb-8 with SP-A persisted after endoglycosidase-F treatment, but was lost after digestion with collagenase as assessed by Western blot after SDS-PAGE. Reactivity of MAb to SP-A was sensitive to beta-elimination, supporting the presence of another blood group antigenic site distinct from the epitope dependent on the asparagine-linked carbohydrate. The finding that the SP-A molecule contains a major blood group epitope has implication for the clinical use of surfactant replacement preparations and diagnostic reagents based on this protein.

Intermolecular cross-links mediate aggregation of phospholipid vesicles by pulmonary surfactant protein SP-A.

As the most abundant glycoprotein component of pulmonary surfactant, SP-A (Mr = 30,000-36,000) plays a central role in the organization of phospholipid bilayers in the alveolar air space. SP-A, isolated from lung lavage, exists in oligomeric forms (N = 6, 12, 18, ...), mediated by collagen-like triple helices and intermolecular disulfide bonds. These protein-protein interactions, involving the amino-terminal domain of SP-A, are hypothesized to facilitate the alignment of surfactant lipid bilayers into unique tubular myelin structures. SP-A reorganization of surfactant lipid was assessed in vitro by quantitating the calcium-dependent light scattering properties of lipid vesicle suspensions induced by SP-A. Accelerated aggregation of unilamellar vesicles required SP-A and at least 3 mM free calcium. The initial rate of aggregation was proportional to the concentration of canine SP-A over lipid:protein molar ratios ranging from 200:1 to 5000:1. Digestion with bacterial collagenase or incubation with dithiothreitol (DTT) completely blocked lipid aggregation activity. Both treatments decreased the binding of SP-A to phospholipids. The conditions used in the DTT experiments (10 mM DTT, nondenaturing Tris buffer, 37 degrees C) resulted in the selective reduction and 14C-alkylation of the intermolecular disulfide bond involving residue 9Cys, whereas the four cysteines found in the noncollagenous domain of SP-A were inefficiently alkylated with [14C]-iodoacetate. HPLC analysis of tryptic SP-A peptides revealed that these four cysteine residues participate in intramolecular disulfide bond formation (138Cys-229Cys and 207Cys-221Cys). Our data demonstrate the importance of the quaternary structure (triple helix and intermolecular disulfide bond) of SP-A for the aggregation of unilamellar phospholipid vesicles.

Surfactant-associated protein inhibits phospholipid secretion from type II cells.

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.