Bradley J. Benson

Subfractionation of lung surfactant. Implications for metabolism and surface activity.

Because previous studies have suggested that lung surfactant is not a simple compartment of homogeneous material, we subfractionated lamellar bodies and components of alveolar lavage from male New Zealand white rabbits, according to differences in sedimentability. We recovered two lamellar body populations at different densities in discontinuous sucrose density gradients; we separated six subfractions of alveolar lavage by differential centrifugation. To determine whether or not precursor-product relationships existed among the subfractions, we injected radioactive palmitate intravenously, killed the rabbits 1-72 h later, and measured phospholipid specific activities. The two populations of lamellar bodies had similar phospholipid composition, fatty acyl composition of phosphatidylcholine and phosphatidylglycerol, and surface activity. Light lamellar bodies had a higher ratio of phospholipid to protein, and labelled with tracer later in time than dense ones. For alveolar lavage subfractions, later labelling with tracer, lower adsorption rate and lower total protein and phosphatidylglycerol content seemed to correlate with decreasing average density and particle size as well as with the disappearance of tubular myelin structure and appearance of predominantly vesicular structure. The subfractions appear to be in a metabolic sequence in which heavier, more dense material is a precursor to lighter, less dense material. The results suggest that subfractions of surfactant are extensively recycled.

Isolation and sequence of a cDNA clone for the rat pulmonary surfactant-associated protein (PSP-A)

Pulmonary surfactant is composed mainly of phospholipid and two groups of apoproteins. One of these apoproteins is a family of glycoproteins (pulmonary surfactant-associated protein A, PSP-A). We have isolated and sequenced a cDNA clone encoding for rat PSP-A and the full amino acid sequence has been deduced from the nucleotide sequence. The sequence of 56 amino acids at the N-terminus of PSP-A isolated from rats treated with silica was determined independently, and there is complete agreement with the sequence deduced from the cDNA. Isolated rat alveolar type II cells contain two species of mRNA for this protein.

Protein composition of rabbit alveolar surfactant subfractions

The goal of this investigation was to characterize the proteins in subfractions of alveolar surfactant obtained by lung lavage and separated by differential centrifugation. It was previously demonstrated that the material in the more sedimentable fraction, which was enriched in tubular-myelin and was surface-active may be a precursor to the less sedimentable, vesicular, inactive material [1]. Separation of the proteins by polyacrylamide gel electrophoresis showed that the more sedimentable subfractions and rabbit surfactant isolated by conventional methods contained proteins with molecular weights comparable to those previously reported for alveolar surface active material (approximately 36 000 and 10 000). The less sedimentable subfractions contained less of these proteins. Immunoblots with anti-dog surfactant apoprotein antibodies, which cross-react with rabbit proteins, supported these observations. Immunoblots also showed that all of the subfractions contained serum proteins and secretory IgA, with the less sedimentable subfractions containing more secretory IgA. These results suggested that changes in protein composition may accompany functional changes in surfactant in the alveoli.

Role of calcium ions in the structure and function of pulmonary surfactant

Pulmonary surfactant isolated by centrifugation in buffers containing ions contains at least three different morphologic structures. The presence of one of these, tubular myelin, is dependent on calcium ions, since chelation of the calcium ions causes disruption of this structure. Addition of EDTA also decreases the ability of the surfactant to absorb rapidly to air-water interfaces and lower surface tension. Titration with calcium ions (2.5 or 5 mM) restores rapid surface adsorption and restores the tubular myelin structural forms. Magnesium ions cannot substitute for calcium ions in these processes. The reversibility of structure and function induced by calcium ions and EDTA is also accompanied by reversible isopycnic density shifts probably related to aggregation and disaggregation of the lipid-protein complex with calcium ions and EDTA, respectively.

Corticosteroid induction of phosphatidic acid phosphatase in fetal rabbit lung.

Summary Prenatal betamethasone therapy is known to reduce the incidence of respiratory distress syndrome in premature infants. We report that administration of an equivalent dose of this corticosteroid to pregnant rabbits increases phosphatidic acid phosphatase (PAPase) activity and the rate of choline incorporation into lecithin in fetal lung. Under these conditions there was no induction of choline phosphotransferase or glycerolphosphate phosphatidyltransferase. These results suggest that PAPase may be a key regulatory enzyme in surfactant synthesis during both normal and glucocorticoid-accelerated lung maturation.

Isolation of a major apolipoprotein of canine and murine pulmonary surfactant Biochemical and immunochemical characteristics

We studied some of the biochemical and immunochemical properties of a major apolipoprotein in isolated pulmonary surfactant from dog and rat lungs. These apolipoproteins were purified by DEAE-cellulose chromatography in buffers containing Triton X-100. Purity of the apolipoproteins was assessed by both fused rocket and crossed immunoelectrophoreses. In addition, the apolipoproteins showed one band with an apparent molecular weight of 72 000-73 000 on SDS-polyacrylamide gel electrophoresis. These proteins are composed of two polypeptide chains of 36 000 daltons. When subjected to isoelectric focusing, the major component of the apolipoprotein had an isoelectric point of about 4.4, with very minor components near 4.6. Even though the apolipoproteins of both species had very similar amino acid compositions, including a relatively high glycine content, no immunologic cross-reactivity was observed. Rocket immunoelectrophoretic analysis of several preparations of dog and rat surfactant using the respective purified apolipoproteins as standards indicated that the apolipoprotein constituted 56.9% +/- 4.6. (S.D., n = 3) and 42.1% +/- 2.1 (S.D., n = 2) of the total protein in dog and rat surfactant, respectively.

An electrophoretic and immunochemical characterization of human surfactant-associated proteins

We have prepared an antiserum against a serum-free extract of alveolar proteinosis lavage that recognizes the same proteins as an antiserum to human surfactant. Using one and two-dimensional gel electrophoresis, protein blotting and immunostaining we have found proteins with Mr of approx. 35 and 60 kDa to be present in every source of human surfactant we have examined. These proteins are immunologically related to those found in the lavage from alveolar proteinosis patients, have the same electrophoretic characteristics and are not found in serum. The 35 kDa protein is a group of at least eight isoforms ranging in relative molecular mass Mr from 32 to 36 kDa with isoelectric points between 4.8 and 5.5. Neuraminidase digestion studies have shown that at least part of this charge heterogeneity may be due to sialic acid residues. The less abundant form, with a Mr of about 60 kDa is also a sialoglycoprotein with similar isoelectric points.

Changes in phospholipid composition of lung surfactant during development in the fetal lamb.

The lung surfactant isolated from pulmonary fluid of fetal sheep changes both in amount and composition during gestation. Total phosphatidylcholine (PC) and its most surface-active components, disaturated PC, are present at very low levels 3-4 weeks prior to term and rise to adult levels 3-4 days before birth. The acidic phospholipids appear with a different time course. Phosphatidylserine reaches elevated levels about 21 days before birth. Phosphatidylinositol begins to increase at about 130 days of gestation. Phosphatidylglycerol is not a component (less than 1%) of the surfactant in this fetal lung fluid. At term, phosphatidylinositol is the major acidic phospholipid found in these fluids.

Role of Apoprotein and Calcium Ions in Surfactant Function

Pulmonary surfactant isolated in the presence of calcium ions contains substantial amounts of the morphologic structure, tubular myelin. Chelation of these calcium ions results in disruption of this structure and attendant loss of surface adsorption. Reassembly studies indicate that ability of the lipids to rapidly form surface films is dependent on the presence of a specific surfactant protein in addition to the calcium ions. The formation of this surface-active complex (apoprotein-lipid-calcium ions) is accompanied by aggregation of the lipid. This increase in aggregation may have important implications in the mechanism of surfactant function.

1164 TRANSPLACENTAL EFFECTS OF A THYROXINE ANALOG ON PHOSPHOLIPID SYNTHESIS IN FETAL RABBIT LUNG

To investigate the possible role of thyroid hormones in fetal lung maturation, we treated pregnant rabbits with 3,5-dimethyl-3′-isopropylthyronine (DIMIT), a thyroxine analog which binds to nuclear receptors in fetal lung. On days 24 and 25 of gestation does received diluent or 0.05-1 mg/kg DIMIT im; fetuses were studied on day 26. We found no effect of DIMIT on viability, body weight, lung weight, lung wet/dry ratio, or fetal plasma cortisol levels. NADPH-cytochrome C reductase, a known T4 inducible enzyme, was increased 64% (p<0.01) in fetal liver with 1 mg/kg DIMIT. Mean±SE values for the rate of 3H-choline incorporation into lecithin (pmol/mg tissue/h) by minced fetal lung and activity of phosphatidic acid phosphatase (PAPase, nmol/mg prot/min) in lung homogenate are shown below (*=p<0.001):Similar results were found for incorporation of 3H-glycerol.We conclude that DIMIT crosses the rabbit placenta and stimulates lecithin synthesis and PAPase activity in fetal lung. Since similar effects occur with glucocorticoid treatment, these two hormones may act in part by controlling the same enzyme.