Biomedical subjects

S Hawgood

Publications and source records attributed to S Hawgood.

At least 55 records · Page 3Linked to original sources

PubMed1989

Proteolytic inactivation of dog lung surfactant-associated proteins by neutrophil elastase.

The adsorption of pulmonary surfactant to an air/fluid interface is influenced by calcium-dependent interactions between its lipid and protein components. The latter include a glycoprotein of 28-36 kDa (SP-A) and two smaller hydrophobic proteins of 5-8 kDa (SP-B, SP-C). Neutrophil elastase and other proteolytic enzymes found in the alveolar washings in a variety of acute lung injuries may cleave the protein components of lung surfactant. To examine the hypothesis that free airspace elastolytic activity may thereby impair surfactant function, we analyzed the effect of neutrophil elastase on surfactant activity in vitro. The adsorption characteristics of dog surfactant and of complexes reassembled from purified surfactant components were examined after incubations with active or heat-inactivated neutrophil elastase. Surfactant preincubated with the active enzyme showed a marked concentration-dependent slowing of adsorption associated with proteolytic cleavage of SP-A. To determine whether elastase also decreases surface activity by affecting the hydrophobic proteins SP-B and SP-C, we studied the effect of incubating elastase with liposomes prepared from surfactant lipid fractions which contain SP-B and SP-C. The addition of intact SP-A to these liposomes incubated with inactive enzyme immediately enhanced adsorption speed. This enhancement was greatly attenuated in liposomes treated with active elastase, suggesting that one or both of the hydrophobic surfactant proteins had been affected by elastase. We conclude that proteolytic cleavage of surfactant proteins reduces adsorption speed in vitro and may disturb surfactant function in vivo.

Animals↗

PubMed1989

Lung surfactant apoprotein SP-A (26-36 kDa) binds with high affinity to isolated alveolar type II cells.

Pulmonary surfactant is synthesized and secreted by alveolar type II cells. These cells recycle surfactant lipids by an internalization process that is enhanced in vitro by the surfactant proteins with molecular masses of 26-36 kDa (SP-A). SP-A also inhibits the secretion of lipid by type II cells. These results suggest that SP-A may play a role in feedback regulation of surfactant pool size and are consistent with the hypothesis that the type II cell surface has receptors for SP-A. The goal of this study is to characterize the binding of radioiodinated SP-A to isolated rat type II cells. Binding of SP-A to type II cells at 4 degrees C has a K1/2 of approximately 5 X 10(-10) M, is saturable, and is inhibited by excess unlabeled SP-A. Binding is dependent on calcium and is reduced by heat treatment of SP-A. The binding of a proteolytic fragment of SP-A that is produced by collagenase treatment is reduced by excess unlabeled SP-A. The binding of the fragment to macrophages and lung fibroblasts is not inhibited by excess unlabeled SP-A. Trypsinization of the type II cell surface reduces the binding of both intact SP-A and the collagenase-resistant fragment. These results show that SP-A binds to type II cells with high affinity and suggest that these cells have receptors that recognize the carboxyl-terminal domain of SP-A.

Animals↗

PubMed1989

Pulmonary surfactant apoproteins: a review of protein and genomic structure.

In recent years, as the complexity of the surfactant system has become more apparent, investigators with an increasingly diverse set of skills have been attracted to the study of this secretory product of the alveolar epithelium. In addition to advancing our knowledge of the mechanisms underlying the mechanical stability of the lung, recent studies of the surfactant system have also contributed information to less organ-specific biological phenomena such as exocytosis, endocytosis, cell differentiation and lipid-protein interactions in biomembranes. Pulmonary surfactant is not composed of a single class of molecules but, rather, is a collection of interrelated macromolecular lipoprotein complexes that differ in composition, structure, and function. The purpose of this review is to describe the structure of the lung-specific proteins that are associated with the phospholipids of surfactant in the alveolar space. The organization of the genes for the surfactant proteins is outlined and the affects of these proteins on the properties of phospholipid membranes are discussed.

Animals↗

PubMed1989

Studies of the structure of lung surfactant protein SP-A.

SP-A, a glycoprotein of pulmonary surfactant, consists of an NH2-terminal domain containing a collagen-like sequence and a COOH-terminal domain with sequence homology to several Ca2(+)-dependent lectins. We have compared the size, thermal stability, and secondary structure of recombinant SP-A, the product of a fibroblast line transfected with a single human gene encoding SP-A, with natural SP-A isolated from canine and human lungs. Our results suggest both recombinant and natural SP-A are assembled as large oligomers. More variability in the degree of oligomerization was observed with recombinant human SP-A than with natural canine SP-A. As shown by collagenase digestion, the full assembly of protein subunits was dependent on an intact collagen-like domain. The cysteines in the noncollagen domain of SP-A form intrachain bonds between residues 135-226 and 204-218. The circular dichroism spectra of both recombinant and natural SP-A were consistent with the presence of a collagen-like triple helix. As determined by the change in ellipticity at 205 nm, the thermal transition temperatures of canine, natural human, and recombinant SP-A were 51.5, 52.3, and 42.0 degrees C, respectively. These results suggest differences in the assembly and stability of the natural and recombinant proteins.

Amino Acid Sequence↗

PubMed1989

Regulation of messenger RNAs for the hydrophobic surfactant proteins in human lung.

The pulmonary surfactant proteins SP-B (8,000 D) and SP-C (4,000 D) accelerate surface film formation by surfactant phospholipids. We used cDNA probes to examine regulation of these proteins in human fetal lung. The mRNAs were detectable at 13 wk gestation and increased to approximately 50% (SP-B) and approximately 15% (SP-C) of adult levels at 24 wk. The mRNAs were detected only in lung of 11 dog tissues examined. When human fetal lung was cultured as explants without hormones, SP-B mRNA increased and SP-C mRNA decreased. Exposure for 48 h to glucocorticoids, but not other steroids, increased both SP-B mRNA (approximately 4-fold) and SP-C mRNA (approximately 30-fold) vs. controls. Half-maximal stimulation occurred with 1 nM dexamethasone and 300 nM cortisol for SP-B mRNA and at three- to fivefold higher concentrations for SP-C mRNA. Both stimulation and its reversal on removal of hormone were more rapid for SP-B than for SP-C. Terbutaline and forskolin increased SP-B mRNA but not SP-C mRNA. Levels of both mRNAs were much higher in type II cells than fibroblasts prepared from explants. Thus, the genes for SP-B and SP-C are expressed in vivo before synthesis of both SP-A (28,000-36,000 D) and surfactant lipids. Glucocorticoid induction of SP-B and SP-C mRNAs in type II cells appears to be receptor mediated but may involve different mechanisms.

Animals↗

PubMed1989

Pulmonary surfactant metabolism.

Recent work suggests that surfactant undergoes a complex sequence of metabolic events during its life cycle in the alveolar airspace. The composition, turnover, and metabolism of surfactant, and the possible "control points" in its metabolic pathway that might be compromised in disease states are discussed.

Humans↗

PubMed1988

Synthesis of surfactant components by cultured type II cells from human lung.

We examined the effect of monolayer culture on surfactant phospholipids and proteins of type II cells isolated from human adult and fetal lung. Type II cells were prepared from cultured explants of fetal lung (16-24 weeks gestation) and from adult surgical specimens. Cells were maintained for up to 6 days on plastic tissue culture dishes. Although incorporation of [methyl-3H]choline into phosphatidylcholine (PC) by fetal cells was similar on day 1 and day 5 of culture, saturation of PC fell from 35 to 26%. In addition, there was decreased distribution of labeled acetate into PC, whereas distribution into other phospholipids increased or did not change. The decrease in saturation of newly synthesized PC was not altered by triiodothyronine (T3) and dexamethasone treatment or by culture as mixed type II cell/fibroblast monolayers. The content of surfactant protein SP-A (28-36 kDa) in fetal cells, as measured by ELISA and immunofluorescence microscopy, rose during the first day and then fell to undetectable levels by the fifth. Synthesis of SP-A, as measured by [35S]methionine labeling and immunoprecipitation, was detectable on day 1 but not thereafter. Levels of mRNAs for SP-A and for the two lipophilic surfactant proteins SP-B (18 kDa) and SP-C (5 kDa) fell with half-times of maximally 24 h. In contrast, total protein synthesis measured by [35S]methionine incorporation increased and then plateaued. In adult cells, the content of SP-A and its mRNA decreased during culture, with time-courses similar to those for fetal cells. We conclude that in monolayer culture on plastic culture dishes, human type II cells lose their ability to synthesize both phospholipids and proteins of surfactant. The control of type II cell differentiation under these conditions appears to be at a pretranslational level.

Adult↗

PubMed1988

Interactions of the low molecular weight group of surfactant-associated proteins (SP 5-18) with pulmonary surfactant lipids.

The interaction of the low molecular weight group of surfactant-associated proteins, SP 5-18, with the major phospholipids of pulmonary surfactant was studied by fluorescence measurements of liposomal permeability and fusion, morphological studies, and surface activity measurements. The ability of SP 5-18 to increase the permeability of large unilamellar lipid vesicles was enhanced by the presence of negatively charged phospholipid. The permeability of these vesicles increased as the protein concentration was raised and the pH was lowered. SP 5-18 also induced leakage from liposomes made both from a synthetic surfactant lipid mixture and from lipids separated from SP 5-18 during its purification from canine sources. When SP 5-18 was added to egg phosphatidylglycerol liposomes, the population of liposomes which became permeable leaked all encapsulated contents, while the remaining liposomes did not leak at all. The extent of leakage was higher in the presence of 3 mM calcium. SP 5-18 also induced lipid mixing between two populations of egg phosphatidylglycerol liposomes in the presence of 3 mM calcium, as monitored by resonance energy transfer between two different fluorescent lipid probes, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine. Negative-staining electron microscopy showed that the addition of SP 5-18 and 3 mM calcium produced vesicles twice the size of control egg phosphatidylglycerol liposomes. In addition, surface balance measurements revealed that the adsorption of liposomal lipids to an air/water interface was enhanced by the presence of SP 5-18, negatively charged phospholipids, and 3 mM calcium. These observations suggest a similar lipid dependence for the interactions observed in the fluorescence and adsorption experiments.

Animals↗

PubMed1988

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.

Animals↗

PubMed1987

Divalent cation and hydrogen ion effects on the structure and surface activity of pulmonary surfactant.

The structure and surface activity of the extracellular fraction of pulmonary surfactant known as tubular myelin are Ca2+ dependent. Previous studies have demonstrated surfactant-specific proteins with monomeric molecular weights of 28,000-36,000 (SP28-36) are associated with this fraction. In reassembled lipoprotein mixtures, SP28-36 promotes the Ca2+-induced aggregation and surface activity of surfactant lipids, but the detailed interactions between Ca2+, SP28-36, and surfactant lipids have not been established. In this study, we investigated the effect of various cations on the aggregation of surfactant lipid liposomes in the presence of SP28-36. SP28-36 reduced the threshold ion concentration for liposome aggregation from greater than 10 to 0.5 mM for Ca2+, Ba2+, and Sr2+ but not Mg2+ or Mn2+. The liposome aggregation was reversed by ethylenediaminetetraacetic acid and not associated with leakage of carboxyfluorescein. SP28-36 promoted similar liposome aggregation at pH less than 5 in the absence of divalent cations. Surfactant lipids adsorbed slowly to an air-fluid interface in all ionic conditions unless SP28-36 was present. Both Ca2+ and H+ induced rapid lipid adsorption in the presence of SP28-36. The surface activity of native surfactant had a similar ion dependence. Electron micrographs of native surfactant showed typical tubular myelin structures at pH 7.4 only in the presence of Ca2+. At pH 4.4 in the absence of Ca2+, similar but not identical structures were seen. In the reconstituted system, SP28-36 in the presence of Ca2+ induced the formation of larger multilayered structures including parallel bilayers and small areas of squares and triangles with dimensions similar to structures found in the native material.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

The major lung surfactant protein, SP 28-36, is a calcium-dependent, carbohydrate-binding protein.

SP 28-36, a major protein of pulmonary surfactant, has striking amino acid sequence homology with soluble mannose-binding proteins isolated from rat liver and contains residues common to the carbohydrate-binding domains of other mammalian lectins. We have used carbohydrate-affinity chromatography to investigate carbohydrate-binding properties of SP 28-36 isolated from canine and human (alveolar proteinosis patients) lung lavage. SP 28-36 binds to immobilized D-mannose, L-fucose, D-galactose, and D-glucose. The protein binds only weakly to N-acetyl-D-galactosamine and N acetyl-D-glucosamine. Binding is Ca2+-dependent. The threshold Ca2+ concentration is 0.6 mM and maximal binding occurs with 1 mM Ca2+. Bound protein is quantitatively recovered by elution with 2 mM EDTA. Ba2+, Sr2+, and Mn2+, but not Mg2+, can substitute for Ca2+. Unlike some other mammalian lectins, SP 28-36 binds to carbohydrate at pH 5.0. Recombinant human SP 28-36 isolated from the media of Chinese hamster ovary cells, transfected with a DNA construct encoding SP 28-36, has similar carbohydrate-binding activity to the native proteins. Mannose affinity chromatography of the culture medium of Chinese hamster ovary cells results in an efficient purification of the secreted recombinant human SP 28-36.

Animals↗

PubMed1987

Surfactant apoprotein Mr = 26,000-36,000 enhances uptake of liposomes by type II cells.

The alveolar type II cell which synthesizes and secretes surfactant also plays a major role in the reuptake of surfactant lipids. In a recent in vivo study we found that the subfractions of natural surfactant that contained the surfactant protein with molecular weights of 26,000-36,000 (SP-26-36) were preferentially taken up into lamellar bodies of type II cells to a greater extent than were fractions that did not contain SP-26-36. Because the subfractions of natural surfactant in that study differed in other properties than the presence or absence of SP-26-36, the current study was undertaken to determine whether purified SP-26-36 enhanced the uptake of surfactant-like lipids by freshly isolated type II cells. SP-26-36 increased the uptake of label in radioactive surfactant-like lipids by up to 10-fold, and the effect of SP-26-36 was dependent on time, protein concentration, and temperature. The enhancement was inhibited by heat-treating the protein, by a polyclonal antibody against SP-26-36, and by metabolic inhibitors. The distribution of radioactivity in cell-associated phospholipids differed if cells were incubated with or without SP-26-36. If SP-26-36 was present during the incubation, greater than 96% of the radioactivity remained associated with phosphatidylcholine. In the absence of SP-26-36, only 85% of the radioactivity remained associated with phosphatidylcholine and 7% of the label appeared in phosphatidylglycerol. We hypothesize that SP-26-36 may act as a ligand to direct surfactant lipids to type II cells, perhaps to different metabolic pathways, and to regulate recycling and surfactant homeostasis.

Animals↗

PubMed1987

Sialic acid of lung surfactant apoprotein, SP 28-36, is not required for the Ca2+-mediated interactions between surfactant lipids and SP 28-36.

We examined whether removal of sialic acid from the lung surfactant apoprotein (SP 28-36) affected certain properties of reassembled surfactant lipid-SP 28-36 complexes. SP 28-36 was treated with neuraminidase and then added to liposomes made from extracted surfactant lipids. We found that in the presence of Ca2+ the asialoprotein was as effective as the native SP 28-36 in binding to surfactant lipids, causing aggregation and promoting rapid surface film formation.

Apoproteins↗

PubMed1987

Nucleotide and amino acid sequences of pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28-36 in surfactant lipid adsorption.

Pulmonary surfactant is a lipid-rich material that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. The turnover of surfactant phospholipids in the alveolar space is fast, and several lines of evidence suggest there is rapid formation and replenishment of the phospholipid surface film during normal respiration. Specific proteins may regulate these dynamic surface properties. The predominant surfactant protein is a well-characterized, lipid-associated glycoprotein, SP 28-36 (28-36 kDa). A second group of very hydrophobic proteins has recently been shown to affect the surface activity of surfactant phospholipids. We have isolated this group of hydrophobic proteins, herein called SP 5-18 (5-18 kDa), from canine surfactant and have shown by NH2-terminal sequence analysis that at least two proteins, SP 5-8 and SP 18, are present in this group. We have derived the full amino acid sequence of SP 18 from the nucleotide sequence of the cDNAs identified with oligonucleotide probes that were based on the NH2-terminal amino acids of SP 18. The protein isolated from extracellular surfactant appears to be a fragment of a much larger precursor protein (40 kDa). The amino acid sequence of SP 18 is markedly hydrophobic and contains two possible bilayer-spanning domains. We have shown that SP 18 and the glycoprotein SP 28-36 have a cooperative, calcium-dependent action in promoting the formation of phospholipid surface films.

Amino Acid Sequence↗

PubMed1987

Low molecular weight human pulmonary surfactant protein (SP5): isolation, characterization, and cDNA and amino acid sequences.

Pulmonary surfactant is a lipid-protein complex that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. A group of hydrophobic surfactant-associated proteins has been shown to be essential for rapid surface film formation by surfactant phospholipids. We have purified a hydrophobic surfactant protein of approximately 5 kDa that we term SP5 from bronchopulmonary lavage fluid from a patient with alveolar proteinosis and shown that it promotes rapid surface film formation by simple mixtures of phospholipids. We have derived the full amino acid sequence of human SP5 from the nucleotide sequence of cDNAs identified with oligonucleotide probes based on the NH2-terminal sequence of SP5. SP5 isolated from surfactant is a fragment of a much larger precursor protein (21 kDa). The precursor contains an extremely hydrophobic region of 34 amino acids that comprises most of the mature SP5. This hydrophobicity explains the unusual solubility characteristics of SP5 and the fact that it is lipid-associated when isolated from lung.

Amino Acid Sequence↗

PubMed1987

Pulmonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type II cells.

Pulmonary surfactant is synthesized and secreted by alveolar type II cells. Radioactive phosphatidylcholine has been used as a marker for surfactant secretion. We report findings that suggest that surfactant inhibits secretion of 3H-labeled phosphatidylcholine by cultured rat type II cells. The lipid components and the surfactant protein group of Mr 26,000-36,000 (SP 26-36) inhibit secretion to different extents. Surfactant lipids do not completely inhibit release; in concentrations of 100 micrograms/ml, lipids inhibit stimulated secretion by 40%. SP 26-36 inhibits release with an EC50 of 0.1 microgram/ml. At concentrations of 1.0 microgram/ml, SP 26-36 inhibits basal secretion and reduces to basal levels secretion stimulated by terbutaline, phorbol 12-myristate 13-acetate, and the ionophore A23187. The inhibitory effect of SP 26-36 can be blocked by washing type II cells after adding SP 26-36, by heating the proteins to 100 degrees C for 10 min, by adding antiserum specific to SP 26-36, or by incubating cells in the presence of 0.2 mM EGTA. SP 26-36 isolated from canine and human sources also inhibits phosphatidylcholine release from rat type II cells. Neither type I collagen nor serum apolipoprotein A-1 inhibits secretion. These findings are compatible with the hypothesis that surfactant secretion is under feedback regulatory control.

Animals↗

PubMed1987

Surfactant protein of molecular weight 28,000-36,000 in cultured human fetal lung: cellular localization and effect of dexamethasone.

We have examined the effect of explant culture and hormones on the major surfactant associated protein of Mr 28,000-36,000 (SP 28-36) in human fetal lung. Explants of 16- to 23-week gestation lung were maintained for up to 5 days in culture. Polyclonal antibodies raised to SP 28-36 purified from alveolar proteinosis lung lavage were used in immunofluorescence experiments (n = 11). There was no specific fluorescence seen in frozen sections of preculture tissue. In explants cultured without serum or hormones, fluorescence was seen in most epithelial cells lining potential airspaces. In cultures treated with 10 nM dexamethasone and 2 nM T3 much brighter fluorescence was seen in virtually all epithelial cells. Immunofluorescence studies on cell monolayers prepared from explants confirmed that SP 28-36 is found in the cytoplasm of type II cells but not in fibroblasts. The pattern of fluorescence was consistent with the presence of SP 28-36 on rough endoplasmic reticulum. SP 28-36 mRNA was measured in isolated cell populations using a 32P-labeled cDNA probe. mRNA levels were manyfold higher in type II cell preparations (purity 78-92%) than in fibroblasts (purity 81-97%). A competitive enzyme linked assay was developed to quantify SP 28-36. The SP 28-36 content of five lungs before culture (17-23 weeks) was less than 0.02 microgram/mg DNA. During explant culture without hormones the SP 28-36 content increased exponentially. Exposure to dexamethasone accelerated the increase in SP 28-36 content. T3, alone or in the presence of dexamethasone, did not influence SP 28-36 content. We conclude that SP 28-36 content is very low in human fetal lung before 24 weeks gestation. Explant culture and treatment with dexamethasone synchronize development of type II cells from epithelial precursors, and induce synthesis of SP 28-36 in type II cells. These findings provide evidence of concomitant regulation by glucocorticoids of the phospholipid synthetic enzymes and the major protein of pulmonary surfactant.

Cells, Cultured↗

PubMed1986

The role of calcium in the secretion of surfactant by rat alveolar type II cells.

Beta adrenergic agonists, tetradecanoylphorbol acetate, and the ionophore A23187 all stimulate surfactant secretion in type II cells isolated from rats. We found that combinations of these agonists cause augmented secretion, suggesting that the agonists may effect different steps in the secretory process. Previous studies have shown that cAMP is likely to be an intracellular 'second messenger' in type II cells. A23187, which has been reported to increase cAMP in some cell systems, did not increase the cAMP content of type II cells. We investigated the possible role of Ca2+ as another 'second messenger' by studying cellular 45Ca fluxes and the effect of extracellular calcium depletion on secretion. Depletion of extracellular calcium for as long as 3 h did not alter stimulated secretion, although basal secretion was increased. Secretagogues did not stimulate 45Ca influx from extracellular sources. A23187 and, to a lesser extent, terbutaline caused an acceleration of 45Ca efflux from type II cells. The addition of terbutaline or tetradecanoylphorbol acetate to A23187 further accelerated 45Ca efflux, suggesting that these agonists may act on separate calcium pools or by different mechanisms on the same calcium pool. Although secretion from type II cells is not inhibited by extracellular calcium depletion, the studies on 45Ca efflux suggest that Ca2+ plays a role in the regulation of surfactant secretion from isolated type II cells.

Animals↗