Thermodynamic studies of bovine lung surfactant extract mixing with cholesterol and its palmitate derivative.

Langmuir film behavior of bovine lipid extract surfactant (BLES), mixed with cholesterol (CHOL) and cholesterol palmitate (CHOLP), has been studied by surface pressure (pi)-area (A) measurements. Associative interactions, observed for both systems, were less favored at lower BLES content. The presence of unsaturated phospholipids and surfactant proteins in BLES favored the association. Miscibility of BLES was better with CHOLP than with CHOL at all compositions, indicating more compact packing of the BLES-CHOLP than of the BLES-CHOL system. The most stable mixtures were found at 30-40 mol% CHOL and at low pi and at 20-25 mol% CHOLP but at higher pi. These results suggest that BLES-CHOL miscibility is better at low pi and low CHOL concentrations, while BLES-CHOLP miscibility is better at high pi and high CHOLP concentrations.

Comparison of inhibitory effects of oxygen radicals and calf serum protein on surfactant activity.

The effects of the reactive oxygen species (ROS) superoxide anion (O2*-) and hydroxyl radical (*OH) on the surface tension lowering properties of bovine lipid extract surfactant (BLES) were compared to the effects of calf serum protein (CSP) in a captive bubble surfactometer (CBS). O2*- was generated from xanthine/xanthine oxidase (X/XO), and *OH was generated by the Fenton reaction. ROS were demonstrated by electron spin resonance (ESR) using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap. Lipid peroxidation was measured using the thiobarbituric acid method. *OH had broad inhibitory effects on surface tension parameters, including adsorption, minimum surface tension, percentage film area change and film compressibility. O2*- showed inhibitory effects on adsorption, film area change and film compressibility but had no significant effect on minimum surface tension. Both O2*- and *OH treatment were associated with a large 'squeezeout' plateau around 20-25 mN/m in the surface tension-area relation, indicating poor film organization during the compression phase. At the concentrations used, ROS were associated with lipid peroxidation of BLES, which also demonstrated radical scavenging properties. Calf serum protein produced inhibitory effects on adsorption, minimum surface tension and percentage film area change that were quantitatively similar to those produced by *OH. The effects on film compression were significantly greater and qualitatively different from those seen with either O2*- or *OH. We conclude that the inhibition of BLES surface activity by ROS and inhibitory proteins can be distinguished in the captive bubble surfactometer and, particularly, by changes in the film compressibility modulus.

Molecular cloning and expression of pulmonary lipid phosphate phosphohydrolases.

Pulmonary lipid phosphate phosphohydrolase (LPP) was shown previously to hydrolyze phosphatidic acid and lysophosphatidic acid in purified rat lung plasma membranes. To better investigate the nature of pulmonary LPP isoforms and their role in the lung, LPPs were cloned by RT-PCR from both adult rat lung and type II cell RNA. The RT-PCR generated LPP1 (849 bp), up to three LPP1 variants, and LPP3 (936 bp) cDNAs. The three LPP1 variants include LPP1a (852 bp) and two novel isoforms, LPP1b (697 bp) and LPP1c (1004 bp). The pulmonary LPP1 and LPP3 isoforms are essentially identical to the previously cloned rat liver and intestinal LPPs, respectively, and the LPP1a isoform has 80% sequence identity to the human homolog. The LPP2 isoform was not detected in lung by RT-PCR. Northern analyses revealed that the mRNAs for LPP1 and LPP3 increase in fetal rat lung in late gestation to day 1 after birth. These mRNAs decrease somewhat during the neonatal period but increase slightly during postnatal development. Expression of LPP1, LPP1a, and LPP3 cDNAs in HEK 293 cells established that they encode functional LPP. In contrast, the novel isoforms LPP1b and LPP1c contain frameshifts that would result in premature termination, producing putative catalytically inactive polypeptides of 30 and 76 amino acids, respectively. Further investigation of the LPP1b isoform revealed that it was present across a variety of tissues, although at lower levels than LPP1/1a. Transient mammalian expression of LPP1b failed to increase phosphatidate phosphohydrolase activity in HEK 293 cells.

Pulmonary lipid phosphate phosphohydrolase in plasma membrane signalling platforms.

Lipid phosphate phosphohydrolase (LPP) has recently been proposed to have roles in signal transduction, acting sequentially to phospholipase D (PLD) and in attenuating the effects of phospholipid growth factors on cellular proliferation. In this study, LPP activity is reported to be enriched in lipid-rich signalling platforms isolated from rat lung tissue, isolated rat type II cells and type II cell-mouse lung epithelial cell lines (MLE12 and MLE15). Lung and cell line caveolin-enriched domains (CEDs), prepared on the basis of their detergent-insolubility in Triton X-100, contain caveolin-1 and protein kinase C isoforms. The LPP3 isoform was predominantly localized to rat lung CEDs. These lipid-rich domains, including those from isolated rat type II cells, were enriched both in phosphatidylcholine plus sphingomyelin (PC+SM) and cholesterol. Saponin treatment of MLE15 cells shifted the LPP activity, cholesterol, PC+SM and caveolin-1 from lipid microdomains to detergent-soluble fractions. Elevated LPP activity and LPP1/1a protein are present in caveolae from MLE15 cells prepared using the cationic-colloidal-silica method. In contrast, total plasma membranes had a higher abundance of LPP1/1a protein with low LPP activity. Phorbol ester treatment caused a 3.8-fold increase in LPP specific activity in MLE12 CEDs. Thus the activated form of LPP1/1a may be recruited into caveolae/rafts. Transdifferentiation of type II cells into a type I-like cell demonstrated enrichment in caveolin-1 levels and LPP activity. These results indicate that LPP is localized in caveolae and/or rafts in lung tissue, isolated type II cells and type II cell lines and is consistent with a role for LPP in both caveolae/raft signalling and caveolar dynamics.

Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant.

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir. The extent to which surfactant components including the main component, dipalmitoylphosphatidylcholine (DPPC), are adsorbed into the monolayer, and how surfactant protein SP-A affects their adsorptions, is not clear. Transport of cholesterol to the surface region from dispersions of bovine lipid extract surfactant [BLES(chol)] with or without SP-A at 37 degrees C was studied by measuring surface radioactivities of [4-(14)C]cholesterol-labeled BLES(chol), and the Wilhelmy plate technique was used to monitor adsorption of monolayers. Results showed that transport of cholesterol was lipid concentration dependent. SP-A accelerated lipid adsorption but suppressed the final level of cholesterol in the surface. Surfactant adsorbed from a dispersion with or without SP-A was transferred via a wet filter paper to a clean surface, where the surface radioactivity and surface tension were recorded simultaneously. It was observed that 1) surface radioactivity was constant over a range of dispersion concentrations; 2) cholesterol and DPPC were transferred simultaneously; and 3) SP-A limited transfer of cholesterol. These results indicate that non-DPPC components of pulmonary surfactant can be adsorbed into the monolayer. Studies in the transfer of [1-(14)C]DPPC-labeled BLES(chol) to an equal or larger clean surface area revealed that SP-A did not increase selective adsorption of DPPC into the monolayer. Evaluation of transferred surfactant with a surface balance indicated that it equilibrated as a monolayer. Furthermore, examination of transferred surfactants from dispersions with and without prespread BLES(chol) monolayers revealed a functional contiguous association between adsorbed monolayers and reservoirs.

Surfactant protein interactions with neutral and acidic phospholipid films.

The captive bubble tensiometer was employed to study interactions of phospholipid (PL) mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) at 50 microg/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37 degrees C. All surfactant proteins enhanced lipid adsorption to equilibrium surface tension (gamma), with SP-C being most effective. Kinetics were consistent with the presence of two adsorption phases. Under the conditions employed, SP-A did not affect the rate of film formation in the presence of SP-B or SP-C. Little difference in gamma(min) was observed between the acidic POPG and the neutral POPC systems with SP-B or SP-C with and without SP-A. However, gamma(max) was lower with the acidic POPG system during dynamic, but not during quasi-static, cycling. Considerably lower compression ratios were required to generate low gamma(min) values with SP-B than SP-C. DPPC-POPG-SP-B was superior to the neutral POPC-SP-B system. Although SP-A had little effect on film formation with SP-B, surface activity during compression was enhanced with both PL systems. In the presence of SP-C, lower compression ratios were required with the acidic system, and with this mixture, SP-A addition adversely affected surface activity. The results suggest specific interactions between SP-B and phosphatidylglycerol, and between SP-B and SP-A. These observations are consistent with the presence of a surface-associated surfactant reservoir which is involved in generating low gamma during film compression and lipid respreading during film expansion.

Covalent modification of the non-catalytic sites of the H(+)-ATPase from chloroplasts with 2-azido-[alpha-(32)P]ATP and its effect on ATP synthesis and ATP hydrolysis.

Incubation of the isolated H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (beta-Tyr362) and non-catalytic (beta-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 microM 2-azido-[alpha-(32)P]ATP, that with the intermediate dissociation constant (site 5) at 2 microM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[alpha-(32)P]ATP was possible. CF(0)F(1) with non-covalently bound 2-azido-[alpha-(32)P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by Delta pH/Delta phi. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF(0)F(1). Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between beta- and alpha-subunits which is abolished by covalent derivatization of beta-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F(0)-part and the F(1)-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three beta-Tyr385.

Surface activity in situ, in vivo, and in the captive bubble surfactometer.

For studies of the mechanical effects of lung surfactants, the captive bubble surfactometer (CBS) combines the advantages of the continuous film of Pattle's bubbles with the feasibility of the Langmuir-Wilhelmy balance to produce surface tension-area hysteresis loops. The CBS allows the compression of films to very low and stable surface tensions of 1-2 mN/m. Such low and stable surface tensions are in line with results obtained from pressure-volume studies on excised lungs. In addition, the CBS is useful to test other essential physical properties of the surfactant system, including: (1) rapid film formation (within seconds) through adsorption from the hypophase; (2) low film compressibility with a fall in surface tension to very low (<2 mN/m) values during surface compression; and (3) effective replenishment of the surface film on expansion by the incorporation of surfactant material from material associated with the surface (the surface associated surfactant reservoir). Morphological observations of films fixed in situ or in vitro reveal frequently their multilayered structure, which is consistent with the concept of the surface reservoir. The deviation of the bubbles from a Laplacian shape at very low surface tension and the morphological observations suggest that the surfactant film cannot be considered a simple monolayer.

Surface activity in vitro: role of surfactant proteins.

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Our group has investigated the roles of the surfactant proteins (SP-) SP-A, SP-B, and SP-C using a captive bubble tensiometer. These studies show that SP-C>SP-B>SP-A in enhancing surfactant lipid adsorption (film formation) to the equilibrium surface tension of approximately 22-25 mN/m from the 70 mN/m of saline at 37 degrees C. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area reduction results in the lowering of surface tension (gamma) from approximately 25 mN/m (equilibrium) to values near 0 mN/m. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphosphatidylcholine (DPPC). The results are consistent with DPPC enrichment occurring through two mechanisms, selective DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve gamma near 0 mN/m with DPPC/PG samples containing SP-B or SP-A plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression-expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low gamma values. Addition of SP-A to samples containing SP-B but not SP-C limits the increase in gamma(max) during expansion. It is concluded that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SP-A can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.

Formation of folds and vesicles by dipalmitoylphosphatidylcholine monolayers spread in excess.

Lipid monolayers exist in several biological systems, including the stratum corneum of the skin, the fluid tear film of the eye, the Eustachian tube of the ear, and airway and alveolar pulmonary surfactants. In this paper, the monolayer-to-bilayer transition was studied using dipalmitoylphosphatidylcholine (DPPC) as the model. Depositing DPPC organic solvent solutions in excess at an air:buffer interface led to the formation of elongated structures which could be imaged on carbon grids by transmission electron microscopy. The structures appeared to be DPPC folds protruding into the sol. The structures were frequently ordered with respect to one another, suggesting that they arose during lateral compression due to excess DPPC and are characteristic of a type of monolayer collapse phase. In some cases, series of short folds in an extended line and series of vesicles in line or parallel to the folds were observed. This suggests the elongated folds are unstable and can resolve by forming vesicles. Fold formation occurred at defined lipid concentrations above which more vesicles were observed. Surfactant protein-A did not influence fold or vesicle formation but bound to the edges of these structures preferentially. It is concluded that DPPC monolayers can form bilayers spontaneously in the absence of surfactant apoproteins, other proteins or agents.

Surfactant levels after reversible tracheal occlusion and prenatal steroids in experimental diaphragmatic hernia.

BACKGROUND/PURPOSE: In normal lungs, fetal tracheal occlusion (TO) induces lung growth but decreases the number of type II cells; this is remedied if TO is released (TR) before delivery. In the current study, the effects of TO with or without TR on pulmonary structure and surfactant were assessed in the ovine model in which lung hypoplasia was induced by creation of a diaphragmatic hernia (CDH). METHODS: A left-sided CDH was created in fetal lambs at 80 days gestation; TO was done at 108 days; and TR at 129 days. All ewes were given 1 dose of glucocorticoids at 135 days. At 136 days, the fetus was delivered. Lung weight to body weight ratio, mean terminal bronchiole density, type II cell density, bronchoalveolar lavage fluid (BAL) phosphatidylcholine (PC), BAL surfactant protein A (SP-A) and B (SP-B), and lung tissue SP-A and SP-B were assessed in CDH, CDH with TO, CDH with TO and TR, and controls. RESULTS: CDH lungs were hypoplastic and structurally immature, but had increased type II cell density. TO with or without TR caused lung growth with normalization of lung parenchymal architecture and type II cell density. Although the BAL SP-A and BAL SP-B were similar in all 4 groups, the BAL PC was low in CDH with or without TO or TR. Also, lung tissue SP-B levels were low in CDH with or without TO or TR. However, lung tissue SP-A levels were normal in CDH, but low in CDH with TO with or without TR. CONCLUSIONS: Despite the finding that lung morphology was improved in CDH with TO with or without TR animals, surfactant content and composition remained abnormal. Although surfactant secreted early by the fetus into alveolar spaces contained normal levels of BAL SP-A and BAL SP-B, the low levels of BAL PC and low lung tissue stores of SP-B indicate that these experimental lambs may experience respiratory insufficiency soon after birth. This implies that prophylactic surfactant at birth might be beneficial for CDH.

Covalent modification of the catalytic sites of the H+-ATPase from chloroplasts and 2-nitreno-ADP. Modification of the catalytic site 1 (tight) and catalytic sites 1 and 2 together impairs both uni-site and multi-site catalysis of ATP synthesis and ATP hydrolysis.

After isolation and purification, the H+-ATPase from chloroplasts, CF0F1, contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-32P]ADP leads to tight binding of azidonucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-32P]ADP, the covalently bound label was found exclusively at beta-Tyr-362. Incubation conditions with 2-azido-[alpha-32P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. For measurements of the degree of inhibition by covalent modification, CF0F1 was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K+/valinomycin diffusion potential. The rate of ATP synthesis was 50-80 s(-1), and the rate of ATP hydrolysis was 15 s(-1) measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF0F1. Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF0F1. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with derivatised CF0F1 and with non-derivatised CF0F1. ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.

Characterization of the pulmonary N-ethylmaleimide-insensitive phosphatidate phosphohydrolase.

Phosphatidate phosphohydrolase (PAPase) is a key enzyme involved in glycerolipid synthesis where it converts phosphatidic acid to diacylglycerol. Previous studies performed in lung have demonstrated the existence of 2 different forms of PAPases, namely PAP-1 and PAP-2. The former pulmonary Mg+2-dependent enzyme is N-ethylmaleimide (NEM)-sensitive, heat labile, and is involved in phospholipid biosynthesis. However, the function of the latter lung isozyme is unknown. PAP-2 activity was selectively assayed using NEM in the absence of Mg+2. Studies employing this assay and adult rat lung microsomal preparations demonstrated that PAP-2 activity was inhibited by amphiphilic amines, sphingoid bases, products of the PAP-2 reaction (monoacylglycerol [MAG] and diacylglycerol [DAG]), and substrate analogs such as lysophosphatidic acid (lyso-PA), ceramide-1-phosphate, and to a lesser extent, sphingosine-1-phosphate. Purified lung plasma membranes, prepared using discontinuous sucrose and Percoll gradients, showed that PAP-2 activity was enriched 6.9 +/- 1.6-fold over the whole homogenate and was between the enrichment for plasma membrane markers, 5'-nucleotidase (14.7 +/- 0.3) and Na+, K(+)-ATPase (4.0 +/- 0.2). Both phosphatidic acid and lysophosphatidic acid were good substrates for PAP-2 activity in this purified plasma membrane fraction. In contrast, sphingosine-1-phosphate was a relatively poor substrate. PAP-2 activity was slightly enriched in isolated type II cells and low in isolated rat lung fibroblasts. This study shows lung contains PAP-2 activity in plasma membranes and type II cells where it could play a role in signal transduction.

Three-dimensional structure of rat surfactant protein A trimers in association with phospholipid monolayers.

Surfactant protein A (SP-A) is a C-type lectin found primarily in the lung and plays a role in innate immunity and the maintenance of surfactant integrity. To determine the three-dimensional (3D) structure of SP-A in association with a lipid ligand, we have used single particle electron crystallography and computational 3D reconstruction in combination with molecular modeling. Recombinant rat SP-A, containing a deletion of the collagen-like domain, was incubated with dipalmitoylphosphatidylcholine:egg phosphatidylcholine (1:1, wt/wt) lipid monolayers in the presence of calcium, negatively stained, and examined by transmission electron microscopy. Images of SP-A-lipid complexes with different angular orientations were used to reconstruct the 3D structure of the protein. These results showed that SP-A subunits readily formed trimers and interacted with lipid monolayers exclusively via the globular domains. A homology-based molecular model of SP-A was generated and fitted into the electron density map of the protein. The plane of the putative lipid-protein interface was relatively flat and perpendicular to the hydrophobic neck region, and the cleft region in the middle of the trimer had no apparent charge clusters. Amino acid residues that are known to affect lipid interactions, Glu(195) and Arg(197), were located at the protein-lipid interface. The molecular model indicated that the hydrophobic neck region of the SP-A did not interact with lipid monolayers but was instead involved in intratrimeric subunit interactions. The glycosylation site of SP-A was located at the side of each subunit, suggesting that the covalently linked carbohydrate moiety probably occupies the spaces between the adjacent globular domains, a location that would not sterically interfere with ligand binding.

Ovine surfactant protein cDNAs: use in studies on fetal lung growth and maturation after prolonged hypoxemia.

cDNAs for ovine surfactant-associated protein (SP) A, SP-B, and SP-C have been cloned and shown to possess strong similarity to cDNAs for surfactant apoproteins in other species. These reagents were employed to examine the effect of fetal hypoxia on the induction of surfactant apoprotein expression in the fetal lamb. Postnatal lung function is dependent on adequate growth and maturation during fetal development. Insulin-like growth factor (IGF) I and IGF-II, which are present in all fetal tissues studied, possess potent mitogenic and proliferative actions, and their effects can be modulated by IGF-specific binding proteins (IGFBPs). Hypoxia can lead to increases in circulating cortisol and catecholamines that can influence lung maturation. Therefore, the effects of mild hypoxia in chronically catheterized fetal lambs at gestational days 126-130 and 134-136 (term 145 days) on the expression of pulmonary surfactant apoproteins and IGFBPs were examined. Mild hypoxia for 48 h resulted in an increase in plasma cortisol that was more pronounced at later gestation, and in these animals, there was a twofold increase in SP-A mRNA. SP-B mRNA levels also increased twofold, but this was not significant. SP-C mRNA was not altered. No significant changes in apoprotein mRNA were observed with the younger fetuses. However, these younger animals selectively exhibited reduced IGFBP-5 mRNA levels. IGF-I mRNA was also reduced at 126-130 days, although this conclusion is tentative due to low abundance. IGF-II levels were not affected at either gestational age. We conclude that these data suggest that mild prolonged fetal hypoxia produces alterations that could affect fetal cellular differentiation early in gestation and can induce changes consistent with lung maturation closer to term.

Characterization of nucleotide binding sites of the isolated H(+)-ATPase from spinach chloroplasts, CF(0)F(1).

Soluble purified CF(0)F(1) from chloroplasts was either oxidized or reduced and then incubated with [alpha-(32)P]ATP in the presence or in the absence of Mg(2+). Depending on the conditions of incubation, the enzyme showed different tight-nucleotide binding sites. In the presence of EDTA, two sites bind [alpha-(32)P]ATP from the reaction medium at different rates. Both sites promote ATP hydrolysis, since equimolar amounts of [alpha-(32)P]ATP and [alpha-(32)P]ADP are bound to the enzyme. In the presence of Mg(2+), only one site appears during the first hour of incubation, with characteristics similar to those described in the absence of Mg(2+). However, after this time a third site appears also permitting binding of ATP from the reaction medium, but in this case the bound ATP is not hydrolyzed. Covalent derivatization by 2-azido-[alpha-(32)P]ATP was used to distinguish between catalytic and noncatalytic sites. In the presence of Mg(2+), there are at least three distinct nucleotide binding sites that bind nucleotide tightly from the reaction medium: two of them are catalytic and one is noncatalytic.

Alveolar environment influences the metabolic and biophysical properties of exogenous surfactants.

Several factors have been shown to influence the efficacy of exogenous surfactant therapy in the acute respiratory distress syndrome. We investigated the effects of four different alveolar environments (control, saline-lavaged, N-nitroso-N-methylurethane, and hydrochloric acid) on the metabolic and functional properties of two exogenous surfactant preparations: bovine lipid extract surfactant and recombinant surfactant-associated protein (SP) C drug product (rSPC) administered to each of these groups. The main difference between these preparations was the lack of SP-B in the rSPC. Our results demonstrated differences in the large aggregate pool sizes recovered from each of the experimental groups. We also observed differences in SP-A content, surface area cycling characteristics, and biophysical activities of these large aggregate forms after the administration of the two exogenous surfactant preparations. We conclude that the alveolar environment plays a critical role, influencing the overall efficacy of exogenous surfactant therapy. Thus further preclinical studies are warranted to investigate the specific factors within the alveolar environment that lead to the differences observed in this study.

Effects of antenatal endotoxin and glucocorticoids on the lungs of preterm lambs.

OBJECTIVE: We hypothesized that the proinflammatory response to intra-amniotic endotoxin would induce lung maturation in preterm lambs. STUDY DESIGN: Ewes were randomly assigned to receive 20 mg Escherichia coli endotoxin by intra-amniotic injection, maternal betamethasone (0.5 mg/kg), or sodium chloride solution. Preterm lambs were delivered at 125 days' gestation and underwent ventilation to assess lung function. Lung gas volume, surfactant concentrations, and inflammation were subsequently evaluated, with data analyzed by analysis of variance. RESULTS: Fetal endotoxin exposure 6 days before delivery increased compliance by 59%, increased lung gas volume 2.3-fold, increased concentrations of surfactant lipids, increased surfactant A and B protein levels, and increased messenger ribonucleic acid expressions for surfactant proteins (all P <.01, vs control group). Betamethasone exposure resulted in less consistent effects. White blood cell counts were increased in fetal membranes and lungs after endotoxin exposure, but there was no severe inflammation. CONCLUSION: A single fetal exposure to endotoxin resulted in large improvements in postnatal lung function and increases in surfactant concentrations after preterm delivery. These effects were qualitatively larger than those achieved with betamethasone.

Covalent modification of the catalytic sites of the H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ADP: modification of the catalytic site 2 (loose) and the catalytic site 3 (open) impairs multi-site, but not uni-site catalysis of both ATP synthesis and ATP hydrolysis.

The H(+)-ATPase from chloroplasts, CF(0)F(1), was isolated and purified. The enzyme contained one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]AD(T)P leads to a tight binding of the azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and after UV-irradiation, the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-(32)P]ADP, the covalently bound label was found exclusively at beta-Tyr-362, i.e. binding occurs only to catalytic sites. Incubation conditions with 2-azido-[alpha-(32)P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, either to catalytic site 2 or to catalytic site 3. For measurements of the degree of inhibition by covalent modification, CF(0)F(1) was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential. The rate of ATP synthesis was 120 s(-1), and the rate of ATP hydrolysis was 20 s(-1), both measured under multi-site conditions. Covalent modification of either catalytic site 2 or catalytic site 3 inhibited both ATP synthesis and ATP hydrolysis, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that modification of one catalytic site, either site 2 or site 3, is sufficient to completely block multi-site ATP synthesis and ATP hydrolysis. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with covalently modified CF(0)F(1) and with non-modified CF(0)F(1). The result was that uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent modification of either catalytic site 2 or site 3. The results indicate cooperative interactions between catalytic nucleotide binding sites during multi-site catalysis, whereas neither uni-site ATP synthesis nor uni-site ATP hydrolysis require interaction with other sites.

Developmental and glucocorticoid regulation of surfactant protein mRNAs in preterm lambs.

Glucocorticoid treatment increases content of surfactant protein (SP) A and SP-B in lung tissue and lavage fluid of preterm lambs. To investigate this process, we determined the ontogeny and glucocorticoid induction of SP mRNAs. In separate treatment protocols, each with its own controls, sheep were injected with betamethasone 15 h, 48 h, or weekly for 1-4 doses before preterm delivery. Using ovine SP cDNAs, we found an increase equal to or more than threefold in basal levels of all three SP mRNAs between 125 days and term. After betamethasone treatment, SP-B and SP-C mRNA levels increased by 15 h and all SP mRNAs were elevated after 24 h (>/=2-fold); mRNA levels in fetuses delivered 1-3 wk after betamethasone were not different from control. We conclude that in vivo betamethasone rapidly induces a coordinated increase in SP mRNAs, which is fully reversible within 7 days despite repetitive doses of betamethasone. Similar increases in mRNA and protein contents for SP-A and SP-B suggest that glucocorticoid regulation of these SPs in vivo is primarily pretranslational.