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.

Exposure of rabbit fetal lung to glucocorticoids in vitro does not enhance transcription of the gene encoding pulmonary surfactant-associated protein-B (SP-B).

We have investigated the ontogeny and hormonal regulation of both synthesis rates and cellular accumulation of the mRNA for surfactant-associated protein B (SP-B) in rabbit fetal lung. The developmental pattern for SP-B mRNA synthesis increased as a function of gestational age and paralleled that for SP-B mRNA levels except on days 22-26 of gestation where relatively higher levels of gene transcription were observed. Time-course studies with explants from 26- and 30-day fetal lung maintained in culture revealed a gradual increase in mRNA levels and a much smaller increase in gene transcription relative to adult values. Within 48 h of exposure of 26-day explants to dexamethasone at 10(-8) M there was a rapid increase in SP-B mRNA levels to 7-fold adult levels. A similar overall although somewhat slower and attenuated pattern was observed with 30-day explants. Dexamethasone at 10(-8) M had no effect on SP-B gene transcription with explants of either gestational age. We conclude that the major effect of dexamethasone treatment in vitro on SP-B mRNA levels appears to be post-transcriptional and there are small but distinct differences in the effects of glucocorticoids on SP-B mRNA levels with explant cultures from early and late stages of fetal lung maturation.

Low-molecular-weight hydrophobic proteins from bovine pulmonary surfactant.

Pulmonary surfactant stabilizes the lung by reducing the surface tension in the terminal air spaces. Lipid extract surfactant contains approx. 1% (w/w) low-molecular-weight hydrophobic proteins SP-B (15 kDa: nonreduced) and SP-C (3.5 kDa) and with the remainder being mainly phospholipids. The hydrophobic proteins were purified from bovine lipid extract surfactant using delipidation by phospholipase C digestion followed by hydroxyapatite chromatography. The phospholipase C step removed most of phosphatidylcholine resulting in a 10-fold enrichment of hydrophobic proteins relative to phospholipid. Chromatography of this preparation on a hydroxyapatite column resulted in the elution of phospholipids followed by SP-C and then SP-B. The column chromatography was repeated to remove residual phospholipids and yield purified SP-B and SP-C. The final recovery of SP-B from the lipid extracts was about 15-20% and that of SP-C was 5-10%. The bovine surfactant proteins were reconstituted with phospholipids and examined for their ability to lower the surface tension with a pulsating bubble surfactometer. Reconstituted surfactant preparations containing SP-B and dipalmitoylphosphatidylcholine plus dioleoylphosphatidylglycerol were capable of reducing the surface tension to near zero values at minimum bubble radius while the reconstitutes with SP-C only lowered the surface tension to approx. 20 mN/m. A more rapid decrease in surface tension was observed with reconstituted samples containing both hydrophobic proteins. These results indicate that both SP-B and SP-C can promote the adsorption and spreading of surfactant lipids at the air/liquid interface. In addition, SP-B appears to facilitate the squeeze-out of unsaturated phospholipids leading to an enrichment of dipalmitoylphosphatidylcholine in the monolayer.

cDNA sequence and alternative mRNA splicing of surfactant-associated protein C (SP-C) in rabbit lung.

An 784 base pair (bp) copy DNA (cDNA) for the low molecular weight hydrophobic surfactant-associated protein C (SP-C) has been isolated from a lambda gt11 cDNA library constructed from fetal rabbit lung mRNA. The cDNA, which coded for a 193 amino-acid proprotein with 6 bp 5' and 193 bp 3' untranslated segments, possesses considerable nucleic acid and predicted amino-acid homology with previously reported SP-C cDNAs. The predicted amino-acid sequence of the 35 amino-acid mature polypeptide shares 94-97% identity with human, rat and mouse SP-C and is 88-91% homologous to the mature proteins from bovine, porcine and canine lung. The last 12 amino acids of mature SP-C are highly hydrophobic and invariant. Alignment of the rabbit and human nucleic acid sequences required introduction of a 27 bp gap in the rabbit sequence at a site corresponding to the exon-intron junction of the 5th exon of the human genomic sequence. Since previous studies have identified differential splicing at the 5' and 3' ends of the human 5th exon, we investigated the potential existence of alternative splicing of rabbit SP-C mRNA. Reverse transcription (RT) of total RNA followed by polymerase chain reaction (PCR) was used to establish the relative abundance of alternative splicing products from fetal and adult lung and from rabbit kidney, placenta and liver. The relative abundance of the 250, 280 and 350 bp bands observed was the same in lung and other tissues. PCR amplification of genomic rabbit DNA indicated that the 350 bp fragment corresponds to the unspliced nascent transcript. The lack of developmental or tissue-specific abundance patterns implies the absence of secondary influences on SP-C mRNA polymorphism. Indeed, free energy of formation calculations predicted the presence of hairpin structures favouring formation of the more abundant 250 bp form. These observations plus the absence of any effect of alternative splicing on SP-C protein structure led us to conclude a physiological role is unlikely.

On the surface activity of surfactant-associated protein C (SP-C): effects of palmitoylation and pH.

The effect of palmitoylation on the surface activity of bovine surfactant-associated protein C (SP-C) in lipid mixtures was investigated. Native and chemically depalmitoylated SP-C were reconstituted with dipalmitoylphosphatidylcholine/egg phosphatidylglycerol (7:3) using two different procedures, one of which included lyophilization and sonication. When tested using a pulsating bubble surfactometer, no significant changes in the surface activity of these mixtures were observed upon the hydrolysis of the palmitates. Since the purification and deacylation procedures of SP-C included the use of acid and alkali, the effect of pH was examined. The surface activity of the mixtures was found to vary with pH. At low pH values (approx. 2.5), surface tensions between 3 and 10 mN/m at minimum bubble radius were reached within 5 pulsations, while at neutral and slightly alkaline pH, surface tension reduction was much slower and near zero (< 5 mN/m) values at minimum bubble radius were not reached by the fiftieth pulsation. Protein-free lipid samples that were exposed to acid exhibited enhanced surface activity over similar non-treated samples. It is therefore concluded that low surface tension measurements recorded for acidic samples are secondary to a pH effect and do not reflect the surface activity at physiological conditions.

Pool sizes of the precursors for phosphatidylcholine synthesis in developing rat lung.

1. Pulmonary maturation in the rat is accompanied by a 30% postnatal increase in the pool size of choline, a 4-fold overall prenatal and postnatal decrease in the level of cholinephosphate, a 3-fold decrease in CDPcholine levels and a 2-fold increase in the content of phosphatidylcholine. 2. The level of 1,2-diacyl-sn-glycerol in rat lung increases 5-fold during the fetal and neonatal periods. Only minor alterations were noted in the fatty acid composition. 3. These results are consistent with an increase in the relative rates of the cholinephosphate cytidylyl-transferase and cholinephosphotransferase steps of phosphatidylcholine production during pulmonary maturation. The relative rate of the step catalyzed by phosphatidate phosphohydrolase may also be increased.

Role of bovine pulmonary surfactant-associated proteins in the surface-active property of phospholipid mixtures.

The surfactant-associated proteins, SP-A, SP-B and SP-C have been isolated from bovine pulmonary surfactant. The biophysical roles of SP-B and SP-C in reconstituted surfactants, with various phospholipid mixtures subjected to different thermal treatments, have been examined using a pulsating bubble surfactometer. The phospholipid mixtures were: (A) dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylcholine (PC)/egg phosphatidylglycerol (PG) (6:2:2, w/w); (B) DPPC/PG (9:1); and (C) DPPC/PG (7:3). Thermal treatments involved mixing SP-B or SP-C, at room temperature, with lipids in chloroform/methanol (9:1, v/v) and removing the solvent under N2 by (1) evaporation at room temperature; (2) evaporation at 45 degrees C; or (3) incubation at 45 degrees C overnight prior to evaporation at 45 degrees C. In all cases, 45 degrees C solvent evaporation was the most effective treatment. DPPC/egg PG (7:3) was the most favourable lipid composition. With either a static or a pulsating bubble, SP-C promoted a rapid decrease in surface tension with little change thereafter. This implies that SP-C is effective in enhancing phospholipid adsorption but does not play an important role in the removal of non-DPPC lipid from the monolayer. While SP-B was not as effective in facilitating phospholipid absorption, samples containing this protein could achieve near zero surface tension upon pulsation. A very low surface tension could also be attained during the initial pulsation of DPPC/PG plus SP-B mixtures which had been allowed to adsorb until equilibrium. This observation indicates that SP-B promotes the removal of PG from the monolayer. SP-A alone had only a slight effect on the surface activity of the DPPC/PG (7:3) mixture, and did not accelerate adsorption of samples containing SP-C. However, SP-A facilitated phospholipid adsorption and may also enhance the removal of PG from monolayers in the presence of SP-B.

Purification and characterization of a phospholipase A2 associated with rabbit lung microsomes: some evidence for its mitochondrial origin.

Phospholipase A2 (EC 3.1.1.4) activity appeared to be unevenly distributed among the subcellular fractions of rabbit lung homogenates. The mitochondrial/lysosomal fraction, which possessed the highest specific activity, was the second most abundant source of enzyme, following the 1000 x g pellet. Crude microsomes, which were the poorest source of enzyme, had a specific activity intermediate between that of crude mitochondria and of cytosol. Despite these observations, in view of the putative role of microsomal phospholipase A2 in remodelling phosphatidylcholines for pulmonary surfactant biosynthesis, the purification of phospholipase A2 from microsomal membranes was investigated. The activity was solubilized from rabbit lung microsomes with 1 M KCl and resolved into two distinct peaks by ion-exchange chromatography. The larger peak (95% of the recovered activity) was subjected to a combination of hydroxyapatite and gel-filtration chromatography, resulting in a purification factor in excess of 70,000 relative to the microsomal membranes. There was no indication for the removal of endogenous inhibitor(s) during the purification. Application of the same purification protocol to a 1 M KCl extract of lung mitochondria resulted in phospholipase A2 profiles in each of the four columns employed that had exactly the same elution characteristics as those generated by the microsomal extracts. The purified enzyme is specific for the sn-2 ester bond of phosphatidylcholine, requires Ca2+ for activity and has an alkaline pH optimum. It is heat-labile and susceptible to treatment by p-bromophenacyl bromide and by 2-mercaptoethanol but remains unaffected by NaF, diisopropylfluorophosphate and thiol reagents.

Contamination of commercial phosphatidylcholine by phospholipase A.

During the course of a study involving the assay of a membrane-bound phospholipase A2 it was observed that a commercial preparation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine used as substrate had intrinsic lipolytic activity at pH 8.5. Further investigation revealed a Ca2+-dependent phospholipase A largely susceptible to treatment by the alkylating reagent p-bromophenacyl bromide or by heat (15 min at 120 degrees C). Complete separation of enzyme and phospholipid could be achieved by thin-layer chromatography. Such a contamination was not observed in a chemically identical phosphatidylcholine obtained from a different supplier. These observations may be relevant to investigators using commercial preparations of phospholipids in a variety of studies, including intracellular phospholipase A2 determination.

Effect of pulmonary surfactant protein B (SP-B) and calcium on phospholipid adsorption and squeeze-out of phosphatidylglycerol from binary phospholipid monolayers containing dipalmitoylphosphatidylcholine.

The pulsating bubble technique was used to study the surface activity of binary phospholipid mixtures containing dipalmitoyl-phosphatidylcholine (DPPC) and an unsaturated acidic phospholipid such as egg phosphatidylglycerol (egg PG), 1-palmitoyl-2-oleoyl-PG (POPG) or egg phosphatidic acid (egg PA) in the presence of surfactant-associated protein B (SP-B) and calcium. The relative surface activities were DPPC/egg PG/SP-B (7:3:1%) greater than DPPC/POPG/SP-B (7:3:1%) greater than DPPC/egg PA/SP-B (7:3:1%). The Wilhelmy surface plate technique was utilized to investigate the interaction between pure SP-B in the bulk phase (0.9% NaCl/1.5 mM CaCl2) and preformed DPPC or phosphatidylglycerol (PG) monolayers. Although SP-B injected into the bulk phase reduces the surface tension of a clean surface, no evidence was obtained for the insertion of SP-B into surface monolayers at equilibrium surface tension. Surface radioactivity measurements and the Wilhelmy surface plate technique were also used to study the potential interactions between liposomes of DPPC/POPG (7:3) with or without SP-B and surface monolayers of [14C]DPPC or [14C]POPG. No exchange of phosphatidylcholine (PC) or PG was found between the monolayer and liposomes. We also compared the adsorption of pure POPG or 1-palmitoyl-2-oleoyl- phosphatidylcholine (POPC) and binary mixed liposomes with DPPC in the presence or absence of SP-B and calcium. DPPC/POPG/SP-B (7:3:1%) was found to be more surface active than pure POPG plus 1% SP-B in the presence of calcium. Injection of SP-B into the bulk phase promoted the adsorption of DPPC/POPG liposomes to a greater extent than POPG liposomes. The enhanced adsorption was dependent on the presence of calcium. In contrast to PG, DPPC/POPC/SP-B (7:3:1%) was less surface active than pure POPC plus 1% SP-B either in the presence or absence of calcium. Our findings suggested that the molecular composition and organization of mixed monolayers play an important role in the surface activity of the surfactant.

Comparative studies on the biophysical activities of the low-molecular-weight hydrophobic proteins purified from bovine pulmonary surfactant.

Two low-molecular-weight hydrophobic proteins with nominal molecular weights Mr = 15,000 and Mr = 3,500 have been isolated from the lipid extracts of bovine pulmonary surfactant by several methods, including (a) dialysis plus silicic acid chromatography, (b) elution from Waters SEP-PAK silica cartridges with a variety of solvent mixtures, and (c) ultrafiltration. As detailed in the text, these proteins have been designated surfactant-associated protein-BC (SP-BC) (15 kDa: nonreduced), and SP-C (3.5 kDa). The biophysical activities of reconstituted surfactant containing these proteins and the phospholipids present in lung surfactant have been compared with the biophysical activities of bovine lipid extract surfactant on a pulsating bubble surfactometer using a phospholipid concentration of 10 mg/ml. At this concentration, unmodified lipid extract surfactant reduces the surface tension of the pulsating bubble to near 0 within 10 pulsations at 20 cycles per min. Similar biophysical properties were observed with modified lipid extract surfactant in which the relative concentration of hydrophobic protein had been reduced from 1 to 0.4% (W/W) of the phospholipids by addition of dipalmitoylphosphatidylcholine (DPPC) or DPPC plus phosphatidylglycerol. Reconstituted surfactants, which contained partially delipidated SP-BC (15 kDa: nonreduced) obtained by method (a) at a relative concentration of 0.1%, were also capable of reducing the surface tension to near 0 mN/m. Preparations of SP-BC (15 kDa: nonreduced) obtained by method (b), which had been subjected to very low pH levels during isolation and were extensively delipidated, exhibited full biophysical activity only at higher protein concentrations and with prolonged pulsation. Extensively delipidated samples of SP-BC obtained by method (c) exhibited impaired biophysical activities, even when prepared with neutral organic solvents. Reconstituted surfactant samples containing SP-C (3.5 kDa) obtained by any of the methods listed above were only able to reduce the surface tension at minimum bubble radius to approx. 20 mN/m. The biophysical activity of SP-C (3.5 kDa) was not significantly affected by low pH or extensive delipidation. Reconstituted samples containing mixtures of SP-BC (15 kDa: nonreduced) and SP-C (3.5 kDa) were more effective than samples containing either protein alone. Furthermore, with samples containing both hydrophobic proteins the final surface tensions at maximum bubble radius were attained within a few bubble pulsations.(ABSTRACT TRUNCATED AT 400 WORDS)

Lysophosphatidylcholine sensitizes lipid extracts of pulmonary surfactant to inhibition by serum proteins.

Interactions between serum protein and lysophospholipid inhibitors of pulmonary surfactant were examined in vitro using a pulsating bubble surfactometer. In previous studies a particular batch of Lipid Extract Surfactant (LES) was observed to be unusually sensitive to inhibition by fibrinogen. This sample was found to contain an abnormally high concentration of lysophosphatidylcholine (lysoPC). Addition of exogenous lysophospholipid to LES at similar concentrations sensitized the surfactant to inhibition by fibrinogen. Sensitization to inhibition by lysoPC is also observed with fetal bovine serum. Under the conditions used, inhibition by bovine serum albumin was not affected. Whereas only small amounts of lysoPC (1 mol% added) maximally sensitize LES to inhibition by fibrinogen, co-addition of equal amounts of palmitic acid can partially offset this effect at low lysoPC concentrations (less than 5 mol%). Lipid Extract Surfactant was digested with phospholipase A2 to mimic the generation of endogenous lysoPC at the expense of surfactant lipids. Digestion of 2-3% of the phosphatidylcholine to lysophosphatidylcholine vastly sensitized the surfactant to inhibition by fibrinogen. These results suggest that the degradation of surfactant phospholipids by phospholipase A2 to lysophospholipids could contribute to the development and progression of adult and neonatal respiratory distress syndromes.

Effect of pulmonary surfactant protein A (SP-A) and calcium on the adsorption of cholesterol and film stability.

The effect of exogenous cholesterol on the stability of surface films at 37 degrees C from various surfactants was studied with the pulsating bubble surfactometer. Addition of cholesterol (5%, w/w) to bovine lipid extract surfactant (bLES) or mixtures of dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoyl-phosphatidylglycerol /SP-B (7:3:1%) dispersed in 1.5 mM CaCl2/0.9% NaCl resulted in unstable surface films. Although 10% cholesterol only partially impaired the surface activity of bLES, it virtually abolished that of the reconstituted surfactant. The inhibitory effects of cholesterol were significantly repressed by SP-A (10%, w/w of lipid) and 3 mM CaCl2 or 5 mM CaCl2 without SP-A. Adsorption of cholesterol from various surfactants into the air/water interface was examined by measuring the surface radioactivity of [14C]cholesterol. Cholesterol alone dispersed in 1.5 mM CaCl2/0.9% NaCl could not adsorb to the interface, but it adsorbed readily when mixed with bLES. Cholesterol adsorption was markedly suppressed by SP-A in 3 mM CaCl2/0.9% NaCl or 5 mM CaCl2/0.9% NaCl without SP-A. Electron microscopy revealed striking ultrastructural differences between bLES/5% cholesterol/10% SP-A in 3 mM CaCl2/0.9% NaCl and bLES/5% cholesterol in 3 or 5 mM CaCl2/0.9% NaCl. The former exhibited large multilayer and small unilamellar vesicles, while the latter displayed condensed patches of aggregates. Adsorption studies showed aggregated patches adsorbed more rapidly than vesicles but attained lower equilibrium surface pressures. These results indicate SP-A and calcium limit the adsorption of surfactant cholesterol to the air-water interface.

Pulmonary phosphatidic acid phosphohydrolase. Developmental patterns in rabbit lung.

1. The developmental patterns of the phosphatidic acid phosphohydrolase activities in developing rabbit lung were determined using both aqueously dispersed phosphatidic acid (PAaq) and membrane-bound phosphatidic acid (PAmb) as the substrates. 2. The specific activities and the total activities of the PAmb-dependent phosphohydrolase activities in the microsomes and to a lesser extent in the homogenates increased between 26 and 30 days gestation (term 31), but decreased in the adult. The PAaq-dependent activities demonstrated a smaller increase during late gestation and a decrease in the adult. 3. There was little change in either the Paaq- or the Pamb-dependent activities in the cytosol between 25 and 30 days gestation. The total activities per g lung were increased in the adult. 4. Fractionation of adult cytosol on Bio-Gel A5m revealed PAaq-dependent activities in the void volume (Vo) (50% total), a peak with an apparent molecular mass (Mr) = 150 kdaltons (25% total) and a peak with Mr = 110 kdaltons (25% total). The PAaq-dependent peak with Mr = 150 kdaltons was not detected in the fetal cytosols. 5. Gel filtration revealed PAmb-dependent activity in the Vo (15% total), a major peak with an apparent Mr = 390 kdaltons (44% total) and minor peaks with Mr = 240 kdaltons (16% total) and Mr = 110 kdaltons (24% total). Little change was observed during development. 6. Thermal denaturation studies on he PAmb-dependent activities in the cytosols produced biphasic curves with a rapidly inactivated component and a relatively heat-stable component. The thermal denaturation profiles for the PAmb-dependent activities remained relatively unaltered throughout fetal development. The thermal denaturation profiles of the PAaq-dependent activities in the fetal cytosols were also biphasic. In contrast, the inactivation profiles of the PAaq-dependent activities in adult cytosol were monophasic.

Pulmonary phosphatidic acid phosphatase. Properties of membrane-bound phosphatidate-dependent phosphatidic acid phosphatase in rat lung.

1. The membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activity of rat lung has been investigated in cytosol and microsomal fractions using as a substrate [32P]phosphatidate bound to heat inactivated rat liver microsomes. Both activities demonstrated broad pH optima with a maximum of 7.4--8 for the cytosol and a maximum of 6.5--7.5 with microsomal preparations. 2. At low concentrations (0--5 mM) Mg2+ produced a slight stimulation of the cytosol activity but at higher concentrations an inhibition was observed. Low concentrations (1.0--2.0 mM) of EDTA abolished the cytosol activity and reduced the microsomal activity to half. In both cases, the addition of Mg2+ in the presence of EDTA resulted in an activity which was more than 2-fold greater than that observed in the absence of chelator or divalent cation. 3. The cytosol activity was relatively resistant to the addition of ionic and nonionic detergents. In general, the addition of a number of phosphate esters increased rather than decreased the release of 32Pi, indicating a relative specificity for phosphate groups associated with a hydrophobic environment. The addition of aqueous dispersions of phosphatidate, lysophosphatidic acid or phosphatidylglycerophosphate markedly reduced the hydrolysis of membrane-bound [32P]phosphatidate. The cytosol activity was slightly inhibited by the addition of phosphatidylcholine. 4. In an attempt to estimate the relative contributions of the cytosol and microsomal activities in vivo, these activities were assayed using [32P]phosphatidate endogenously generated on rat lung microsomes. With the 32P-labelled microsomes, the hydrolysis remained linear over the 45 min of the experiment. Addition of high speed supernatant produced a rapid release of 32Pi during the first 10 min followed by a more gradual release similar to that oberved with the microsomes alone. The cytosol activity remained greater than the microsomal activity at all times studied. 5. When [14C]phosphatidate-labelled microsomes were incubated in the presence of nonradioactive CDPcholine, the addition of cytosol markedly stimulated the incorporation of radioactivity into phosphatidylcholine. This observation suggests that the phosphatidic acid phosphatase activity associated with the cytosol has a role in phosphatidylcholine (and presumably surfactant) biosynthesis in rat lung.

The role of Mg2+-dependent phosphatidate phosphohydrolase in pulmonary glycerolipid biosynthesis.

Rat lung microsomes washed with increasing concentrations of NaCl show a displacement of protein from microsomes to the wash supernatant. Among the proteins removed from the microsomal surface was the Mg2+-dependent phosphatidate phosphohydrolase, while the Mg2+-independent activity remained associated with the microsomes. The Mg2+-dependent activity could be quantitatively assayed in the wash supernatant. Microsomes washed with increasing concentrations of NaCl showed a progressive impairment in the synthesis of labelled neutral lipid and phosphatidylcholine from [14C]glycerol 3-phosphate with a concomitant increase in the labelling of phosphatidic acid. The impairment was sigmoidal and correlated highly with the decrease in Mg2+-dependent phosphatidate phosphohydrolase activity. When Mg2+-dependent phosphatidate phosphohydrolase from wash supernatant was incubated with microsomes previously washed with high salt concentrations, the labelling of neutral lipid and phosphatidylcholine was returned to control levels. Labelling of neutral lipids and phosphatidylcholine could be restored upon addition of a cytosolic Mg2+-dependent phosphatidate phosphohydrolase isolated by gel filtration. Mg2+-independent phosphatidate phosphohydrolase isolated from cytosol was incapable of restoring the labelling of neutral lipids and phosphatidylcholine. These findings confirm that the Mg2+-dependent phosphatidate phosphohydrolase of rat lung is involved in pulmonary glycerolipid biosynthesis. The role of the Mg2+-independent phosphatidate phosphohydrolase activity remains unknown.

Calcium interactions in pulmonary surfactant.

The surfactant properties of natural bovine pulmonary surfactant, its lipid extracts and acetone precipitates of lipid extracts have been examined with an artificial alveolus model, the pulsating-bubble surfactometer. At bulk concentrations of 0.4% (wt./vol.) phospholipid in saline, all three preparations exhibited surfactant activity, i.e., were capable of reducing the surface tension of the pulsating bubble to approx. 27 dynes/cm at maximum bubble radius and to near zero at minimum bubble radius. At a concentration of 0.1% (wt./vol.) in saline, only natural surfactant was effective. Acetone-precipitated surfactant at 0.1% (wt./vol.) achieved these criteria in the presence of 5 mM calcium, but 15-20 mM calcium was required to restore the surfactant activity of lipid extract surfactant. Chemical analysis revealed that lipid extraction decreases the protein content but does not alter the endogenous calcium levels. A calcium requirement for natural surfactant could only be demonstrated after repeated treatment with chelators for divalent cations. Surfactant activity was restored by low levels of calcium or high levels of magnesium. Paradoxically, a calcium requirement could not be demonstrated by treating acetone-precipitated lipid extract with chelators. The subtle differences noted between natural, lipid extract and acetone-precipitated lipid extract surfactant with the pulsating-bubble assay show that the latter preparations do not represent simplified model systems for the natural product.

Adsorption, compression and stability of surface films from natural, lipid extract and reconstituted pulmonary surfactants.

A pulsating bubble surfactometer was used to study the surface activities and surface film stabilities of bovine pulmonary surfactants (10 mg/ml) and a reconstituted surfactant (10 mg/ml). Pulmonary surfactants were natural surfactant (NS), lipid extract surfactant [LES(chol)] and lipid extract surfactant without neutral lipids (LES). NS is composed of phospholipids, neutral lipids and surfactant-associated proteins (SP-A, SP-B and SP-C). Both LES(chol) and LES are organic solvent extracts of NS. LES(chol) retains all the components of NS except SP-A. Reconstituted surfactant was dipalmitoylphosphatidylcholine (DPPC): 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG): SP-B/7:3:1%. All three pulmonary surfactants attained the equilibrium surface tension almost instantaneously at 37 degrees C. The adsorption rates of NS and LES(chol) at 24 degrees C were similar to those at 37 degrees C, while LES exhibited a lower adsorption rate at 24 degrees C. Reconstituted surfactant adsorbed slower than any of the pulmonary surfactants. Film stability was studied by recording the spontaneous increase in the pressure gradient of a static bubble at the minimum size (Rmin) once near zero surface tension was attained. The order of surface film stabilities were: reconstituted surfactant > > NS > LES > LES(chol). Surface films of NS and LES could be stabilized by prolonged pulsation, while film stability of LES(chol) was only moderately affected by pulsation. These results indicate that SP-A in NS promotes formation of some unique structure, possibly tubular myelin, which induces selective adsorption of lipids into the surface.

Fetal utilization of maternally derived ketone bodies for lipogenesis in the rat.

When D-beta-[3-14C]hydroxybutyrate was injected via the femoral vein into pregnant Sprague-Dawley rats at 21 days of gestation, D-beta-[3-14C]hydroxybutyrate was enzymatically detected in fetal plasma within 5 min. The time course of the incorporation of DL-beta-[3-14C]hydroxybutyrate into fetal lipids was studied. Lipid extracts of brown adipose tissue exhibited the greatest relative incorporation followed by pancreas, liver and lung. Less radioactivity was incorporated into brain and placenta. The incorporation into fetal lipids was several-fold greater than into maternal lipids. The labelling of the individual phospholipids was similar in the different tissues with phosphatidylcholine accounting for more than 50%. 75% of the radioactivity in brown adipose tissue was in the triacylglycerol fraction. In brain, liver and placenta, approximately half of the neutral lipid radioactivity was in cholesterol. Experiments in which D-beta-[3-14C]hydroxybutyrate was directly injected into fetuses in utero confirmed that this substrate was directly used by the fetuses without maternal intervention. These studies demonstrate that the rat placenta is permeable to beta-hydroxybutyrate and suggest that this ketone body is rapidly used by the fetus for the synthesis of fatty acids and cholesterol.

Structural relationship between the two small hydrophobic apoproteins in bovine pulmonary surfactant.

Lipid extracts of bovine pulmonary surfactant contain two very hydrophobic surfactant-associated proteins (SP) designated SP-B (15 kDa nonreduced) and SP-C (3.5 kDa). These two low molecular weight apoproteins were delipidated and purified on silica SEP-PAK cartridges using various reagents. Dansylation studies revealed that the 15 kDa apoprotein has three N-termini: Phe, Leu and Ile, while the 3.5 kDa apoprotein has two N-termini: Leu and Ile. In either protein, only a very small amount of N-Ile is present. Quantitative N-terminal dansylation analysis of the 15 kDa protein indicated that Phe and Leu (plus Ile) are present in a 1:1 ratio. Carboxy-terminal analysis showed that the 15 kDa protein contains C-terminal Gly, and the 3.5 kDa protein contains C-terminal Leu. Gas-phase amino terminal sequencing of the 15 kDa protein revealed almost exclusively the Phe-polypeptide (SP-B). These results suggest that the 15 kDa apoprotein is not an oligomer of SP-B and SP-C. The reason that analysis of SP-B reveals N-terminal Leu and Ile by dansylation which cannot be confirmed by amino acid sequencing is not known.