Biomedical subjects

F Possmayer

Publications and source records attributed to F Possmayer.

At least 73 records · Page 4Linked to original sources

PubMed1993

Compliance of the respiratory system in newborn infants pre- and postsurfactant replacement therapy.

Surfactant administration causes a rapid and dramatic improvement in gas exchange, but paradoxically, studies have failed to show an improvement in the mechanical properties of the lung. We have measured dynamic and static (passive flow-volume technique) compliance before and after a single dose of bovine lipid extract surfactant in 22 premature infants with RDS. This had no effect on the measured dynamic compliance. In contrast, surfactant significantly increased static compliance from 0.41 +/- 0.02 to 0.55 +/- 0.04 mL/cm H2O/kg. This improvement was the result of a substantial recruitment of lung volume after surfactant administration. This led us to reduce ventilator pressures, which produced an increase in both dynamic and static compliance, but did not recruit additional volume. We conclude that surfactant causes a substantial increase in static compliance due to volume recruitment, which is consistent with reports of increase in the measured FRC. However, despite this improvement, the compliance is still below our normal range.

Female↗

PubMed1993

Surfactant-associated proteins (SP-A, SP-B) are increased proportionally to alveolar phospholipids in sheep silicosis.

In silicosis, a profile of surfactant overproduction associated with type II epithelial cell hypertrophy and hyperplasia has been documented. In addition, enhanced accumulation of surfactant-associated protein A (SP-A) has been seen in the rat model of acute silicosis by 2 independent groups, but the exact role of these surfactant changes in silicosis are incompletely understood. In this study we measured in lung lavage surfactant total phospholipids and surfactant-associated proteins A and B (SP-A, SP-B). In addition, the surface tension reducing activities of lipid extracts of bronchoalveolar lavage fluids (BALFs) in the sheep silicosis model were examined using a pulsating bubble surfactometer. Two groups of animals (n = 18) were investigated: a saline-exposed (PBS-PBS) group and a silica-exposed (Si-PBS) group. Total surfactant phospholipids in the silicotic sheep increased 2-fold over the control sheep values (p < 0.05), as previously reported. In addition, we found a substantial rise in total surfactant-associated proteins, with significant increase in SP-A (1.16 +/- 0.22 micrograms/ml Si-PBS group vs. 0.70 +/- 0.07 micrograms/ml PBS-PBS group, p < 0.05) and a parallel but not significant increase in SP-B (2.68 +/- 0.90 micrograms/ml Si-PBS group vs. 1.10 +/- 0.30 micrograms/ml PBS-PBS group). The surface-tension-reducing activities of alveolar fluid lipid extracts did not differ significantly between the groups (Si-PBS group at maximal bubble radius [Rmax]: 27.0 +/- 1.6 and at minimal bubble radius [Rmin]: 1.0 +/- 0.7 milli Newton/m, vs. PBS-PBS group Rmax: 27.0 +/- 2.2 and Rmin: 0.7 +/- 0.3 milli Newton/m at 4 min pulsation). The ratios of SP-A and SP-B to lipid phosphorus levels document a proportional enhancement of surfactant-associated proteins and phospholipids, thus suggesting a co-ordinated upregulation of both surfactant-associated proteins and phospholipids in this model of silicosis. However, on an individual basis, these changes were not related, suggesting a more complex model of regulation. This study documents significant increases of the surfactant apoproteins proportional to changes in phospholipids in the lung of silicotic sheep. In spite of these alterations of surfactant components, organic solvent lipid extracts of BAPLFs surfactant remained equally effective in reducing surface tension.

Animals↗

PubMed1993

Alterations in pulmonary surfactant composition and activity after experimental lung transplantation.

Pulmonary surfactant facilitates breathing by reducing the surface tension at the air/liquid interface. We examined the effect of experimental lung transplantation on the phospholipid pool sizes of alveolar surfactant large and small aggregates, the composition of the large aggregates, the surface tension-reducing ability of lipid extract surfactant, and the leakage of serum proteins into the lung. A double-lung block from the donor animal was stored for 2 or 12 h after perfusion with either Euro-Collins solution or University of Wisconsin solution. The right donor lung was lavaged immediately after the storage period to determine the effects of storage on pulmonary surfactant. The left donor lung was transplanted. The recipient animal, containing its own native right lung and the transplanted left lung, was reperfused for 6 h. After the reperfusion period, the transplanted left lung and the native right lung were lavaged. After an ischemic time of 12 h, impaired gas exchange was observed in the transplanted lung as well as the native lung during the 6 h of reperfusion. This impaired gas exchange was associated with several significant changes in pulmonary surfactant: (1) total serum protein in the lung lavage was increased, (2) the small to large surfactant aggregate ratio was increased, (3) sphingomyelin content was increased and phosphatidylglycerol content was decreased in large aggregates, and (4) the surfactant-associated protein A content was decreased in large aggregates. No significant differences were observed between the results obtained with Euro-Collins and University of Wisconsin solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1993

Optimizing alveolar expansion prolongs the effectiveness of exogenous surfactant therapy in the adult rabbit.

We evaluated four ventilator patterns after the administration of 80 mg/kg bovine lipid extract surfactant (LES) to anesthetized, paralyzed, saline-lavaged New Zealand white rabbits. Two ventilator types were compared: high frequency oscillatory ventilation (HFO) versus conventional mechanical ventilation (CMV), each at high (HI) and low (LO) end-expiratory lung volumes (EELV); n = 6, each group; treatment duration = 4 h. Target PaO2 ranges were > 350 mm Hg for groups with high EELV (i.e., HFO-HI and CMV-HI) and 70 to 100 mm Hg for those with low EELV (i.e., HFO-LO and CMV-LO). Ventilator pressures were limited to < or = 39/9 cm H2O in the CMV-HI group. Five of six CMV-HI-treated animals did not maintain target PaO2 levels. Both ventilator type and strategy influenced outcome significantly. Animals managed with HFO had higher mean arterial pressures (p = 0.004), lower mean airway pressures (Paw) (p < 0.00008) and HCO3- requirements (p < 0.02), larger inflation (p = 0.003) and deflation (p < 0.00001) respiratory system volumes at 10 cm inflation pressure, and higher lung lamellar body (p = 0.0006) and lavage fluid (p = 0.003) phospholipid quantities than did CMV-treated animals. The deflation P-V curve (p = 0.0004), lamellar body (p < 0.00001) and lavage fluid (p = 0.0002) phospholipid levels were superior after the high EELV strategy. We conclude that ventilator pattern strongly influences exogenous surfactant efficacy. Benefits arise from keeping EELV high enough to prevent atelectasis and using small (approximately 2 ml/kg) tidal volumes to prevent overdistension.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗

PubMed1993

99mTc-DTPA clearance in preterm lambs. Effect of surfactant therapy and ventilation.

Surfactant therapy and high-frequency oscillatory ventilation (HFO) may minimize damage to the pulmonary epithelium of surfactant-deficient newborns. Using pulmonary clearance of insufflated, aerosolized 99mTc-DTPA (molecular weight 492) as an index of lung epithelial permeability, we examined the effects of 300 mg bovine lipid extract surfactant (S) administered at birth to preterm lambs ventilated by either HFO or conventional mechanical ventilation (CMV). Four groups of lambs, delivered by cesarean section at 129 to 133 days of gestation, were studied: (1) HFO + S, (2) CMV + S, (3) HFO, and (4) CMV. 99mTc-DTPA clearance was assessed at 2, 4, and 5.5 h after birth. Surfactant treatment improved oxygenation and lung pressure-volume relationships, with oxygenation best maintained by the combination of HFO + S. All groups had similar biexponential clearance curves at the three time points, however, and there was no significant difference in the mean rates of clearance (k) between the four groups at 2 h (k = 6.03 +/- 0.60 [SEM], 7.04 +/- 1.46, 5.67 +/- 0.91, and 7.23 +/- 0.97 %/min for Groups 1, 2, 3, and 4, respectively), 4 h (k = 6.95 +/- 0.77, 5.60 +/- 0.51, 6.39 +/- 0.64, and 6.78 +/- 1.71 %/min), and 5.5 h (k = 7.43 +/- 0.78, 6.08 +/- 0.80, 7.86 +/- 0.90, and 7.95 +/- 0.66 %/min). These data suggest that neither surfactant nor HFO significantly alters pulmonary epithelial permeability to a small radiolabeled molecule in preterm lambs.

Analysis of Variance↗

PubMed1992

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.

Amino Acid Sequence↗

PubMed1992

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.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1992

Pulmonary SP-A enhances adsorption and appears to induce surface sorting of lipid extract surfactant.

The effect of surfactant concentration and supplementation with surfactant-associated protein A (SP-A) on the surface activity of lipid extract surfactant (LES) was examined using a captive bubble technique. Adsorption of LES is strongly concentration dependent over the range of 50-1,000 micrograms/ml. Addition of SP-A to LES at low concentrations in the presence of calcium dramatically increases the rate of adsorption. In quasistatic cycling experiments, samples containing SP-A require less compression to achieve low surface tensions even during the first compression cycle. Calculated film compressibilities at 15 mN/m indicate that SP-A alters the surfactant monolayer behavior such that in a small number of cycles the compressibility is indistinguishable from that of pure DPPC. Furthermore, SP-A reduces the incidence of bubble "clicking," suggesting a stabilization of the monolayer at low surface tensions. In dynamic cycling experiments, SP-A reduces compression of the film area required to achieve a low surface tension of approximately 1 mN/m. SP-A eliminated the plateau just below 25 mN/m normally observed during the compression phase with low concentrations of LES and the shoulder observed at approximately 35 mN/m during expansion. In the presence of SP-A and, to a lesser extent with high concentrations of LES, there is a marked lag in the increase in surface tension during the initial part of the dynamic expansion loop, with surface tensions remaining near 1 mN/m for approximately 10% of the increase in bubble area. The results indicate that SP-A enhances phospholipid adsorption during dynamic cycling and may enhance elimination of non-DPPC lipids during cycling.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗

PubMed1991

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.

Animals↗

PubMed1991

The role of palmitic acid in pulmonary surfactant: enhancement of surface activity and prevention of inhibition by blood proteins.

The surface activity of two surfactant preparations, Lipid Extract Surfactant (LES) and Survanta, was examined during adsorption and dynamic compression using a pulsating bubble surfactometer. At low surfactant phospholipid concentrations (1-2.5 mg/ml), Survanta reduces surface tension at minimum bubble radius faster than LES: however, with continued pulsation LES obtains a lower surface tension. Addition of surfactant-associated protein A (SP-A) to LES significantly reduces the time required to reduce surface tension. Survanta is completely unresponsive to the addition of SP-A in that no further reduction of surface tension is observed. Addition of various blood components has been previously shown to inactivate surfactants in vitro. Addition of fibrinogen to Survanta causes an increase in surface tension when measured in the absence of calcium. When assayed in the presence of calcium, inhibition by fibrinogen is not observed possibly due to aggregation of this protein. Albumin and alpha-globulin strongly inhibit Survanta at physiological serum concentrations both in the presence and absence of calcium. The surface activity of Survanta is also inhibited by lysophosphatidylcholine (lyso-PC). The role of palmitic acid in the surface activity of pulmonary surfactant was examined by adding palmitic acid to LES. At low phospholipid concentrations addition of palmitic acid (10% w/w of the surfactant phospholipid) greatly enhances the surface activity of LES. Maximal enhancement of surface activity and adsorption was observed at or above 7.5% added palmitic acid (w/w of surfactant lipid). LES supplemented with palmitic acid is more resistant to inhibition by fibrinogen, albumin, alpha-globulin and lyso-PC than LES alone, however, the counteraction of blood protein inhibition is not as pronounced as that observed with SP-A.

Albumins↗

PubMed1991

Rat liver and lung mitochondria do not incorporate radioactivity from glycerol-3-phosphate or CDP-choline into glycero-3-phosphocholine.

The proposed formation of glycero-3-phosphocholine (GPC) from glycerol-3-phosphate (GP) and CDP-choline catalysed by the enzyme GPC-synthetase has been examined in liver and lung subcellular fractions. Previous observations on the incorporation of radioactive GP into the GPC-spot on paper chromatograms have been interpreted as evidence for the GPC-dependent synthesis of phosphatidylcholine. Although we could reproduce this incorporation of GP, we could not detect any incorporation of radioactive CDP-choline into the GPC-spot using the same paper chromatographic system. TLC separation of the substrate and products showed no detectable formation of GPC with either radioactive substrate. These results strongly suggest that the previously reported formation of GPC in liver and lung was due to an inaccurate identification of the true radioactive products. We demonstrate that the major radioactive product formed in liver mitochondria is glucose. A small amount of radioactive glycerol was also detected. Lung mitochondria incorporate radioactive GP into glycerol and into another unidentified compound or compounds. It is concluded that the occurrence of the GPC dependent formation of phosphatidylcholine is unlikely.

Animals↗

PubMed1991

Surfactant analysis and replacement therapy: a future tool of the lung transplant surgeon?

From 1965 to 1974 extensive research was carried out concerning the effects of experimental lung reimplantation and allografting on the surface tension properties of pulmonary surfactant. Since then, surfactant has been more rigorously examined in terms of its composition and function, and the potential roles of three surfactant-associated proteins have been established. Furthermore, surfactant replacement therapy for neonatal respiratory distress syndrome has come of age. The efficacy of surfactant treatment for adult respiratory distress syndrome is currently under clinical scrutiny, and experimental work on alterations in surfactant after lung transplantation has resumed after a 15-year hiatus. This article reviews current knowledge of the pulmonary surfactant system, as well as previous studies of the changes in surfactant after experimental lung transplantation. The experience in surfactant replacement therapy for the neonatal and adult respiratory distress syndromes is briefly described. Suggestions are made concerning the potential experimental and clinical applications of surfactant analysis and replacement therapy in lung transplantation.

Adult↗

PubMed1991

Levels of surfactant-associated protein messenger ribonucleic acids in rabbit lung during perinatal development and after hormonal treatment.

The levels of messenger RNAs for the surfactant-associated proteins, SP-A, SP-B, and SP-C, have been examined in the developing rabbit lung in vivo. Northern blot analysis detected SP-C mRNA by day 22 of gestation (term 31 days) and SP-A mRNA and SP-B mRNA on day 26, while solution hybridization assays detected all three mRNAs on day 22 of gestation. Both techniques revealed that the mRNA levels increased rapidly during the last quarter of gestation. The mRNA levels determined by solution hybridization were highly correlated during development, with average molar ratios of 1.0:1.1:2.1 for SP-A, SP-B, and SP-C, respectively. We also examined the effect of accelerating fetal pulmonary maturation by maternal administration of either 17 beta-estradiol or betamethasone (9 alpha-fluoro-16 beta-methylprednisolone) on day 26 of gestation. These treatments increased SP-A mRNA levels 8- to 12-fold, resulting in levels 3- to 4-fold greater than in the adult. SP-B mRNA levels increased by approximately 2-fold to near adult levels, while SP-C mRNA was lowered somewhat by 17 beta-estradiol and significantly to less than half by betamethasone. No differences in the levels of surfactant apoprotein mRNAs or in choline incorporation into total or disaturated phosphatidylcholine were noted between male and female fetuses. These observations are consistent with the accepted view that the genes for the surfactant-associated proteins are independently regulated. However, the various factors affecting these mRNAs result in a coordination of mRNA levels during normal perinatal development.

Animals↗

PubMed1991

Bovine surfactant replacement therapy in neonates of less than 30 weeks' gestation: a randomized controlled trial of prophylaxis versus treatment.

The influence of the timing of surfactant replacement therapy for the treatment of neonatal respiratory distress syndrome was evaluated in a study of 182 neonates of less than 30 weeks' gestation who were randomly assigned prior to delivery to one of three study groups: control (dummy instillation of air given at birth), early surfactant (surfactant given at birth), or late surfactant (surfactant given at less than 6 hours of age). Subjects in the late surfactant group could avoid treatment if they had a clear chest roentgenogram and required no supplemental oxygen at a mean airway pressure of less than 7 cm of water. All treated neonates were eligible to receive up to three additional doses during the first 5 days of life. The three groups were comparable with respect to birth weight, gestational age, and other perinatal parameters with the exception of a lower cord arterial pH and 1-minute Apgar score in the early surfactant group. Of the 60 neonates randomly assigned to late treatment, 29 (48%) were deemed surfactant sufficient and thereby avoided treatment; the other 31 received their first dose at a mean age of 2.9 hours. There was a significant improvement in gas exchange during the first week of life in both surfactant groups compared with the control group, reflected by differences in fraction of inspired oxygen, arterial/alveolar PO2, and ventilation index (peak pressure x rate on the ventilator) (P less than .001). Surfactant therapy also resulted in a lower incidence of pulmonary air leak and severe chronic lung disease (defined as requirement for respiratory support beyond 36 weeks post-conceptional age). There were no differences between early and late surfactant groups in any of these parameters. The only statistically significant difference between the surfactant groups was that the early group had a higher incidence of mild chronic lung disease (respiratory support beyond 28 days of age) than the late treatment group (P less than .005). Neonates in the late treatment group were extubated earlier and had a shorter neonatal intensive care unit stay than control neonates (P less than .05), whereas those in the early group were not significantly different from control neonates in these parameters. It is concluded that replacement therapy with bovine lung surfactant extract in neonates of less than 30 weeks' gestation results in decreased oxygen and ventilatory requirements during the first week of life and a lower incidence of pulmonary air leak and severe chronic lung disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

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.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1990

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.

Animals↗

PubMed1990

Pulmonary surfactant-associated protein A enhances the surface activity of lipid extract surfactant and reverses inhibition by blood proteins in vitro.

Although a monolayer of dipalmitoylphosphatidylcholine, the major component of pulmonary surfactant, is thought to be responsible for the reduction of the surface tension at the air-liquid interface of the alveolus, the participation of unsaturated and anionic phospholipids and the three surfactant-associated proteins is suggested in the generation and maintenance of this surface-active monolayer. We have examined the effects of surfactant-associated protein A (SP-A) purified from bovine lavage material on the surface activity of lipid extract surfactant (LES), an organic extract of pulmonary surfactant containing all of the phospholipids and SP-B and SP-C, but lacking SP-A. Measurements of the surface tension during dynamic compression were made on a pulsating bubble surfactometer. Addition of SP-A to LES reduces the number of pulsations required to attain surface tensions near zero at minimum bubble radius. This increase in surface activity is dependent upon the presence of Ca2+ in the assay mixture. Maximal enhancement is observed at or below 1% of the lipid concentration (w/w). The addition of two blood proteins, fibrinogen and albumin, at physiological concentrations to LES causes severe inhibition of surface activity. Addition of SP-A in the presence of Ca2+ completely counteracts the inhibition by fibrinogen. The amount of SP-A required for full reversal of this inhibition was less than 0.5% of the lipid concentration. Complete reversal of inhibition by albumin was also observed, even though there was a approximately 5000-fold molar excess of inhibitor. Addition of lysophosphatidylcholine also inhibits LES; however, SP-A has no effect on this inhibition.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1990

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.

1,2-Dipalmitoylphosphatidylcholine↗