Lipid compositional analysis of pulmonary surfactant monolayers and monolayer-associated reservoirs
2003; Elsevier BV; Volume: 44; Issue: 3 Linguagem: Inglês
10.1194/jlr.m200380-jlr200
ISSN1539-7262
Autores Tópico(s)Neuroscience of respiration and sleep
ResumoPulmonary surfactant is a lipid:protein complex containing dipalmitoyl-phosphatidylcholine (DPPC) as the major component. Recent studies indicate adsorbed surfactant films consist of a surface monolayer and a monolayer-associated reservoir. It has been hypothesized that the monolayer and its functionally contiguous reservoir may be enriched in DPPC relative to bulk phase surfactant. We investigated the compositional relationship between the monolayer and its reservoir using paper-supported wet bridges to transfer films from adsorbing dishes to clean surfaces on spreading dishes. Spreading films appear to form monolayers in the spreading dishes. We employed bovine lipid extract surfactant [BLES(chol)] containing [3H]DPPC and either [14C]palmitoyl, oleoyl-phosphatidylcholine (POPC), [14C]dipalmitoyl-phosphatidylglycerol (DPPG), [14C]palmitoyl, oleoyl-phosphatidylglycerol (POPG), or [14C]cholesterol. Radiolabeled phosphatidylglycerols were prepared using phospholipase D. The studies demonstrated that the [3H]DPPC-[14C] POPC ratios were the same in the prepared BLES dispersions as in Langmuir-Blodgett films, indicating a lack of DPPC selectivity during film formation. Furthermore, identical 3H-14C isotopic ratios were observed with DPPC and either 14C-labeled POPC, DPPG, POPG, or cholesterol in the original dispersions, the bulk phases in adsorption dish D1, and monolayers recovered from spreading dish D2. These relationships remained unperturbed with 2-fold increases in bulk concentrations in D1 and 10-fold variations in D1-D2 surface area.These results indicate adsorbed surfactant monolayers and their associated reservoirs possess similar lipid compositions and argue against selective adsorption of DPPC. Pulmonary surfactant is a lipid:protein complex containing dipalmitoyl-phosphatidylcholine (DPPC) as the major component. Recent studies indicate adsorbed surfactant films consist of a surface monolayer and a monolayer-associated reservoir. It has been hypothesized that the monolayer and its functionally contiguous reservoir may be enriched in DPPC relative to bulk phase surfactant. We investigated the compositional relationship between the monolayer and its reservoir using paper-supported wet bridges to transfer films from adsorbing dishes to clean surfaces on spreading dishes. Spreading films appear to form monolayers in the spreading dishes. We employed bovine lipid extract surfactant [BLES(chol)] containing [3H]DPPC and either [14C]palmitoyl, oleoyl-phosphatidylcholine (POPC), [14C]dipalmitoyl-phosphatidylglycerol (DPPG), [14C]palmitoyl, oleoyl-phosphatidylglycerol (POPG), or [14C]cholesterol. Radiolabeled phosphatidylglycerols were prepared using phospholipase D. The studies demonstrated that the [3H]DPPC-[14C] POPC ratios were the same in the prepared BLES dispersions as in Langmuir-Blodgett films, indicating a lack of DPPC selectivity during film formation. Furthermore, identical 3H-14C isotopic ratios were observed with DPPC and either 14C-labeled POPC, DPPG, POPG, or cholesterol in the original dispersions, the bulk phases in adsorption dish D1, and monolayers recovered from spreading dish D2. These relationships remained unperturbed with 2-fold increases in bulk concentrations in D1 and 10-fold variations in D1-D2 surface area. These results indicate adsorbed surfactant monolayers and their associated reservoirs possess similar lipid compositions and argue against selective adsorption of DPPC. It is generally agreed that the alveolar surface is covered by a continuous thin layer of water that supports a surface active film of pulmonary surfactant (1Bastacky J. Lee C.Y. Goerke J. Koushafar J.H. Yager D. Kenaga L. Speed T.P. Chen Y. Clements J.A. Alveolar lining layer is thin and continuous: low-temperature scanning electron microscopy of rat lung.J. Appl. Physiol. 1995; 79: 1615-1628Google Scholar, 2Manabe T. Freeze-fracture study of alveolar lining layer in adult rat lung homogenates.J. Ultrastruct. Res. 1979; 69: 86-97Google Scholar) [for review see (3Goerke J. Pulmonary surfactant: Functions and molecular composition.Biochim. Biophys. Acta. 1998; 1408: 79-89Google Scholar, 4Perez-Gil J. Keough K.M.W. Interfacial properties of surfactant proteins.Biochim. Biophys. Acta. 1998; 1408: 203-217Google Scholar, 5Possmayer F. Physicochemical aspects of pulmonary surfactant.in: Polin R.A. Fox W.W. Fetal and Neonatal Physiology. W. B. Saunders Company, Philadelphia, PA1997: 1259-1275Google Scholar, 6Possmayer F. Nag K. Rodriguez K. Qanbar R. Schürch S. Surface activity in vitro: role of surfactant proteins.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 209-220Google Scholar, 7Veldhuizen R. Nag K. Orgeig S. Possmayer F. The role of lipids in pulmonary surfactant.Biochim. Biophys. Acta. 1998; 1408: 90-108Google Scholar)]. Through its ability to reduce the surface tension of this air-water interface, pulmonary surfactant stabilizes the terminal air spaces. Considerable evidence has accumulated indicating that surfactant films are composed of more than a single monolayer. Pattle (8Pattle R. Properties, function and origin of the alveolar lining layer.Nature. 1955; 175: 1125-1126Google Scholar) first proposed that the surfactant film overlying the alveolar lining layer consists of a monomolecular layer and underlying material that serves as a reservoir. Using electron microscopy, Weibel and Gil (9Weibel E.R. Gil J. Electron microscopic demonstration of an extracellular duplex lining layer of the alveoli.Resp. Physiol. 1968; 4: 42-57Google Scholar) observed the presence of lamellar layers of phospholipids with three to six repeating distances of 38–51 Å on the alveolar epithelial surface of rat lungs. Studies by Manabe (2Manabe T. Freeze-fracture study of alveolar lining layer in adult rat lung homogenates.J. Ultrastruct. Res. 1979; 69: 86-97Google Scholar) and, more recently, Bastacky et al. (1Bastacky J. Lee C.Y. Goerke J. Koushafar J.H. Yager D. Kenaga L. Speed T.P. Chen Y. Clements J.A. Alveolar lining layer is thin and continuous: low-temperature scanning electron microscopy of rat lung.J. Appl. Physiol. 1995; 79: 1615-1628Google Scholar) using scanning electron microscopic studies indicate that the alveolar lining layer is continuous and its surface contains many lipidic structures. In vitro studies involving surface films adsorbed from surfactant dispersions have also provided evidence indicating the surface monolayer is accompanied by a functional continuous reservoir (10Schürch S. Green F.H.Y. Bachofen H. Formation and structure of surface films: captive bubble surfactometry.Biochim. Biophys. Acta. 1998; 1408: 180-202Google Scholar, 11Schürch S. Bachofen H. 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DPPC, the main component of pulmonary surfactant, possesses a bilayer gel-to-liquid crystal transition temperature of 41°C and an overall cylindrical shape (31Cullis P.R. de Kruijff B. Lipid polymorphism and the functional roles of lipids in biological membranes.Biochim. Biophys. Acta. 1979; 559: 399-420Google Scholar, 32Hope M.J. Mui B. Ansell S. Ahkong Q.F. Cationic lipids, phosphatidylethanolamine and the intracellular delivery of polymeric, nucleic acid-based drugs (review).Mol. Membr. Biol. 1998; 15: 1-14Google Scholar), characteristics that favor the ability of DPPC to sustain high surface pressures of close to 70 mN/m at physiological temperatures. These properties allow spread monolayers of DPPC to reduce surface tension to low values, approaching 0 mN/m on the Langmuir-Wilhelmy surface balance, whereas spread monolayers of unsaturated phospholipids collapse near the equilibrium surface tension of ∼24 mN/m (3Goerke J. Pulmonary surfactant: Functions and molecular composition.Biochim. Biophys. Acta. 1998; 1408: 79-89Google Scholar, 5Possmayer F. Physicochemical aspects of pulmonary surfactant.in: Polin R.A. Fox W.W. Fetal and Neonatal Physiology. W. B. Saunders Company, Philadelphia, PA1997: 1259-1275Google Scholar, 6Possmayer F. Nag K. Rodriguez K. Qanbar R. Schürch S. Surface activity in vitro: role of surfactant proteins.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 209-220Google Scholar, 30Notter R.H. Wang Z. Pulmonary surfactant: physical chemistry, physiology, and replacement.Rev. Chem. Engin. 1997; 13: 1-118Google Scholar, 33Goerke J. Lung surfactant.Biochim. Biophys. Acta. 1974; 344: 241-261Google Scholar). However, at physiological temperatures, liposomes of DPPC adsorb very slowly. Since natural and lipid extract surfactants adsorb very rapidly, the other surfactant components appear critical for adsorption of DPPC into the surface film. The ability of surfactant films to attain low surface tension approaching 0 mN/m during compression (8Pattle R. Properties, function and origin of the alveolar lining layer.Nature. 1955; 175: 1125-1126Google Scholar, 34Clements J. Surface tension of lung extracts.Proc. Soc. Exp. Biol. Med. 1957; 95: 170-172Google Scholar, 35Hildebran J.N. Goerke J. Clements J.A. Pulmonary surface film stability and composition.J. Appl. Physiol. 1979; 47: 604-611Google Scholar, 36Goerke J. Gonzales J. Temperature dependence of dipalmitoyl phosphatidylcholine monolayer stability.J. Appl. Physiol. 1981; 51: 1108-1114Google Scholar) has long been attributed to the formation of a monolayer highly enriched in DPPC [as reviewed in refs. (3Goerke J. Pulmonary surfactant: Functions and molecular composition.Biochim. Biophys. Acta. 1998; 1408: 79-89Google Scholar, 4Perez-Gil J. Keough K.M.W. Interfacial properties of surfactant proteins.Biochim. Biophys. Acta. 1998; 1408: 203-217Google Scholar, 5Possmayer F. Physicochemical aspects of pulmonary surfactant.in: Polin R.A. Fox W.W. Fetal and Neonatal Physiology. W. B. Saunders Company, Philadelphia, PA1997: 1259-1275Google Scholar, 6Possmayer F. Nag K. Rodriguez K. Qanbar R. Schürch S. Surface activity in vitro: role of surfactant proteins.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 209-220Google Scholar, 33Goerke J. Lung surfactant.Biochim. Biophys. Acta. 1974; 344: 241-261Google Scholar, 37Schüurch S. Qanbar R. Bachofen H. Possmayer F. The surface-associated surfactant reservoir in the alveolar lining.Biol. Neonate. 1995; 67: 61-76Google Scholar, 38Schürch S. Bachofen H. Possmayer F. Surface activity in situ, in vivo, and in the captive bubble surfactometer.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 195-207Google Scholar)]. It has been suggested that DPPC enrichment during film compression arises through squeeze-out of the more fluid, non-DPPC lipids (39Bangham A.D. Morley C.J. Phillips M.C. The physical properties of an effective lung surfactant.Biochim. Biophys. Acta. 1979; 573: 552-556Google Scholar, 40Clements J.A. Functions of the alveolar lining.Am. Rev. Respir. Dis. 1977; 115: 67-71Google Scholar, 41Watkins J.C. The surface properties of pure phospholipid to those of lung extracts.Biochim. Biophys. Acta. 1968; 152: 293-306Google Scholar). Repeated compression-expansion cycles during breathing could result in monolayers highly enriched in DPPC by this mechanism. The presence of a surface monolayer highly enriched in DPPC is consistent with the properties of lung (42Horie T. Hildebrandt J. Dynamic compliance, limit cycles, and static equilibria of excised cat lung.J. Appl. Physiol. 1971; 31: 423-430Google Scholar). Recent physicochemical measurements have demonstrated that surface area reductions required to attain surface tensions near 0 mN/m during initial film compression of adsorbed surfactant films can be lower than that predicted by the DPPC content of surfactant (37Schüurch S. Qanbar R. Bachofen H. Possmayer F. The surface-associated surfactant reservoir in the alveolar lining.Biol. Neonate. 1995; 67: 61-76Google Scholar, 43Schürch S. Bachofen H. Goerke J. Possmayer F. A captive bubble method reproduces the in situ behavior of lung surfactant monolayers.J. Appl. Physiol. 1989; 67: 2389-2396Google Scholar, 44Qanbar R. Cheng S. Possmayer F. Schürch S. Role of the palmitoylation of surfactant-associated protein C in surfactant film formation and stability.Am. J. Physiol. 1996; 271: L572-L580Google Scholar, 45Nag K. Munro J.G. Inchley K. Schürch S. Petersen N.O. Possmayer F. SP-B refining of pulmonary surfactant phospholipid films.Am. J. Physiol. 1999; 277: L1179-L1189Google Scholar, 46Rodriguez Capote K. Nag K. Schürch S. Possmayer F. Surfactant protein interactions with neutral and acidic phospholipid films.Am. J. Physiol. Lung Cell. Mol. Physiol. 2001; 281: L231-L242Google Scholar). These observations led to the proposal that the surface monolayer may become enriched in DPPC during adsorption (3Goerke J. Pulmonary surfactant: Functions and molecular composition.Biochim. Biophys. Acta. 1998; 1408: 79-89Google Scholar, 4Perez-Gil J. Keough K.M.W. Interfacial properties of surfactant proteins.Biochim. Biophys. Acta. 1998; 1408: 203-217Google Scholar, 5Possmayer F. Physicochemical aspects of pulmonary surfactant.in: Polin R.A. Fox W.W. Fetal and Neonatal Physiology. W. B. Saunders Company, Philadelphia, PA1997: 1259-1275Google Scholar, 6Possmayer F. Nag K. Rodriguez K. Qanbar R. Schürch S. Surface activity in vitro: role of surfactant proteins.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 209-220Google Scholar, 37Schüurch S. Qanbar R. Bachofen H. Possmayer F. The surface-associated surfactant reservoir in the alveolar lining.Biol. Neonate. 1995; 67: 61-76Google Scholar, 38Schürch S. Bachofen H. Possmayer F. Surface activity in situ, in vivo, and in the captive bubble surfactometer.Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2001; 129: 195-207Google Scholar). We have previously used a filter paper-supported wet bridge technique (47Heyn S.P. Egger M. Gaub H.E. Lipid and lipid-protein monolayer spread from a vesicle suspension: a microfluorescence film balance study.J. Phys. Chem. 1990; 94: 5073-5078Google Scholar, 48Schindler H. Exchange and interactions between lipid layers at the surface of a liposome solution.Biochim. Biophys. Acta. 1979; 555: 316-336Google Scholar) to transfer lipid from the surface of adsorbing dishes containing surfactant dispersions to the surface of spreading dishes (13Yu S-H. Possmayer F. Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant.J. Lipid Res. 2001; 42: 1421-1429Google Scholar). The small quantities of material present in the transferred films made it difficult to measure lipid compositions accurately. Nevertheless, we obtained evidence indicating both [14C]DPPC and [14C]cholesterol were incorporated into the surface monolayer (13Yu S-H. Possmayer F. Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant.J. Lipid Res. 2001; 42: 1421-1429Google Scholar). We also obtained evidence indicating the underlying reservoir was functionally contiguous with the surface monolayer. However, the lipid compositions of the adsorbed monolayer and its associated reservoir have not been directly determined. In the present studies, we have used [3H]DPPC and [14C]POPC-labeled BLES(chol) to compare the relative amounts of these radiolipids in the bulk phase, the surface monolayer, and the surface-associated reservoir. In addition, [14C]DPPG and [14C]POPG, which are not commercially available, were prepared enzymatically and used for similar experiments. [3H]DPPC and [14C]cholesterol mixed with the bovine surfactant extract were also studied. Taken together, our results indicate that the [3H]DPPC content of the adsorbed monolayers is not enriched relative to the surface-associated reservoir or lipid extract surfactant in the bulk phase. [4-14C]cholesterol, [2-3H]DPPC, and 1-palmitoyl-2-oleoyl[1-14C]PC were purchased from New England Nuclear (Boston, MA). Phospholipase D prepared from Savoy cabbage was a gift from Drs. D. R. Voelker and M. K. Storey (Anna Perahia Adatto Clinical Research Center, Denver, CO). Bio-Sil A (100-200 mesh) was from Bio-Rad (Richmond, CA). Unless indicated, all other chemicals and reagents were from BHD (Poole, UK). Concentrations of 3H and 14C radioactivities were verified with a scintillation counter (LS 6000 5C; Beckman, Fullerton, CA) using Beckman ReadySolve HP scintillation fluid. Distilled water purified through a Millipore (Danvers, MA) Milli-Q four-cartridge system was used in all experiments. BLES(chol) was obtained from natural bovine pulmonary surfactant, kindly provided by BLES Biochemicals, through chloroform-methanol extraction by the method of Bligh and Dyer (49Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar), as described previously (12Yu S-H. Possmayer F. Effect of pulmonary surfactant protein A and neutral lipid on accretion and organization of dipalmitoylphosphatidylcholine in surface films.J. Lipid Res. 1996; 37: 1278-1288Google Scholar). BLES(chol) retains all lipid components of surfactant and surfactant proteins, SP-B and SP-C, but not SP-A or SP-D. BLES(chol) differs from BLES® used clinically, which has the neutral lipids removed (20Yu S. Harding P.G. Smith N. Possmayer F. Bovine pulmonary surfactant: chemical composition and physical properties.Lipids. 1983; 18: 522-529Google Scholar). [14C]DPPG and [14C]POPG were prepared from the corresponding [14C]PCs through transphosphorylation catalyzed by phospholipase D using a modification of the method of Comfurius and Zwaal (50Comfurius P. Zwaal R.F. The enzymatic synthesis of phosphatidylserine and purification by CM-cellulose column chromatography.Biochim. Biophys. Acta. 1977; 488: 36-42Google Scholar). Briefly, 100 μg [14C]PC in 200 μl diethylether was mixed with 100 μg phospholipase D in 100 μl buffer of 200 mM sodium acetate-CaCl2, pH 5.6, and 100 μl 50% glycerol in the same buffer. The mixture was shaken vigorously with a Multi-Mixer (Lab Line Instruments, Inc., Melrose Park, IL) at room temperature for about 1 h until the yield of [14C]PG was at least 80%, as detected by TLC using a solvent system (chloroform-methanol-water-triethylamine; 30:34:8:35, v/v/v/v) described by Touchstone et al. (51Touchstone J.C. Chen J.C. Beaver K.M. Improved separation of phospholipids in thin layer chromatography.Lipids. 1980; 15: 61-62Google Scholar). The reaction was stopped with 100 μl of 200 mM EDTA. Diethylether was evaporated and the [14C]phospholipids were extracted with chloroform-methanol following the procedure of Bligh and Dyer (49Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). The [14C]PGs were purified by silicic acid column chromatography. The column was washed with 10 vol of chloroform. The [14C]phospholipids dissolved in chloroform were applied into the column and eluted with a step-gradient of chloroform-methanol. The [14C]PGs were eluted with chloroform-methanol, 9:1 (v/v). Radiolabeled purity was confirmed by TLC, as above. The desired amount of BLES(chol) was mixed with [3H]DPPC and either [14C]POPC, [14C]DPPG, [14C]POPG, or [14C]cholesterol in chloroform-methanol, 9:1 (v/v). The solvent was evaporated under N2, and the residue was hydrated with 150 mM NaCl-1.5 mM CaCl2 and 5 mM Hepes, pH 7.4 (1 mg/100μl). The suspension was shaken with a Multi-Mixer at room temperature for about 100 min and incubated at 37°C for 2–3 h. Ten microliter samples were transferred to a counting vial as a sample of the initial dispersion before injection into dish 1 (D1, see the next section). The specific radioactivity of the [3H]DPPC used was 0.5–1.5 μCi/mg lipid, and that of [14C]POPC, 0.25–0.5 μCi/mg; [14C]DPPG, 0.5 μCi/mg; [14C]POPG, 0.5 μCi/mg; and cholesterol, 0.5–1 μCi/mg lipid. Two Teflon dishes, dish 1 (D1) and dish 2 (D2), containing 150 mM NaCl-1.5 mM CaCl2 and 5 mM Hepes (pH 7.4) in a 37°C water bath, were connected with a 1 × 2.5 cm2 strip of ashless filter paper that was suspended with a Teflon tape-wrapped wire (Fig. 1). A 5 mm-wide platinum plate dipped into D2 served to monitor surface tension. The surface area of D1 was 2, 2.5 or 20 cm2 and that of D2 was 10, 12.5, or 20 cm2, providing surface area ratios for D1-D2 of 2:1, 1:1, and 1:10. After 30 min of equilibration, a 37°C preincubated sample of [3H]DPPC and [14C]POPC, [14C]DPPG, [14C]POPG, or [14C]cholesterol-labeled BLES(chol) was injected into D1. The final concentration of BLES(chol) in D1 was 0.15 mg/ml for D1-D2 = 2:1, 0.25 mg/ml for D1-D2 = 1:1, and 0.5 mg/ml for D1-D2 = 1:10. The filter paper was removed within 15 min after the equilibrium surface tension (∼24 mN/m) in D2 was reached. All experiments were performed in a temperature-controlled box at 37 ± 0.5°C. The surface monolayer in D2 was transferred with a pipette into a test tube, and lipids were extracted with chloroform-methanol, 1:1 (v/v) (49Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar). The chloroform layer was transferred to a counting vial, and solvent was evaporated under N2. Twenty microliters of the dispersion from the bulk (D1) were taken and transferred to a counting vial at the end of each experiment. Five microliters of scintillation fluid was added into all vials, and the values of [3H]DPPC-[14C]lipid were obtained from the scintillation counter. Radioactive counting was continued to preset errors of <1%. L-B films were deposited from adsorbed dispersions of [3H]DPPC- and [14C]POPC-labeled BLES(chol) on 1 × 1 cm2 microscope glass cover slips at 37°C as described previously (12Yu S-H. Possmayer F. Effect of pulmonary surfactant protein A and neutral lipid on accretion and organization of dipalmitoylphosphatidylcholine in surface films.J. Lipid Res. 1996; 37: 1278-1288Google Scholar). Surface tension was maintained at 24 mN/m during deposition. The films were eluted with chloroform-methanol, 1:1 (v/v), into a counting vial. Solvent was evaporated under N2, and the residues were dissolved in 5 ml scintillation fluid. Values of [3H]DPPC-[14C]POPC were obtained from scintillation counting. Initial experiments investigated the transfer of radiolabeled PCs from the bulk phase to the air-water interface during adsorption. L-B films were deposited from dispersions of [3H]DPPC- and [14C]POPC-labeled BLES(chol). Table 1 reveals that the eluted L-B films contained DPPC to POPC ratios similar to those in the initial dispersions and in samples recovered from the bulk phase. In previous studies, we have observed that the L-B films deposited in this manner contained more radioactivity than could arise from a single monolayer (12Yu S-H. Possmayer F. Effect of pulmonary surfactant protein A and neutral lipid on accretion and organization of dipalmitoylphosphatidylcholine in surface films.J. Lipid Res. 1996; 37: 1278-1288Google Scholar). In addition, X-ray autoradiography revealed the presence of highly intense radioactive areas, suggesting lipid aggregates. These observations indicated that L-B films prepared from adsorbed BLES(chol) films include both the surface monolayer and associated material. The present data indicate that the relative concentrations of DPPC and POPC in the adsorbed films are same as in the bulk phase and are representative of the original injected dispersions.TABLE 1[3H]DPPC-[14C]POPC isotopic ratios in L-B films deposited from [3H]DPPC and [14C]POPC labeled dispersions of BLES(chol)Initial Dispersionsa"Initial Dispersions" indicates samples hydrated and incubated at 37°C prior to injection into adsorbing dish D1. Three individual samples were prepared and each was tested once.L-B FilmsbL-B films were eluted with chloroform-methanol, 1:1 (v/v), and radioactivity determined by scintillation counting.% of Initial DispersionBulk PhasecBulk phase samples were taken from the subphase of the adsorbing dish after deposition of the L-B films.% of Initial Dispersion1.8191.826(100.4)1.821(100.1)2.2342.258(101.1)2.258(101.1)2.7122.760(101.8)2.669(98.4)101.1 ± 0.40 (n = 3)99.9 ± 0.78 (n = 3)a "Initial Dispersions" indicates samples hydrated and incubated at 37°C prior to injection into adsorbing dish D1. Three individual samples were prepared and each was tested once.b L-B films were eluted with chloroform-methanol, 1:1 (v/v), and radioactivity determined by scintillation counting.c Bulk phase samples were taken from the subphase of the adsorbing dish after deposition of the L-B films. Open table in a new tab Table 2 displays [3H]DPPC-[14C]POPC ratios in surface films recovered from spreading dish D2 that arise after injection of [3H]DPPC-[14C]POPC-labeled BLES dispersions into adsorbing dish D1. With surface areas D1 = D2 (Table 2), the 3H-14C ratios of the recovered surfactant were within a percentage point of that of the original dispersion and of BLES(chol) recovered from the bulk phase of D1 after equilibrium surface tension had been achieved. This is within the counting error of the procedures used. Th
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