EDITORIAL FOCUS
Content-dependent activity of lung surfactant protein B in mixtures with lipids

Z. Wang1, J. E. Baatz2, B. A. Holm3,4, and R. H. Notter1,5,6

1 Departments of Pediatrics, 5 Environmental Medicine, and 6 Chemical Engineering, University of Rochester, Rochester 14642; 3 Departments of Pediatrics and 4 Obstetrics and Gynecology, State University of New York at Buffalo, Buffalo, New York 14214; and 2 Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B approx  SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels <= 0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions <1 mN/m in pulsating bubble studies at low phospholipid concentration (1 mg/ml). Mixtures of PPL or SL with SP-B (0.5%) also had minimum surface tensions <1 mN/m at 1 mg/ml, whereas DPPC/SP-B (0.5%) reached <1 mN/m at 2.5 mg/ml. Physiological activity also was strongly dependent on SP-B content. The ability of instilled SL/SP-B mixtures to improve surfactant-deficient pressure-volume mechanics in excised lavaged rat lungs increased as SP-B content was raised from 0.1 to 0.75% by weight. This study emphasizes the crucial functional activity of SP-B in lung surfactants. Significant differences in SP-B content between exogenous surfactants used to treat respiratory disease could be associated with substantial activity variations.

surfactant proteins; lung surfactant; exogenous surfactants; calf lung surfactant extract


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOGENOUS PULMONARY SURFACTANT is a complex mixture of lipids and three biophysically active proteins: surfactant protein (SP)-A, SP-B, and SP-C. The physiological necessity of surfactant in respiratory function has led to extensive research on the biophysics, cell and molecular biology, and physiology of this complex material (11, 20, 34, 38). Although lipids (primarily phospholipids) comprise the great majority of lung surfactant in a quantitative sense, the apoprotein constituents of the surfactant system are highly active and crucial in a functional sense. This is also the case in most of the exogenous surfactant preparations used in the therapy of the neonatal and acute respiratory distress syndromes. The hydrophobic surfactant proteins SP-B and SP-C both have strong molecular interactions with lipids. Because of its combination of polar and nonpolar residues and overall amphipathic structure, SP-B can interact with both phospholipid head groups and fatty chains and is particularly active in enhancing surface active behavior in endogenous and exogenous lung surfactants (13, 25, 34, 44, 46, 48, 56, 58). However, the content-dependent effects of SP-B on the surface and physiological activity of lipids have not been fully quantitated.

A better understanding of SP-B's content-dependent actions in mixtures with lipids is directly relevant for the function of animal-derived clinical exogenous surfactants, which vary significantly in their compositional levels of this apoprotein (4, 19, 39, 47). The present study examines the effects of ascending amounts of bovine SP-B in increasing the adsorption and dynamic surface activity of 1,2 dipalmitoyl-sn-3-phosphocholine (DPPC), mixed synthetic lipids (SL), and purified surfactant phospholipids (PPL) isolated by gel permeation chromatography from calf lung surfactant extract (CLSE). Adsorption is measured in an apparatus with a stirred subphase to remove diffusion resistance, and dynamic surface tension lowering is assessed during cycling at rapid physiological rates in a pulsating bubble surfactometer. Surface pressure-area (pi -A) isotherms and dynamic respreading are also defined for solvent-spread phospholipid-SP-B films at the air-water interface in a Wilhelmy surface balance. In addition to surfactant biophysical studies, the content-dependent activity of SP-B combined with synthetic lipids is examined in a well-characterized excised rat lung model used in the quality control manufacture of several Food and Drug Administration-approved clinical exogenous surfactants (Survanta, Infasurf) (3). Biophysical and excised lung experiments are used to study the activity of SP-B contents incrementally varying from 0.05 to 0.75% by weight in mixtures with lipids.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Synthetic phospholipids and lipids. Synthetic DPPC, egg phosphatidylcholine (egg PC), egg phosphatidylglycerol (egg PG), egg phosphatidylinositol (egg PI), egg phosphatidylethanolamine (egg PE), and bovine brain sphingomyelin (Sph) for activity studies were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol was reagent grade from Sigma (St. Louis, MO). DPPC was >99% pure as supplied and gave a single well-defined spot on thin-layer chromatography with solvent system C of Touchstone et al. (51). Egg PC, egg PG, egg PI, egg PE, and Sph gave broader single spots on thin-layer analysis, consistent with their content of multiple fatty chain derivatives. Activity experiments utilized either pure DPPC or SL, the latter roughly based on the lipid composition of lavaged lung surfactant (SL = 40:40:10:5:2.5:2.5 molar ratio DPPC/egg PC/egg PG/egg PI/egg PE/Sph + 5% cholesterol).

CLSE and PPL. Mixed phospholipids were isolated by gel permeation chromatography from solvent-extracted, lavaged calf lung surfactant (18). Intact lungs from freshly killed calves (Gold Medal Packing, Rome, NY) were lavaged with 2-3 l of cold 0.15 M NaCl. Lavage fluid was immediately centrifuged at 12,000 g to obtain whole surfactant, which was then extracted with chloroform-methanol (5) to obtain CLSE. The composition of CLSE by weight was 93% phospholipid, 4.5% cholesterol, 1.5% protein (a mixture of SP-B and SP-C), and 1% other. The phospholipid class distribution was ~85% PC, 5% PG, 4% PI (including phosphatidylserine), 3% PE, 1% Sph, and 2% other (51). ELISA testing using a monoclonal antibody to bovine SP-B (rabbit derived) has reported a level of 0.9% SP-B by weight relative to phospholipid in CLSE material similar to that used here (39). Mixed lung surfactant phospholipids (PPL) were isolated from CLSE by column chromatography with an elution solvent of 1:1 (vol/vol) chloroform-methanol plus 5% 0.1 N HCl (18). Two passes through a Sephadex LH-20 column (Pharmacia-LKB Biotechnology, Piscataway, NJ) separated phospholipids away from hydrophobic surfactant apoproteins and neutral lipids. Acid remaining in PPL fractions was removed by a second chloroform-methanol extraction. Final PPL fractions had a protein content below the limits of detection of both the Folin-phenol reagent assay of Lowry et al. (27) and the amido black assay of Kaplin and Pedersen (26) modified by the addition of 5-15% sodium dodecyl sulfate (SDS).

Purified SP-B. SP-B was purified from CLSE on a 450-ml Silica C8 column with 7:1:0.4 methanol-chloroform-0.1 N HCl or 7:1:0.4 methanol-chloroform-H2O + 0.1% trifluoroacetic acid as elution solvents. Eluted fractions from the first and second column passes were screened by an ultraviolet detector (Pharmacia Biotech, Uppsala, Sweden) at 254 nm, and final pooled SP-B isolates were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and NH2-terminal amino acid analysis. Fractions for SDS-PAGE were dried, resuspended in NuPage sample buffer (Novex, San Diego, CA), and applied to 4-10% gradient acrylamide NuPage gels. Electrophoresis was at 200 V for 40 min with a 2-(N-morpholino)ethanesulfonic acid buffer (MES; Novex) containing (in mM) 50 MES, 50 Tris base, 3.5 SDS, and 1 EDTA, pH 7.7. Silver staining for detection of protein bands was according to Morrissey (29). NH2-terminal sequence analysis (7 cycles) was done with an Applied Biosystems Procise amino acid analyzer (pure SP-B yielded a single sequence of Phe-Pro-Ile-Pro-Ile-Pro-Tyr). SP-B fractions were also analyzed by Western blots using rabbit anti-bovine SP-B antibody. Proteins for Western blots were transferred from gels to 0.22-µm nitrocellulose using a Novex electrotransfer unit with current preset at 3 mA/cm2 and a 60-min transfer time. After transfer, the nitrocellulose was blocked in 25 mM Tris base buffer, pH 7.7, containing 0.05% vol/vol Tween 20 and 5% wt/vol nonfat dry milk (Blotto), followed by three 15-min washes with 25 mM Tris base and 0.05% Tween 20, pH 7.7. The nitrocellulose was then incubated in primary polyclonal antibody (rabbit anti-bovine SP-B antibody) in Blotto (1:10,000 dilution) for 60 min, washed several times with Tris-Tween buffer, incubated for 30 min with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2,500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), washed with 25 mM Tris base (pH 7.7), and incubated with Luminol reagent (Santa Cruz Biotechnology) for 60 s followed by removal of excess reagent and film development.

Lipid-SP-B mixture formulation. DPPC, PPL, or SL were combined with SP-B as follows. Appropriate amounts of lipids dissolved in chloroform were initially mixed with SP-B in chloroform-methanol and evaporated to dryness under nitrogen. Amounts of phospholipid were determined by phosphate assay (7), and levels of SP-B protein were based on the Folin-phenol reagent assay of Lowry et al. (27) modified by the addition of 15% SDS. Dried lipid-SP-B mixtures were redissolved in 9:1 vol/vol hexane-ethanol (HPLC grade) for Wilhelmy balance film studies or were dispersed at desired concentrations in buffered saline (10 mM HEPES, 1.5 mM CaCl2, 150 mM NaCl, pH 7.0) by probe sonication for adsorption, pulsating bubble, or excised lung studies. Dispersion was with a Heat Systems sonicator (model W-220F) at 40 W of power applied for three to five 15-s bursts on ice. Highly pure distilled and deionized water (Milli-Q UV Plus system; Millipore, Bedford, MA) was used in all dispersions.

Adsorption methods. Adsorption was measured at 37 ± 0.5°C for surfactant dispersions in a Teflon dish containing a 35-ml buffered subphase (10 mM HEPES, 1.5 mM CaCl2, 150 mM NaCl, pH 7.0) (33, 35). The subphase was agitated continuously by a Teflon-coated bar and magnetic stirrer to minimize diffusion resistance. At time zero, a bolus of surfactant dispersion (2.5 mg phospholipid/5 ml buffer) was injected into the stirred subphase, and surface pressure (surface tension lowering below that of the pure subphase) was measured as a function of time by a sandblasted platinum slide connected to a force transducer. Final subphase concentration for adsorption studies was uniform at 0.063 mg phospholipid/ml (2.5 mg/40 ml of total subphase).

Pulsating bubble surfactometer methods. The ability of surfactant mixtures to lower surface tension during rapid cycling at 37°C was measured using a pulsating bubble surfactometer (General Transco, Largo, FL; formerly Electronetics, Amherst, NY) (15). An air bubble, communicating with ambient air, was formed in a buffered suspension of surfactant held in a 40-µl plastic sample chamber. The sample chamber was mounted on the instrument pulsator unit, and the bubble was repetitively oscillated between maximum and minimum radii of 0.55 and 0.4 mm at a rate of 20 complete cycles of compression-expansion per minute. The pressure drop across the air-water interface was measured by a precision pressure transducer. Surface tension at minimum bubble radius (minimum surface tension) was calculated as a function of time from the spherical form of the Laplace equation (16). Surfactant dispersions were studied in the pulsating bubble apparatus at concentrations of both 1 and 2.5 mg phospholipid/ml to increase the sensitivity of measurements to discern the relative activity of the different mixtures studied.

Wilhelmy balance methods. pi -A isotherms were measured for solvent-spread interfacial films in a modified Wilhelmy surface balance incorporating a Teflon trough and ribbon barrier (49). Surfactants dissolved in hexane-ethanol (9:1, vol/vol) were spread dropwise from a syringe at the surface of a buffered subphase (10 mM HEPES, 1.5 mM CaCl2, 150 mM NaCl, pH 7.0) in the balance trough. Surfactant films were spread to a uniform initial "surface excess" concentration of 15 Å2/molecule to examine film behavior during dynamic cycling in the collapse regime. Typical volumes of spreading solvent were 0.4 ml, with a 10-min pause allowed for solvent evaporation before dynamic cycling. Surface pressure pi  (the amount by which surface tension was lowered below that of the pure subphase) was measured during cycling from the force on a sand-blasted platinum Wilhelmy slide dipped into the ribbon-confined interface. A second slide outside the ribbon barrier assessed film leakage, which was absent in all isotherm data reported. Cycling was between maximum and minimum areas of 448 and 103 cm2 (compression ratio 4.35:1) at 5 min per cycle at 23 ± 1°C. Respreading of molecules squeezed out of the interfacial film during compression was assessed from collapse plateaus on the pi -A compression curves for cycles 1, 2, and 7, as defined previously by Notter and co-workers (34, 37, 57). A value of zero for the ratio of compression isotherm collapse plateaus on cycle 2/ cycle 1 or cycle 7/cycle 1 indicated no respreading between the designated compressions, whereas a collapse plateau ratio of one indicated complete respreading of film material.

Excised lung pressure-volume methods. Physiological activity was determined by measuring quasistatic pressure-volume (P-V) deflation mechanics in an excised rat lung model at 37°C (3). The thermodynamic consistency of P-V measurements in this model has been documented in multiple studies (see Ref. 34 for review). Adult Sprague-Dawley male rats (400-550 g; Charles River, Wilmington, MA) were given a lethal intraperitoneal bolus of pentobarbital sodium, and lungs were excised and degassed under vacuum. The lungs were attached to an artificial trachea in an environmental chamber and rapidly inflated with air (Harvard small animal respirator, 24.7 ml/min). Inflation was to 30 cmH2O, followed by a 10-15 min period of stress relaxation to standardize total lung capacity (TLC) and rule out air leakage. The lungs were then deflated from TLC at a slow rate of 2.47 ml/min to define normal surfactant-sufficient P-V mechanics. Endogenous surfactant was removed by 15 lavages with cool 0.15 M NaCl, followed by degassing, reinflation to 30 cmH2O, stress relaxation, and a second deflation curve to define the surfactant-deficient state. A surfactant mixture in 0.15 M NaCl was then instilled via the trachea at a uniform total dose of 100 mg phospholipid/kg rat body wt, and a third P-V deflation curve was measured with identical methods to determine the degree of restoration of mechanics toward normal. The concentration of instilled surfactant mixtures was constant at 25 mg/ml. Percent volume recovery at selected transpulmonary pressures was calculated as 100(Vsurf - Vdef)/(Vnorm - Vdef), where Vnorm is the volume of the freshly excised surfactant-sufficient lungs, Vdef is the volume of the lavaged surfactant-deficient lungs, and Vsurf is the volume after surfactant instillation at the pressure of interest.

Statistical analyses. All results are expressed as means ± SE. Statistical analyses used the Student's t-test for comparisons of discrete data points, and functional data were analyzed by one-way analysis of variance with Scheffé's procedure identifying points of significant difference. Differences were considered statistically significant if the probability of the null hypothesis was <0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The adsorption of mixtures of DPPC/SP-B, SL/SP-B, and PPL/SP-B at 37°C is shown in Fig. 1, A-C, respectively. At fixed SP-B content, the rate of adsorption was generally most rapid for mixtures containing PPL and SL, followed by DPPC. However, the variation of adsorption as a function of SP-B content was very similar in all three sets of mixtures. Very low amounts of SP-B (<= 0.1% by weight) had a measurable effect in improving adsorption relative to lipids alone. However, the rate and magnitude of adsorption increased continuously as the weight percentage of SP-B was incrementally raised up to 0.75%. PPL/SP-B containing 0.75% apoprotein by weight reached equilibrium surface pressures of 48.5 ± 0.5 mN/m (surface tensions of just under 22 mN/m) after 20 min of adsorption (Fig. 1C). CLSE, which contains all the components in PPL/SP-B plus cholesterol and SP-C, reached the same final equilibrium adsorption surface pressure but at a faster rate (Fig. 1C). Mixtures of SL/SP-B (0.75%) and DPPC/SP-B (0.75%) reached slightly lower final adsorption surface pressures than PPL/SP-B (0.75%). Mixtures of lipids with 0.5% SP-B by weight adsorbed less rapidly than corresponding mixtures containing 0.75% SP-B. However, final surface pressures for mixtures containing 0.5% SP-B were only slightly lower than for those containing 0.75% SP-B (Fig. 1, A-C).


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Fig. 1.   Effects of increasing contents of surfactant protein (SP)-B on the adsorption of dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and column-purified lung surfactant phospholipids (PPL). A: DPPC/SP-B mixtures; B: SL/SP-B mixtures; C: PPL/SP-B mixtures. SP-B had consistent content-dependent effects in enhancing adsorption in mixtures with DPPC, SL, and PPL. Also shown for comparison in C is calf lung surfactant extract (CLSE), which contains all of the hydrophobic components of lavaged calf lung surfactant. Adsorption measurements were made in a Teflon dish with a stirred subphase at 37°C (see MATERIALS AND METHODS). Final subphase surfactant concentration was uniform at 0.063 mg phospholipid/ml. Data are means ± SE for n = 3-5.

The surface tension lowering of mixtures of DPPC/SP-B, SL/SP-B, and PPL/SP-B during dynamic compression in pulsating bubble experiments is shown in Figs. 2-4. The pattern of activity in these dynamic studies also showed successive improvements as the content of SP-B was raised from 0.05 to 0.75% by weight relative to phospholipid. As in the adsorption results above, overall dynamic surface activity was generally ordered as PPL/SP-B approx  SL/SP-B > DPPC/SP-B in terms of the time course and/or magnitude of surface tension lowering. This order of dynamic surface activity was found at both phospholipid concentrations studied (1 and 2.5 mg/ml). PPL/SP-B mixtures containing 0.75% by weight of apoprotein to phospholipid reached minimum surface tensions <1 mN/m almost as rapidly as CLSE at a low phospholipid concentration of 1 mg/ml (Fig. 4A). Mixtures of SL or DPPC with 0.75% SP-B also reached very low minimum surface tensions <1 mN/m at 1 mg phospholipid/ml (Figs. 2A, 3A). Minimum surface tension values <1 mN/m were also attained by mixtures of PPL/SP-B (0.5%) and SL/SP-B (0.5%) at a phospholipid concentration of 1 mg/ml, and by DPPC/SP-B (0.5%) at a higher phospholipid concentration of 2.5 mg/ml (Figs. 2-4).


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Fig. 2.   Effects of SP-B on dynamic surface tension lowering in mixtures with DPPC. A: 1 mg phospholipid/ml; B: 2.5 mg phospholipid/ml. Surface tension lowering in DPPC/SP-B mixtures on a pulsating bubble surfactometer (20 cycles/min, 50% area compression, 37°C) was successively improved as apoprotein content increased from 0.05 to 0.75% by weight. Data are means ± SE for n = 3-5.



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Fig. 3.   Effects of SP-B on dynamic surface tension lowering in mixtures with SL. A: 1 mg phospholipid/ml; B: 2.5 mg phospholipid/ml. Surface tension lowering during dynamic cycling is shown for mixtures of SL/SP-B from pulsating bubble studies. Experimental details are the same as in the legend to Fig. 2. The composition of SL is given in MATERIALS AND METHODS. Data are means ± SE for n = 3-5.



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Fig. 4.   Effects of SP-B on dynamic surface tension lowering in mixtures with PPL. A: 1 mg phospholipid/ml; B: 2.5 mg phospholipid/ml. Surface tension lowering during dynamic cycling on the pulsating bubble apparatus is shown for PPL/SP-B mixtures. Experimental details are the same as in Figs. 2 and 3. Data are means ± SE for n = 3-5.

In addition to investigating surface activity in dispersed phospholipid/SP-B mixtures, apoprotein effects were also studied directly in spread interfacial films in the Wilhelmy balance. In particular, dynamic respreading during cycling was examined in films of DPPC/SP-B and PPL/SP-B spread to an initial surface concentration of 15 Å2/molecule. This surface concentration substantially exceeds monolayer coverage of the interface and emphasizes film properties associated with compression past collapse. Representative surface excess pi -A isotherms for DPPC/SP-B films at apoprotein contents of 0, 0.1, 0.25, 0.5, and 0.75% by weight relative to phospholipid are shown in Fig. 5. As SP-B content is raised, there is a successively larger shift to the right in the second and/or seventh compression curves on these representative isotherms, indicative of increased film respreading. Respreading calculations for DPPC/SP-B and PPL/SP-B films based on collapse plateau ratios from multiple isotherms at different apoprotein contents are given in Table 1. PPL/SP-B films had significantly better respreading than corresponding DPPC/SP-B films at fixed apoprotein content, reflecting the much better respreading of the complete mix of lung surfactant phospholipids relative to DPPC alone. Very low SP-B contents of <= 0.1% by weight improved respreading in films with DPPC, whereas effects on PPL film respreading were small. However, all phospholipid/SP-B films exhibited a similar conceptual pattern of increased dynamic respreading as SP-B content was incrementally increased to 0.75% (Table 1).


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Fig. 5.   Surface pressure-area (pi -A) isotherms for mixtures of DPPC with bovine SP-B. A-E: pi -A isotherms for solvent-spread films containing SP-B contents of 0.1-0.75% by weight relative to DPPC. Graphs show the compression curves for cycles 1, 2, and 7, and the expansion curve for cycle 1. Respreading ratios calculated from multiple isotherms at fixed SP-B content are given in Table 1. All films were spread to an initial concentration of 15 Å2/molecule at the air-water interface in a Wilhelmy balance at 23°C (see MATERIALS AND METHODS).


                              
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Table 1.   Effects of SP-B on the respreading of DPPC and PPL in dynamically cycled surface films

Physiological activity experiments addressed the effects of tracheally instilled SL/SP-B mixtures on P-V deflation mechanics in excised, lavaged rat lungs. The lipid mixture in these excised lung studies had a broad distribution of lipid components superficially related to those in lavaged lung surfactant (SL = 40:40:10:5:2.5:2.5 DPPC/egg PC/egg PG/egg PI/egg PE/Sph + 5% cholesterol). As found in biophysical studies, the activity of SL/SP-B mixtures in improving surfactant-deficient P-V deflation mechanics increased as apoprotein content was raised from 0.1 to 0.75% (Fig. 6, Table 2). Small but measurable improvements in surfactant-deficient mechanics were present for SL/SP-B mixtures with low apoprotein contents of 0.1-0.2% by weight relative to phospholipid. SL/SP-B (0.3% by weight) had a significantly increased activity in improving surfactant-deficient mechanics, and activity at low transpulmonary pressures was increased further in SL/SP-B (0.5%) and SL/SP-B (0.75%) mixtures. The activity of SL/SP-B (0.75% by weight) in lavaged excised rat lungs approached but did not fully equal that of CLSE (Fig. 6, Table 2).


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Fig. 6.   Effects of SL/SP-B mixtures on pressure-volume deflation mechanics in surfactant-deficient rat lungs. Volume fraction is relative to total lung capacity. Lungs were excised from adult rats, and mechanics were measured during quasistatic deflation in the normal (surfactant-sufficient) state, after depletion of endogenous surfactant by lavage (surfactant-deficient), and after tracheal instillation of SL/SP-B mixtures or CLSE (100 mg/kg rat body wt) (see MATERIALS AND METHODS). SP-B contents are weight % relative to phospholipid. Calculated % volume recoveries at selected transpulmonary pressures are given in Table 2. Data for each mixture are means ± SE for n = 3.


                              
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Table 2.   Percent volume recoveries at selected transpulmonary deflation pressures for SL/SP-B mixtures in surfactant-deficient rat lungs


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study has examined the content-dependent effects of surfactant protein SP-B on the adsorption, dynamic surface behavior, and pulmonary mechanical activity of lipids. Biophysical and physiological studies on three different lipid/SP-B mixtures were consistent in demonstrating substantial activity differences as a function of apoprotein content. At fixed SP-B content, adsorption and dynamic surface tension lowering for the mixtures studied were generally ordered as PPL/SP-B approx  SL/SP-B > DPPC/SP-B. However, all mixtures gave similar patterns of surface activity changes with SP-B content. Very low SP-B contents <= 0.1% by weight had a measurable effect in increasing the adsorption, dynamic surface tension lowering, and film respreading of DPPC, SL, and/or column-purified lung surfactant phospholipids (Figs. 1-4, Table 1). However, surface activity successively increased as SP-B content was incrementally raised to 0.75% by weight relative to phospholipid. The adsorption and dynamic surface tension lowering of PPL/SP-B (0.75%) were only slightly less than CLSE, a highly active surfactant extract containing the complete mix of lipids and hydrophobic proteins from lavaged calf lung surfactant (18, 21, 33, 34, 38) (Figs. 1C and 4). Mixtures of SL/SP-B (0.75%) and DPPC/SP-B (0.75%) also adsorbed rapidly and reached low minimum surface tensions <1 mN/m during dynamic cycling at a low phospholipid concentration of 1 mg/ml in pulsating bubble studies (Figs. 1-3). Mixtures of PPL, SL, or DPPC with 0.5% SP-B had reduced rates of adsorption and dynamic surface tension lowering, but did reach minimum surface tensions <1 mN/m during prolonged cycling (Figs. 1-4).

Wilhelmy balance experiments on dynamic respreading in cycled interfacial films also displayed the content-dependent effects of SP-B in mixtures with phospholipids (Fig. 5, Table 1). Variations in respreading as a function of SP-B content in interfacial films at room temperature in the Wilhelmy balance were conceptually similar to those in adsorption and pulsating bubble studies on surfactant dispersions at 37°C. Even very low SP-B contents <= 0.1% by weight impacted the respreading of DPPC, and this film property then improved further as levels of SP-B were increased from 0.1 to 0.75% (Table 1). Previous studies have also documented the very poor respreading of pure DPPC in cycled surface films (e.g., Refs. 37, 52), which may make the effects of low levels of SP-B more apparent. The complete mix of rigid and fluid surfactant phospholipids in PPL has much higher film respreading than DPPC (57). Despite this, low SP-B contents of 0.1-0.25% by weight also generated measurable respreading improvements in films with PPL, with larger increases as apoprotein content was raised further (Table 1).

The content-dependent activity of SP-B was also apparent in physiological measurements on the effects of instilled SL/SP-B mixtures in excised, lavaged rat lungs. The ability of SL/SP-B mixtures to improve surfactant-deficient P-V deflation mechanics increased as SP-B content increased from 0.1 to 0.75% by weight (Fig. 6, Table 2). SL/SP-B mixtures with apoprotein contents of 0.75% had very substantial mechanical activity, although the degree of P-V restoration did not fully equal that of CLSE. Mixtures of SL/SP-B (0.5 and 0.3%) also had significant activity in the excised lungs. The mechanical activity of SL/SP-B (0.3%) appeared to be greater relative to SL/SP-B (0.2%) than found in adsorption and dynamic surface activity studies on corresponding SL/SP-B mixtures (Fig. 6 vs. Figs. 1B and 3). However, physiological data were consistent with biophysical findings in showing an overall pattern of increasing activity for SL/SP-B mixtures as apoprotein content increased. The excised rat lung model in our experiments has previously been used with success to correlate the pulmonary activity of lung surfactants with their surface activity and inhibition (e.g., 17, 21, 22, 38). The rat lung model has also been shown in multiple studies to respond to the instillation of whole and extracted lung surfactant materials that improve in vivo pulmonary function in other animal models of surfactant deficiency and dysfunction (see Ref. 34 for review).

The physiological range of SP-B contents in endogenous lung surfactant from animals of different species and age is not known precisely. CLSE used in comparison studies here has been reported to have a content of SP-B of ~0.9% by weight relative to phospholipid (39). Our data indicate that lower SP-B contents of 0.75% (and to a lesser extent 0.5%) also generate significant improvements in the adsorption, dynamic surface activity, and physiological activity of lipids. If molecular weights of 750 and 7,500 are used for phospholipids and monomeric SP-B, respectively, a weight content of 0.5% SP-B relative to phospholipid is equivalent to a molar ratio of 5 × 10-4. This indicates that SP-B significantly affects phospholipid activity when one molecule of peptide monomer is present among 2,000 phospholipid molecules. If dimer SP-B rather than monomer SP-B is assumed to be the active functional form, the number of phospholipid molecules per molecule of active protein is twice as large. The ability of SP-B to enhance the surface activity of phospholipids at such molecular ratios is striking and is consistent with prior research indicating the crucial functional relevance of this surfactant apoprotein in endogenous and exogenous lung surfactants.

Although both of the hydrophobic lung surfactant proteins are known to enhance phospholipid activity, multiple studies indicate that SP-B is more effective than SP-C on a weight and molar basis (13, 25, 34, 40, 41, 44, 46, 48, 56, 58). SP-B has been shown to be more active than SP-C in increasing both the adsorption and dynamic surface tension lowering of phospholipids (13, 44, 46, 48, 56, 58). The improved adsorption of mixtures of phospholipids with SP-B is consistent with the finding that this apoprotein has a fourfold higher capacity for binding lipid vesicles to interfacial films on a weight basis compared with SP-C (40, 41). Mixtures of phospholipids with SP-B vs. SP-C also have an improved ability to resist inhibition by serum albumin (48, 56). These biophysical findings correlate directly with physiological activity studies. Exogenous surfactants containing lipids combined with SP-B have been found to give greater improvements in pulmonary mechanics and function in animals than corresponding mixtures of lipids with SP-C (45). Supplementation with SP-B or related synthetic peptides has also been shown to improve the surface and physiological activity of the clinical exogenous surfactant Survanta, which contains SP-C but has only very low levels of SP-B (28, 39, 55). The high surface and physiological activity of SP-B vs. SP-C is consistent with the lethal nature of congenital SP-B deficiency in humans (14, 19, 31, 32). Lethal respiratory failure also occurs in "knockout" mice lacking the SP-B gene (9), and compliance abnormalities exist even in mice that are SP-B (+/-) heterozygotes (8, 50).

Although not a primary focus of study, our experiments did address the interactions of SP-B with different phospholipids. The finding that mixtures of DPPC/SP-B had substantial surface and physiological activity, which increased with SP-B content, indicates the ability of this amphipathic apoprotein to interact strongly with disaturated phosphatidylcholine molecules. However, the fact that PPL/SP-B mixtures consistently had better surface activity suggests that the complete mix of phospholipids in lung surfactant is important in interacting with SP-B. This mix of lung surfactant phospholipids includes unsaturated as well as saturated phosphatidylcholines plus multiple anionic phospholipids (34). The detailed roles and importance of specific phospholipid molecules in interacting with SP-B require more comprehensive investigation in future research.

The current study focused on defining quantitative improvements in activity associated with different contents of SP-B in dispersions and films and did not address functional molecular biophysical mechanisms. Substantial prior research has investigated the molecular biophysical interactions of SP-B with phospholipids. SP-B is known to increase phospholipid aggregation, disrupt and fuse phospholipid bilayers and vesicles, and promote phospholipid insertion into surface films (6, 12, 40-42). Human SP-B monomer contains not only hydrophobic amino acids but also polar residues, including 10 basic Arg/Lys residues that are positively charged at neutral pH. This may facilitate specific interactions of SP-B with anionic phospholipids as well as zwitterionic phosphatidylcholines in lung surfactants. Due to its overall amphipathic structure, SP-B likely occupies a relatively peripheral position in phospholipid bilayers where it can interact with both head groups and hydrophobic chains (1, 10, 40, 42, 43, 53). SP-B can order the head-group region of lipid bilayers and increase the gel-to-liquid crystal transition temperatures of fluid phospholipids (1, 2, 30, 53, 54). SP-B also broadens phase transition width, indicating an ability to disrupt or reduce order in at least a portion of the bilayer (1, 2, 23, 24, 30, 43). The extensive molecular biophysical interactions between SP-B and phospholipids support a crucial role for this apoprotein in surfactant biophysical function consistent with the activity findings here.

In summary, this study shows the striking content-dependent impact of SP-B on the surface and physiological activity of lipids. Even low SP-B contents of <= 0.1% by weight had measurable effects in increasing the adsorption, dynamic surface tension lowering, and/or film respreading of DPPC, mixed synthetic lipids, and column-purified lung surfactant phospholipids. The magnitude of surface activity improvements associated with SP-B increased significantly as apoprotein content was incrementally raised to 0.75% by weight in phospholipid mixtures. Physiological studies in lavaged excised rat lungs also showed that SP-B had content-dependent activity in improving the ability of instilled lipid-SP-B mixtures (0.1-0.75%) to reverse surfactant-deficient P-V deflation mechanics. Activity results were consistent with the ability of SP-B to influence the biophysical function of phospholipids in a molar ratio of 1:2,000 in endogenous and exogenous lung surfactants.


    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-56176.


    FOOTNOTES

Address for reprint requests and other correspondence: R. H. Notter, Dept. of Pediatrics (Neonatology, Box 850), Univ. of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00431.2001

Received 2 November 2001; accepted in final form 18 March 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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