Surface Dilatational Behavior of Pulmonary Surfactant Components

Surface Dilatational Behavior of Pulmonary Surfactant. Components Spread on the Surface of a Captive Bubble. 3. Dipalmitoyl Phosphatidylcholine, Surfa...
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Langmuir 2003, 19, 7521-7527

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Surface Dilatational Behavior of Pulmonary Surfactant Components Spread on the Surface of a Captive Bubble. 3. Dipalmitoyl Phosphatidylcholine, Surfactant Protein C, and Surfactant Protein B N. Wu¨stneck,* R. Wu¨stneck, and U. Pison Humboldt-Universita¨ t Berlin, Charite´ , Campus Virchow-Klinikum, Anaesthesiologie, Augustenburger Platz 1, D-13353 Berlin, Germany Received February 3, 2003. In Final Form: June 13, 2003 Spread monolayers containing dipalmitoyl phosphatidylcholine (DPPC), surfactant protein C (SP-C), and surfactant protein B (SP-B) were characterized by dynamic and equilibrium surface pressure/area isotherms, surface dilatational rheological measurements, and transient stress relaxation. The maximum surface pressure which can be realized by monolayer compression depends significantly on the compression rate. The surface rheological behavior is strongly influenced by the presence of SP-B, which reduces the characteristic time of stress relaxation. Therefore the surface pressure of a layer containing SP-B (DPPC + SP-B or DPPC + SP-B + SP-C) changes within some seconds after a transient surface pressure jump to a constant value which corresponds to an equilibrium surface pressure plateau at about 51 mN/m, whereas DPPC + SP-C relaxes within tens of seconds to a steady-state value of surface pressure. In the frequency range of human breathing, the surface dilatational viscosity is weak and nearly independent of surface pressure and frequency for all systems investigated. The surface rheological behavior of the phospholipid/surfactant layers in this range is mainly elastic, with maximum elasticity at a surface pressure slightly below the equilibrium surface pressure plateau, whereas the compressibility of the pulmonary film in the surface pressure range of breathing crosses a stationary compressibility maximum. This study suggests that the presence of SP-B is essential for expiration and the presence of both proteins, SP-B and SP-C, is essential for the inspiration process of the breathing cycle.

Introduction Respiration is accompanied by gas transfer across a thin lipid/protein film that floats on the alveolar lining layer. This film is composed of components of the so-called pulmonary surfactant. Pulmonary surfactant is a complex mixture of lipids and four surfactant-associated proteins (SP-A, SP-B, SP-C, SP-D). Biological functions of pulmonary surfactant are complex. Pulmonary surfactant is one of the first barriers participating in innate immune defense of the lung and possibly other mucosal surfaces. Several studies have shown that the surfactant proteins SP-A and SP-D interact with a number of viruses, bacteria, and fungi1-3 and with inhaled glycoconjugate allergens,4,5 such as pollen grains. Another main function of pulmonary surfactant is to reduce the interfacial work and to prevent agglutination and collapse of the alveoli. Although the composition of the interfacial film in the lung is not known, it is usually accepted that it contains different saturated and unsaturated phospholipids as well as the two extraordinary hydrophobic surfactant proteins SP-B and SPC.6 It is also widely accepted that pulmonary surfactant should provide rapid film formation within a few seconds * Corresponding author. E-mail: [email protected]. (1) Reid, K. B. Biochim. Biophys. Acta 1998, 1408, 290-295. (2) Crouch, E. C. Am. J. Respir. Cell Mol. Biol. 1998, 19, 177-201. (3) Haagsman, H. P. Biochim. Biophys. Acta 1998, 1408, 264-277. (4) Curstedt, T.; Jornvall, H.; Robertson, B.; Bergman, T.; Berggren, P. Eur. J. Biochem. 1987, 168, 255-262. (5) Malhotra, R.; Haurum, J.; Thiel, S.; Jensenius, J. C.; Sim, R. B. Biosci. Rep. 1993, 13, 79-90. (6) Schu¨rch, S.; Bachofen, H.; Possmayer, F. Alveolar lining layer: functions, composition, structures. In Complexity in Structure and Function of the Lung; Hlastala, M. P.; Robertson, H. T., Eds.; Lung Biology in Health and Disease, Vol. 121; Marcel Dekker: New York, 1998; pp 35-73.

through adsorption from the hypophase, “low film compressibility” of about 0.01 m/mN with a fall in surface tension to very low values of ≈0 mN/m upon surface compression, and effective replenishment of the surface film on expansion by the incorporation of reserve material associated with the surface.6,7 The two demands, low interfacial work and low compressibility of the surface layer at the same time, are contradictory. Recently we focused on the investigation of the surface rheological behavior of pulmonary surfactant layers. We suggest that not only the ordinary surface tension, γ, is important in minimizing the interfacial work to enable breathing at a low work load, but also the dynamic layer behavior in cases of surface deformation. There are two principal modes of interfacial deformation, shearing and compression/dilatation. The relevant surface rheological behaviors to evaluate breathing are surface dilatational rheological parameters, that is, dilatational elasticity and viscosity, as the respiration process is in first approximation accompanied by compression and expansion of the alveolar lining layer. These parameters characterize the spontaneous and the delayed response of a stressed layer.8 A fact that is often neglected is that these parameters are not constants of matter. Actually they change with deformation velocity. Therefore the understanding of the respiration process on the basis of surface rheological treatment requires knowledge of surface dilatational (7) Schu¨rch, S.; Bachofen, H.; Possmayer, F. Comp. Biochem. Physiol., A 2001, 129, 195-207. (8) Since the lung is full of converging and diverging passages, it is also possible that for some aspects surface shear rheology becomes important. This may be the case when taking into account adsorption of the lung surfactant components and various lateral transport processes.

10.1021/la030045o CCC: $25.00 © 2003 American Chemical Society Published on Web 07/24/2003

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elasticity and viscosity in the deformation range of respiration frequency. In the past, we characterized surface dilatational rheological properties of pulmonary surfactant by investigating both spread pure phospholipid layers9 and layers of dipalmitoyl phosphatidylcholine (DPPC) containing one of the hydrophobic pulmonary surfactant proteins, SP-C or SP-B.10,11 The methods used were transient stress relaxation experiments,9,12 yielding characteristic times of surface pressure relaxation, and harmonic drop10,11 or bubble oscillation,13 yielding dilatational elasticities and viscosities depending on surface pressure and surface area oscillation frequency.14 In the present paper, we shall use mixtures that contain DPPC and both hydrophobic surfactant proteins, SP-C and SP-B, to test if our previous findings can be generalized for such a particular film, which resembles the behavior of the thin surfactant film floating on the alveolar lining layer of the lung in many aspects. We will use an improved captive bubble device15 to determine surface pressure/ area isotherms, but also for bubble oscillation experiments in the frequency range of respiration and for transient stress relaxation measurements in the range of high surface pressure to characterize saturated highly compressed films. Materials and Experimental Details Materials. Chloroform (Ultra-Resi analyzed) was obtained from J.T. Baker (Griesheim, Germany), and methanol (LiChrosolv gradient grade) from Merck (Darmstadt, Germany). L-Dipalmitoyl phosphatidylcholine (DPPC) was purchased from Sigma and used without further purification (99% purity). SP-B and SP-C were isolated from butanol lipid extracts of sheep lung washings using gel exclusion chromatography on an LH-60 column (Pharmacia) with acidified chloroform/methanol solvent as a mobile phase.16,17 DPPC, SP-B, and their mixtures were dissolved in chloroform/methanol (1:1, v/v). Water was purified by means of a Milli-Q Plus water system (Millipore, Eschborn, Germany). The mixtures of DPPC, SP-B, and SP-C were prepared by mixing stock solutions of each component. The films were spread on pure water. Based on the assumption that the SP-B to SP-C ratio in pulmonary surfactant is 1:3 (by weight),17 experiments were performed with a DPPC + SP-B + SP-C mixture containing 17 wt % of SP-B and 5.7 wt % of SP-C, which corresponds to 0.25 mol % of SP-B dimer (relative to DPPC) and 3 mol % SP-C. The molecular weight of ovine SP-B is 17 380 (dimer, 158 amino acid residues), and that of SP-C is 4200 (dipalmitoylated form, 35 amino acid residues). Experimental Details. The captive bubble technique in combination with the axisymmetric drop shape analysis for captive bubbles (ADSA-CB)18 was used as a microfilm balance,19 (9) Wu¨stneck, R.; Wu¨stneck, N.; Grigoriev, D. O.; Pison, U.; Miller, R. Colloids Surf., B 1999, 15, 275-288. (10) Wu¨stneck, R.; Wu¨stneck, N.; Moser, B.; Karageorgieva, V.; Pison, U. Langmuir 2002, 18, 1119-1124. (11) Wu¨stneck, R.; Wu¨stneck, N.; Moser, B.; Pison, U. Langmuir 2002, 18, 1125-1130. (12) Joos, P.; van Uffelen, M.; Serrien, G. J. Colloid Interface Sci. 1992, 152, 521-533. (13) Wu¨stneck, N.; Wu¨stneck, R.; Fainerman, V. B.; Miller, R.; Pison, U. Colloids Surf., B 2001, 21, 191-205. (14) Wu¨stneck, R.; Moser, B.; Muschiolik, G. Colloids Surf., B 1999, 15, 263-273. (15) Wu¨stneck, R.; Wu¨stneck, N.; Vollhardt, D.; Miller, R.; Pison, U. Mater. Sci. Eng., C 1999, 8-9, 57-64. (16) Bu¨nger, H.; Kaufner, L.; Pison, U. J. Chromatogr., A 2000, 870, 363-369. (17) Bu¨nger, H.; Kru¨ger, R. P.; Pietschmann, S.; Wu¨stneck, N.; Kaufner, L.; Tschiersch, R.; Pison, U. Protein Expression Purif. 2001, 23, 319-327. (18) Prokop, R. M.; Jyoti, A.; Cox, P.; Frndova, H.; Policova, Z.; Neumann, A. W. Colloids Surf., B 1999, 13, 117-126. (19) Kwok, D. Y.; Vollhardt, D.; Miller, R.; Li, D.; Neumann, A.W. Colloids Surf., A 1994, 88, 15-58.

Wu¨ stneck et al. for harmonic oscillation,14 and for transient stress relaxation experiments.20 The captive bubble device was described in detail earlier.15 All glass vessels and the measuring cell of the captive bubble device were cleaned in KOH-saturated 2-propanol. The surface tension of pure water (Millipore) used here was 72.7 ( 0.2 mN/m at 23 °C as determined by the captive bubble technique. The mixtures were spread at the air bubble surface. For spreading, a 0.5 µL Hamilton syringe was used. The tip of the syringe was inserted through a membrane in the wall of the measuring cell to touch the bubble surface. The amount of spreading solution was reduced to 0.1 µL to avoid influences of the spreading solvent on the surface behavior of the monolayer. The spreading amount was chosen to realize starting surface pressures of 0 mN/m and to obtain the whole π/A isotherm beginning from the formation of a two-dimensional “gaseous” monolayer up to collapse monolayer region. Measurements of π/A Isotherms. For π/A isotherm determination, the internal pressure of the measuring cell was continuously increased or decreased by using a syringe pump. The surface tension and the bubble surface were determined by ADSA-CB from bubble profiles recorded every second with a CCD camera. The usual compression rate was 7.4 × 10-4 nm2 s-1. Harmonic Captive Bubble Experiments. For these experiments, a piezo transmitter was connected to the measuring cell and inserted into the liquid phase. The transmitter was controlled by an arbitrary frequency generator (Grundig AFG 100, Germany), which allows oscillation of the bubble volume in a frequency range of 0.001-1 kHz. For our measurements, we used frequencies in the range of 1 × 10-2 to 2.5 × 10-1 Hz and bubble volume amplitudes of 0.5 µL. This frequency range covers the human respiration frequency (4 × 10-2 to 2 × 10-1 Hz),21 including the range of frequencies where a maximum of the rheological parameters was found for DPPC and DPPC + SP-C (≈1.7 × 10-2 Hz).13,20 Amplitudes of 0.5-1.5 µL were found to avoid surface structure destruction by drop oscillation experiments.11 The surface tension and the bubble area were determined using ADSA-CB and recording 10 bubble profiles per second. The rheological parameters, dilatation surface elasticity and viscosity, were obtained by recording bubble profiles at 9 different levels of surface pressure and 9 different frequencies. Every set of experiments was done at 23 °C ((0.1). Surface pressure was stepwise adjusted with a compression speed of 3 × 10-2 nm2/ molecule‚s up to one of the nine levels of π. The elasticity was determined by the ratio between the surface pressures via surface area amplitude. The viscosity was determined by using the phase shift between the oscillation of surface area and pressure.14 A spline-fit was used to create planes of elasticity and viscosity via frequency and surface pressure, which are based on a symmetric 9 × 9 frequency/surface pressure matrix. The confidence intervals (95% confidence level) of the elasticities were about 1-4%; those of the corresponding viscosities were in the order of 10-15%. Transient Stress Relaxation Experiments. Transient stress relaxation experiments were carried out to discover rearrangement processes, which occur when the steady state of a surface layer is rapidly changed by a sudden surface deformation. These experiments were carried out in the squeeze-out film region, which corresponds to a surface pressure plateau in the equilibrium π/A isotherm. Before measurement, the layers were cycled by consecutive fast compression and expansion in the surface pressure range 0-70 mN/m. Cycling was often used by different authors. It finally leads to layers that are more comparable to native pulmonary layers. The repeated compression to high surface pressures is connected with monolayer folding or with formation of liposome-like aggregates.15 Because in this case the actual surface coverage becomes arbitrary, we decided to establish comparable starting conditions and to use the actual change of the bubble surface. The transient stress relaxation experiments were started at a surface pressure of 51 mN/m by (20) Wu¨stneck, R.; Enders, P.; Wu¨stneck, N.; Pison, U.; Miller, R. PhysChemComm 1999, 11. (21) Roussos, C.; Campbell, E. J. M. Respiratory muscle energetics. In Handbook of Physiology, Section 3: The Respiratory System, Volume III; Macklem, P. T., Mead, J., Eds.; American Physiological Society: Bethesda, 1986; pp 481-509.

Pulmonary Surfactant Components on a Bubble

Figure 1. Equilibrium π/A isotherms of spread layers of DPPC (white circles), DPPC + 2 mol % SP-C (black triangles), DPPC + 2 mol % SP-B (white squares), and DPPC + 0.25 mol % SP-B + 3 mol % SP-C (white diamonds). a sudden change of the inner pressure in the measuring cell. The resulting changes of the bubble area were not more than 1020% to avoid surface destruction. The amplitude of the suitable surface pressure jump has to be determined previously, and it depends on the mechanical properties of the layer. The surface pressure increases or decreases when the surface is transiently compressed or dilated, respectively. The decay of π was recorded after the pressure jump. There is usually a spontaneous response, which can be used to calculate an elasticity parameter, and a delayed one that can be described by an exponential function, which yields a relaxation time. This time constant is characteristic for the readjustment of a new steady state of the film. For further details, see ref 22. In practice, the delayed response includes different relaxation processes with different characteristic times. Therefore a distribution for these processes was assumed that yields a main relaxation time and a homogeneity parameter and describes the stress relaxation more properly than a singleexponential approach.22 For discussing respiration processes, extremely slow relaxation processes are less important. In the present case, we restricted the time of our experiments to 120400 s.

Results π/A Isotherms in Equilibrium. Surface compression/ dilatation changes the interfacial concentration of surface layer components. The resulting surface tension meets requirements of equilibrium conditions if the velocity of matter exchange between the surface and the bulk and also rearrangement processes within the surface layer exceed the velocity of surface deformation. Most of the π/A isotherms published claim to satisfy such requirements. Collected isotherms of DPPC, individual mixtures of DPPC and the surfactant proteins SP-B and SP-C, and the mixture DPPC + SP-B + SP-C are shown in Figure 1. The isotherm of DPPC is in good agreement with those reported in the literature by use of different experimental techniques.9-11,23 It shows the typical liquid expanded (LE)/ liquid condensed (LC) transition plateau, which is found at 23 °C at 10 mN/m.24 The isotherms for the mixtures of DPPC with the two proteins are comparable to those previously reported11,13and agree well with those found by Taneva et al.,25,26 although a direct comparison is complicated by the known influence of protein preparation procedures.27-29 (22) Wu¨stneck, R.; Enders, P.; Ebisch, Th.; Miller, R. Thin Solid Films 1997, 298, 39-46. (23) Albrecht, O.; Gruler, H.; Sackmann, E. J. Phys. 1978, 39, 301313. (24) Vollhardt, D.; Fainerman, V. B. Adv. Colloid Interface Sci. 2000, 115, 103-151. (25) Taneva, S.; Keough, K. M. Biophys. J. 1994, 66, 1149-1157. (26) Taneva, S.; Keough, K. M. Biochemistry 1997, 326, 912-922.

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Figure 2. π/A isotherms of a spread mixed layer of DPPC + 0.25 mol % SP-B + 3 mol % SP-C at different speeds of compression/expansion: 2.2 × 10-3 nm2/molecule‚s (black squares), 8.75 × 10-3 nm2/molecule‚s (gray circles), and 0.875 × 10-3 nm2/molecule‚s (white circles). Inserted images show the bubble at a surface coverage of 1.3 nm2/molecule at the beginning of compression and at 0.5 nm2/molecule at the beginning of the protein squeeze-out plateau.

In comparison to the DPPC isotherm, the increase of surface pressure is significantly shifted to lower surface coverage for the mixtures DPPC + protein. In contrast to SP-C, the surface pressure of DPPC + 2% SP-B reaches 4 mN/m already at 1.4 nm2/molecule. This is the consequence of the different molecular weight and dimensions of these proteins.10 The isotherm for DPPC + 2% SP-C shows a slightly pronounced LE/LC plateau and a plateau at 51 mN/m. The isotherm for DPPC + 2% SP-B shows no pronounced LE/LC plateau at all at 10 mN/m but a well distinct change of slope at 44 mN/m corresponding to the isotherm of pure SP-B,11 and additionally a plateau at 51 mN/m. The surface pressure did not exceed 54 mN/m although the DPPC/protein layers were strongly compressed to a state where the nominal surface coverage definitely becomes smaller than the cross section of a DPPC molecule. The extension of the plateau at 51 mN/m directly corresponds to the protein content in the layer. The isotherm of the mixture DPPC + SP-B + SP-C is found between the curves of the DPPC mixtures with the individual hydrophobic surfactant proteins. Obviously a weakly pronounced LE/LC transition for the DPPC + SP-B + SP-C mixture is observed too, but it is shifted to a surface pressure just between those of DPPC + SP-B and DPPC + SP-C. π/A Isotherms in Nonequilibrium. Figure 2 shows three π/A isotherms of a spread DPPC + SP-B + SP-C layer for compression and expansion at different compression rates. Obviously, a higher compression speed enables attainment of higher maximum surface pressures, that is, higher than 51 mN/m. The plateau region degenerates into a range of changed surface pressure slopes. At the point where the compression changes into expansion, the surface pressure rapidly drops. This decay is expansion speed dependent too. The expansion isotherms are remarkably shifted to lower surface pressures when increasing expansion rates, that is, depending on the maximum surface pressure reached. These isotherms can be reproduced by repeating compression and expansion when the layer collapse is avoided. (27) Taneva, S. G.; Stewart, J.; Taylor, L.; Keough, K. M. W. Biochim. Biophys. Acta 1998, 1370, 138-150. (28) Perez-Gil, J.; Keough, K. M. W. Biochim. Biophys. Acta 1998, 1408, 203-217. (29) Wu¨stneck, N.; Wu¨stneck, R.; Perez-Gil, J.; Pison, U. Biophys. J. 2003, 84, 1940-1949.

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Figure 3. Surface pressure π (black circles), surface area A (white diamonds), and volume V (black triangles) of an oscillating air bubble with a spread mixed layer of DPPC + 0.25 mol % SP-B + 3 mol % SP-C depending on time, with starting surface pressure π ) 51 mN/m at 0.50 nm2/molecule.

Harmonic Bubble Oscillation Experiments. Harmonic oscillation experiments only lead to defined surface rheological parameters when the layer structure is undestroyed, that is, when both the oscillation of surface area and the resulting oscillation of the surface pressure remain sinusoidal. It has to be shown for each system investigated separately if this requirement is met.14 Figure 3 shows the oscillation of bubble volume, surface area, and pressure at a starting surface pressure of 51 mN/m for the mixture DPPC + SP-B + SP-C, that is, at the beginning of the plateau region of π in Figure 1. The oscillations are sinusoidal within the range of experimental error. Measurements at a starting surface pressure exceeding 51 mN/m were not possible, because surface pressure drops immediately after stopping the compression and starting the volume oscillation. Therefore we had to restrict our experiments to a starting surface pressure of 51 mN/m. The surface dilatational elasticities and viscosities for a mixed DPPC + SP-B + SP-C layer are given in Figures 4 and 5. The surface dilatational elasticity (Figure 4) is

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almost independent of frequency in the range investigated, in contrast to the viscosity that significantly changes with frequency (Figure 5). There are considerable high values of both  and η even at relatively low surface pressures of about 5 mN/m.  and η are higher than those found for DPPC previously and for the mixtures DPPC + SP-B or DPPC + SP-C.10,11 At the surface pressure of 10-20 mN/ m, the elasticity passes a stationary plateau, which corresponds to the LE/LC surface pressure plateau in the π/A isotherms. Starting from π ≈ 20 mN/m, the elasticity increases with the surface pressure, striving for a maximum ( ) 150 mN/m) at a surface pressure of 50 mN/m. In the range of about π ≈ 50 mN/m, the elasticity only slightly depends on frequency. The viscosity is comparably low at about 40-50 mN‚s/m over a wide range of frequencies and film pressures below π ) 30 mN/m. It strongly increases at higher surface pressures with two maxima, but only at low frequencies, that is, 8 × 10-3 and 2 × 10-2 Hz. In the range of breathing frequencies, the viscosity remains almost constant at a low level of about 50 mN‚s/m. Transient Stress Relaxation Experiments. Some typical results of stress relaxation experiments are given in Figure 6. These figures show the surface pressure response after a transient compression or expansion of the bubble surface, respectively. The starting surface pressures after the pressure jump depend on the experimental conditions and will not be discussed here. We will focus on the recovery behaviors of the systems, which differ remarkably. For better orientation, the plateau value of π is shown by a horizontal line in the graphs. The stress relaxation of the spread DPPC layer only slowly decreases or increases after the surface pressure jump and does not return to the equilibrium plateau value of π within the time of the experiment (Figure 6A). The addition of SP-C changes this behavior remarkably (Figure 6B). The layers are obviously very stable in the case of compression to high surface pressures, which was previously reported by different authors.30,31 Characteristic for such layers is that π also does not return to the equilibrium value of ≈51 mN/m, neither in the case of layer compression nor in that of previous layer expansion. The recovery is obviously accelerated by the presence of SP-B (Figure 6C,D). In the case of DPPC + SP-B + SP-C, the surface pressure relaxes in all cases investigated with rapid recovery of the plateau

Figure 4. Surface dilatational elasticity  of spread DPPC + 3 mol % SP-C + 0.25 mol % SP-B layers depending on surface pressure and frequency.

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Figure 5. Surface dilatational viscosity η of spread DPPC + 3 mol % SP-C + 0.25 mol % SP-B layers depending on surface pressure and frequency. Table 1. Main Relaxation Times Determined after Transient Layer Compression or Expansion Using the Decay of the Surface Pressure main relaxation time τmax [s]

Figure 6. Surface pressure decay after a transient bubble volume change of spread layers: DPPC (A), DPPC + 2 mol % SP-C (B), DPPC + 2 mol % SP-B (C), and DPPC + 3 mol % SP-C + 0.25 mol % SP-B (D). Curves at the top show the relaxation after film compression. Curves below show the recovery of the film pressure after expansion. The equilibrium value of π is shown by a horizontal line.

equilibrium surface pressure in both cases, layer compression and expansion (Figure 6D), for the lipid/protein ratio chosen. There are of course also differences between the recovery behavior for layer compression and expansion. Such differences can be explained by the formation of different surface structures in both cases. Analogous results are known from the literature and were reported for many different monolayers.24 Table 1 contains the calculated mean values for the relaxation time τ. The values were calculated by 10 different relaxation experiments with different starting values of π after the pressure jump. The homogeneity parameters22 were in the range 1.5 -3.5, which proves the relaxation processes to be quite narrowly distributed, (30) Schu¨rch, S.; Bachofen, H.; Goerke, J.; Green, F. H. Y. Biochim. Biophys. Acta 1992, 1103, 127-136. (31) Schu¨rch, S.; Green, F. H. Y.; Bachofen, H. Biochim. Biophys. Acta 1998, 1408, 180-202.

system

compression

dilatation

DPPC DPPC + 2 mol % SP-C DPPC + 2 mol % SP-B DPPC + 3 mol % SP-C + 0.25 mol % SP-B

29 35 2.4 2.5

22 16 4.8 4.3

that is, the Poisson distribution is a sufficient approximation. The main values of the relaxation time for DPPC agree with those reported previously in ref 9 and are in good agreement with those for the system DPPC + SP-C. In contrast, the relaxation times after compression for DPPC + SP-B and DPPC + SP-C + SP-B are about 2.5 s, that is, 1 order of magnitude smaller than those for DPPC alone and DPPC + SP-C. Discussion Pulmonary surfactant is a complex lipid/protein mixture optimized by evolution. To understand some aspects of the interfacial behavior of pulmonary surfactant, we used a model layer, which contains the main hydrophobic components of pulmonary surfactant, that is, DPPC, SPB, and SP-C. We characterized spread model layers by dynamic and equilibrium surface pressure/area isotherms, by transient stress relaxation experiments, and by surface dilatational rheological parameters in a wide range of film pressure and frequency, including the human breathing frequency. The results show that the shape of the π/A isotherms of the lipid/protein layers strongly depends on the velocity of surface deformation. There is a plateau of surface pressure at ≈51 mN/m in the case of quasi-equilibrium deformation. A surface pressure higher than 51 mN/m was not observed for quasi-equilibrium deformation for the surfactant layers investigated even when the area per molecule became smaller than the cross section of a vertically orientated DPPC molecule. Only in cases of fast surface compression (nonequilibrium conditions) could

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surface pressures higher than 51 mN/m be reached. When the surface compression is stopped, the surface stress relaxes to the equilibrium value (51 mN/m) in the presence of SP-B in the surface layer. This relaxation process is characterized by a very short relaxation time, which is in the range of frequency of normal breathing. Only for layers without SP-B was a steady-state surface pressure higher than 51 mN/m detected. The π/A isotherms resulting from fast compressed and fast expanded DPPC + SP-B + SP-C films agree well with surface tension cycling experiments widely investigated with adsorbed pulmonary films.6,7,31-35 Quasi-static surface pressures higher than 51 mN/m result for DPPC or DPPC + SP-C films. For these films, the characteristic time for stress relaxation is higher by 1 order of magnitude than that of DPPC + SP-B + SP-C films. This may explain the stable steady-state surface pressures for DPPC and DPPC + SP-C films near the collapse pressure (≈70 mN/m).25,26,36-38 The presented results support our previous findings about the dilatational behavior of the main phospholipids9,20 and the hydrophobic proteins10,11,13 of the pulmonary surfactant. Our main finding is that in the frequency range of human breathing the surface dilatational viscosity of our DPPC + SP-B + SP-C model layers is low and nearly independent of surface pressure and frequency. The viscosities are decisively smaller than the corresponding elasticity parameters. Therefore, in the frequency range of human breathing the main rheological behavior of the phospholipid/surfactant layer is almost elastic. The maximum elasticities of the mixture DPPC + SP-B + SP-C at a surface coverage slightly below the plateau at 51 mN/m are about 150 mN/m and clearly differ from those for DPPC + SP-C11,13 and DPPC + SP-B,10 which were found to increase to about 300 mN/m, or 200 mN/m for DPPC.13 This indicates that the film compressibility is strongly increased for the mixture that contains both proteins, SP-C and SP-B. A minimum of elasticity, that is, a maximum of film compressibility, is realized in the plateau region where the amplitude of surface oscillation becomes very small. This effect is observed by the decrease of elasticity and viscosity at 51 mN/m. An extraordinary effect of SP-B was also observed by Pastrana-Rios et al.,39 who observed that SP-B was partially excluded from a surface film during compression at a surface pressure even slightly below the plateau, whereas in contrast, SP-C either remains at the surface at high pressures or leaves accompanied by lipids as a result of the formation of intermolecular sheet structures. These differences between the interaction of DPPC and the two hydrophobic proteins also explain the different stress relaxation behavior observed, the different value of surface pressure that is approached after relaxation, and the two maxima of dilatational viscosity at different frequencies observed for the mixed layers DPPC + SP-B + SP-C slightly below 51 mN/m. (32) Possmayer, F.; Nag, K.; Rodriguez, K.; Qanbar, R.; Schu¨rch, S. Comp. Biochem. Physiol., A 2001, 129, 209-220. (33) Keough, K. M. W. In Pulmonary Surfactant: from molecular biology to clinical practice; Robertson, B., Van Golde, L. M. G., Batenburg, J. J., Eds.; Elsevier Science: New York, 1992; p 112. (34) Schu¨rch, S.; Lee, M.; Gehr, P. Pure Appl. Chem. 1992, 64, 1745. (35) Schu¨rch, S.; Goerke, J.; Clements, J. A. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 3417-3421. (36) Tabak, S. A.; Notter, R. H.; Ultman, J. S.; Dinh, S. M. J. Colloid Interface Sci. 1977, 60, 117-120. (37) Watkins, J. C. Biochim. Biophys. Acta 1968, 152, 293. (38) Possmayer, F.; Nag, K.; Rodriguez, K.; Qanbar, R.; Schu¨rch, S. Comp. Biochem. Physiol., A 2001, 129, 209-220. (39) Pastrana-Rios, B.; Taneva, S.; Keough, K. M. W.; Mautone, A. J.; Mendelsohn, R. Biophys. J. 1995, 69, 2531-2535.

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We did not consider the temperature dependence here, but it was previously shown that increasing temperature does not qualitatively change the surface dilatational behavior.10,11,13,20 Therefore it can be assumed that the results presented will also hold for the range of higher temperatures, whereas both elasticity and viscosity decrease simultaneously. A controversially discussed question is, what is the region of surface pressure in which breathing actually occurs? Undisputed is the fact that for native surfactant lipid extracts, the equilibrium surface tension for adsorbed films is between 22 and 25 mN/m at 37 °C when the bulk concentration is higher than approximately 50 µg/mL of total phospholipid.40 This value of the corresponding surface pressure agrees quite well with the plateau observed for the spread layers investigated and the more or less pronounced plateaus observed by cycling experiments.6,7,35 This suggests the formation of a highly saturated lipid/protein layer at the interface air/solution by adsorption. Starting from this point, different authors came to quite different assumptions about the surface pressure range of breathing.32,41,42 It is mainly discussed at surface pressures exceeding 55 mN/m and reaching values of π of nearly 70 mN/m, which corresponds to zero surface tension.7 Such films are metastable per definition; they are below equilibrium surface tension.7 On the other hand, a main demand of reducing the mechanical work of breathing, which is determined by both tissue forces and interfacial forces, is a high surface layer compressibility, which is the inverse of elasticity. This demand is ideally fulfilled by the so-called “collapse” plateau, where a layer folding would explain the experimental results found for those films.13,42,43 Bachofen et al.44 suggested that changes of surface tension of approximately 15 mN/m occur when deep breaths between 40 and 80% TLC (total lung capacity) are taken, whereas during normal breathing, however, variations of surface tensions are small, barely exceeding a range of 5 mN/m. We cannot exactly determine surface rheological dilatational parameters for DPPC + SP-B + SP-C film in this range of surface pressure because the surface pressure drops immediately after stopping the compression, although peaks of π ≈ 68 mN/m can be reached when increasing oscillation amplitudes. Furthermore, we know from experiments with DPPC layers that the amplitudes of oscillation become very high for small variations at a surface pressure of about 62 mN/ m,13 which corresponds to a low compressibility for surface pressures higher than 51 mN/m. During further compression, the surface elasticity increases again and drops rapidly at film pressures higher than 65 mN/m.13 This however contradicts the demand of low interfacial work. The interfacial work becomes low for highly compressible films, that is, in a range of the isotherm where the surface pressure becomes constant. This is the case for the collapse plateau but also for the plateau at 51 mN/m. Our results show that both dilatation elasticity and viscosity become maximum just below the plateau at 51 mN/m, but they decrease starting from 51 mN/m. Considering that layers containing SP-B relax very fast to the equilibrium pressure (40) Schu¨rch, S.; Schu¨rch, D.; Curstedt, T.; Robertson, B. J. Appl. Physiol. 1994, 77, 974-986. (41) Schu¨rch, S.; Bachofen, H.; Weibel, E. R. Respir. Physiol. 1985, 62, 1-45. (42) Ding, J.; Takamoto, D. Y.; von Nahmen, A.; Lipp, M.; Yee, K.; Lee, C.; Waring, A. J.; Zasadzinski, J. A. Biophys. J. 2001, 80, 22622272. (43) Takamoto, D. Y.; Lipp, M.; von Nahmen, A.; Yee, K.; Lee, C.; Waring, A. J.; Zasadzinski, J. A. Biophys. J. 2001, 81, 153-169. (44) Bachofen, H.; Schu¨rch, S. Comp. Biochem. Physiol. 2001, 129, 183-193.

Pulmonary Surfactant Components on a Bubble

when the compression stops, it seems to be a realistic suggestion that the surface pressure oscillates around the equilibrium at 51 mN/m during the dynamic process of breathing. The zero surface tension seems to be realized only in cases of very strong expiration for a short period, whereas a large increase in lung volume and surface area by deep sighing breaths is equilibrated rather by reentering of surface-associated aggregates or by refolding of bilayers.42,43 Actually, we cannot suggest the surface structure by observing the surface pressure plateau in the surface pressure/area isotherm or an increase of layer compressibility only. Surface pressure may remain constant by both decrease of surface concentration by a squeeze-out of some surface-active components of the layer6,7 or by bilayer formation as assumed in refs 42, 43, and 45. Besides the limitations of the model layers investigated, our data suggest that in the frequency range of human (45) Exerowa, D.; Kruglyakov, P. M. In Studies in Interface Science 5, Foam and Films; Mo¨bius, D., Miller, R., Eds.; Elsevier Science: New York, 1998.

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breathing the rheological behavior of the thin surfactant film that floats on the alveolar lining layer is highly compressible and its replenishment is accelerated in the presence of SP-B. This study suggests that the presence of SP-B is essential for expiration (compression) and the presence of both proteins SP-B and SP-C is essential for the inspiration (dilatation) process of the breathing cycle. Normal breathing is supported by high film compressibility of the thin lipid/protein layer lining the alveoli of mammalian lungs. Breathing takes place at a nearly constant film pressure around the protein squeeze-out plateau where the compressibility of the films is very high but decreases when the surface pressure becomes higher or lower than the equilibrium value. Acknowledgment. The financial support by the Deutsche Forschungsgemeinschaft (Grant Pi 165/7-3) is gratefully acknowledged. LA030045O