Surface Dilatational Behavior of Pulmonary Surfactant Components

and viscosity of two of these pulmonary surfactant components, dipalmitoyl ... We conclude that films containing the pulmonary surfactant components D...
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Langmuir 2002, 18, 1119-1124

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Surface Dilatational Behavior of Pulmonary Surfactant Components Spread on the Surface of a Pendant Drop. 1. Dipalmitoyl Phosphatidylcholine and Surfactant Protein C R. Wu¨stneck,† N. Wu¨stneck,‡ B. Moser,† V. Karageorgieva,† and U. Pison*,‡ Universita¨ t Potsdam, Institut fu¨ r Physik, Am Neuen Palais 10, D-14415 Potsdam, Germany, and Humboldt-Universita¨ t Berlin, Charite´ Campus Virchow-Klinikum, Anaesthesiologie, Augustenburger Platz 1, D-13344 Berlin, Germany Received May 8, 2001. In Final Form: October 21, 2001 Surface rheological characteristics of the components that are part of the film floating on the alveolar lining fluid in mammalian lungs are not well established. We measured the surface dilatational elasticity and viscosity of two of these pulmonary surfactant components, dipalmitoyl phosphatidylcholine (DPPC) and pulmonary surfactant protein C (SP-C). Pure DPPC or DPPC with 2 mol % SP-C was spread on the surface of a pendant phosphate buffered saline drop. Harmonic drop oscillation experiments were carried out below the surface pressure of protein squeeze-out in a frequency range of 0.006-0.25 Hz and at 20, 30, and 40 °C. We found that the dilatational elasticity of DPPC and DPPC/SP-C films increased up to a surface pressure of 40-45 mN/m. The dilatational viscosity of DPPC and DPPC/SP-C films dropped in the frequency range of human respiration (0.4-0.2 Hz). Dilatational elasticity and viscosity decrease with temperature. We conclude that films containing the pulmonary surfactant components DPPC and SP-C are in general viscoelastic but at breathing frequencies spontaneously elastic. SP-C stabilizes DPPC films, forming an alveolar lining layer of low compressibility at surface pressures below the pressure of protein squeeze-out and of high compressibility in the squeeze-out region.

Introduction Pulmonary surfactant has unusual surface properties that reduce the mechanical work of breathing and prevent lung collapse.1 During respiration, the surface layer is compressed and expanded. This dynamic process should be described in physical terms using surface rheological parameters. Surface rheological behavior includes surface dilatational and surface shear properties. Surface dilatational parameters describe compression/expansion processes, such as pulmonary surfactant layer deformation during breathing. Shear parameters describe processes with constant surface area. Surface dilatational behavior of L-dipalmitoyl phosphatidylcholine (DPPC), the most abundant phospholipid component of pulmonary surfactant, was described at the solution/air interface by various authors.2-11 However, * Corresponding author. Ulrich Pison, MD, Medizinische Fakulta¨t Charite´, Humboldt-Universita¨t Berlin, Campus VirchowKlinikum, Klinik fu¨r Anaesthesiologie und operative Intensivmedizin, Augustenburger Platz 1, D-13353 Berlin, Germany. Phone: +49 (0)30 450-559678. Fax: +49 (0)30 450-551900. E-mail: [email protected]. † Universita ¨ t Potsdam. ‡ Humboldt-Universita ¨ t Berlin. (1) Nag, K.; Perez-Gil, J.; Cruz, A.; Keough, K. M. W. Biophys. J. 1996, 71, 246. (2) Kretzschmar, G.; Li, J. B.; Miller, R.; Motschmann, H.; Mo¨hwald, H. Colloids Surf., A 1996, 114, 277. (3) Kra¨gel, J.; Kretzschmar, G.; Li, J. B.; Loglio, G.; Miller, R.; Mo¨hwald, H. Thin Solid Films 1996, 284/285, 361. (4) Joos, P.; van Uffelen, M.; Serrien, G. J. Colloid Interface Sci. 1992, 152, 521. (5) Wu¨stneck, R.; Wu¨stneck, N.; Grigoriev, D. O.; Pison, U.; Miller, R. Colloids Surf., B 1999, 15, 275. (6) Panaiotov, I.; Ivanova, T.; Proust, J.; Boury, F.; Denizot, B.; Keough, K.; Taneva, S. Colloids Surf., B 1996, 6, 243. (7) Watkins, J. C. Biochim. Biophys. Acta 1968, 152, 293. (8) Tabak, S. A.; Notter, R. H.; Ultman, J. S.; Dinh, S. M. J. Colloid Interface Sci. 1977, 60, 117. (9) Taneva, S.; Keough, K. M. W. Biochemistry 1997, 36, 912. (10) Taneva, S.; Keough, K. M. W. Biophys. J. 1994, 66, 1149.

surface rheological behavior of pulmonary surfactant is still not well established due to a number of reasons. First, pulmonary surfactant is a complex mixture of several components, some of which are not easily available. Second, measuring devices that produce reliable rheological data at physiologically relevant surface pressures are rare. Third, rheological data are not constants of matter but depend on deformation and velocity of deformation. Rheological studies of pulmonary surfactant have not considered these aspects in the past. Recently, we described the surface dilatational behavior of two of the main pulmonary surfactant components, DPPC and surfactant protein C (SP-C), by using a captive bubble device.12 Although we covered a wide frequency range (0.006-0.02 Hz), human body temperature and respiration frequencies were not included. In the present study, we therefore analyzed surface dilatational behavior of these films at different temperatures and in the frequency range from 0.006 to 0.25 Hz, that is, a range that covers human body temperature as well as respiration frequency (0.04-0.2 Hz).13 Experiments were performed with a pendant drop microfilm balance14 and using a new microdroplet injection system15,16 to obtain π/A isotherms of DPPC and DPPC/SP-C films and to perform harmonic drop oscillation experiments.17,18 The latter were used to (11) Possmayer, F.; Nag, K.; Rodriguez, K.; Qanbar, R.; Schu¨rch, S. Comp. Biochem. Physiol., A 2001, 129, 209. (12) Wu¨stneck, N.; Wu¨stneck, R.; Fainerman, V. B.; Miller, R.; Pison, U. Colloids Surf., A 2000, 164, 267. (13) 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, MD, 1986; pp 481-509. (14) Kwok, D. Y.; Vollhardt, D.; Miller, R.; Li, D.; Neumann, A. W. Colloids Surf., A 1994, 88, 51. (15) Do¨ring, M. Feinmechanik & Messtechnik 1991, 99, 11. (16) Schober, A.; Gu¨nther, R.; Schwienhorst, A.; Do¨ring, M.; Lindemann, B. F. BioTechniques 1993, 15, 324. (17) Benjamins, J.; Cagna, A.; Lucassen-Reynders, E. H. Colloids Surf. 1996, 114, 245.

10.1021/la010685w CCC: $22.00 © 2002 American Chemical Society Published on Web 01/26/2002

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experimentally determine the surface dilatational behavior of DPPC and DPPC/SP-C films. Materials and Experimental Details Materials. Chloroform and methanol were p.a. grade and purchased from Baker (J.T. Baker B.V. Deventer-Holland). Water was double distilled. DPPC was purchased from Sigma and used without further purification (99% purity). SP-C was isolated from butanol lipid extracts of sheep lung washings using size exclusion chromatography on Sephadex LH-60 (Pharmacia Biotech) with acidified chloroform/methanol as the mobile phase.19 DPPC, SP-C, and their mixtures were dissolved in chloroform/methanol (1:1, v/v). Mixtures of DPPC with 2 mol % SP-C were prepared from stock solutions of each component. The films were spread on 10 mM phosphate buffered saline (pH 7, 150 mM NaCl). Microfilm Balance and Harmonic Drop Oscillation Experiments. The pendant drop technique in combination with the axisymmetric drop shape analysis (ADSA) was used as a film microbalance to obtain π/A isotherms and for harmonic drop oscillation experiments. This method was described in detail earlier.5,20 For drop creation, a poly(tetrafluoroethylene) capillary was used. In our harmonic drop oscillation experiments, we changed the pendant drop volume periodically, which caused compression and expansion of the spread surface layer at the drop surface. The oscillation of surface area (S) yielded an oscillation of the surface pressure (π) around a certain value, which is characteristic for a certain monolayer steady state. The periodic oscillation of surface pressure produces anisotropic effects, which yield the surface dilatational moduli. These moduli encompass not only the storage elasticity but also the dissipative component, which is affected by the retarded flow. The temporal retardation can be determined from the phase angle and results in an intrinsic surface dilatational viscosity, if there is no mass transfer between the bulk and the interface. The complex modulus is

 ) || cos θ + i|| sin θ ) r + ii

(1)

with r being the real and i being the imaginary part of the complex modulus and θ being the phase angle. The dilatational elasticity is given by

|| ) -

dγ d ln A

(2)

The phase angle yields the dilatational viscosity

η)

|| sin θ ω

(3)

ω ) 2πf is the circular frequency of the area oscillation. Further details are given in the literature.12,18 In a first set of harmonic drop oscillation experiments, we determined the suitable drop volume amplitude that yields harmonic oscillations of both the drop area and surface pressure. We found that drop volume amplitude of (0.5 µL caused disharmonic oscillations of the surface pressure, especially at surface pressures higher than 50 mN/m. Drop volume amplitude of (0.25 µL caused harmonic oscillation of both the drop area and surface pressure, with drop area deformation of less than 10%. In a second set of harmonic drop oscillation experiments, we used a drop volume amplitude of (0.25 µL to measure dilatational elasticity and viscosity of DPPC and DPPC/SP-C films. Elasticity and viscosity were obtained by measurements at nine different levels of surface pressure and for nine different frequencies in the range between 0.006 and 0.25 Hz. Each set of experiments was done at 20, 30, and 40 °C ((0.1). Surface pressure was adjusted stepwise with a compression speed of 3 × 10-2 nm2/ (18) Wu¨stneck, R.; Moser, B.; Muschiolik, G. Colloids Surf., B 1999, 15, 263. (19) Hawgood, S.; Benson, B. J.; Schilling, J.; Damm, D.; Clements, J. A.; White, R. T. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 66. (20) Wu¨stneck, R.; Wu¨stneck, N.; Vollhardt, D.; Miller, R.; Pison, U. Mater. Sci. Eng., C 1999, 8-9, 57.

Figure 1. Image of a pendant drop (0.04 cm3) and an injected chloroform/methanol droplet recorded by using a stroboscope. The volume of the small droplet is 412 pL. The frequency of droplet injection was 100 Hz. (molecule s) up to one of the nine levels of π. Then, the drop volume was kept constant for 10 min to obtain steady-state conditions, and oscillations at the nine different frequencies were carried out, allowing breaks of 10 min for reaching steady-state conditions between the various frequencies. A new surface layer was spread to obtain dilatational elasticity and viscosity at each of the nine surface pressure levels. A spline-fit based on a symmetric frequency/surface pressure matrix was used to create planes of elasticity and viscosity as a function of frequency and surface pressure. 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%. The Microdroplet Injection System. A microdroplet injection system (microdrop-Gesellschaft fu¨r Mikrodosiertechnik, Norderstedt, Germany) was used for the application of spreading solutions onto the pendant drop surface. Small droplets in the order of some picoliters were injected by compressing a small capillary with a piezo crystal.15,16 The microdroplets were injected over a distance of about 1 cm on the pendant drop without bringing the drop surface into contact with the capillary. A CCD camera and a stroboscope diode monitored droplet formation and spreading (Figure 1). To create single droplets of defined volume, an injection period of about 900 µs/drop was used. The volume of a single droplet was determined by the drop volume difference of a pendant water drop in a chloroform-saturated atmosphere before and after the injection of 999 chloroform/methanol droplets and using ADSA. The spreading amount for adjusting a definite surface coverage was determined from the average drop volume. The mean volume of a single droplet of chloroform/methanol (1:1, v/v) was 412.8 ( 4.3 pL. The atmosphere of the measuring cell was exchanged after spreading to remove evaporated chloroform, and any changes of the drop volume after removing the chloroform atmosphere were corrected before further measurements, as described earlier.20

Experimental Results π/A Isotherms. The π/A isotherms for pure DPPC and for DPPC + 2 mol % SP-C were different (Figure 2). Isotherms for DPPC had slightly pronounced phase transition regions. The DPPC isotherms were very similar at 20, 30, and 40 °C for a surface coverage of 40-45 mN/ m, DPPC elasticity dropped. The surface dilatational viscosity of DPPC had a sharp maximum at about 0.008 Hz. The viscosity dropped at frequencies between 0.04 and 0.2 Hz, which correspond to those of human respiration. Thus, in the frequency range of human respiration the DPPC film showed almost pure elastic behavior. Increasing temperature from 20 to 40 °C decreased DPPC elasticity and DPPC viscosity. Elasticity and viscosity of DPPC/SP-C films are given in Figures 6 and 7. Although the dependencies of both elasticity and viscosity on π and f for DPPC/SP-C films showed some differences compared to pure DPPC films, many similarities were found. The most obvious difference was that in the presence of SP-C the dilatational elasticity decreased much faster when the temperature was increased from 20 to 30 and 40 °C (results for 30 °C not shown). The dilatational viscosity of a DPPC/SP-C film was lower compared to that of a DPPC film, but both films had very low viscosities in the frequency range that resembles human respiration. Discussion

Figure 2. π/A isotherms of spread DPPC (a) layers and DPPC + 2% SP-C (b) monolayers at 20 °C (blue diamonds), 30 °C (red squares), and 40 °C (yellow triangles).

At a temperature of 20 °C, the squeeze-out plateau was in the range of π ≈ 50 mN/m, and at 30 and 40 °C it was at 45 mN/m. The slopes of the isotherms shifted to the right from 20 °C over 30 °C to 40 °C. Harmonic Drop Oscillation Experiments. A typical example of a harmonic drop oscillation experiment is shown in Figure 3. The drop volume oscillation of (0.25 µL caused a harmonic oscillation of surface pressure and surface area even for a starting surface pressure of about 54.5 mN/m. The surface pressure slightly decreased during the first oscillations. Then, however, the oscillations became stable and the surface pressure oscillated around a constant level of π. Such behavior indicates an undestroyed surface layer structure. To obtain dilatational elasticity and viscosity of DPPC and DPPC/SP-C films, we carried out a series of harmonic drop oscillation experiments with each of the films and for various temperatures. Elasticity and viscosity of DPPC films are shown in Figures 4 and 5. The surface dilatational elasticity of DPPC increased more or less continuously up to a surface pressure of about 40-45 mN/m. The most pronounced increase of elasticity (from about 200 mN/m up to more than 300 mN/m) occurred at frequencies of 0.04-0.1 Hz. Temperature influenced DPPC elasticity only moder-

For the first time, the surface rheological behavior of two main components of pulmonary surfactant, DPPC and SP-C, was characterized in the frequency range of human breathing. We used carefully tested equipment for the measurement of dynamic film behavior and found that in the frequency range of human respiration the surface dilatational behavior of DPPC/SP-C films is spontaneously elastic and negligibly influenced by viscosity. Experimental determination of rheological film behavior requires careful design and testing of the measuring device, since different measuring devices may yield different results for same surface layers. We used a pendant drop microfilm balance and a new microdroplet injection system to study the rheological behavior of pulmonary surfactant films. The pendant drop microfilm balance has two limitations. One limitation is due to extremely low surface tensions of the lipid surface film; the other, to possible disharmonic oscillation of highly elastic films at high amplitudes of surface deformation. The first limitation was carefully evaluated by measuring π/A isotherms of DPPC and DPPC/SP-C. Insoluble phospholipid monolayers can be compressed to a state where the nominal area per molecule becomes smaller than the molecular cross section. In such cases, film pressure (σ0 - σ, the difference between the surface tension of the subphase liquid and the tension realized by the monolayer) strongly increases and a surface tension of nearly zero results. When the surface tension becomes nearly zero, the wetting of the capillary strongly increases and the film may spread over the capillary surface even when it is hydrophobic.5 Therefore, our surface rheological investigation was limited to a level of surface pressure of 55 mN/m. The second limitation of the pendant drop microfilm balance for rheological experiments is given by possible disharmonic oscillation of the surface pressure at high amplitudes of surface deformation probably indicating ruptures of the film. The range of oscillation amplitudes that does not cause disharmonic oscillation of surface pressure has to be determined experimentally for each system.18 We found that an amplitude of drop volume oscillation of 0.25 µL is sufficient to guarantee evaluation of undestroyed DPPC and DPPC/SP-C layers even at a starting surface pressure of 54.5 mN/m. The new microdroplet injection system used in this study accurately spreads extremely small amounts of spreading

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Figure 3. Harmonic oscillation of a DPPC monolayer spread on phosphate buffer (pH ) 7) at 20 °C: drop volume (V, blue diamonds), area (S, yellow triangles), and surface pressure (π, red diamonds). Before drop volume oscillation, the monolayer was compressed to π ) 54.4 mN/m. Amplitude of the drop volume oscillation: (0.25 µL.

liquid in the range of some picoliters onto the surface of a pendant drop, thus creating defined, insoluble layers. Such an accurate high-speed liquid handling system allows extending the application range of the pendant drop microfilm balance. The results of the π/A isotherms determined by using the new spreading technique for DPPC confirm our previous results using a pendant drop microfilm balance5 and a captive bubble device12,22 and also agree well with those of others using a conventional Langmuir film balance.6,21 It was pointed out by different authors5,18,23-25 that one reason for differences between π/A isotherms determined by using a conventional Langmuir film balance and the pendant drop technique is caused by different spreading conditions which have to be used according to the special features of these techniques. Wege et al.26 even reported effects due to the point of contacting the drop surface by a capillary. Such effects were not observed with our new spreading technique. In addition, because phosphate buffered saline was used in the present study and pure water was used in our previous studies with the captive bubble12 and the pendant drop,5 we believe that the subphase does not influence rheology of the interface that consists of DPPC and SP-C. The π/A isotherms of DPPC/SP-C layers exhibit a wellpronounced squeeze-out plateau at film pressures of 4550 mN/m. The squeeze-out process is nearly completed when the minimum area per molecule equals that of the cross section of vertically orientated lipid molecules (≈0.4 nm2/molecule). After the proteins are squeezed out, the film pressure rapidly increases even for small surface deformations. The occurrence of the squeeze-out plateau found in our study using a spreading technique for DPPC/ SP-C is comparable to the plateau that was observed for (22) Putz, G.; Walch, M.; van Eijk, M.; Haagsman, H. P. Biophys. J. 1998, 75, 2229. (23) Kwok, D. Y.; Tadros, B.; Deol, H.; Vollhardt, D.; Miller, R.; Cabrerizo-Vilchez, M. A.; Neumann, A. W. Langmuir 1996, 12, 1851. (24) Jyoti, A.; Prokop, R. M.; Li, J.; Vollhardt, D.; Kwok, D. Y.; Miller, R.; Mo¨hwald, H.; Neumann, A. W. Colloids Surf. 1996, 116, 173. (25) Wu¨stneck, R.; Siegel, S.; Ebisch, Th.; Miller, R. J. Colloid Interface Sci. 1998, 203, 83. (26) Wege, H. A.; Holgado-Terriza, J. A.; Ga´lvez-Ruiz, M. J.; Cabrerizo-Vı´lchez, M. A. Colloids Surf., B 1999, 12, 339.

Figure 4. Surface dilatational elasticity, , via surface pressure, π, and oscillation frequency, f. DPPC spread on phosphate buffer, pH ) 7, at 20 °C (a) and 40 °C (b).

native pulmonary surfactant extracts using rapid film formation by adsorption in a captive bubble device.27 We do not know yet how many layers the pulmonary sur-

Pulmonary Surfactant Components on a Pendant Drop

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Figure 5. Surface dilatational viscosity, η, via surface pressure, π, and oscillation frequency, f. DPPC spread on phosphate buffer, pH ) 7, at 20 °C (a) and 40 °C (b).

Figure 6. Surface dilatational elasticity, , via surface pressure, π, and oscillation frequency, f. DPPC + 2% SP-C spread on phosphate buffer, pH ) 7, at 20 °C (a) and 40 °C (b).

factant film consists of in vivo and how many layers would be formed using adsorption of native surfactant in a captive bubble device. However, the comparable data between measurements at the pendant drop and in the captive bubble28 indicate that the Laplacian shape of the interface in the captive bubble and at the surface of the pendant drop is consistent with the shape of an interface which is modified by one monolayer up to very high surface pressures. Surface dilatational elasticity and viscosity of spread DPPC and DPPC/SP-C films quantitatively confirm previous findings for films spread on water at 20 °C in a smaller range of frequency (0.006-0.02 Hz) but up to surface pressures of 70 mN/m using a captive bubble device.12 It was shown that at 20 °C the surface dilatational behavior of such layers is viscoelastic with two maxima of surface dilatational elasticity and viscosity, one before the protein squeeze-out and one after the squeeze-out region, at about 0.008 Hz.12 Due to the limitations of the pendant drop, we found only one maximum before the squeeze-out region in the present study. However, in both studies the elasticities and viscosities increased with film pressure up to the protein squeeze-out plateau. In extension to our previous study, we demonstrate now that the surface dilatational viscosity of DPPC and DPPC/SP-C

films strongly drops in the range of breathing frequencies even at high surface pressures while the elasticity is only negligible influenced by the frequency of deformation. Thus, in the frequency range of breathing the surface mechanical behavior of such films is spontaneously elastic. Nearly all statements about pulmonary surfactant’s film compressibility (i.e., its elastic behavior) are based on the inversed first derivative of a quasi-static π/A isotherm or other relation of surface tension versus surface concentration. This, however, is only a crude approximation to the real rheological behavior and is not comparable with direct surface rheological measurements.29,30 It actually ignores the fact that any rheological property depends on stress and strain and their velocities. On the basis of our experimental measurements of pure DPPC and DPPC/ SP-C films, we are able to describe the rheological behavior of such films in more precise physical terms and demonstrate the influence of temperature and the presence of protein on lipid film behavior. The temperature remarkably influences the surface dilatational behavior of spread DPPC and DPPC/SP-C films without changing the rheological behavior qualitatively. Both elasticity and viscosity moderately decrease

(27) Schu¨rch, S.; Schu¨rch, D.; Curstedt, T.; Robertson, B. J. Appl. Physiol. 1994, 77, 974. (28) Schu¨rch, S.; Green, F. H. Y.; Bachofen, H. Biochem. Biophys. Acta 1998, 1408, 180.

(29) Wu¨stneck, R.; Enders, P.; Wu¨stneck, N.; Pison, U.; Miller, R. PhysChemComm 1999, 11. (30) Wu¨stneck, R.; Enders, P.; Ebisch, Th.; Miller, R.; Siegel, S. J. Colloid Interface Sci. 1998, 206, 33. (31) Pe´rez-Gil, J.; Keough, K. M. W. Biochim. Biophys. Acta 1998, 1408, 203.

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constant. For fast deformations, the influence of viscosity vanishes without altering the main feature of surface dilatational behavior, that is, the surface dilatational viscosity is very low at higher frequencies. In general, our results suggest that the investigated films are more compressible and flexible at higher temperatures and that in the range of breathing frequencies elastic film properties are dominant. The presence of 2% SP-C yields elasticities of DPPC films that are only slightly influenced by frequency at high film pressures. In addition, the elasticity of DPPC/ SP-C films is lower compared to that of pure DPPC films, indicating that mixed films are more compressible than pure DPPC films, and compressibility increases with increasing temperatures (compressibility equals the inverse elasticity). Such behavior supports the concept that more flexible film structures are formed in the presence of proteins.31 It also explains the shift of the squeeze-out plateau to a lower film pressure. However, surfactantassociated proteins may not necessarily decrease film compressibility of phospholipid films, as suggested by Schu¨rch and co-workers.27 In summary, the rheological behavior of DPPC and DPPC/SP-C layers is spontaneously elastic with nearly vanishing dilatation viscosities at frequencies of human respiration (0.04-0.2 Hz). This rheological behavior was found in carefully tested equipment for the measurement of dynamic film deformation under stress and strain and their velocities. Whether and how the presence of the second pulmonary hydrophobic surfactant protein, SP-B, modifies the rheological behavior of a film spread on a pendant drop needs further investigation.

Figure 7. Surface dilatational viscosity, η, via surface pressure, π, and oscillation frequency, f. DPPC + 2% SP-C spread on phosphate buffer, pH ) 7, at 20 °C (a) and 40 °C (b).

when the temperature is increased from 20 to 30 °C. In the temperature range of 30-40 °C, the elasticity of DPPC films further decreases, while the viscosity almost remains

Acknowledgment. The financial support by the Deutsche Forschungsgemeinschaft (Grant Pi 165/7, Wu¨ 187/6, Wu¨ 187/8) is gratefully acknowledged. Many thanks go to Klaus Dannenberg (Virchow-Klinikum, Werkstatt) for helping us to build the experimental setups and to Anne Gale for proofreading of the manuscript. LA010685W