Multilayer structures in lipid monolayer films containing surfactant protein C: effects of cholesterol and POPE

Abstract

The influence of cholesterol and POPE on lung surfactant model systems consisting of DPPC/DPPG (80:20) and DPPC/DPPG/surfactant protein C (80:20:0.4) has been investigated. Cholesterol leads to a condensation of the monolayers, whereas the isotherms of model lung surfactant films containing POPE exhibit a slight expansion combined with an increased compressibility at medium surface pressure (10-30 mN/m). An increasing amount of liquid-expanded domains can be visualized by means of fluorescence light microscopy in lung surfactant monolayers after addition of either cholesterol or POPE. At surface pressures of 50 mN/m, protrusions are formed which differ in size and shape as a function of the content of cholesterol or POPE, but only if SP-C is present. Low amounts of cholesterol (10 mol %) lead to an increasing number of protrusions, which also grow in size. This is interpreted as a stabilizing effect of cholesterol on bilayers formed underneath the monolayer. Extreme amounts of cholesterol (30 mol %), however, cause an increased monolayer rigidity, thus preventing reversible multilayer formation. In contrast, POPE, as a nonbilayer lipid thought to stabilize the edges of protrusions, leads to more narrow protrusions. The lateral extension of the protrusions is thereby more influenced than their height.

FIGURE 1

Compression isotherms of DPPC/DPPG (80:20) (a) and DPPC/DPPG/SP-C (80:20:0.4) (b) monolayers containing an increasing amount of cholesterol (shaded arrow). The concentrations of cholesterol are 0, 1, 5, 10, and 30 mol % of the total lipid amount. FLM images were taken at the surface pressures marked by black arrows.

FIGURE 2

Fluorescence light microscopy images of DPPC/DPPG (80:20) (a–i) and of DPPC/DPPG/SP-C (80:20:0.4) (m–x) monolayers containing different amounts of cholesterol. Images were taken at surface pressures marked in Fig. 1. The concentrations of cholesterol are: 0 mol % (first row), 1 mol % (second row), 10 mol % (third row), and 30 mol % (fourth row). The image size is 130 × 130 μm².

FIGURE 3

Compression isotherms of (a) DPPC/DPPG (80:20) and (b) DPPC/DPPG/SP-C (80:20:0.4) monolayers containing an increasing amount of POPE (shaded arrow). The concentrations of POPE are 0, 1, 5, 10, and 30 mol % of the total lipid amount. FLM images were taken at the surface pressures marked by black arrows.

FIGURE 4

Fluorescence light microscopy images of DPPC/DPPG (80:20) (a–i) and of DPPC/DPPG/SP-C (80:20:0.4) (m–x) monolayers containing different amounts of POPE. Images were taken at surface pressures marked in Fig. 2. The concentrations of POPE are: 0 mol % (first row), 1 mol % (second row), 10 mol % (third row), and 30 mol % (fourth row). The image size is 130 × 130 μm².

FIGURE 5

Statistical analysis of FLM images at a surface pressure of 5 mN/m. (a) The areas of bright and dark domains were calculated by fitting the data to the sum of two Gaussian distributions. The Gaussian curve with an area of 78% represents the l_e phase, whereas the other one marks the dark domains with a total area of 22%. (b and c) Histograms of fluorescence light intensity of (b) DPPC/DPPG (80:20) and (c) DPPC/DPPG/SP-C (80:20:0.4) monolayers with various amounts of cholesterol/POPE. Model surfactant system in black, cholesterol-doped in light shaded, and POPE-doped in dark shaded.

FIGURE 6

SFM images and height profiles of DPPC/DPPG/SP-C films containing different amounts of cholesterol/POPE. (a) 0 mol % of cholesterol/POPE, (b) 10 mol % of cholesterol, (c) 30 mol % of cholesterol, (d) 10 mol % of POPE, and (e) 30 mol % of POPE. The line scans exhibit the height profile. Protrusions are visualized as single steps of lipid bilayers. The image size is 5 × 5 μm².

FIGURE 7

SFM images and histograms of the gray-scale analysis of model surfactant monolayers. (a) DPPC/DPPG/SP-C (80:20:0.4), (b) DPPC/DPPG/SP-C/ cholesterol (80:20:0.4:30), and (c) DPPC/DPPG/SP-C/POPE (80:20:0.4:30). The insets show magnifications of the protrusions with an image size of 2 × 2 μm². The histogram analysis of the full-scale images represent fits to a Gaussian distribution. The image size is 25 × 25 μm². Insets are 2 × 2 μm².

FIGURE 8

Proposed model of cholesterol/POPE interactions with the model surfactant system DPPC/DPPG/SP-C (80:20:0.4). POPE is enriched in the rims of the protrusions, whereas cholesterol stabilizes the bilayers.