Neutral lipids induce critical behavior in interfacial monolayers of pulmonary surfactant

Abstract

We have shown previously that lateral compression of pulmonary surfactant monolayers initially induces separation of two phases but that these remix when the films become more dense (1). In the studies reported here, we used fluorescence microscopy to examine the role of the different surfactant constituents in the remixing of the separated phases. Subfractions containing only the purified phospholipids (PPL), the surfactant proteins and phospholipids (SP&PL), and the neutral and phospholipids (N&PL) were obtained by chromatographic separation of the components in extracted calf surfactant (calf lung surfactant extract, CLSE). Compression of the different monolayers produced nonfluorescent domains that emerged for temperatures between 20 and 41 degreesC at similar surface pressures 6-8 mN/m higher than values observed for dipalmitoyl phosphatidylcholine (DPPC), the most prevalent component of pulmonary surfactant. Comparison of the different preparations showed that the neutral lipid increased the total nonfluorescent area at surface pressures up to 25 mN/m but dispersed that total area among a larger number of smaller domains. The surfactant proteins also produced smaller domains, but they had the opposite effect of decreasing the total nonfluorescent area. Only the neutral lipids caused remixing. In images from static monolayers, the domains for N&PL dropped from a maximum of 26 +/- 3% of the interface at 25 mN/m to 4 +/- 2% at 30 mN/m, similar to the previously reported behavior for CLSE. During continuous compression through a narrow range of pressure and molecular area, in N&PL, CLSE, and mixtures of PPL with 10% cholesterol, domains became highly distorted immediately prior to remixing. The characteristic transition in shape and abrupt termination of phase coexistence indicate that the remixing caused by the neutral lipids occurs at or close to a critical point.

Figure 1

Surface pressure–area isotherm with epifluorescence micrographs of DPPC monolayers. Chloroform solutions containing 1% (mol/mol) Rh–DPPE were spread at 20 °C on HSC. Images were obtained from static films following compression at 2.8 Ų/(phospholipid/min) to the desired pressure. Representative images are given at the specified surface pressures. Scale bar is 100 μm. Isotherms were obtained in separate experiments for DPPC without the addition of Rh–DPPE during compression at 1 Ų/(phospholipid/min).

Figure 2

Images and isotherms for compression of PPL monolayers. Conditions are identical to those for Figure 1, except that the scale bar represents 50 μm and the isotherm was obtained in a separate experiment without added Rh–DPPE during compression at 1 Ų/(phospholipid/min).

Figure 3

Images and isotherms for compression of SP&PL monolayers. Conditions are identical to those for Figure 2.

Figure 4

Images and isotherms for compression of N&PL monolayers. Conditions are identical to those for Figure 2.

Figure 5

Variation of the shape of domains in PPL with time at 25 mN/m. Films of PPL containing 1% (mol/mol) Rh–DPPE were spread to an initial area of 150 Ų/phospholipid and compressed at 2.8 Ų/(phospholipid/min) to 25 mN/m. Images of the static films were then recorded at the following number of hours after cessation of compression: A, 0; B, 2.5; C, 3.5; D, 6.5.

Figure 6

Variation of total area of condensed domains with surface pressure for the different preparations of surfactant components. Total area of the domains is expressed as the fraction of the total image area analyzed. At least three images were analyzed at each surface pressure for each of four experiments. (See Methods for details). Values are mean ± SD.

Figure 7

Density per area of domains in different preparations of surfactant components. The preparations were spread and compressed to specific surface pressures. Images were recorded from static films at the pressures indicated. The number of domains was counted from three images at each surface pressure for each preparation in four experiments. Values are mean ± SD.

Figure 8

Size distribution of domains in monolayers of surfactant constituents. Results are expressed as the fraction of domains analyzed that occurred within specific ranges of size. The different preparations produced different maximum sizes and required the following different box car intervals in area for this analysis: 5 μm² for CLSE and N&PL; 10 μm² for SP&PL; 50 μm² for PPL. Results at each surface pressure were averaged from at least three images for each of four experiments.

Figure 9

Temperature dependence of the surface pressure at which domains first emerged. Films of the different preparations spread at the temperatures indicated to 150 Ų/phospholipid were then compressed continuously at 2.8 Ų/(phospholipid/min). Values are mean ± SD; n = 3.

Figure 10

Fluorescence images of films containing N&PL. Films were spread in chloroform and compressed at 2.8 Ų/(phospholipid/min) until microscopy suggested the beginning of the shape transition. Images were then recorded from static films at the surface pressures A, 30 mN/m, B, 31 mN/m, and C, 31 mN/m, with compression of the film from Figure 10B only to overcome the small decay in surface pressure after the cessation of compression.

Figure 11

Remixing of separated phases in monolayers of CLSE. The film containing 1% (mol:mol) Rh–DPPE was spread in chloroform to 150 Ų/phospholipid, compressed at 2.8 Ų/(phospholipid/min) to 30 mN/m, and then held at constant area for 3 min before recording image A. The subsequent images were then recorded during continuous compression at 2.8 Ų/(phospholipid/min) at the following intervals in time (and molecular area) after reaching 34 mN/m: B, 0 s (0 Ų/phospholipid); C, 4 s (0.2 Ų/phospholipid); D, 13 s (0.6 Ų/phospholipid).

Figure 12

Fluorescence images of films containing mixtures of cholesterol:PPL (1:9, mol:mol). Images were recorded during continuous compression at 2.8 Ų/(phospholipid/min) of films spread in chloroform to initial molecular areas of 125 Ų/phospholipid. Images correspond to the following molecular areas (Ų/phospholipid): A, 68.0; B, 64.3; C, 64.2; D, 63.8; E, 60.9; F, 58.0.