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°C 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. † © 1999 American Chemical Society * To whom correspondence should be addressed at Mail Code OHSU, Portland,.. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 December 08. The phase behavior of biological phospholipid films and membranes may be important in at least two respects. First, the different physical characteristics of the different phases can alter processes such as lateral diffusion. Second, phase separation can maintain components in distinct compartments and provide a means of organizing biological processes (2). Both of these issues may be important for the function of pulmonary surfactant. This complex mix of lipids and proteins coats the thin liquid layer that lines the lung's alveolar air spaces. When compressed by decreasing alveolar surface areas during normal exhalation, the surfactant film achieves very high densities, well above equilibrium values. Measurements in situ show that the surfactant films lower the surface tension of the air-liquid interface to very low levels (3) and in doing so stabilize the alveoli. Only films with the physical characteristics of a highly ordered phase se...

Distribution of Coexisting Solid and Fluid Phases Alters the Kinetics of Collapse from Phospholipid Monolayers

“…During compression of CLSE films, the l o phase appears at a π approximately 10 mN/m above the LE-TC transition for DPPC. 47 The l o -l d coexistence region for CLSE, with its multiple components, also extends over a much broader range of π than for DPPC. 49 Consequently, at 37 °C, where the main transition for DPPC begins at ~35 mN/m, the l o phase in CLSE appears just before the onset of collapse.…”

Synthetic Pulmonary Surfactant Preparations: New Developments and Future Trends

“…However, cholesterol is well recognized as a component that provides mechanical stability to cell membranes [48] and an important structural element to modulate their lateral structure, including segregation of specialized domains such as lipid rafts [49,50]. Recent results have shown that physiological proportions of cholesterol modulate the lateral organization and distribution of proteins and lipids in pulmonary surfactant membranes [51] and films [52,53], as well as the dynamical behaviour of surfactant films subjected to compression-expansion cycling [54]. Presence of cholesterol could be particularly important to permit surfactant films to reach and sustain the lowest surface tensions at 100% humidity [55], a condition thought to exist in the alveolar spaces but poorly reproduced in in vitro models.…”

Structure– Function Relationships of Hydrophobic Proteins SP-B and SP-C in Pulmonary Surfactant

“…Phase coexistence at σ e in calf lung surfactant extract (CLSE) can be minimal or nonexistent. During compression at 20°C, initial separation of two phases terminates before reaching σ e (34). Remixing immediately follows dramatic changes in the shape of the ordered domains and in the extent of the interfacial boundary, consistent with behavior caused by low line tension between two immiscible fluid phases close to a critical point.…”

“…Remixing immediately follows dramatic changes in the shape of the ordered domains and in the extent of the interfacial boundary, consistent with behavior caused by low line tension between two immiscible fluid phases close to a critical point. This remixing requires the presence of cholesterol (34). Critical points between fluid phases are now well documented in monolayers containing mixtures of cholesterol and phospholipids (38).…”

“…The multiple components also affect the progression of the transition as well as its onset. Although the Gibbs phase rule for a single component film confines coexistence to a single surface tension, the presence of multiple components releases these constraints, and completion of the transition for films that include the surfactant phospholipids requires compression through a broad range of surface tensions (33,34). Consequently at σ e and 37°C, the ordered phase in complete …”