Comparative Studies On Bovine And Rat Pulmonary Surfactants Using AFM

Abstract

Recent studies employing a variety of microscopic techniques have revealed that phospholipid (PL) phase separations and transitions may play important roles in determining the biophysical properties of pulmonary surfactant. Most of these microscopic studies used model systems which were composed of simple mixtures of PL with or without hydrophobic surfactant proteins and cholesterol. The present work compared modified natural (lipid extract) bovine and rat surfactants using atomic force microscopy (AFM). AFM revealed PL phase separation upon compression of both surfactant monolayers, and a monolayer-to-multilayer transition at surface pressure 40-50 mN/m. Similar to bovine surfactant, the tilted-condensed (TC) phase in rat surfactant consisted of domains both on micrometer and nanometer scales. Upon film compression, the microdomains were dissociated into nanodomains, thus forming a more homogeneous two-phase mixture. Differences between rat and bovine surfactants were: (1) more TC domains were formed at lower surface pressures in rat than in bovine surfactant; and (2) an interesting domain-in-domain structure was exclusively observed in rat surfactant. These structural differences were attributed to the higher cholesterol content of rat surfactant (∼ 10 vs ∼2.5 wt%). To further investigate the effects of cholesterol on the structure of surfactant films, we have studied cholesterol-depleted bovine surfactant (∼ 0%) prepared by repetitive acetone extraction. Removal of cholesterol from bovine surfactant induced significant variations in film structure. More importantly, the film structure can be effectively restored by recombining cholesterol with the cholesterol-depleted bovine surfactant. Recombinant bovine surfactant with 10% cholesterol showed domain-in-domain structures similar to those found with rat surfactant. These interspecies studies of the micro- and nano- structures of natural pulmonary surfactants add insight into the biophysical interpretation of phospholipid phase transition and separation, in particular the role of cholesterol.