Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant

Shou-Hwa Yu,Fred Possmayer

doi:10.1016/s0022-2275(20)30274-1

Abstract

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir. The extent to which surfactant components including the main component, dipalmitoylphosphatidylcholine (DPPC), are adsorbed into the monolayer, and how surfactant protein SP-A affects their adsorptions, is not clear. Transport of cholesterol to the surface region from dispersions of bovine lipid extract surfactant [BLES(chol)] with or without SP-A at 37°C was studied by measuring surface radioactivities of [4-14C]cholesterol-labeled BLES(chol), and the Wilhelmy plate technique was used to monitor adsorption of monolayers. Results showed that transport of cholesterol was lipid concentration dependent. SP-A accelerated lipid adsorption but suppressed the final level of cholesterol in the surface. Surfactant adsorbed from a dispersion with or without SP-A was transferred via a wet filter paper to a clean surface, where the surface radioactivity and surface tension were recorded simultaneously. It was observed that 1) surface radioactivity was constant over a range of dispersion concentrations; 2) cholesterol and DPPC were transferred simultaneously; and 3) SP-A limited transfer of cholesterol. These results indicate that non-DPPC components of pulmonary surfactant can be adsorbed into the monolayer. Studies in the transfer of [1-14C]DPPC-labeled BLES(chol) to an equal or larger clean surface area revealed that SP-A did not increase selective adsorption of DPPC into the monolayer. Evaluation of transferred surfactant with a surface balance indicated that it equilibrated as a monolayer. Furthermore, examination of transferred surfactants from dispersions with and without prespread BLES(chol) monolayers revealed a functional contiguous association between adsorbed monolayers and reservoirs. —Yu, S-H., and F. Possmayer. Dipalmitoylphosphatidylcholine and cholesterol in monolayers spread from adsorbed films of pulmonary surfactant.

Highlights

Pulmonary surfactant forms a surface film that consists of a monolayer and a monolayer-associated reservoir
surfactant proteins (SP)-A is able to impede the interaction of cholesterol with DPPC in DPPC/SP-A/cholesterol spread monolayers and decrease the surface area reduction required to reach near zero surface tension [18]
Using [26-14C]cholesterol-labeled bovine lipid extract surfactant (BLES)(chol), we have shown that SP-A decreased the detectable surface radioactivity of [14C]cholesterol in the surface films formed from dispersions of BLES plus exogenous SP-A and cholesterol [23]

Summary

MATERIALS AND METHODS

Materials [4-14C]cholesterol and [1-14C]DPPC were purchased from New England Nuclear (Boston, MA). All reagents (analytical grade) were obtained from BDH (Poole, UK). Distilled water purified through a Millipore (Danvers, MA) Milli-Q four-cartridge system was used in all experiments. SP-A was purified by HPLC as described previously [6]. 10 mg of bovine pulmonary surfactant was suspended in 1 ml of 6 M urea-0.05% trifluoroacetic acid (TFA) and filtered. SP-A was eluted with a linear gradient of 2-propanol in 0.05% TFA at a flow rate of 0.8 ml/min. BLES(chol) was produced by chloroform-methanol extraction of bovine pulmonary surfactant, using the method of Bligh and Dyer [26]. BLES(chol) retains all lipid components of the sur factant and surfactant proteins, SP-B and SP-C, but not SP-A or SP-D [6]

Preparation of samples

Studies of transfer of surfactant to a clean surface

Surface properties of the transferred surfactants

RESULTS AND DISCUSSION

Further characterization of the transferred surfactant