Phospholipid packing and hydration in pulmonary surfactant membranes and films as sensed by LAURDAN

Abstract

The efficiency of pulmonary surfactant to stabilize the respiratory surface depends critically on the ability of surfactant to form highly packed films at the air-liquid interface. In the present study we have compared the packing and hydration properties of lipids in native pulmonary surfactant and in several surfactant models by analyzing the pressure and temperature dependence of the fluorescence emission of the LAURDAN (1-[6-(dimethylamino)-2-naphthyl]dodecan-1-one) probe incorporated into surfactant interfacial films or free-standing membranes. In interfacial films, compression-driven changes in the fluorescence of LAURDAN, evaluated from the generalized polarization function (GPF), correlated with changes in packing monitored by surface pressure. Compression isotherms and GPF profiles of films formed by native surfactant or its organic extract were compared at 25 or 37 °C to those of films made of dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), DPPC/phosphatidylglycerol (PG) (7:3, w/w), or the mixture DPPC/POPC/palmitoyloleoylphosphatidylglycerol (POPG)/cholesterol (Chol) (50:25:15.10), which simulates the lipid composition of surfactant. In general terms, compression of surfactant films at 25 °C leads to LAURDAN GPF values close to those obtained from pure DPPC monolayers, suggesting that compressed surfactant films reach a dehydrated state of the lipid surface, which is similar to that achieved in DPPC monolayers. However, at 37 °C, the highest GPF values were achieved in films made of full surfactant organic extract or the mixture DPPC/POPC/POPG/Chol, suggesting a potentially important role of cholesterol to ensure maximal packing/dehydration under physiological constraints. Native surfactant films reached high pressures at 37 °C while maintaining relatively low GPF, suggesting that the complex three-dimensional structures formed by whole surfactant might withstand the highest pressures without necessarily achieving full dehydration of the lipid environments sensed by LAURDAN. Finally, comparison of the thermotropic profiles of LAURDAN GPF in surfactant model bilayers and monolayers of analogous composition shows that the fluorophore probes an environment that is in average intrinsically more hydrated at the interface than inserted into free-standing bilayers, particularly at 37 °C. This effect suggests that the dependence of membrane and surfactant events on the balance of polar/non-polar interactions could differ in bilayer and monolayer models, and might be affected differently by the access of water molecules to confined or free-standing lipid structures.

Interfacial behavior and structural properties of a clinical lung surfactant from porcine source

“…Thermotropic phase transitions in NPLS, ENPLS and Surfacen suspensions were also characterized by following the temperature-dependent shift of the fluorescence of a trace (1% with respect to phospholipid, mol/mol) of the fluorescent probe LAURDAN, as previously described [33]. LAURDAN was incorporated into surfactant samples as a small dimethylsulfoxide aliquot, and the final phospholipid concentration was of 10 μg/mL.…”

“…The fluorescence spectra of all samples were recorded in an Aminco-Bowman Series 2 luminescence spectrometer equipped with thermostated cells, using an excitation wavelength of 370 nm and recording the emission between 400 and 550 nm, within a temperature range of 10 to 60°C. The results were expressed as a generalized polarization function (GPF), defined as GPF =(I B − I R )/I B −I R being I B and I R the intensities at the blue and red edges of the emission spectrum, respectively [33,34].…”

Surfactant and its role in the pathobiology of pulmonary infection

“…Phosphatidylglycerol (PG) and phosphatidylinositol (PI) account for 8–15% of the phospholipid portion [8, 11, 18]. Other minor phospholipids and neutral lipids are also detectable [8, 19, 21]. Although proteins make up a small fraction of surfactant, the four surfactant-associated proteins, surfactant protein (SP)-A, SP-B, SP-C, and SP-D [2, 6], all perform important roles in regulating surfactant function.…”

“…The lipid portion of surfactant is primarily responsible for the surface activity of the film [4, 7, 19], though it is not the sole component contributing to this function. The film is enriched in DPPC [11], the main component conferring reduced surface activity although other saturated molecular species of PC also may exert similar properties [25]; DPPC becomes a highly packed monomolecular film with a squeeze out of other unsaturated lipids during compression phases of the respiratory cycle to achieve the low surface tensions needed to protect alveoli from atelectasis during end-expiration [7, 19].…”

Laurdan Monitors Different Lipids Content in Eukaryotic Membrane During Embryonic Neural Development

“…This sensitivity arises from the greater than 50-nm red shift of the emission maximum in polar versus non polar environments, so that simple fluorescence intensity measurements at two properly selected wavelengths provide information on the membrane polarity. Several studies have shown that laurdan spectroscopic properties reflect local water content in the membrane (54–61) and indirectly, membrane fluidity. Laurdan is a molecule whose spectroscopic properties are influenced by both the composition and dynamics of its local surroundings (62–64).…”