Pulmonary Surfactant Membranes Research Articles

Understanding the Retention of Vaping Additives in the Lungs: Model Lung Surfactant Membrane Perturbation by Vitamin E and Vitamin E Acetate.

Deviations from the normal physicochemical and functional properties of pulmonary surfactants are associated with the incidence of lung injury and other respiratory disorders. This study aims to evaluate the alteration of the 2D molecular organization and morphology of pulmonary surfactant model membranes by the electronic cigarette additives α-tocopherol (vitamin E) and α-tocopherol acetate (vitamin E acetate), which have been associated with lung injury, termed e-cigarette or vaping-use-associated lung injury (EVALI). The model membranes used contained a 7:3 molar ratio of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to which α-tocopherol and α-tocopherol acetate were added to form mixtures of up to 20 mol % additive. The properties of the neat tocopherol additives and DPPC/POPG (7:3) mixtures with increasing molar proportions of additive were evaluated by surface pressure-area isotherms, excess area calculations, Brewster angle microscopy, grazing incidence X-ray diffraction, X-ray reflectivity, and atomic force microscopy. The addition of either additive alters the essential phase balance of the model pulmonary surfactant membrane by generating a greater proportion of the fluid phase. Despite this net fluidization, both tocopherol additives have space-filling effects on the liquid-expanded and condensed phases, yielding negative excess areas in the liquid-expanded phase and reduced tilt angles in the condensed phase. Both tocopherol additives alter the stability of the fluid phase, pushing the eventual collapse of this phase to higher surface pressures than the model membrane in the absence of an additive.

Pulmonary surfactant and drug delivery: Vehiculization of a tryptophan-tagged antimicrobial peptide over the air-liquid interfacial highway

This work evaluates interaction of pulmonary surfactant (PS) and antimicrobial peptides (AMPs) in order to investigate (i) if PS can be used to transport AMPs, and (ii) to what extent PS interferes with AMP function and vice versa. This, in turn, is motivated by a need to find new strategies to treat bacterial infections in the airways. Low respiratory tract infections (LRTIs) are a leading cause of illness and death worldwide that, together with the problem of multidrug-resistant (MDR) bacteria, bring to light the necessity of developing effective therapies that ensure high bioavailability of the drug at the site of infection and display a potent antimicrobial effect. Here, we propose the combination of AMPs with PS to improve their delivery, exemplified for the hydrophobically end-tagged AMP, GRR10W4 (GRRPRPRPRPWWWW-NH2), with previously demonstrated potent antimicrobial activity against a broad spectrum of bacteria under various conditions. Experiments using model systems emulating the respiratory interface and an operating alveolus, based on surface balances and bubble surfactometry, served to demonstrate that a fluorescently labelled version of GRR10W4 (GRR10W4-F), was able to interact and insert into PS membranes without affecting its biophysical function. Therefore, vehiculization of the peptide along air–liquid interfaces was enabled, even for interfaces previously occupied by surfactants layers. Furthermore, breathing-like compression-expansion dynamics promoted the interfacial release of GRR10W4-F after its delivery, which could further allow the peptide to perform its antimicrobial function. PS/GRR10W4-F formulations displayed greater antimicrobial effects and reduced toxicity on cultured airway epithelial cells compared to that of the peptide alone. Taken together, these results open the door to the development of novel delivery strategies for AMPs in order to increase the bioavailability of these molecules at the infection site via inhaled therapies.

Dimerization of the pulmonary surfactant protein C in a membrane environment.

Surfactant protein C (SP-C) has several functions in pulmonary surfactant. These include the transfer of lipids between different membrane structures, a role in surfactant recycling and homeostasis, and involvement in modulation of the innate defense system. Despite these important functions, the structures of functional SP-C complexes have remained unclear. SP-C is known to exist as a primarily α-helical structure with an apparently unstructured N-terminal region, yet there is recent evidence that the functions of SP-C could be associated with the formation of SP-C dimers and higher oligomers. In this work, we used molecular dynamics simulations, two-dimensional umbrella sampling, and well-tempered metadynamics to study the details of SP-C dimerization. The results suggest that SP-C dimerizes in pulmonary surfactant membranes, forming dimers of different topologies. The simulations identified a dimerization motif region V21xxxVxxxGxxxM33 that is much larger than the putative A30xxxG34 motif that is commonly assumed to control the dimerization of some α-helical transmembrane domains. The results provide a stronger basis for elucidating how SP-C functions in concert with other surfactant proteins.

Pathological cardiolipin-promoted membrane hemifusion stiffens pulmonary surfactant membranes

Lower tract respiratory diseases such as pneumonia are pervasive, affecting millions of people every year. The stability of the air/water interface in alveoli and the mechanical performance during the breathing cycle are regulated by the structural and elastic properties of pulmonary surfactant membranes (PSMs). Respiratory dysfunctions and pathologies often result in, or are caused by, impairment of the PSMs. However, a gap remains between our knowledge of the etiology of lung diseases and the fundamental properties of PSMs. For example, bacterial pneumonia in humans and mice has been associated with aberrant levels of cardiolipin, a mitochondrial-specific, highly unsaturated 4-tailed anionic phospholipid, in lung fluid, which likely disrupts the structural and mechanical integrity of PSMs. Specifically, cardiolipin is expected to significantly alter PSM elasticity due to its intrinsic molecular properties favoring membrane folding away from a flat configuration. In this paper, we investigate the structural and mechanical properties of the lipidic components of PSMs using lipid-based models as well as bovine extracts affected by the addition of pathological cardiolipin levels. Specifically, using a combination of optical and atomic force microscopy with a surface force apparatus, we demonstrate that cardiolipin strongly promotes hemifusion of PSMs and that these local membrane contacts propagate at larger scales, resulting in global stiffening of lung membranes.

Are Plant-Based Carbohydrate Nanoparticles Safe for Inhalation? Investigating Their Interactions with the Pulmonary Surfactant Using Langmuir Monolayers.

Nanoparticle carriers show promise for drug delivery, including by inhalation, where the first barrier for uptake in the lungs is the monolayer pulmonary surfactant membrane that coats the air/alveoli interface and is critical to breathing. It is imperative to establish the fate of potential nanocarriers and their effects on the biophysical properties of the pulmonary surfactant. To this end, the impact of the nanoparticle surface charge on the lateral organization, thickness, and recompressibility of Langmuir monolayers of model phospholipid-only and phospholipid-protein mixtures was investigated using native and modified forms of nanophytoglycogen, a carbohydrate-based dendritic polymer extracted from corn as monodisperse nanoparticles. We show that the native (quasi-neutral) and anionic nanophytoglycogens have little impact on the phase behavior and film properties. By contrast, cationic nanophytoglycogen alters the film morphology and increases the hysteresis associated with the work of breathing due to its electrostatic interaction with the anionic phospholipids in the model systems. These findings specifically highlight the importance of surface charge as a selection criterion for inhaled nanoformulations.

Evaluation of the permeability and potential toxicity of polycyclic aromatic hydrocarbons to pulmonary surfactant membrane by the parallel artificial membrane permeability assay model

Polycyclic aromatic hydrocarbons (PAHs) can penetrate and accumulate in the pulmonary surfactant (PS) membranes, leading to abnormalities of biological macromolecules and the destruction of membrane structure and properties. In the present study, the bioavailability, apparent permeability, effective permeability and residual coefficient of 10 PAHs on PS membrane was assessed by the parallel artificial membrane permeability assay (PAMPA). The influence of various forces on permeability is obtained by analyzing the correlation between parameters and physicochemical properties. Research shows that octanol-water partition coefficient (Kow) cannot directly predict permeability, and permeability has no significant relationship with polarity. Dispersion, induction, coupling/polarization promote permeation, while hydrogen bonded acid and n-n electron pair inhibit permeation. Further surface pressure-area (π-A) isotherms test and Brewster angle microscope observation manifested that there are huge differences in the transmembrane ability and effects on the membrane of PAHs with different structures. This work has considerable significance that will help to evaluate the bioavailability and human health risk of PAHs.

Nebulised surface-active hybrid nanoparticles of voriconazole for pulmonary Aspergillosis demonstrate clathrin-mediated cellular uptake, improved antifungal efficacy and lung retention

BackgroundIncidence of pulmonary aspergillosis is rising worldwide, owing to an increased population of immunocompromised patients. Notable potential of the pulmonary route has been witnessed in antifungal delivery due to distinct advantages of direct lung targeting and first-pass evasion. The current research reports biomimetic surface-active lipid-polymer hybrid (LPH) nanoparticles (NPs) of voriconazole, employing lung-specific lipid, i.e., dipalmitoylphosphatidylcholine and natural biodegradable polymer, i.e., chitosan, to augment its pulmonary deposition and retention, following nebulization.ResultsThe developed nanosystem exhibited a particle size in the range of 228–255 nm and drug entrapment of 45–54.8%. Nebulized microdroplet characterization of NPs dispersion revealed a mean diameter of ≤ 5 μm, corroborating its deep lung deposition potential as determined by next-generation impactor studies. Biophysical interaction of LPH NPs with lipid-monolayers indicated their surface-active potential and ease of intercalation into the pulmonary surfactant membrane at the air-lung interface. Cellular viability and uptake studies demonstrated their cytocompatibility and time-and concentration-dependent uptake in lung-epithelial A549 and Calu-3 cells with clathrin-mediated internalization. Transepithelial electrical resistance experiments established their ability to penetrate tight airway Calu-3 monolayers. Antifungal studies on laboratory strains and clinical isolates depicted their superior efficacy against Aspergillus species. Pharmacokinetic studies revealed nearly 5-, 4- and threefolds enhancement in lung AUC, Tmax, and MRT values, construing significant drug access and retention in lungs.ConclusionsNebulized LPH NPs were observed as a promising solution to provide effective and safe therapy for the management of pulmonary aspergillosis infection with improved patient compliance and avoidance of systemic side-effects.

Towards the Molecular Mechanism of Pulmonary Surfactant Protein SP-B: At the Crossroad of Membrane Permeability and Interfacial Lipid Transfer

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.

Non-linear van't Hoff behavior in pulmonary surfactant model membranes

Pulmonary surfactant exhibits phase coexistence over a wide range of surface pressure and temperature. Less is known about the effect of temperature on pulmonary surfactant models. Given the lack of studies on this issue, we used electron paramagnetic resonance (EPR) and nonlinear least–squares (NLLS) simulations to investigate the thermotropic phase behavior of the matrix that mimics the pulmonary surfactant lipid complex, i.e., the lipid mixture composed of dipalmitoyl phosphatidylcholine (DPPC), palmitoyl–oleoyl phosphatidylcholine (POPC) and palmitoyl–oleoyl phosphatidylglycerol (POPG). Irrespective of pH, the EPR spectra recorded from 5°C to 25°C in the DPPC/POPC/POPG (4:3:1) model membrane contain two spectral components corresponding to lipids in gel-like and fluid-like phases, indicating a coexistence of two domains in that range. The temperature dependence of the distribution of spin labels between the domains yielded nonlinear van't Hoff plots. The thermodynamic parameters evaluated were markedly different for DPPC and for the ternary DPPC/POPC/POPG (4:3:1) membranes and exhibited a dependence on chemical environment. While enthalpy and entropy changes for DPPC were always positive and presented a quadratic behavior with temperature, those of the ternary mixture were linearly dependent on temperature and changed from negative to positive values. Despite that, enthalpy-entropy compensation takes place in the two systems. The thermotropic process associated with the coexistence of the two domains is entropically–driven in DPPC and either entropically– or enthalpically–driven in the pulmonary surfactant membrane depending on the pH, ionic strength and temperature. The significance of these results to the structure and function of the pulmonary surfactant lipid matrix is discussed.

Permeability of Pulmonary Surfactant Membranes is Modulated by Proteins SP-B and SP-C

Permeability of Pulmonary Surfactant Membranes is Modulated by Proteins SP-B and SP-C

Surface Activity as a Crucial Factor of the Biological Actions of Ole e 1, the Main Aeroallergen of Olive Tree (Olea europaea) Pollen.

Aeroallergens are airborne substances-mainly proteins-capable of triggering Th2-immune responses in respiratory allergies. They enter into the body through the upper airways, reaching the mucosa afterward. Mucosae lining at the luminal side consists of an epithelial barrier completely covered by mucus and pulmonary surfactant. Both pulmonary surfactant and plasma membrane of the epithelial cells represent two physiological phospholipid-based barriers where allergens first impact before triggering their biological effects. The interaction of allergens with lipids at relevant physiological surfaces could promote structural changes on the molecule, resulting on a potential modification of its allergenic properties. In this work, we have first described the surface and phospholipid interaction capabilities of the clinically relevant aeroallergen Ole e 1, the main allergen of olive tree pollen. By using epifluorescence microscopy of Langmuir transferred films, we observed that lipid-packed ordered domains may function as a preferential location for allergen to accumulate at the air-liquid interface, an effect that is abolished in the presence of cholestenone. The possible implications of phospholipid-interfacial effects in the modification of allergen structural and functional properties will be discussed.

Effects of a novel antimycobacterial compound on the biophysical properties of a pulmonary surfactant model membrane

In this work, the interactions of a novel rifabutin's analogue (N'-acetyl-rifabutin, RFB2) with two-dimensional (Langmuir monolayers) and three-dimensional (large unilamellar and multilamellar vesicles) membrane models of the pulmonary surfactant (PS) were evaluated. The main purpose of this study is to obtain detailed information at the molecular level between the interactions of RFB2 with the phospholipids of the PS, under physiological conditions. Therefore, the effects of RFB2 in the monolayer phase behaviour at the air-water interface and in the lipid bilayer of membrane models composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) have been systematically compared. In this context, several biophysical techniques were carried out to establish the interactions of RFB2 with the two-dimensional membrane models of the PS: Langmuir isotherms, Brewster angle microscopy (BAM), and polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS); and with three-dimensional membrane models of the PS: derivate spectrophotometry partition coefficient (Kp), dynamic light scattering (DLS), small and wide angle X-ray scattering (SAXS and WAXS). The results gathered by the different biophysical techniques and the PS membrane model used provide detailed information about the strong interactions of RFB2 with the polar head groups of the PS phospholipids and permit to establish the impact of the RFB2-PS membrane interactions, justifying an often unexplored biophysical approach to the drug's pharmacokinetics and toxicological effect.

Hydrophobic Pulmonary Surfactant Proteins SP-B and SP-C Induce Pore Formation in Planar Lipid Membranes: Evidence for Proteolipid Pores

Pulmonary surfactant is a complex mixture of lipids and specific surfactant proteins, including the hydrophobic proteins SP-B and SP-C, in charge of stabilizing the respiratory surface of mammalian lungs. The combined action of both proteins is responsible for the proper structure and dynamics of membrane arrays in the pulmonary surfactant network that covers the respiratory surface. In this study, we explore the possibility that proteins SP-B and SP-C induce the permeabilization of phospholipid membranes via pore formation. To this end, electrophysiological measurements have been carried out in planar lipid membranes prepared with different lipid/protein mixtures. Our main result is that channel-like structures are detected in the presence of SP-B, SP-C, or the native mixture of both proteins. Current traces show a high variety of conductance states (from pS to nS) that are dependent both on the lipid composition and the applied potential. We also show that the type of host lipid crucially determines the ionic selectivity of the observed pores: the anionic selectivity observed in zwitterionic membranes is inverted to cationic selectivity in the presence of negatively charged lipids. All those results suggest that SP-B and SP-C proteins promote the formation of proteolipid channels in which lipid molecules are functionally involved. We propose that proteolipidic membrane-permeabilizing structures may have an important role to tune ionic and lipidic flows through the pulmonary surfactant membrane network at the alveolar surfaces.

Alteration of Surface Properties of Dipalmitoyl Phosphatidylcholine by Benzo[<I>a</I>]pyrene: A Model of Pulmonary Effects of Diesel Exhaust Inhalation

Effects of benzo[a]pyrene (BaP) on the surface properties of dipalmitoyl phosphatidylcholine (DPPC) being the basic component of pulmonary surfactant and lipid membranes were studied experimentally and analyzed by molecular dynamics simulations. Isotherms and surface compressibility of mixed BaP/DPPC monolayers on water were determined using Langmuir-Wilhelmy film balance. It was demonstrated that BaP induced concentration-dependent deviations from the initial surface activity of the phospholipid film and a noticeable decrease of its fluidity. Results obtained by molecular dynamics simulations allow to explain the observed effects indicating a migration of BaP molecules towards hydrophobic part of the interfacial layer of DPPC and the concentration-dependent restriction of flexibility of the hydrocarbon chains of phospholipid molecules. The results of the study may be related to the physicochemical disturbance of the pulmonary surfactant system after breathing with air contaminated by diesel exhaust components. It is speculated that such phenomena may have an input to undesired health effects.

Interplay of mycolic acids, antimycobacterial compounds and pulmonary surfactant membrane: A biophysical approach to disease

This work focuses on the interaction of mycolic acids (MAs) and two antimycobacterial compounds (Rifabutin and N′-acetyl-Rifabutin) at the pulmonary membrane level to convey a biophysical perspective of their role in disease. For this purpose, accurate biophysical techniques (Langmuir isotherms, Brewster angle microscopy, and polarization-modulation infrared reflection spectroscopy) and lipid model systems were used to mimic biomembranes: MAs mimic bacterial lipids of the Mycobacterium tuberculosis (MTb) membrane, whereas Curosurf® was used as the human pulmonary surfactant (PS) membrane model. The results obtained show that high quantities of MAs are responsible for significant changes on PS biophysical properties. At the dynamic inspiratory surface tension, high amounts of MAs decrease the order of the lipid monolayer, which appears to be a concentration dependent effect. These results suggest that the amount of MAs might play a critical role in the initial access of the bacteria to their targets. Both molecules also interact with the PS monolayer at the dynamic inspiratory surface. However, in the presence of higher amounts of MAs, both compounds improve the phospholipid packing and, therefore, the order of the lipid surfactant monolayer. In summary, this work discloses the putative protective effects of antimycobacterial compounds against the MAs induced biophysical impairment of PS lipid monolayers. These protective effects are most of the times overlooked, but can constitute an additional therapeutic value in the treatment of pulmonary tuberculosis (Tb) and may provide significant insights for the design of new and more efficient anti-Tb drugs based on their behavior as membrane ordering agents.

Phase-field model for the morphology of monolayer lipid domains

Phase-separated domains exist in multicomponent lipid monolayers and bilayers. We present here a phase-field model that takes into account the competition between lipid dipole-dipole interactions and line tension to define the domain morphology. A dynamic equation for the phase-field is solved numerically showing stationary non-circular shapes like starfish shapes. This phase-field model could be applied to study the dynamic properties of complex problems like phase segregation in pulmonary surfactant membranes and films.

Fluorescence and Infrared Spectroscopy for the Study of Structure and Lipid Packing/Hydration in Pulmonary Surfactant Membranes and Lamellar Body -Like Particles

Pulmonary surfactant is a lipid-protein complex that reduces surface tension at the air-liquid interface in the alveoli preventing their collapse at end-expiration. Lung surfactant is synthesized and stored in special organelles of alveolar type II (AT II) cells called lamellar bodies (LBs). Upon exocytosis of LBs, surfactant still maintains its packed structure that we call lamellar body-like particles (LBPs), which disintegrate in an efficient and cooperative manner once they reach the air-liquid interface to form the surface active film.In the present study we compared the packing and hydration properties of native lung surfactant membranes (NS) isolated from porcine bronchoalveolar lavage and surfactant in LBPs secreted by primary cultures of rat AT II cells, taking advantage of the spectral properties of the fluorescent probe LAURDAN. The thermotropic profiles of LAURDAN are significantly different in NS and LBPs, indicating the possible existence of additional levels of structural complexity in LBPs that could impose restrictions to temperature-dependent hydration as associated with potential phase transitions in membranes.Infrared spectra provide valuable information about structural reorganizations, at both the headgroup and the acyl chain regions of membranes, associated with lipid phase transitions. The differences in the thermotropic profiles of LAURDAN in NS and LBPs correlate with important differences in the thermotropic profile exhibited by the IR spectra of the same materials, suggesting the existence of significant structural differences between both types of surfactant organizations.

Effects of Hidrophobic Surfactant Proteins SP-B and SP-C on the Mechanical Properties and Structural Stability of Phospholipid Bilayers

The respiratory surface is stabilized by pulmonary surfactant, a complex mixture of lipids and proteins, whose main function is reducing surface tension at the alveolar air-liquid interface in order to facilitate the work of breathing. It is composed by around 90% lipids and 8-10% specific proteins, including the hydrophobic polypeptides SP-B and SP-C. A combined action of both proteins is essential for a proper organization of functional membrane arrays in surfactant complexes.SP-B and SP-C have dramatical effects on membrane structure and dynamics. In the present study we compare some structural, mechanical and dynamical properties of model POPC vesicles in the absence and presence of them, either in their physiological combined proportion or each protein by itself. Structural effects caused by hydrophobic surfactant proteins were noticed both by optical microscopy of giant proteolipid vesicles and by electron microscopy of 100 nm diameter extruded vesicles. Also, impermeable POPC membranes became permeable when supplemented with any of these proteins. Significant differences were noticed between the effect of SP-B and SP-C on giant vesicles: suspensions containing only SP-B were stable but those containing only SP-C were quite dynamic, undergoing frequent fluctuations, reorganizations and ruptures as observed under the microscope. In order to investigate the physical explanation of these phenomena, mechanical studies have been carried out with giant phospholipid and proteolipid vesicles.The differences found between the effect of each protein separately and their physiological mixture support the concept that SP-B and SP-C mutually modulate their membrane-perturbing properties, which may be crucial for the structure, arrangement and dynamics of pulmonary surfactant membranes.

A combined action of pulmonary surfactant proteins SP-B and SP-C modulates permeability and dynamics of phospholipid membranes

Proteins SP-B and SP-C are essential to promote formation of surface-active films at the respiratory interface, but their mechanism of action is still under investigation. In the present study we have analysed the effect of the proteins on the accessibility of native, quasi-native and model surfactant membranes to incorporation of the fluorescent probes Nile Red (permeable) and FM 1-43 (impermeable) into membranes. We have also analysed the effect of single or combined proteins on membrane permeation using the soluble fluorescent dye calcein. The fluorescence of FM 1-43 was always higher in membranes containing SP-B and/or SP-C than in protein-depleted membranes, in contrast with Nile Red which was very similar in all of the materials tested. SP-B and SP-C promoted probe partition with markedly different kinetics. On the other hand, physiological proportions of SP-B and SP-C caused giant oligolamellar vesicles to incorporate FM 1-43 from the external medium into apparently most of the membranes instantaneously. In contrast, oligolamellar pure lipid vesicles appeared to be mainly labelled in the outermost membrane layer. Pure lipidic vesicles were impermeable to calcein, whereas it permeated through membranes containing SP-B and/or SP-C. Vesicles containing only SP-B were stable, but prone to vesicle-vesicle interactions, whereas those containing only SP-C were extremely dynamic, undergoing frequent fluctuations and ruptures. Differential structural effects of proteins on vesicles were confirmed by electron microscopy. These results suggest that SP-B and SP-C have different contributions to inter- and intra-membrane lipid dynamics, and that their combined action could provide unique effects to modulate structure and dynamics of pulmonary surfactant membranes and films.

Pulmonary Surfactant Membranes of Hibernating Ground Squirrels Possess Increased Fluidity but are Capable of Maintaining an Ordered Membrane Structure at Low Temperatures

Pulmonary surfactant, a mixture of lipids and proteins, regulates the surface tension at the air-liquid interface of the lung. Reduced body temperature during hibernation is accompanied in 13-lined ground squirrels (Ictidomys tridecemlineatus) by an increase in fluid monounsaturated phosphatidylcholine (PC) species (e.g. PC 16:0/16:1, PC 16:0/18:1) and phosphatidylglycerol (PG) species (e.g. PG 16:0/18:1, PG 18:0/18:2), but fewer disaturated PC and PG species. Previously we speculated that altered surfactant lipid composition during metabolic depression states such as torpor or hibernation will reduce the phase transition temperature (Tm) of the mixture, enabling pulmonary surfactant to remain fluid over a broader range of temperatures and thereby maintaining respiratory function. Here we analyze thermodynamic properties and behavior of surfactant from hibernating and summer-active 13-lined ground squirrels in relation to natural porcine surfactant, using differential scanning calorimetry and LAURDAN fluorescence spectroscopy. In addition we conducted epifluorescence studies to visualize changes in phase coexistence of surfactant films of hibernating and summer-active animals. Surfactant membranes of hibernators showed gel-to-fluid transitions at lower Tm with reduced enthalpy relative to membranes from summer-active squirrels. Both exhibited lower enthalpy than porcine surfactant. LAURDAN fluorescence and epifluoresence suggested possible structural re-arrangements of surfactant membrane lipids and films, respectively, in hibernators. These exhibited a similarly dehydrated and condensed highly packed ordered phase as for summer active squirrels, despite differences in composition and Tm. In conclusion, pulmonary surfactant composition changes in hibernating squirrels to increase overall fluidity, but to maintain an ordered membrane structure at low temperature.