The Role of Lipids in Pulmonary Surfactant

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Biochimica et Biophysica Acta 1408 (1998) 90^108

Review The role of lipids in pulmonary surfactant

Ruud Veldhuizen a, Kaushik Nag b, Sandra Orgeig c, Fred Possmayer d;*

a Lawson Research Institute and Departments of Medicine and Physiology, University of Western Ontario, St. Joseph's Health Centre, London, ON N6A 4V2, Canada b Department of Obs/Gyn, MRC Group in Fetal and Neonatal Health and Development, University of Western Ontario, London Health Sciences Centre, University Campus, London, ON N6A 5A5, Canada c Department of Physiology, University of Adelaide, Adelaide, S.A. 5005, Australia d Departments of Obs/Gyn and Biochemistry, MRC Group in Fetal and Neonatal Health and Development, University of Western Ontario, London Health Sciences Centre, University Campus, 339 Windermere Road, London, ON N6A 5A5, Canada

Received 17 April 1998; received in revised form 6 July 1998; accepted 6 July 1998

Abstract

Pulmonary surfactant is composed of approx. 90% lipids and 10% protein. This review article focusses on the lipid components of surfactant. The first sections will describe the lipid composition of mammalian surfactant and the techniques that have been utilized to study the involvement of these lipids in reducing the surface tension at an air-liquid interface, the main function of pulmonary surfactant. Subsequently, the roles of specific lipids in surfactant will be discussed. For the two main surfactant phospholipids, phosphatidylcholine and phosphatidylglycerol, specific contributions to the overall surface tension reducing properties of surfactant have been indicated. In contrast, the role of the minor phospholipid components and the neutral lipid fraction of surfactant is less clear and requires further study. Recent technical advances, such as fluorescent microscopic techniques, hold great potential for expanding our knowledge of how surfactant lipids, including some of the minor components, function. Interesting information regarding surfactant lipids has also been obtained in studies evaluating the surfactant system in non-mammalian species. In certain non-mammalian species (and at least one marsupial), surfactant lipid composition, most notably disaturated phosphatidylcholine and cholesterol, changes drastically under different conditions such as an alteration in body temperature. The impact of these changes on surfactant function provide insight into the function of these lipids, not only in non-mammalian lungs but also in the surfactant from mammalian species. ß 1998 Elsevier Science B.V. All rights reserved.

Keywords: Pulmonary surfactant; Surfactant lipid; Surfactant composition; Dipalmitoylphosphatidylcholine; Phosphatidylcholine; Phosphatidylinositol; Phosphatidylglycerol; Neutral lipid; Non-mammalian surfactant; Surface tension; Alveolar stability

Contents

1. Introduction ...... 91

2. Composition ...... 92

Abbreviations: DPPC, dipalmitoylphosphatidylcholine; FRC, functional residual capacity; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine; SM, sphingomyelin; TLC, total lung capacity * Corresponding author. Fax: (519) 6633388; E-mail: [email protected]

BBADIS 61765 30-10-98 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 91

3. Methodology ...... 93 3.1. Techniques for studying surfactant activity ...... 93 3.2. Reconstitution studies and in vivo alterations in surfactant composition ...... 94

4. The roles of speci¢c lipids in surfactant function ...... 95 4.1. Phosphatidylcholines ...... 95 4.2. Acidic phospholipids ...... 96 4.3. Minor phospholipid components ...... 97 4.4. Neutral lipids ...... 97

5. Recent contributions from novel approaches ...... 98

6. Non-mammalian surfactants ...... 102

7. Future directions ...... 105

Acknowledgements ...... 105

References ...... 106

1. Introduction presence of the disaturated lecithin, dipalmitoylphosphatidylcholine (DPPC) in lung tissue had been re- A major function of pulmonary surfactant is to ported in 1946 [6]. Although Pattle initially intuited reduce surface tension at the air-water interface of that pulmonary surfactant was an insoluble protein the terminal airways, thereby decreasing the tendency ¢lm [7], further studies soon established that phos- for alveolar collapse. It was surfactant's ability to pholipids comprised the major surface tension reduc- reduce surface tension and thereby increase compli- ing component and DPPC was a major constituent ance (i.e., elasticity) of the excised lung that led to its [8^10]. initial discovery by von Neergaard in the 1920s [1]. The rediscovery of pulmonary surfactant did not This was truly a `premature' discovery [2,3] which go unnoticed by physicians concerned with RDS. In exceeded the scienti¢c and medical communities' ca- 1959, Avery and Mead were able to report saline pacity to appreciate its signi¢cance. Von Neergaard extracts of lungs from premature infants succumbing clearly understood that the lining layer of the lung to RDS were surfactant de¢cient compared to in- possessed special surface properties superior to those fants dying of other causes [11]. This discovery of adsorbed serum. Whether he understood that spurred enormous interest in the topic of this dedi- phospholipids, or even lipids, were involved is not cated issue. clear. Von Neergaard also recognized the importance This review will discuss the roles of the lipid com- of low surface tension for the newborn lung. ponents of pulmonary surfactant. Although accom- Surfactant's capacity for reducing surface tension plished in cooperation with the surfactant-associated also led to its rediscovery in the mid-1950s by Pattle proteins (SP-A, SP-B, and SP-C), it is the lipids that [4] and Clements [5]. These early surfactologists not are ultimately responsible for surface tension reduc- only recognized the ability of pulmonary surfactant tion to low value during inhalation and expiration to adsorb and spread rapidly to form an insoluble (see chapters by Goerke, Schu«rch and by Perez-Gil surface ¢lm, but also demonstrated that reductions in and Keough for further details on the roles of sur- surface area resulted in a corresponding fall in surfactant proteins). Furthermore, although it has never face tension to surprising if not unprecedented low been demonstrated chemically, considerable circum- values. By this time, the ability of lipids to act as stantial evidence implies that a surface monolayer surface tension reducing agents was well recognized. highly enriched in DPPC is responsible for the crit- Lecithin, or phosphatidylcholine (PC), had been dis- ical reduction in surface tension to values near 0 mN/ covered as a component of egg yolk in 1847. The m that facilitates lung expansion at birth and allows

BBADIS 61765 30-10-98 92 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 regular breathing throughout life. The roles of the chain with no double bonds. Virtually no other di- other lipid components of pulmonary surfactant are saturated PC species have been observed. Thus, less well understood. This is particularly true with about 40% of the total phospholipid in mammalian surfactants recovered from non-mammalian species. surfactant is composed of DPPC. Recent studies on This review will try to present our best `educated surfactant from neonatal calves reported DPPC ac- guesses' as to these roles and suggest potential ways counted for only 32% of the total phospholipid [12]. of limiting the guesswork in the future. Due to lim- Although high DPPC levels have long been consid- ited space, other chapters in this dedicated issue and ered a hallmark of pulmonary surfactant, DPPC ac- previous reviews will be used liberally to document counts for 10^20% of the phosphatidylcholine con- evidence for some concepts and as sources of original tent of brain myelin and erythrocyte membranes references. In addition, in many cases only the most [13,14]. recent, key reference will be cited. Readers can use The discrepancy between disaturated PC levels giv- these publications to ¢nd references to earlier work. en here and some previous reports suggesting higher levels may have arisen in part from the use of di¡erent techniques to measure disaturated lipids. For ex- 2. Composition ample, it has been reported that the osmium tetroxide method of Mason [15] also detects some The overall lipid and phospholipid compositions monoenoic (one double bond) species. This leads to of surfactants isolated from a number of experimen- arti¢cially high estimates of disaturated PC [16]. It tally used species (mouse, rat, rabbit, and sheep), of should also be recognized that not all of the DPPC bovine surfactant (which is used clinically), and of in lungs is in surfactant. In rat and ovine lungs, only human surfactant are listed in Table 1. The authors 25 and 40%, respectively, of the DPPC is surfactant are more impressed with the similarities than the associated [13,14]. di¡erences. It is noteworthy that the lipid composi- The remaining PC in surfactant is composed pri- tion of lamellar bodies, the storage form of surfac- marily of molecular species containing monoenoic tant, is very similar to those given in Table 1. In all and dienoic fatty acids at the 2-position, with only species, PC comprises approx. 80%, about half of minor amounts of short chains or polyunsaturated which is DPPC. Palmitic acid is a 16 carbon acyl acyl groups. The acidic phospholipids, phosphatidyl-

Table 1 Lipid composition of extracellular surfactant in selected mammalian species Phospholipid composition (% total) [% disaturated] Cholesterol (% chol/PL) PC LPC SM PG PI PS PE LBPA Mus musculus 72.3 0 3.3 18.1 1.9 9.7a Mousea Rattus norvegicus 82.3 0.3 0.8 7.5 1.8 0.1 5.1 Tr 7.1e Ratb [49.3] [32.3] [2.2] O. cuniculus 80.6 Tr 1.5 7.2 4 1.9 4.4 Tr Rabbitb [52.8] [38.7] [2.5] Ovis spp. 81 Tr 1.7 7.9 2.6 Tr 4.8 2 Ovinec Bos spp. 79.2 Tr Tr 11.3 1.8 Tr 3.5 2.6 3 Bovined [49.9] [33.3] Homo sapiens 80.5 Tr 2.7 9.1 2.6 0.9 12.3 7.3e Humanc [47.7] PC, phosphatidylcholine; LPC, lysophosphatidylcholine; SM, sphingomyelin; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; LBPA, lyso-bis-phosphatidic acid; PL, phospholipid; chol, cholesterol; Tr, trace. (In some cases, the authors have added `trace' on the basis of their experience.) Data from: aLangman et al., 1996 [71]; bAkino, 1992 [14]; cPossmayer, 1984 [13]; dYu et al., 1983 [78]; eDaniels et al., 1995 [65].

BBADIS 61765 30-10-98 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 93 glycerol (PG) and phosphatidylinositol (PI), account tional aspects of pulmonary surfactant have emerged for 8^15% of the total surfactant phospholipid pool from biophysical techniques foreign to most bio- with most species. Adult surfactants are depicted in chemists. In order to appreciate current investiga- Table 1, so PG is high while PI is low. With surfac- tions on the roles of surfactant lipids, it is imperative tant from fetal and neonatal lung, PI levels are ele- to understand the kind of information derived from vated and PG correspondingly lower. With some an- these techniques and, just as importantly, their limi- imals, such as the rhesus monkey, PI remains high tations. The reader can ¢nd further information in into adult life. PG possesses a signi¢cant amount of previous reviews [19^21]. disaturated lipids, although not as much as PC. The Langmuir-Wilhelmy surface balance, designed However, this accounts for only a small part of the by Clements for his initial studies [5,22], consists of a total disaturated phospholipid pool. In contrast, PI trough whose surface area can be varied, and a Wil- contains very low amounts of disaturated compo- helmy dipping plate which monitors surface tension nents, although this remains somewhat controversial [19,20]. This apparatus is well suited for examining [14]. area-surface tension characteristics with dilute ¢lms, The remaining phospholipids are present in rather that is, surfactant ¢lms with surface tensions between small amounts. Except for the human, phosphatid- the 70 mN/m observed with a clean saline surface at ylethanolamine (PE) is low with only 3^5% being 37³C and the equilibrium surface tension of approx. present. The other phosphorus-containing phospho- 25 mN/m for phospholipids. The equilibrium surface lipids are present at very low levels. While they could tension is the lowest surface tension pure or mixed represent contaminants arising from membrane lip- phospholipid ¢lms can achieve, given su¤cient lipids ids, the levels are quite consistent from one prepara- and a long enough time. Identical equilibrium sur- tion to another and from species to species. The low face tensions are observed whether the phospholipid phosphatidylserine (PS) level is not surprising in view is spread from above or adsorbed from below. Sur- of the known role of PS in triggering blood clotting factant apoproteins increase adsorption rate, but to reactions and in apoptosis [17]. the same equilibrium surface tension. Less information is available concerning the neu- Lateral compression of insoluble ¢lms decreases tral lipid components of pulmonary surfactant. In the surface area such that surface tension can fall those mammalian species reported, cholesterol ap- below equilibrium. Such ¢lms are inherently unstable pears to be the major component (80^90%). Small by de¢nition and therefore in a metastable state. The amounts of monoacylglycerol, diacylglycerol, and tri- high surface pressure required to attain low surface acylglycerol are present with most species. Cholester- tensions can result in loss of ¢lm material, not only ol esters appear to be low in most species [13,18]. by ¢lm collapse but also onto or under the te£on Most surfactants appear to have some free fatty barriers. This loss, termed `leakage', is dependent acids, mainly palmitate. on the sample and is more serious with increased levels of £uid lipids, i.e., lipids above their corresponding gel-to-liquid transition temperatures for bi- 3. Methodology layers, and for surfactants with neutral lipids. Such ¢lms cannot pack in an ordered fashion and are less 3.1. Techniques for studying surfactant activity stable than ¢lms of phospholipids below their corresponding bilayer gel-to-liquid crystalline transition Pulmonary surfactant is a unique biological system temperatures. The Langmuir-Wilhelmy balance is in that a major function involves the rapid formation particularly suited for studying spread ¢lms. At of a highly surface-active ¢lm with an equilibrium present, it is the only technique where precise surface tension of approx. 25 mN/m (see below). amounts of ¢lm material can be deposited, where Furthermore, during breathing this ¢lm is subject surface radioactivity can be monitored conveniently, to dynamic compression, resulting in lateral packing and where £uorescent probes can be used to study of the ¢lm to produce further lowering to near 0 mN/ ¢lm structure. m. As a result, most of the information on the func- The pulsating bubble surfactometer introduced by

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Enhorning in 1977 [23] consists of an air bubble con- ble in terms of controlling surface area reduction, nected by a capillary to the atmosphere. With this speed of compression, and manipulation of other arti¢cial alveolus model, a bubble created within a bubble characteristics. One example of new informa- suspension of the surfactant sample is pulsated, nor- tion obtained with this apparatus is evidence for a mally at 20 cycles/min at 37³C, and the pressure surface-associated surfactant reservoir which selec- across the bubble monitored [19]. The law of Young tively replenishes the surface ¢lm with DPPC-rich and Laplace, which states that the pressure across material during surface area expansion (see below). the bubble equals twice the surface tension divided by the radius, is used to calculate surface tension. 3.2. Reconstitution studies and in vivo alterations in The pulsating bubble monitors adsorption of surfac- surfactant composition tant during bubble expansion and its ability to ob- tain low surface tension during ¢lm compression gen- In general, knowledge regarding the role of lipids erated during the 50% surface area change which in surfactant function arises from two approaches: occurs during reduction of bubble volume. The sur- reconstitution studies where puri¢ed or synthetic lip- face tension at maximum bubble radius is indicative id and protein components are combined, and stud- of surfactant adsorption during surface expansion, ies where physiological conditions occur or are im- while the surface at minimum radius indicates the posed which result in alteration of the surfactant ¢lm's ability to achieve low surface tensions during system in vivo. Surfactant reconstitution studies can compression. These parameters are important deter- employ simple mixtures, providing insight into the minants of the quality of the surfactant and, in gen- contributions made by individual components under eral, re£ect the biological activity of the surfactant in speci¢c conditions. Physiological conditions leading vivo. The small amounts of sample required, the rel- to surfactant alteration include not only acute lung atively short time needed, and the relatively straight- injury but also transplantation after prolonged stor- forward technical requirements recommend this age, mechanical ventilation, and physiological states assay. It functions best at relatively high concentra- such as exercise. As will be discussed later, with some tions (1^10 mg/ml) and short pulsation periods be- non-mammalian species, temperature adaptation re- cause with the time bubble radius can change during sults in signi¢cant alterations in overall surfactant pulsation. However, at low surface tensions (i.e., composition. high surface pressures), surface material can be With acute lung injury and a number of other pushed onto the capillary so the true surface area conditions small decreases in DPPC as a proportion reduction is less than 50%. Initiating pulsation while of PC and increases in the PI/PG ratio are often the capillary walls are wetted can also lead to in- observed. Increases in the level of so-called `mem- creased surface area [24]. The pulsating bubble is brane phospholipids', PE, PS and sphingomyelin extremely useful for monitoring the quality of surfac- (SM) have also been noted. In some cases, lyso-PC, tant relative to its biological activity, but can be a product of phospholipase A2 degradation, can also problematic for mechanistic studies. increase. Alterations arising during severe lung injury The last technique to be discussed here, the captive are remarkably consistent among various species and bubble tensiometer introduced by Schu«rch [25], con- di¡erent initiating causes [26,27]. These alterations in sists of a gas-tight chamber containing a bubble phospholipid composition are not only indicative of £oating against a hydrophilic agar gel. Increasing lung injury, but could contribute to surfactant dys- the pressure within the chamber compresses the bub- function within the lung. It must be pointed out that ble, thereby decreasing surface area. The hydrophilic studies in which organic solvent lipid extracts were agar roof is incapable of accepting hydrophobic lip- altered by addition of speci¢c lipids produced no ids, resulting in an apparently leak-proof system. The detectable e¡ects on pulsating bubble surfactometer captive bubble tensiometer is more accurate but is tracings (I.L. Metcalfe, F. Possmayer, unpublished very time-consuming compared to the pulsating bub- results; A.M. Cockshutt, F. Possmayer, unpublished ble surfactometer, both experimentally and for data results). However, surfactant isolation unavoidably analysis. However, this apparatus is extremely £exi- results in an averaging of total lung surfactant com-

BBADIS 61765 30-10-98 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 95 position. Even with uniform forms of acute lung in- was ¢rst suggested by Clements in 1957, based on jury, considerable variation is likely. Marked altera- theoretical calculations [5]. DPPC is not only the tions in surfactant lipid composition in individual major component of pulmonary surfactant, but alveoli could lead to localized surfactant dysfunction also the only major component capable of generating without greatly a¡ecting overall composition. In ad- low surface tensions during compression on the dition, to the best of our knowledge, the e¡ect of Langmuir-Wilhelmy balance and the captive bubble combined alterations in surfactant lipids has not tensiometer. With the captive bubble tensiometer, been examined. area reductions of 10^12% are su¤cient to lower sur- Lung injuries are often, if not universally, accom- face tension of pure DPPC ¢lms from equilibrium panied by increases in alveolar serum protein con- (approx. 25 mN/m) to less than 2 mN/m. Further- tent. Increases in lyso-PC level which did not in£u- more, if surface area is held constant, the ¢lm main- ence the surface activity of bovine lipid extract tains a low surface tension and returns towards equi- surfactants on the pulsating bubble surfactometer librium only after many hours. In situ studies by sensitized this preparation to inhibition by serum Schu«rch, which used the lung as a surface balance proteins. Furthermore, SP-A, which prevents protein by employing £uorocarbon microdroplets to assess inhibition, did not reverse inhibition arising from alveolar surface tension, have revealed surface ten- lyso-PC [28]. sions of 30 mN/m, slightly above equilibrium at total It should also be recognized that interpretation of lung capacity (TLC) [29]. Reducing lung volume to results relating alterations in surfactant lipids to sur- functional residual capacity (FRC) is accompanied factant function in vivo is complicated by the fact by a 20% reduction in surface area and a fall in that lung injury-associated surfactant alterations do surface tension to near 1 mN/m. When held at not occur in isolation. The acute lung injury changes FRC, surface tension remains low for several mi- in lipid pro¢le are invariably accompanied by alter- nutes, showing the alveolar surface ¢lm is stable. ations in surfactant apoprotein content, and as indi- The properties of DPPC mentioned above contrast cated above, increases in serum proteins, which can with those of unsaturated PC ¢lms above the gel-to- inhibit surfactant. liquid crystalline transition temperature of bilayers composed of the same lipids. Since such £uid ¢lms cannot pack in an ordered fashion, compression 4. The roles of speci¢c lipids in surfactant function can lower surface tension only to 15^20 mN/m, and when dynamic compression ceases, surface tension Surfactant is a complex mixture of proteins and returns towards equilibrium. Such surface tensions lipids. In the lung this complex functions to reduce are incapable of stabilizing the lung at low lung vol- the surface tension at the air-liquid interface. For umes [29^31]. Thus it is implied that the lung con- some of the major lipid classes a role in this process tains a ¢lm highly enriched in DPPC. As indicated has been indicated, but for most of the minor lipid earlier, the alveolar surface monolayer cannot be components their exact contribution must still be es- sampled, so chemical evidence for a DPPC ¢lm is tablished. It should also be noted that whereas most lacking. of the experimental focus has been on surface ten- Vesicles of pure DPPC, below the phase transition sion-reducing properties of surfactant, the lipid com- temperature of 41³C, form a surface monolayer only ponents of surfactant could also be involved in other extremely slowly and therefore are incapable of act- metabolic aspects of the surfactant life cycle, for ex- ing directly as an adequate surfactant replacement. ample lamellar body assembly and secretion. These Addition of unsaturated PCs (or other £uid phos- areas require further study. pholipid or neutral lipid components) enhances adsorption [19] (see chapters by Goerke and Pe¨rez-Gil 4.1. Phosphatidylcholines and Keough). However, in the absence of the low molecular weight hydrophobic proteins or some oth- The need for low surface tensions approaching er adsorbance-inducing factor, adsorption of such 0 mN/m to stabilize the lungs during expiration mixtures is still inadequate. Nevertheless, it is

BBADIS 61765 30-10-98 96 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 thought the £uidity resulting from unsaturated phos- issue), washout experiments with the captive bubble pholipids and other £uid components is important tensiometer have provided evidence for a surface-as- for enhancing adsorption to a clean surface. Further- sociated surfactant reservoir, which appears to be more, SP-B and SP-C are very e¡ective in promoting important for feeding DPPC-enriched material into adsorption of surfactant lipids to partially ¢lled the surface monolayer during ¢lm expansion. Re- monolayers so that equilibrium surface tension is plenishing the surface monolayer with DPPC-en- rapidly attained [32]. riched components is particularly important after Studies with the captive bubble tensiometer sug- ¢lm overcompression. This reservoir is dependent gest two mechanisms, selective DPPC adsorption on the palmitoylated SP-C [33^35]. and preferential squeeze-out of unsaturated lipids, Although considered less often, the £uid compo- could be involved in DPPC enrichment of the inter- nents of pulmonary surfactant could be important in facial ¢lm [19] (see chapters by Schu«rch and by other aspects of the surfactant cycle. The processing Pe¨rez-Gil and Keough). For example, bovine lipid and packaging of pure DPPC into lamellar bodies extract surfactant (BLES) lipids are approx. 40% could present di¤culties arising from the sti¡ness disaturated (Table 1). However, in the captive bub- of this phospholipid. The e¡ect of lipid £uidity could ble, BLES ¢lms adsorbed to the equilibrium value of be important for secretion of lamellar bodies, forma- 25 mN/m, require only 30^35% surface area reduction of tubular myelin and/or other precursors of the tion to change surface tensions to near 0 mN/m. surface ¢lm, and in the re-uptake of surfactant com- When SP-A is added, surface area reductions of ponents by the type II cells [36]. It should be evident 20^25% are su¤cient. Perfect squeeze-out of the that the ancillary functions suggested for unsaturated less stable unsaturated lipid components of BLES PC, discussed here, will apply to unsaturated PG and (40% disaturated) would require 60% surface area the other £uidizing phospholipids and neutral lipid reduction. A further 4^5% surface area reduction components of pulmonary surfactant. would be required to pack the remaining DPPC to In summary, it appears likely that the surface ¢lm reduce surface tension from equilibrium to near 0. covering the alveoli is highly enriched in DPPC. Un- This indicates the initial adsorbed ¢lm is highly en- saturated PC and other £uid components could facil- riched in DPPC. With additional compression-de- itate adsorption of surfactant lipids but such a role compression cycles, surface area reduction required has not been proven. to achieve low surface tension declines further to 15^ 20%, a value consistent with a remaining ¢lm even 4.2. Acidic phospholipids more highly enriched in DPPC. The £uid components of pulmonary surfactant, The observation that all pulmonary surfactants such as PC and the other lipids, could also play an possess signi¢cant amounts of PG and/or PI suggests important role in ¢lm respreading. DPPC ¢lms can an important speci¢c role. Mixing £uid acidic phos- be repeatedly cycled between equilibrium and 1 mN/ pholipids with DPPC leads to enhanced adsorption, m. Such cycling occurs reversibly without hysteresis suggesting a potential role in surface tension reduc- [29] (hysteresis implies non-reversible behaviour). tion [37]. A speci¢c interaction between PG and SP-B Nor is hysteresis observed with the lung during quiet has been suggested by £uorescence studies [38] and tidal breathing. However, overcompression of DPPC by the highly surface-active properties observed with ¢lms at low surface tension results not only in hys- DPPC-PG-SP-B mixtures [39]. As suggested above, teresis, but leads to ever-increasing surface tensions DPPC-enriched ¢lms could arise through the prefer- during subsequent cycles. This occurs because DPPC ential adsorption of DPPC during ¢lm formation, forced out of the monolayer into a collapsed state through selective PG squeeze-out during compres- cannot reform the monolayer rapidly (i.e., cannot sion, or both. With DPPC-PG mixtures, SP-B ap- respread). As suggested above, the £uid components pears superior to SP-C both in promoting preferen- of pulmonary surfactant and the surfactant apopro- tial DPPC adsorption and facilitating PG squeeze- teins could be important for respreading. As dis- out [34,39]. Such selective squeeze-out is not ob- cussed further by Schu«rch et al. (pp. 180^202, this served during compression of either spread or ad-

BBADIS 61765 30-10-98 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 97 sorbed phospholipid ¢lms in the absence of SP-B or precise nature of this lipid has not been determined. SP-C [40]. Lysosomes contain lyso-bis-phosphatidic acid and Decreases in the amount of PG have been ob- since lamellar bodies are thought to be modi¢ed ly- served in patients with ARDS and in other acute sosomes, the presence of lyso-bis-phosphatidic acid disease states. As with the decrease in DPPC, it has in surfactant could be related to this origin. It could been suggested the lower PG levels could be involved be that the lamellar body limiting membrane con- in the decreased biophysical activity of recovered ma- tains higher levels of the membrane lipids PE and terial. It has also been inferred that the high PI/PG SM. The limiting membrane does contain high levels ratios noted with surfactant from premature animals of cholesterol (S. Orgeig, T.E. Nichols, unpublished could result in lower activity. However, recent stud- results). The limiting membrane fuses with the plas- ies have suggested di¡erences in SP-A could account ma membrane during exocytosis, but some limiting for the lower in vitro and in vivo activity of pulmo- membrane may be ejected with surfactant. It should nary surfactant recovered from lambs of di¡erent also be recognized that the minor phospholipid com- gestational ages [41] (see chapter by Jobe for further ponents could be involved in signalling events related discussion). Increasing the proportion of PI in sur- to surfactant metabolism. However, this has not been factant by administering inositol to adult rabbits had demonstrated. no signi¢cant e¡ect on surfactant function in vitro or in vivo [42,43]. 4.4. Neutral lipids In summary, acidic phospholipids represent a signi¢cant component of pulmonary surfactant. How- Although present in signi¢cant amounts, the neu- ever, their precise role and why PG rather than PI is tral lipids of surfactant have received far less atten- present in most adult surfactants are not understood tion experimentally than the phospholipids. Neutral and require further study. lipid e¡ects are complex and depend on the system being studied and the technique used for investiga- 4.3. Minor phospholipid components tion. Addition of mono-, di-, and triacylglycerol or free palmitic acid to DPPC or DPPC-PG enhance Analysis of surfactant has revealed the consistent adsorption rate, presumably by introducing minor presence of a number of phospholipids present in packing defects in the membrane structure. Choles- relatively low amounts (Table 1). In general the terol can also enhance adsorption of vesicles primar- role of these lipids is not clear and investigations ily composed of DPPC, presumably by increasing into their contribution to surfactant function have £uidity, and improve ¢lm respreading [44,45]. How- been limited. From a surface tension reduction per- ever, it should be noted that, by increasing £uidity, spective it would be anticipated that these low cholesterol limits the minimum surface tensions ob- amounts of phospholipids would have minimal ef- tainable during compression. This occurs because fects on surfactant activity. The higher amounts of this sterol cannot be squeezed out easily from these lipids observed in lung injury could contribute DPPC-cholesterol spread ¢lms [44,46,47]. Possibly to decreased surfactant activity, but there is no spe- for this reason, cholesterol is removed from most ci¢c evidence for this. The exception is lyso-PC, modi¢ed natural surfactants used clinically for treat- which can make surfactants more susceptible to pro- ing neonates. Whether removing cholesterol a¡ects tein inhibition [28]. physiological function signi¢cantly has not been es- Since a speci¢c surface active role for these minor tablished. lipid components of surfactant appears unlikely, oth- The e¡ect of neutral lipids on the surface activity er explanations for their presence in surfactant have of DPPC and DPPC-PG has been examined using to be sought. The presence of some of these lipids in reconstitution studies by Tanaka et al. [48]. These surfactant could be related to some metabolic as- investigators found the most e¡ective reconstituted pects. For example, small amounts of an acidic phos- preparation contained DPPC, PG, tripalmitate or pholipid thought to be lyso-bis-phosphatidic acid is palmitic acid, and the hydrophobic surfactant pro- present in most surfactants (Table 1), although the teins. DPPC-hydrophobic apoproteins and DPPC-

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PG-hydrophobic apoprotein mixtures adsorbed well body) source of cholesterol which is rapidly mobi- but were not e¡ective in attaining low surface ten- lized by hyperventilation [50]. In addition, trained sions during surface area reduction on the Langmuir- athletes demonstrated a decrease in cholesterol/disa- Wilhelmy balance. Surface activity was markedly im- turated PC ratio during intense activity compared proved by addition of high amounts of neutral lipids with less ¢t individuals who demonstrated an in- (tripalmitin and/or palmitic acid). Mixtures contain- crease in this ratio [51]. The signi¢cance of these ing DPPC-PG-palmitic acid and hydrophobic apo- rapid alterations in surfactant sterol levels is not proteins were also e¡ective in promoting lung expan- known. sion in prematurely delivered rabbit pups of 27 days As indicated above, the other neutral lipids have gestation. not been extensively examined. Surfactant possesses Removing cholesterol from bovine surfactant lipid small amounts of vitamin E which could function as extracts hampers adsorption activity at 25³C but not an internal anti-oxidant. There is also a very small 37³C. Interestingly, SP-A greatly retarded the ability amount of plasmalogen in lung which could also of radioactive cholesterol in [14C]cholesterol-labelled function in this capacity [52]. BLES to attain the air-liquid interface during adsorption to equilibrium [46]. Furthermore, the addition of SP-A markedly enhanced the surface activity 5. Recent contributions from novel approaches of such preparations during compression on the Langmuir-Wilhelmy surface balance and during pul- Recent technical advances hold great potential for sation with the pulsating bubble surfactometer. improving our understanding of what surfactant is These results are consistent with SP-A enhancing and how it functions. For example, the electron DPPC adsorption while limiting cholesterol access spray mass spectrometer tracing of BLES lipids de- to the air-liquid interface. SP-A also appears to picted in Fig. 1, which shows individual lipid molec- bind pure cholesterol ¢lms in such a manner as to ular species, allows for exceptional sensitivity and increase their surface activity during compression accuracy. Mass spectrometer analysis using matrix- [47]. The exact manner in which SP-A in£uences sur- assisted methodology has been applied to SP-B and face activity of the phospholipid-cholesterol mixtures SP-C [53]. The advent of novel microscopic and spec- in BLES is not known, but could be related to the troscopic techniques capable of detecting individual formation of surface-associated phospholipid aggre- lipid-lipid and lipid-protein molecular interactions gates rich in DPPC [46,47]. and associations could provide direct means for an- In contrast to studies conducted with the Lang- swering fundamental questions not accessible by con- muir-Wilhelmy surface balance and the pulsating ventional surface chemistry (i.e., do minor lipid com- bubble surfactometer, cholesterol appears to have ponents play speci¢c roles in forming and minimal e¡ects on surface tension reduction of maintaining the interfacial layer? Do surfactant pro- BLES with the captive bubble tensiometer [49]. teins attain and remain at the surface?). Thus, some of the detrimental e¡ects attributed to During the past decade, £uorescent microscopic cholesterol in lipid extract surfactants may be related techniques have provided direct visual con¢rmation to ¢lm leakage associated with the particular appa- of earlier suggestions by Clements and others (see ratus being used. chapter by Goerke) that DPPC ¢lms undergo phase An intriguing, and as yet unexplained, aspect of transitions during dynamic compression at the air- surfactant lipid composition is the fact that di¡erent water interface. As ¢lms containing DPPC are com- species possess quite di¡erent proportions of neutral pressed, the lipids assemble into gel-like condensed lipids relative to phospholipids. Interestingly, choles- phases, and then with further packing, into a solid- terol levels in lung surfactant can vary rapidly. For like phase. Each phase transition is re£ected by the example, the hyperventilation induced in swimming appearance of a transitory aggregated lipid structure rats lowers the cholesterol/disaturated PC ratio. in one phase coexisting with another. A solid-like Studies with the isolated perfused lung model indi- phase generated below 20 mN/m may represent cate there may be an alternate (i.e., non-lamellar structures capable of withstanding high surface pres-

BBADIS 61765 30-10-98 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 99

Fig. 1. A typical mass spectrum of phospholipid species present in BLES. The mass spectrum was obtained using electrospray ioniza- tion mass spectrometry of organic solutions of BLES. The lipid species identi¢ed as their exact molecular mass and chain distributions are shown in parentheses. DPPC, dipalmitoylphosphatidylcholine; POPC, 1-palmitoyl, 2 oleyl-PC. sures, so as to generate low surface tensions near gested by Pierre Giles DeGennes, Nobel Laureate in 0 mN/m. Condensed structures generated within 1991 for studies on `Soft Matter' [58], the DPPC DPPC ¢lms possess peculiar, yet characteristic, kid- condensed regions can be considered as a form of a ney-shaped structures (Fig. 2A,B) [54]. Such struc- liquid-crystalline structure. Such structures are tures have recently been observed with surfactant formed due to di¡erential tilt and orientation of in- extract preparations [55,56]. These structures are dividual molecules in the ¢lms during packing. These not £uorescent probe-induced artifacts, since they monomolecular model ¢lms can also be used to can also be observed at the air-water interface by study the manner in which other lipids present in non-probe-dependent techniques such as Brewster surfactant interact with DPPC-condensed structures angle microscopy [55]. Similar structures are ob- at the interface. Small amounts of cholesterol change served with solvent or vesicle-spread and also with the DPPC-condensed structures from kidney shapes adsorbed ¢lms [57]. to elaborate ¢ne structures (Fig. 2C^F). When added It should be apparent that surface-£uorescence together with PG, the kidney-shaped structures are studies can provide unique structural detail not ac- modi¢ed to appear stellate (Fig. 2G). When calcium cessible by conventional surface techniques. As sug- is also present, there is increased DPPC condensa-

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Fig. 2. Various phase structures formed in DPPC and mixed lipid ¢lms as observed using £uorescence microscopy at an air-saline interface at 60 mN/m and 25³C. The structures arise due to lipid-lipid interactions, and multiple tilt and orientation of the molecules perpendicular to the plane of the air-water interface. The structures were observed in ¢lms of DPPC (A,B); DPPC with cholesterol (0.2^4 mol%) (C^F); at surface pressures near the gel-£uid phase coexistence regions of such ¢lms (3^14 mN/m). Images G and H indicate DPPC ¢lms containing PG (DPPC-DPPG), 7:3) and unsaturated POPC and cholesterol (DPPC-POPC-cholesterol, 7:2:1) respectively. The black regions indicate the gel-like liquid-condensed (LC) phase and the white the £uid liquid expanded (LE) phase. The LC phases are observed by £uorescence microscopy from the exclusion of £uorescent probes from such regions. Note the high susceptibility of the molecular packing or shape of the gel domains of DPPC in ¢lms to minor amounts of perturbants like cholesterol. The £uorescent probe was 1-palmitoyl, 2-nitrobenzoxadiole amino-dodecanoyl (NBD)-phosphatidylcholine. Scale bar is 25 Wm. (From [54].)

BBADIS 61765 30-10-98 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 101 tion, resulting in formation of multi-sized, circular domains (Fig. 2F). Recent studies with £uorescent- labelled ¢lms have con¢rmed the ability of unsaturated PC to promote DPPC respreading from condensed structures, providing further evidence for the reversible squeeze-out of PC and, in the presence of calcium, of PG during ¢lm compression [56]. Studies with £uorescent-labelled cholesterol have con¢rmed this steroid cannot be readily squeezed out of DPPC ¢lms [44,47,59]. The £uorescence approach also provides direct evidence for surfactant apoprotein interaction with lipids in surfactant ¢lms. SP-C alters DPPC and DPPC- PG packing in solvent-spread and adsorbed ¢lms [35,53,57]. The presence of multilayered structures resulting from molecular squeeze-out in the presence of SP-C has been directly demonstrated by atomic force microscopic analysis of ¢lms compressed to low surface tensions [35]. Such structures are consistent with the role of SP-C acylation in the generation of a surface-associated surfactant reservoir [34]. SP-B and SP-B-like peptides interact with DPPC-DPPG ¢lms di¡erently from SP-C in that they form protein networks at the surface. Such protein networks could provide initiation sites for molecular squeeze-out [60], thus explaining the remarkable ability of SP-B to promote PG squeeze-out [61]. The propensity of SP-A to bind to gel-like condensed regions of DPPC has been directly demonstrated by £uorescence techniques [62]. SP-A:DPPC interactions are highly sensitive to ionic conditions. That calcium and other ions a¡ect SP-A-DPPC interactions has also been shown with transmission electron microscopy. The latter electron microscopic investigations reveal that SP-A interacts with DPPC via its C-terminal carbohydrate recognition domains [63], as has been inferred from site-directed mutagen- esis of SP-A [64]. The £uorescence microscopic investigations indicate that small amounts of surfactant apoproteins remain associated with surfactant ¢lms Fig. 3. Fluorescence micrographs of ¢lms of porcine lipid sur- compressed to near zero, suggesting the proteins factant extract, at high (50 mN/m) (A), intermediate (30 mN/m) could be involved in respreading [53,62] as previously (B) and low (10 mN/m) (C) surface tensions. The dark con- indicated by captive bubble tensiometric studies densed domains seen in B are probably composed primarily of [33,34]. Readers are referred to chapters by Pe¨rez- DPPC, as their total area estimates agree well with the propor- Gil and Keough and by Schu«rch in this dedicated tion of DPPC present in porcine surfactant [54,55]. The bright issue for further details on these aspects. structures with black spots (in C) appear to be multilayered squeezed-out materials from such ¢lms above the air-water in- As indicated above, condensed domains reminis- terface. The £uorescent probe was 1-palmitoyl, 2-nitrobenzoxa- cent of those observed with DPPC ¢lms can be ob- diazole aminododecanoyl (NBD)-phosphatidylcholine.

BBADIS 61765 30-10-98 102 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 served during lateral packing of porcine lipid extract fraction to examine DPPC ¢lms at low surface surfactant (Fig. 3) [54]. The presence of subphase tensions indicate the presence of head group dehy- calcium produces a further increase in condensed dration, which could further stabilize monolayers at phase area, suggesting the possibility that PG, com- low surface tensions and enhance interactions with plexed with calcium, may coexist within the DPPC- hydrophobic areas of SP-B and with calcium [53,56]. rich regions [56]. Further addition of SP-A to such systems results in coalescence of separate DPPC-like domains, resulting in a smaller number of larger con- 6. Non-mammalian surfactants densed regions, but with a similar overall surface area. Although the actual molecular composition of The pulmonary surfactant system, including both these aggregated domains is unknown, it is clear they the lipids and SP-A, is present in the lungs of air- are DPPC-rich. When cholesterol is also present, cal- breathing representatives of all vertebrate groups cium induces formation of novel, still not-understood [65,66]. In fact, it appears surfactant function related multilayer structures at surface tensions near 0 mN/ to air breathing evolved at least 400 million years m (Fig. 3C). Whether such structures are related to ago in the lungs of primitive air-breathing ¢sh and the surface-associated reservoir [33] or to DPPC-rich has been highly conserved [66]. This suggests surfac- cholesterol-poor surface ¢lm-associated aggregates tant could have `older' and perhaps some as yet un- arising during ¢lm formation [46,47] is not known. de¢ned functions. Furthermore, relatively large However, it appears these structures could be capa- amounts of surfactant have also been found in the ble of ¢lm replenishment during surface area expan- swim bladders of modern teleost ¢sh such as the sion [55,56]. Recent investigations using X-ray dif- gold¢sh [67]. The role of surfactant in the swim blad-

Table 2 Phospholipid pro¢les for lavage material for di¡erent species Phospholipid composition (% total) [% disaturated] Cholesterol (% chol/PL) PC LPC SM PS/PI PE PG C. auratusa 75 0 15.9 5.1 4.7 1.2 32.8 Gold¢sh [27.7] L. osseusb 76.4 3 5.9 7.9 4.6 1.4 17.5 Gar ¢sh [18.5] N. forsteric 59.6 0 15.3 10.2 14.4 0.5 24.2 Australian lung¢sh [9.0] P. annectensc 80.2 0.4 5.6 7.9 3 3 5.2 African lung¢sh [25.4] B. marinusb 62.6 6.1 8.6 9 13.1 13 Cane toad [5.6] N. depressusd 70.7 0 9.9 19.4 0 0 6.1 Sea turtle [39.5] C. porosusd 67.6 0 4.7 25 2.5 0 6.7 Crocodile [50.0] C. atroxb 68.6 17.7 11.7 1.4 0 0 7.9 Rattlesnake [63.6] C. nuchalisb 73.8 0 2.5 23.7 0 0 8 Lizard [62.8] S. crassicaudatae 70.7 0 7.7 13.5 2.1 6 6.4 Dunnart [56.9] Amounts of phospholipids are given as the percentage of total phospholipids. PC, phosphatidylcholine; LPC, lysophosphatidylcholine; SM, sphingomyelin; PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; chol, cholesterol; PL, phospholipid. Data from: aDaniels and Skinner, 1994 [67]; bDaniels et al., 1995 [65]; cOrgeig and Daniels, 1995 [79]; dDaniels et al., 1996 [80]; eLangman et al., 1996 [71]. All samples collected at room temperature except sea turtles (32³C) and lizards (37³C).

BBADIS 61765 30-10-98 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 103 der, which is derived from the lungs of air-breathing molecular species method. Thus, the values for disa- ¢sh, is not completely known. However, as the swim turated PC in Table 2 could be elevated by approx. bladder is not required for air breathing, this ¢nding 20% relative to Table 1.) Nevertheless, it is clear suggests that surfactant is requisite for an in£ating/ there is a signi¢cant e¡ect of body temperature which de£ating organ with an air-liquid interface. a¡ects disaturated phospholipid as a percentage of Early work on mammalian pulmonary surfactant total in di¡erent species. The compositional variation focussed on lipid components, in particular DPPC. among groups may re£ect a balance between temper- The relative lack of this so-called `active ingredient' ature-dependent £uidity of surfactant phospholipids in the lungs of non-mammalian vertebrates led to the due to the need to maintain homeoviscosity (similar concept that these surfactants were less important for viscosity at di¡erent temperatures) and the requisite airway stabilization and may function as an anti-ad- low surface tensions during expiration. Only homeo- herent (also known as an `anti-glue') factor and as an therms and some heliothermic reptiles experience anti-oedemic factor, rather than as an anti-atelectatic body temperatures which approach the 41³C gel-to- agent. Studies on non-mammalian surfactants not liquid crystalline transition temperatures of DPPC only provide interesting insights into the evolution- bilayers. It is generally thought that only these ani- ary aspects of pulmonary surfactant, but similarities mals require (or can tolerate) high disaturated PC and di¡erences between these surfactants could prove levels. However, lipid extracts of mammalian surfac- valuable in identifying new roles for speci¢c compo- tant with high disaturated PC levels display £uid nents. Furthermore, non-mammalian species have characteristics at 20^25³C and show similar surface provided valuable model systems for studying surfac- activities at 25³ and 37³C on the pulsating bubble tant and its functions under di¡erent physiological surfactometer [61,68]. This suggests the possibility conditions, such as alterations in body temperature. that simply being in the £uid state at body temper- Table 2 presents the phospholipid composition of atures may not be su¤cient to meet the demands on a number of non-mammalian surfactants. While not the surfactant system in poikilotherms. Unfortu- identical, there are obvious similarities to each other nately, the nature of these demands, which impose and to mammalian surfactants. Interesting di¡eren- relatively low disaturation levels, must still be de- ces are the higher levels of SM in many species and ¢ned. the extremely high levels of lyso-PC in snakes. Most A major di¡erence between species in Tables 1 and of the PS/PI appears to be PI but PG tends to be low 2 is the cholesterol content. In mammals, cholesterol or absent. The exception appears to be the anurans relative to phospholipid varies from approx. 3% in (frogs and toads), which demonstrate a similar PG/ the cow to approx. 10% in the rat and sheep. A PI pattern to mammals. This may be an example of similar 3^10% range is observed for most non-mam- convergent evolution. Depending on the species, PE malian species, but this can be greater in speci¢c can be higher or lower than with mammals. species. Surfactant from gold¢sh swim bladders and The major di¡erences between surfactants from the lungs of ancient air-breathing ¢sh (actinoptery- mammalian and non-mammalian species are the pro- gians) have high cholesterol (15^35%), as does the portion of disaturated PC and the amount of neutral primitive Australian lung¢sh (N. forsteri). These lipids. Compositional di¡erences also occur between ¢sh also have relatively low (10^30%) disaturated di¡erent non-mammalian species. Among the latter PC levels. These values contrast with the high disa- species, the disaturated PC varies from approx. 10 to turated phospholipid and relatively low cholesterol 30% in ¢sh and amphibians, and up to 60% in rep- values in rat and human surfactant (Table 1) and tiles. (Note that the disaturated PC levels in Table 2 snake and lizard surfactant (Table 2). However, the were determined with the osmium tetroxide techni- African and South American lung¢sh have inter- que of Mason [15], which gives higher values than mediate (25^38%) disaturated lipids and low (5%) the molecular species methods used for the values in cholesterol levels. Intermediate PC disaturation and Table 1. For example, the Mason method indicates low cholesterol levels are also observed with sala- PC from human surfactant is 67.3% disaturated com- manders, turtles and toads. A possible explanation pared to the 44.7% reported by the more accurate for the radically di¡erent lipid pro¢les of surfactant

BBADIS 61765 30-10-98 104 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 from the teleost ¢sh, primitive air-breathing ¢sh, and factant from warm-active dunnarts measured. Hence, Australian lung¢sh (high cholesterol and low disatu- it appears that this species is capable of altering its rated PC) is that it may represent a proto-surfactant surfactant composition in response to £uctuations in which is used infrequently in air breathing, but is body temperature, thereby optimizing its surface ten- important for other functions in the swim bladder. sion modulating ability [72]. Apart from the evolutionary trends in surfactant Since surfactant recovered from non-mammalian composition between vertebrate groups, there are vertebrates is generally less disaturated, the surface also di¡erences within a particular group such as activity tends to be relatively low. Fish, amphibian, the amphibians. For example, temperature can a¡ect bird, and some reptiles have surfactants which ap- surfactant composition via habitat preference, be- pear incapable of attaining low surface tensions dur- cause terrestrial amphibians as a group have a sur- ing surface area reduction on the Langmuir-Wil- factant lipid pro¢le which is di¡erent from aquatic helmy balance, which has normally been used for amphibians. Terrestrial amphibians with variable these studies [73]. Only a few heliothermic lizards body temperatures possess surfactants low in both with very high preferred body temperatures (e.g., disaturated PC (13^20%) and cholesterol (0^6%), Pogona vitticeps and Trachydosaurus rugosa) and while aquatic amphibians with more stable body mammals possess surfactant which demonstrates temperatures have high disaturated PC (26^43%) high surface activity. Non-mammals (other than and cholesterol (8^11%) [69]. With both groups, rel- birds) possess relatively large, highly compliant res- atively similar cholesterol to disaturated PC ratios piratory units known as faveoli. This has led to sug- are observed, implying the proportion of cholesterol gestions that a highly surface-active surfactant capa- to disaturated PC must be kept within certain limits. ble of attaining very low surface tensions may not be Temperature also has profound e¡ects on the re- critical for optimal lung function in these animals. lation between cholesterol and disaturated lipids Certainly the larger size of the faveoli (100^1000 within individual species. For example, decreasing times larger than alveoli of similar sized mammals) the body temperature of the lizard, Ctenophorus nu- would avoid the high collapsing forces thought to chalis, from 37³C to 18³C results in a doubling of induce atelectasis (alveolar collapse) in the 50^100 surfactant cholesterol [70]. Similarly a torpid marsu- Wm mammalian alveoli. Faveoli possess an enhanced pial, the fat-tailed dunnart (Sminthopsis crassicauda- backbone consisting of elastin and collagen and an ta), exhibits a higher ratio of cholesterol to total and inner trabecular system which supports and stabilizes disaturated phospholipid (11% and 22% respectively) the inner connecting units [74]. at 15³C than at its normal active body temperature These considerations have led to the suggestion of 35³C (7% and 18%) [71] (marsupials are a subclass that surfactant has other functions in non-mamma- of mammals but it was thought appropriate to men- lian vertebrates such as acting as an anti-adherent tion this study with non-mammalian species). These (also known as an anti-glue) factor. A water droplet changes occur within a few hours, showing an amaz- between two glass microscope slides spreads readily ing ability of the surfactant system to adapt in these by capillary action because hydrophilic glass binds animals. A possible explanation is that cholesterol water better than water binds itself. The surprisingly functions to modulate the £uidity of the surfactant high force required to separate wetted glass slides is lipids, thereby enhancing surfactant adsorption, par- due to surface tension forces resisting expansion of ticularly at low temperatures. Certainly in the dun- the air-water interface. Air-breathing ¢sh, some liz- nart the alteration in surfactant lipid composition ards, snakes and amphibians can partially or com- during torpor (a form of diurnal hibernation) corre- pletely degas their respiratory units [65,75]. Reopen- lates with surfactant activity. For example, surfac- ing the wet tissue surfaces of interfolded faveoli (or tant from torpid dunnarts is more e¡ective in low- collapsed alveoli) is greatly aided by reducing surface ering surface tension during compression at 20³C tension to the equilibrium value of approx. 25 mN/ than 37³C. Moreover, surfactant from torpid dun- m. A similar phenomenon occurs with the ¢rst narts has a reduced ability to lower surface tension breath in newborn mammals, where by reducing sur- during compression at 37³C when compared to surface tension surfactant facilitates lung expansion

BBADIS 61765 30-10-98 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 105 with air. It should be stressed there is no `glue' for a number of crucial details are still lacking. For ex- the anti-glue factor to counter but only surface ten- ample, how is the DPPC-enriched monolayer gener- sion forces. The anti-glue function can be determined ated during adsorption? What are the precise roles of by completely emptying the lungs of air and measur- PC, PG, and possibly the other minor phospholipid ing the pressure required to open the lungs before constituents in selective DPPC adsorption and in £u- and after lavage. In almost all species examined, id lipid squeeze-out? What is the exact composition there is a greater opening pressure following surfac- of the interfacial ¢lm and what structures does it tant removal [65,75]. However, in control experi- adopt? Does cholesterol contribute to selective ments, where gut tissue is collapsed and in£ated be- DPPC adsorption in the lung? Is cholesterol ex- fore and after lavage, no such increase in opening cluded from the surface, as appears likely, and if pressure is observed. Surfactants contributing to res- so, how does this occur? What is the mechanism piratory unit opening must adsorb rapidly at body by which surfactant desorbs from the interface and temperature, but this action is independent of the what controls its return to the type II cells and the surfactant's ability to attain low surface tension dur- subsequent cellular re-uptake? ing compression. High cholesterol and low disatura- Are the minor lipid components, the major lipid tion should enhance adsorption and respreading at components, or the surfactant apoproteins involved low temperatures. in signal transduction processes responsible for con- Another function suggested for both non-mamma- trolling alveolar surfactant levels? Are surfactant lip- lian and mammalian surfactants is that of an anti- ids involved in other signalling events? oedema factor [76]. It has been suggested this func- What is the reason for the di¡erences in neutral tion could be important in birds which have minute lipid content between mammalian species, and what rigid air capillaries [73]. The theoretical basis of this is the signi¢cance of the observed alterations in al- anti-oedema function is that water molecules at the veolar cholesterol content during activity? Why do air-liquid interface experience lower average attrac- some non-mammalian species have surfactants with tive forces into the bulk phase, resulting in a de- very similar phospholipid pro¢les, but di¡erent cho- creased pressure di¡erence across the interface, as lesterol/disaturation patterns? Do non-mammalian indicated by the Laplace equation. However, water surfactants lower surface tension in the faveoli to in the bulk phase would experience association with low values like mammalian surfactants? ions and proteins as indicated by the Starling equa- We anticipate these and other issues will keep sci- tions, and to date this anti-oedema function of sur- entists in many disciplines occupied for a consider- factant has not been experimentally proven for nor- able time. We speculate that, during these investiga- mal mammalian lungs. Nevertheless, considerable tions, they may discover other important biological evidence is available for the ability of surfactant to functions for surfactant still not suspected. We pre- prevent £ooding of the lungs injured through surfac- dict a better understanding of surfactant function tant de¢ciency arising from prematurity, lavage, or will prove valuable not only in contributing to our other insults which tend to increase serum protein understanding of the biology of surfactant, but also levels [19,76]. Thus it appears likely that pulmonary as a basis for important improvements in therapy for surfactant could act synergistically with epithelial so- a number of respiratory diseases. dium channels to maintain wetted but not £ooded alveoli [77]. Acknowledgements

7. Future directions Support was received from the Medical Research Council of Canada and from the Australian Re- Since the rediscovery of pulmonary surfactant by search Council. The authors would like to express Pattle and Clements in the 1950s, information arising their gratitude to Dr. C.B. Daniels for helpful dis- from a number of scienti¢c disciplines has greatly cussion and to Mary Possmayer for excellent edito- expanded our understanding. However, it is evident rial assistance and advice. They would also like to

BBADIS 61765 30-10-98 106 R. Veldhuizen et al. / Biochimica et Biophysica Acta 1408 (1998) 90^108 express their thanks to Dr. John Clements for his mayer, Cellular distribution of enzymes involved in phospha- role in founding the ¢eld featured in this dedicated tidylcholine synthesis in developing rat lung, Can. J. Bio- issue, for his many original contributions to this fas- chem. 61 (1983) 107^114. [17] R.F. Zwaal, A.J. Schroit, Pathophysiologic implications of cinating area, and for his assistance and support in membrane phospholipid asymmetry in blood cells, Blood 89 our continuing endeavour to understand the surfac- (1997) 1121^1132. tant system of the lung. [18] R.J. King, Isolation and chemical composition of pulmonary surfactant, in: B. Robertson, L.M.G. van Golde, J.J. Baten- burg (Eds.), Pulmonary Surfactant, Elsevier, Amsterdam, 1984, pp. 1^15. References [19] F. Possmayer, Physicochemical aspects of pulmonary surfactant, in: R.A. Polin, W.W. 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