The Changing State of Surfactant Lipids: New Insights from ...

AMER. ZOOL., 38:305-320 (1998)

The Changing State of Surfactant Lipids: New Insights from AncientAnimals1

CHRISTOPHER B. DANIELS,2 SANDRA ORGEIG,* PHILIP G. W O O D , * LUCY C. SULLIVAN,*

OLGA V. LOPATKO,* AND ALLAN W. SMITSf

* Department of Physiology, University of Adelaide, Adelaide, South Australia 5005, Australia and^Department of Biology, Quinnipiac College, Hamden, Connecticut 06410 USA

SYNOPSIS. Pulmonary surfactant is a mixture of phospholipids (including disatu-rated phospholipids), cholesterol and proteins lining the air-liquid interface withinthe lung. Surfactant acts to reduce surface tension, thereby increasing lung com-pliance and also preventing edema. The saccular lungs, or other gas-holding struc-tures, of nonmammals have 7-70% more surfactant/cm2 of surface than lungs ofmammals. Nonmammalian surfactant acts as an antiglue that decreases the infla-tion pressures of collapsed lungs by reducing the adherence of apposing epithelialsurfaces. The autonomic nervous system appears to be the primary system con-trolling release of surfactant in nonmammals. The lipid composition is highly con-served within the vertebrates, except that surfactant of teleost fish is dominatedby cholesterol whereas tetrapod surfactant consists primarily of disaturated phos-pholipids (DSP). The dipnoan Neoceratodus forsteri demonstrates a "fish-type"surfactant profile while the other derived dipnoans demonstrate a surfactant pro-file similar to that of tetrapods. Homology of the surfactant protein SP-A withinthe vertebrates points to a single evolutionary origin for the system and indicatesthat fish surfactant is a "protosurfactant". Amongst the tetrapods, the relativeproportions of DSP and cholesterol vary in response to lung structure, habitat,and body temperature (Tb) but not in relation to phytogeny. The cholesterol contentof surfactant is elevated in species with simple saccular lungs, in aquatic species,and in species with low Tb. The DSP content is highest in complex lungs, partic-ularly of aquatic species or species with high Tb. The cholesterol content of sur-factant also increases in response to acute decreases in Tb in lizards and torpidmarsupials, presumably to maintain fluidity of the lipid mixture.

INTRODUCTION inspiration. In the mammalian lung, inter-The environment consists of matter in f a c i a l t e n s i o n i s overcome by pulmonary

one of three physical states: solid, liquid or surfactant which disrupts cohesive forcesgas. The unequal forces of attraction be- between water molecules. Phospholipidstween molecules in different phases result (PL>> particularly disaturated phospholipidsin surface tension at the boundary between ( D S P ) Csuch a s dipalrrutoylphosphatidyl-the phases. The issue of phase-phase inter- choline (DPPC)] and also unsaturated phos-actions is most clearly pronounced within pholipids and neutral lipids such as choles-the lung, because lungs are cyclically in- terol> comprise 80-90% by mass of mam-flating gas-filled structures lined with fluid. m a l i a n surfactant (King, 1984). It is the lip-In a lung lined only with an aqueous fluid, ids> particularly DPPC, which control thehigh interfacial tension would cause respi- surface tension. However, the mammalianratory surfaces to stick together on expira- bronchoalveolar lung is very different totion and impede expansion of the lung on t h e basic saccular "bag" lung of nearly all

other vertebrates. Whether the surfactantlipids have the same composition and func-

1 From the Symposium The Biology of Lipids: in- tions in the lungs of nonmammalian verte-tegration of Structure and Function presented at the brates regardless of the large differences in

querque, New Mexico. lutionary divergence has been the subject of2 E-mail: [email protected] our recent research. In particular, we have

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306 C. B. DANIELS ETAL.

attempted to elucidate the evolution of thesurfactant system and its role in the terres-trial radiation of the vertebrates. Our workon the surfactant system of noneutherianmammals, birds, and ectotherms has fo-cussed on four hypotheses. First, that thesurfactant system is conserved throughoutthe vertebrate radiation. Second, the func-tion of surfactant as an antiglue appears tobe a critical function of nonmammalian sur-factant. Third, temperature profoundly in-fluenced the evolution of the surfactant sys-tem, and fourth, that temperature and theAutonomic Nervous System both controlsurfactant release in nonmammalian verte-brates. Following a brief review of themammalian surfactant system, we discussthe significance of these four new insightsinto the origin, function, and evolution ofthe vertebrate surfactant system.

THE MAMMALIAN SURFACTANT SYSTEM

Morphology

The alveolar type II cells are generallyregarded as the main location for the syn-thesis storage and release of surfactant andthe subsequent reuptake of "used" surfac-tant so that the components can be reuti-lized (Wright and Clements, 1989). Thephospholipids and proteins are assembled inthe endoplasmic reticulum and the Golgiapparatus and stored in lamellar bodies un-til exocytosis (Wright and Clements, 1989).The source(s) of alveolar cholesterol remainunknown, although cholesterol is probablynot derived solely from lamellar bodies(Orgeig et al, 1995). The lamellar bodiesconsist of a dense proteinaceous core withlipid bilayers arranged in parallel, stackedlamellae surrounded by a limiting mem-brane (Wright and Clements, 1989). Afterthe lamellar bodies have been released intothe alveolar space, they swell and unravelinto another highly characteristic form ofsurfactant termed tubular myelin. In cross-section, tubular myelin appears as a cross-hatched structure that consists of an arrayof 40 A-wide squares composed of lipid bi-layers (King, 1984). The surface film is de-rived from the tubular myelin. The resultantmonolayer is thought to lower the surface

tension of the liquid lining the lung (Wrightand Clements, 1989).

The surfactant lipidsThe ability to vary surface tension with

changing surface area, and the profoundhysteresis effect which results in major dif-ferences between surface tension on defla-tion compared with inflation (see below), isusually attributed to the interactions be-tween DPPC and the other lipids, particu-larly unsaturated phospholipids and choles-terol (King, 1984). At low lung volumes,the lipid monolayer at the air-liquid inter-face is thought to be highly enriched inDPPC (Goerke and Clements, 1985). TheseDPPC molecules can be compressed tightlytogether because of the fully saturated fattyacid chains. Only DPPC has the necessaryrigid acyl chains to form the metastablepacking for a monolayer under dynamiccompression (Goerke and Clements, 1985).The DPPC monolayer prevents air and liq-uid from coming into contact, thereby de-creasing surface tension markedly (West,1985; Possmayer, 1991). Upon expirationthe dynamic compression of the monolayerresults in the "squeezing out" of unsatu-rated phospholipids and cholesterol fromthe monolayer, and the monolayer is en-riched in DPPC (Goerke and Clements,1985).

However, in order to generate low sur-face tensions, the monolayer must be belowthe gel-to-liquid-crystalline temperature(usually referred to as the phase transitiontemperature) (Goerke and Clements, 1985).Because DPPC has a phase transition tem-perature of 41°C, a pure DPPC film is be-low the phase transition point and enters thegel form at mammalian body temperatures.However, below the phase transition tem-perature, DPPC adsorbs to a surface veryslowly, in fact, much more slowly than pul-monary surfactant (Fleming and Keough,1988). The unsaturated phospholipids andpossibly cholesterol are probably requiredto convert the DPPC from its gel form tothe more spreadable disordered state, oncethe compression forces are lifted (during in-flation) (Fleming and Keough, 1988). Uponinspiration, unsaturated phospholipids andcholesterol insert into the DPPC monolayer

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THE EVOLUTION OF SURFACTANT 307

to aid in the respreading. Only in the liquid-crystalline state can the lipids disperse tocoat the surface of the expanding fluid layer(Fleming and Keough, 1988).

Cholesterol appears to have many func-tions in pulmonary surfactant. At 10% byMol, cholesterol is the second largest lipidcomponent (King, 1984). Cholesterol mayplay an important role in controlling theviscosity of alveolar surfactant and increasethe ability of DPPC to respread after thecollapse of the monolayer (Fleming andKeough, 1988). Cholesterol may also en-hance the rate of adsorption of the newlyreleased surfactant in vivo (Fleming andKeough, 1988).

The surfactant proteins

Four surfactant proteins have been de-scribed. Termed surfactant protein-A (SP-A), SP-B, SP-C and SP-D these proteins aresynthesised in alveolar type II cells, are allassociated with purified surfactant and con-tribute to the biophysical activities of sur-factant (Possmayer, 1990). Both the secre-tion and the reuptake of surfactant phos-pholipids into type II cells appears to beregulated by SP-A (Wright et al, 1987).Both SP-A and SP-B are essential for theformation and structural integrity of surfac-tant components (Suzuki et al., 1989). Sur-factant proteins (particularly SP-A) are im-portant for the conversion of lamellar bod-ies to tubular myelin and from tubular my-elin to the phospholipid surface film(Venkitaraman et al, 1990). Surfactant pro-tein A, with SP-B and SP-C, also enhancesthe rate of formation of a phospholipid sur-face film at air-liquid interfaces (Venkitar-aman et al, 1990). A binding site on SP-Acomplements those found on the surface ofbacterial cells (Possmayer, 1990), and SP-A may therefore play a role in combatinglung infections. In fact, SP-A has beenfound to enhance the ability of alveolarmacrophages to destroy bacteria (Tenner etal., 1989). Surfactant protein D is a hydro-philic glycoprotein which, in vitro, appearsto counteract the inhibitory effect of SP-Aon surfactant secretion by type II cells(Possmayer, 1990).

The control of surfactant synthesis andrelease

Perhaps the most significant trigger forrelease of surfactant in mammals is directmechanical stimulation of type II cells,which are preferentially located in cornersand crevices where they may be subject tomaximal mechanical torsion which stimu-lates the release of the lamellar bodies(Wirtz and Dobbs, 1990). Hence, the typeII cells respond rapidly to changes inbreathing pattern by increasing release ofsurfactant (and possibly reuptake) (Goerkeand Clements, 1985), and also by alteringthe composition of the surfactant (Orgeig etal, 1995). Type II cells cultured directlyonto fibronectin-coated elastic membranesreleased surfactant for up to 30min follow-ing a single stretch (Wirtz and Dobbs1990). The mechanical stimulation of sur-factant release has been demonstrated invivo with a change in ventilatory pattern,particularly in response to an increase intidal volume above resting levels (reviewedby Wright and Clements, 1989). Ventilatorypattern may also be responsible for changesin surfactant composition. For example, thecholesterol/DSP ratio changes with breath-ing pattern in rats (Orgeig et al., 1995).

The mammalian surfactant system is alsoaffected by input from the Autonomic Ner-vous System. The type II cells have p2 (ad-renergic) receptors. Although these cells arenot directly innervated, stimulation of thesereceptors increases the release of DPPC.While acetylcholine receptors exist on typeII cells, cholinergic agents do not appear toact directly on isolated type II cells to stim-ulate release (reviewed by Wright and Cle-ments, 1989).

Surfactant functions

The primary functions of surfactant aregenerally regarded to include, improvinglung compliance, maintaining alveolar sta-bility, preventing pulmonary edema, aidingthe muco-ciliary escalator, aiding the im-mune system, and acting as an antiglue.Von Neergaard (1929) first demonstratedthat the surface forces at the gas-liquid in-terface of the lung contribute substantiallyto the retractive pressure, and hence static

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compliance, of the lung. He concluded thatthe surface tension of the alveoli had to belower than that of other physiological fluidsbecause of the accumulation of a surfaceactive material at the alveolar-air interface.Subsequent experiments using variousforms of surface balance demonstrated thatthe surface tension of a fluid coated withmammalian surfactant decreased rapidly oninitial compression, then more gradually toa lower limiting value of approximately 0-1 dynes/cm. Upon re-expansion, surfacetension increased rapidly at first and thenremained constant at an upper limiting val-ue of approximately 46—50 dynes/cm(Goerke and Clements, 1985). Plotting sur-face tension as a function of lung areayields a characteristic curve such that at anygiven volume, the surface tension duringinflation is always greater than that duringdeflation. The difference in the inflation anddeflation curves is known as surface hys-teresis. This ability to vary surface tensionduring the regular cycling of respiratorysurface area provides the lung with stabili-ty.

By the law of Young and La Place, thepressure to maintain an alveolus of smallradius would be much greater than that inan alveolus of larger radius, under condi-tions of constant surface tension. Hence, thesmaller alveolus would empty into the larg-er one. However, because surfactant variessurface tension with the radius of curvatureof each alveolus, the pressures within allalveoli are maintained at similar values,permitting alveoli of different sizes to co-exist (Goerke and Clements, 1985). More-over, varying surface tension with relativealveolar surface area has additional bene-fits. At low lung volumes the low surfacetension prevents alveoli from collapsing. Athigh lung volumes the large surface ten-sions set a limit on the expansion of thealveoli, thereby preventing them from rup-turing (Goerke and Clements, 1985; West,1985). Surfactant also assists the narrowestairways to remain open, thereby reducingthe resistance to airflow (Enhorning andHolm, 1993). Surfactant, also controls fluidbalance in the lung (Guyton et al, 1984).The entire alveolar surface is covered witha fluid, termed the hypophase. Surfactant

acts to lower the surface tension of the al-veolar fluid, reducing the negative hydro-static pressure in the hypophase therebykeeping the alveoli relatively dry (Guytonetal, 1984).

However, the alveoli, being interdepen-dent units, do not necessarily stretch uponinflation, but instead expand in a complexmanner, best described as unpleating or un-folding (Sanderson et ah, 1976). Moreover,the many fluid-filled corners and crevices inthe alveoli open and close as the lung in-flates and deflates. Sanderson et al (1976)determined that the work done in openingthe surfactant-filled crevices is proportionalto the surface tension of the fluid, and tothe area of the exposed walls in the case ofa parting of parallel surfaces originally at adistance x from one another. If the area ofthe walls is A, the work (W) required topart them will be the integral of the force(F) attracting each toward its companion,such that

and as

then

F = dW/dx, (1)

F = 2-y(x)A/x, (2)

dW = (2-y(x)A/x) dx, (3)

where 7(x) allows for a variable surfacetension. Therefore, the force attracting thetwo surfaces to each other is proportionalboth to the area of contact and the surfacetension of the fluid between the surfaces,and inversely proportional to the distanceby which they were originally separated.Surfactant can exhibit antiglue properties ifit lines the interface between apposed epi-thelial surfaces within regions of a col-lapsed lung. As the two apposing surfacespeel apart, the lipids rise to the surface ofthe hypophase fluid at the expanding gas-liquid interface and lower the surface ten-sion of this fluid, thereby decreasing thework required to separate the two surfaces.However, for surfactant to act as an anti-glue, the alveoli must "fold" in on them-selves, possibly during exhalation, or whenthe ventilatory period is punctuated by pro-tracted non-ventilatory periods at low lung

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volume. However, these conditions rarelyoccur in eutherian mammals.

Surfactant has important additional func-tions besides controlling alveolar surfacetension. Surfactant has been located in theairways (Gil and Weibel, 1970) and mayenhance the function of the muco-cilary es-calator in removing inhaled foreign parti-cles (Allegra et al, 1985). Surfactant is alsoimportant in protecting the lung from infec-tions by inhibiting the proteolytic activationof viruses, inhibiting the capacity of mono-cytes and granulocytes to release H2O2 orpossibly preventing excess immune re-sponse (Pison et al, 1994).

Conclusion

For mammals, damaging or removing thesurfactant system has dramatic effects onthe overall compliance of the lungs, indi-cating that controlling surface tension at thegas-liquid interface is important for optimallung function. However, the removal of sur-factant appears to have only a marginal (ifany) effect on static lung compliance in allnonmammals examined to date (Daniels etal, 1995a, b). Mammalian lung complianceis one to two orders of magnitude less thanthat of reptilian lungs (reviewed in Danielset al., 1993), avian airsacs (Duncker, 1978),anuran lungs (Hughes and Vergara, 1978)and lungs of actinopterygiian fish (Smits etal., 1994). The lungs of most nonmamma-lian vertebrates (excluding the birds) alsohave large gas exchange units (10-1,000times greater than mammalian alveoli)(Wood and Lenfant, 1976) yet have far few-er alveoli than mammalian lungs (/g lungtissue), and often have a large central air-space. Hence, the surfactant of nonmam-mals may not be required to increase com-pliance or maintain alveolar stability in thesame manner as it does in mammals. Fur-thermore, nonmammalian vertebrates maynot require a surfactant to vary surface ten-sion, or to reduce it to very low values. Arethe primary functions of mammalian sur-factant necessarily relevant to nonmam-mals? Moreover, the radically different lungstructure of mammals suggests it is unlikelythat studies into the functions of mamma-lian surfactant will provide important clues

about the evolution of the vertebrate sur-factant system.

INSIGHTS FROM NONMAMMALIANVERTEBRATES

The surfactant system is conservedthroughout the vertebrate radiation

Our recent work on the composition ofsurfactants from representatives of variousvertebrate groups demonstrated that surfac-tant could be obtained from the lungs and/or swimbladders of all animals tested (Dan-iels and Skinner, 1994; Daniels et al., 1989,1995a, b). The lungs of fish and reptilescontained greater (7—70 fold) amounts oflipid per unit of surface area than those ofmammals (mouse and rat) (Smits et al.,1994; Daniels et al, 1989). In addition,DSP, unsaturated phospholipids and choles-terol were the dominant lipids in all surfac-tants (Daniels et al, 1995a). When ex-pressed as a percentage of PL, the goldfishswimmbladder, fish lungs and the lungs ofthe Australian lungfish, Neoceratodus for-steri had surfactant with approximately 3fold greater amounts of cholesterol than didany of the other vertebrate groups (Fig. la)(Smits et al, 1994; Orgeig and Daniels,1995). The opposite trend was apparentwhen considering the percentage of total PLthat is disaturated (%DSP/PL). Teleostswimbladders and the lungs of the acti-nopterygiian fish and N. forsteri containedphospholipids that were 2—4 fold less sat-urated than those of the derived sarcopter-ygiians, the African and South Americanlungfish, and the majority of the amphibiansstudied to date, and 5 fold less than thoseof reptiles and mammals (Fig. lb).

Because surfactant was present in all spe-cies, and because the greatest range in lipidcomposition occurred in the Dipnoi, it ispossible that pulmonary surfactant had asingle origin, coinciding with the origin ofthe vertebrates (Orgeig and Daniels, 1995).A high cholesterol/low DSP mixture maybe the primitive surfactant, now restrictedto the fish and N. forsteri, and to organs nolonger used for gas exchange (swimblad-ders for example). We hypothesised that thelipid balance was subsequently modifiedthrough the evolution of the vertebrates to

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310 C. B. DANIELS ET AL.

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C.aur C.cal N.for A.tig S.int X.lae C.nuc H.sapP.sen Loss P.ann A.tri B.mar C.atr R.nor

FIG. 1. The composition of pulmonary surfactantfrom the lungs of selected vertebrates. A = the Cho-lesterol/Phospholipid ratio (CHOL/PL) expressed as apercentage. B = the Disaturated Phospholipid/Phos-pholipid ratio (DSP/PL) expressed as a percentage.The abbreviations are: C. aur = (Carassius auratus)(goldfish swimbladder), P. sen = Polypterus senega-lensis, C. cal. = Calimoicthys calabaricus, L. oss =Lepisosteus osseus (all three are airbreathing fish), N.for = Neoceratodus forsteri, P. ann = Protopterus an-nectens (both Dipnoan lungfish), A. tig = Ambystomatigrinum, A. tri = Amphiuma tridactylum, S. int = Si-ren intermedia (all three are salamanders), B. mar =Bufo marinus, X. lae = Xenopus laevis (both Anu-rans), C. atr = Crotalus atrox, C. nuc = Ctenophorusnuchalis (both reptiles), R. nor = Rattus norvegicus,H. sap = Homo sapiens (both mammals). Data ex-pressed as Mean ± SEM, n usually between 4 and 9(Daniels et al, 1995a).

match changing circumstances such as ter-restriality, temperature, habitat, lung struc-ture and lung function, to produce a surfac-tant substance high in DSP (Daniels et al,1995a). We have termed the surfactant offish and Neoceratodus the "protosurfac-tant" (Smits et al, 1994; Orgeig and Dan-iels, 1995).

Because of the substantial variability ofsurfactant lipids within a particular groupof vertebrates, lipids are not necessarilysuitable indicators for the origin and evo-lution of the surfactant system. Moreover,species can demonstrate different surfac-tants in different regions of their lungs. Rat-tlesnakes, with their complex anterior fav-eolar lung ("tracheal lung") and their pos-

terior, smooth bag-like, saccular lung, havetwo different types of surfactant; a highDSP/low cholesterol mixture occurs in thefaveolar lung, and a low DSP/high choles-terol mixture is present in the saccular lung(Daniels et al, 1995c). Surfactant compo-sition also changes as the lung matures dur-ing the development of the tiger salamanderfrom an aquatic larval stage to a terrestrialair-breathing adult (Orgeig et al, 1994). Inthis case, increasing lung maturity correlat-ed with a decreased level of phospholipidsaturation, which is a change that is in di-rect opposition to the pattern of lung mat-uration in the fetal-to-neonatal transition inmammals. In both cases the highly fluid na-ture of the high cholesterol mixtures pre-sumably assists in the easy spreading of thesurfactant from isolated clumps of secretorycells to cover the entire surface.

Therefore, we have recently tested thehypothesis of a single evolutionary originof surfactant by analysing SP-A, a compo-nent that may show little structural changeonce the system was established. We dem-onstrated that an SP-A-like protein is pres-ent in surfactant from all vertebrate classes(even from goldfish swimbladders) (Figs. 2,3; Sullivan et al, 1998). Until this study,the presence of pulmonary surfactant pro-teins has only been conclusively confirmedin mammals. SP-A is probably a critical in-gredient in vertebrate surfactant becausethis protein assists in assembling the tubularmyelin. There are similarities in the ultra-structure of the surfactant system amongstall vertebrates. Lamellar bodies and tubularmyelin-like structures have been observedin the lungs of reptiles (Nagaishi et al,1964; Wetzstein et al, 1980; McGregor etal, 1993), birds (Tyler and Pangborn, 1964;Lambson and Cohn, 1968; Lopez et al,1984), and amphibians (Goniakowska-Wi-talinska, 1984, 1986). Lamellar bodies havebeen observed in the three extant species oflungfish (Klika and Lelek, 1967; Hughes,1973; Hughes and Weibel, 1978), in thelungs of primitive air-breathing fish (Klikaand Lelek, 1967; Marquet et al, 1974), andin the swimbladder of the rainbow trout(Brooks, 1970a, b). Thus, surfactant fromnonmammalian vertebrates would appear to

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MW(kD)

106-80-49-

32-27-

18-

1 2 3 4 5 6

mi^^&w wf^ff

-120 kD-60 kD-30 kD

1 2 3 4 5 6 7 8 910 11 12

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FIG. 2. Western blot analysis of lavage protein after incubation with the primary antibody, rabbit anti-humanSP-A, and the secondary antibody, goat anti-rabbit IgG. Molecular weights are labelled. The antibody crossreacted with all species examined. The most predominant cross-reactivity in all cases is at 55-65 kDa. A smallamount of cross reactivity in all species can be seen at 120 kDa. The marsupial, lizard, turtles, African lungfishand goldfish samples demonstrated cross reactivity at 28—35 kDa. The bands possibly correspond to the SP-Amonomer, dimer and tetramer (Sullivan et al., 1998). a: 1 = purified human SP-A, 2 = mouse, 3 = chicken, 4= shingleback lizard, 5 = sea-snake, 6 = cane toad, b: 1 = purified human SP-A, 2 = mouse, 3 = marsupial(Sminthopsis crassicaudata), 4 = crocodile, 5 = freshwater turtle, 6 = sea-turtle, 7 = salamander, 8 = Africanlungfish, 9 = Australian lungfish, 10 = bichir, 11 = gar, 12 = goldfish.

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312 C. B. DANIELS ET AL.

A B

ASAS

FIG. 3. Electronmicrographs of A. the Australian lungfish (N. forsteri), and B. the bichir {P. senegalensis)demonstrating immunogold particles (arrowheads) associated with lipid complexes predominantly in the air-spaces. Bar = 500 nm. LT = lung tissue, AS = air space (Sullivan et al., 1997).

be produced stored and released in a similarmanner to mammalian surfactant.

Acting as an antiglue appears to be thecritical function of nonmammaliansurfactant

We have proposed that the main functionof surfactant in nonmammals may be to actas an antiglue, that prevents adhesion of ad-jacent epithelial surfaces at low lung vol-umes or when the respiratory units fold inon each other during expiration. Usingscanning electron microscopy and comput-erized tomography scanning, Daniels et al.(1994a) developed a model for the breath-ing dynamics of the lizard, Ctenophorusnuchalis. During lung deflation, the epithe-lial tissues, which are strung between theouter lung wall and the inner trabecular net-work, fold in on each other like a concer-tina. This results in large portions of epi-thelial tissue coming into contact, a situa-tion in which the antiglue function of sur-factant may be critical. However, noempirical evidence for an antiglue functionby mammalian surfactant has been present-ed. We demonstrated an antiglue function

in nonmammalian vertebrates by demon-strating an increase in opening pressure(Fig. 4a) (which we define as the pressurerequired to inflate a completely collapsedisolated lung) following lavage for thelungs of reptiles, actinopterygiian fish, andsalamanders (Fig. 4b). Surfactant also per-formed an antiglue function in the goldfishswimbladder (Daniels and Skinner, 1994).Furthermore, in almost all cases, fillingpressure {i.e., the pressure required to con-tinue to inflate the lung after initial lungopening) was extremely low (1—4 cm H2O)and remained unchanged before and afterlavage (Daniels et al, 1995a). Therefore,the surfactant lipids appear to be importantonly during the initial phase of inflating acollapsed lung and not during further infla-tion or deflation.

The pattern and mode of breathing ofnonmammalian vertebrates also indicatethat an antiglue function might be essentialfor these animals, particularly when theyare very cold. The aquatic amphibians Am-phiuma and Siren collapse their lungs com-pletely upon expiration (Martin and Hutch-ison, 1979; Stark-Vanks et al, 1984) as do

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Opening pressure

Filling pressure

Volume

• pre-lavI post-lav

C.aur P.sen C.cal Loss A.tig C.nuc T.ord

FIG. 4. Opening pressure of the lungs of nonmam-malian vertebrates. A = schematic representation ofthe pressure required to open, and then fill a complete-ly collapsed lung with air infused at a constant rate(usually 1 ml/min). Note that initially the pressure in-creases before the lung begins to inflate, then onceopen the lung inflates with little change in pressure. B= The opening pressure of collapsed lungs removedfrom a range of vertebrates before then after surfactantremoval by lavage. Abbreviations are as for Figure 1,and T. ord = Thamnophis ordinoides (Snake). Dataexpressed as Mean ± SEM, n between 4 and 8 (Dan-iels el al., 1995a).

dipnoan lungfish and Polypterus (Bishopand Foxon, 1968; Brainerd et al., 1989).Complete collapse of the lung may also oc-cur at end-expiration in some small frogs(Hughes and Vergara, 1978). Seasnakesalso cycle air by emptying and collapsingthe saccular lung (Seymour et al., 1981).Furthermore, at low body temperatures thelizard C. nuchalis exhibits periods of apneaduring which the lungs collapse and the ep-ithelial surfaces may come into contact(Frappell and Daniels, 1991a, b; Daniels etal. 1994a). The low metabolic rate of coldectothenns decreases the need for frequentventilations, and the lungs collapse for pro-longed periods. Here, surfactant is criticalto decrease the work of separating the con-tacting epithelial surfaces when the occa-sional breath is required. Without an agentto lower the surface tension of the fluid in-tervening between the contacting epithelialsurfaces, inspiration after lung collapse

might be impossible, or at least extremelycostly in terms of relative energy expendi-ture.

Temperature profoundly influences thesurfactant system

Thermal influences on the evolution ofthe surfactant system.—Within any tetrapodclass, the composition of pulmonary surfac-tant demonstrates significant variability.This variation may reflect both varying lifestyles (particularly aquatic or terrerestrialhabits) and variations in preferred bodytemperature. In the former case, the habitatmay exert a selection pressure via the extentof the cycles in environmental temperaturethese animals may face (Daniels et al.,19946, 1996; Orgeig et al., 1994). How-ever, the major phylogenetic trends in sur-factant composition relate largely to overalldifferences in body temperature (and hencethe methods of body temperature regula-tion). Differences among groups in thecomposition of surfactant probably reflectthe temperature-dependent fluidity of sur-factant phospholipids and the need to main-tain homeoviscosity. Because DPPC under-goes a phase-transition from a gel to a liq-uid-crystalline state at 41°C, and realisti-cally only homeotherms and the mostheliothermic of the reptiles are likely tohave body temperatures that approach thisvalue, it follows that only these groups arelikely to be capable of tolerating DSP/PLratios of 40-50% (Daniels et al, 1995a)(Fig. 5). In contrast, advanced dipnoans andamphibians generally have much lowerbody temperatures, and have incorporatedless DSP in their surfactant, with DSP/PLratios of only 15-30% (Daniels et al.,1995a). Cholesterol is not as effective asDSP at reducing surface tension, but is ableto lower the normal phase-transition tem-perature of DSP, thereby maintaining themixture in a fluid, easily adsorbable stateover a much broader range of temperatures(Hadley, 1985). Similarly, unsaturatedphospholipids have a much lower phase-transition temperature than their saturatedcounterparts. The surfactant of fish and am-phibians, having greater cholesterol/DSPratios (20-130%) compared with that of thewarmer reptiles and mammals (10-15%),

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314 C. B. DANIELS ET AL

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C.car N.dep C.porT.nig T.ord C.nuc C.atr

"Cold" "Hot"

FIG. 5. The DSP expressed as a percentage of PL forreptiles with either a relatively low preferred bodytemperature ("Cold") or a relatively high preferredbody temperature ("Hot"). Abbreviations are: C. car= Caretta caretta, N. dep = Natator depressus (bothSea Turtles), C. por = Crocodylus porosus (Croco-dile), T. nig = Tiliqua nigroleutea (Lizard), T. ord =Thamnophis ordinoides (Snake), C. nuc = Ctenopho-rus nuchalis (Lizard), C. atr = Crotalus atrox, (Rattle-snake). Data expressed as Mean ± SEM, n between 3and 6 (Daniels et al.. 19956, 1996).

appear to benefit from this increased fluid-ity (Daniels et al, 1995a, b).

Within groups of vertebrates, thermal in-fluences can exert effects via habitat pref-erence. Terrestriality affects the lipid profileof amphibian surfactant. The fully terrestri-al cane toad (Bufo marinus), has a surfac-tant that is low in both DSP and cholesterol,whereas the fully aquatic salamanders Sirenintermedia and Amphiuma tridactylum havea surfactant that is rich in both cholesteroland DSP (Daniels et al, \994b). The de-crease in the saturation level in terrestrialamphibians may enable the surfactant to re-main in a fluid state over the often lowerand/or more variable environmental tem-perature range that they may experience.However, the relatively high hydrostaticcompression forces experienced by thelungs of aquatic amphibians may require aDSP-rich surfactant capable of forming a"splint", protecting the epithelial surfacesfrom collapse, and subsequently enablingeasy inflation (Fig. 6). In reptiles, the oc-cupation of either a terrestrial or aquatichabitat does not affect surfactant composi-tion, presumably because possession of ribsor shells assists in protecting the lungs fromthe elevated hydrostatic pressures (Danielset al, 1995fc, 1996). While differences insurfactant composition correlate with dif-ferences in lung structure (unicameral or

12

OO 4 n

n40

I 30O 20

^ 10}

JL _L i

X.lae S.int A.tri T.nat

Aquatic

B.mar A.tig S.tho

Terrestrial

FIG. 6. The composition of pulmonary surfactantfrom aquatic or terrestrial amphibians. A = TheCholesterol/PL ratio; B = the DSP/PL ratio; C = Cho-lesterol/DSP ratio. Abbreviations are as in Figure 1; T.nat = Typhlonectes natans, S. tho = Shistometapumthomense (both caecilians). Data expressed as Mean ±SEM, n between 4 and 8 (Daniels et al.. 19946,1995a).

multicameral lungs), the primary influencewas the preferred body temperature (Fig.5). As with the amphibians and lungfish,phylogenetic grouping was not a determi-nant of the surfactant lipid composition(Daniels et al, \995b, 1996).

Effect of temperature within species.—Temperature exerts profound effects on thecomposition and function of surfactant dur-ing normal thermal cycles in heterothermicand ectothermic vertebrates. Animals mayundergo rapid changes in body temperatureduring their daily cycle. Hibernating or tor-pid animals substantially lower their bodytemperatures for protracted periods. Thelevel of surfactant cholesterol increaseswith rapid short-term decreases in bodytemperature in the agamid lizard, Cteno-phorus nuchalis although the amount of al-veolar surfactant did not change (Fig. 7)(Daniels et al, 1990). In this case the de-crease from 37°C to 13°C doubles theCholesterol/PL ratio (Fig. 7) (Daniels et al,

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100

20

15

I

5

Io

37 10 15 20 25 30 35 40 45

Body Temperature ( C)

FIG. 7. Changes in amount of phospholipid and cho-lesterol (/g wet lung mass) harvested by lavage, to-gether with the cholesterol/phospholipid ratio in thelizard Ctenophorus nuchalis maintained at differentbody temperatures for 4 hours. Data expressed asMean ± SEM, Numbers of lizards are included (Dan-iels et al., 1990).

1990). The amount of surfactant in thelungs of the central Australian agamid liz-ard Pogona vitticeps increases as body tem-perature increases (Wood et al, 1995),while cold turtles hibernating for 2-3months, exhibit less lavageable surfactant,which is also higher in unsaturated fatty ac-ids, than warm active animals (Lau andKeough, 1981).

Recently we have been investigating therelationship between torpor, surfactant com-position and lung function in heterothermicmarsupials (Langman et al., 1996; Orgeiget al., 1996). Torpor is a short term reduc-tion in body temperature by mammals, mar-supials and birds, which enables the ani-mals to conserve energy during periods ofreduced ambient temperature. We comparedthe lipid composition of surfactant of themarsupial, the dunnart (Sminthopsis cras-sicaudata Tb = 35 ± 1°C) in its warm-ac-

tive state with the surfactant composition of20-22g mice (Mus musculus) and dunnarts1, 4 and 8 hours into torpor (Tb = 15 ±1°C). We also examined the influence ofsurfactant on the static lung compliance formembers of these groups. The amounts ofPL, cholesterol and DSP increase duringtorpor (as/g Dry Lung) (Langman et al.,1996; Orgeig et al, 1996). The relative pro-portions of cholesterol and DSP as fractionsof total PL increase after 4 and 8 hours,respectively, while the cholesterol/DSP ra-tio increases significantly after 8 hours oftorpor (Langman et al, 1996; Orgeig et al,1996). The latter change indicates an in-crease in the fluidity of the surfactant, andresults in marked alterations to the surfaceactivity of the surfactant (Orgeig et al,1996).

Whether these changes in surfactantcomposition induced by short, medium-term or extended periods of low body tem-perature have any effect on lung function iscontroversial. In the previous section we ar-gued that the antiglue function is critical forcold lizards to reduce the work of inflatingthe collapsed lung but that surfactant doesnot influence any other aspect of lung com-pliance regardless of body temperature. Inaddition, compositional changes in the sur-factant of cold lizards take at least 2 hoursto occur (Daniels et al, 1990), yet the an-tiglue function is required immediately(Daniels et al, 1993). Moreover, for torpiddunnarts, short periods (1 and 4 hours) oftorpor decrease lung tissue compliance(suggesting an increased resistance of thestructural components), but this decrease isabolished by 8 hours (Fig. 8b, c). However,total lung compliance (with the surfactantsystem in place) remains unchangedthroughout torpor (Fig. 8a) (Langman et al,1996; Orgeig et al, 1996). Thus, surfactantcounteracts the negative effect of the de-creased tissue compliance at 1 and 4 hours.As lung compliance is maintained at 1 hourof torpor in the absence of a compositionalchange, the compositional changes ob-served at 4 and 8 hours of torpor probablyrelate to functions of surfactant other thanthat of maintaining lung compliance (Lang-man et al, 1996; Orgeig et al, 1996). Anantiglue function is possibly important in

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.030prelav

x

Q.

O

o

postlav.025 T - ,

saline

hour 8 hour

FIG. 8. Static lung compliance upon inflation, at 40%of total lung capacity, for warm-active (Tb = 35°C)marsupials {Sminthopsis crassicaudata) and animals 1,4 or 8 hours into torpor (Tb = 13-18°C). A = lungcompliance before lavage; B = lung compliance afterlavage; C = lung compliance determined by saline in-fusion (hence having no air-liquid interface). Data ex-pressed as Mean ± SEM, n = 4, * = significantlydifferent compared with the warm active group (Lang-man et al., 1996; Orgeig et al., 1996).

torpid dunnarts if the lungs collapse or thebreathing pattern becomes episodic. How-ever, the function of the thermally inducedchanges in surfactant composition, the rea-sons and significance of the extended timefor the changes, and the effect of an alteredsurfactant composition on surfactant func-tion when the animals rewarm, remain un-known.

Temperature and/or the autonomicnervous system control the surfactantsystem in nonmammalian vertebrates

The processes controlling surfactant re-lease, re-uptake, and composition are notwell understood. The surfactant system islikely to be affected by the Autonomic Ner-vous System, ventilatory pattern (via tor-sion of the type II cells), and temperature(and hence metabolic rate), of the type IIcell (Wood et al, 1995). Regulating surfac-

tant by distortion of the type II cell mayserve well for the continuous breathing pat-tern of mammals, but may not be as appro-priate a mechanism for species that spendlong periods of time in non-ventilatory pe-riods and exhibit extremely variable lungvolumes. With the exception of our recentstudies (Wood et al, 1995, 1997; Wood andDaniels, 1996), surfactant regulation innonmammals has been poorly examined. Inaddition, the effects of altering the rate ofrelease, re-uptake or composition of surfac-tant on its function within the lung or on itssurface activity and physical behavior havenever been examined.

The autonomic nervous system.—Wehave developed an isolated perfused lungpreparation to analyse the effects of select-ed autonomic neurotransmitters on the re-lease of surfactant in the lizard (Wood andDaniels, 1996; Wood et al, 1995, 1997).Perfusing the lungs with 10 7 g/ml adren-aline in Ringers-albumin stimulates PL re-lease in the lizard (Wood et al, 1995,1997). Moreover, plasma levels of adrena-line and noradrenaline increases at higherbody temperatures in P. vitticeps and bothare accompanied by a greater PL content inthe lungs (Wood et al, 1997). Hence cir-culating, adrenally-derived catecholaminesmay act directly on the type II pneumo-cytes. Perfusing the lungs with 10~5 g/mlacetylcholine (a parasympathetic neuro-transmitter) also stimulates PL release, andthe action of this neurotransmitter is abol-ished by the application of the muscarinicantagonist atropine (5 X 10~6 g/ml). How-ever, acetylcholine continues to stimulatesurfactant release in the presence of theganglion blocker hexamethonium (Fig. 9)(Wood et al, 1997). Acetylcholine maypossibly act on neuro-epithelial bodieswithin the lung, or in some other indirectmanner, but most likely acts directly on thetype II cell. Neither adrenergic nor cholin-ergic administration influence the cholester-ol content of the alveolar surfactant (Dan-iels etal, \995b; Wood et al, 1995, 1997).

Temperature and cellular metabolicrate.—Whether the control systems for al-tering the cholesterol and/or DSP content,and the release (and reuptake) of surfactantare the same in the short or long term

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5 3500Q 3000- 2500B- 20005 1500§" 1000Q. 500

o2

*

Control ACh ACh-Hex ACh-Hex-At

FIG. 9. Amount of phospholipid (/g dry lung mass)released into the isolated, lavaged lung of the beardeddragon (Pogona vitticeps) after 3 hours of perfusionwith Ringers-Albumin containing Acetylcholine(Ach), Acetylcholine and Hexamethonium (Ach-Hex),or Acetylcholine, Hexamethonium and Atropine (Ach-Hex-At). * = significantly elevated compared withControl (Ringers only). Data expressed as Mean ±SEM, n = 6 (Wood et al, 1997).

changes in body temperature remain un-known. In addition, it is difficult to deter-mine whether temperature exerts a direct ef-fect on the surfactant system because theremay be a coincidental marked change inbreathing pattern (Frappell and Daniels1991a, b; Crafter et al, 1995) and auto-nomic output (Wood et al., 1995, 1997).Hence, the changes in surfactant composi-tion and lavageable amount we observed inC. nuchalis, P. vitticeps and S. crassicau-data may be induced either by temperaturedirectly or via alterations in breathing pat-tern, which influence the mechanical stim-uli for surfactant release from type II cells,or by both mechanisms.

However, using the isolated perfused liz-ard lung we recently demonstrated that tem-perature exerts an effect on the lizard sur-factant system but changing the ventilatorypattern does not influence the surfactantsystem in any discemable manner (Wood etal., 1995). The mechanism of the tempera-ture change is unknown, although it mayact via altering the metabolic rate of thetype II cells and hence influence the rate ofsynthesis and/or exocytosis of the lamellarbodies (Daniels et al, 19956; Wood et al,1995). That the surfactant system is not in-fluenced by altering the ventilation patterncontrasts profoundly with the mammaliansituation. Our observations may be a resultof differences in alveolar architecture be-tween lizards and mammals, the mechanismof alveolar inflation, or the very low fre-

quency with which lizards breathe (Woodet al, 1995). The torsion properties of liz-ard alveolar type II cells also may be dif-ferent from those of mammals, but isolatedcell culture experiments are required to testthis hypothesis.

We have suggested that in lizards, thecontrol of surfactant composition and se-cretion primarily involves the interactionbetween temperature and the autonomicnervous system (Daniels et al, 19956;Wood et al, 1995, 1997). As body temper-ature decreases, lung volumes in the lizardare reduced and contact between epithelialsurfaces within the lung is increased. Whilethe presence of surfactant is necessary inthis situation to prevent adhesion of sur-faces, stimulating surfactant secretion viaan increase in circulating catecholamines isinappropriate for a cold lizard with a re-quirement to conserve energy because cat-echolamines can increase metabolic rate.We suggest that the release of surfactant viathe cholinergic stimulation of type II pneu-mocytes may become increasingly impor-tant in this situation (Wood et al., 1995;1997). As temperature increases, type II cellmetabolic rate and increased release ofadrenaline and noradrenaline from pulmo-nary nerves and from the adrenal glandsboth promote surfactant release (Wood etal, 1995, 1997). The control mechanismfor the compositional changes in surfactantinduced by changes in temperature remainunknown.

CONCLUSION

Lung structure and function varies wide-ly among the vertebrates. However, despitetheir morphological diversity, all lungs areessentially internal, fluid-lined structureswhich cycle air by changing volume. Inmammals, pulmonary surfactant lowers thesurface tension of the fluid lining, particularlyat very low lung volumes. This action isdue primarily to a disaturated phospholipid,dipalmitoylphosphatidylcholine (DPPC).Cholesterol and unsaturated phospholipidspromote the respreading of the surfactantupon lung inflation by converting DPPC tothe disordered, liquid-crystalline state. Ourwork on the surfactant proteins of noneuth-erian mammals, birds and ectotherms has

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318 C. B. DANIELS ETAL

demonstrated that all vertebrates probablyhave a homologous surfactant system, pres-ent in the stem ancestor of this phylum over400 million years ago. Surfactant compo-sition, however, ranges from a very highcholesterol/very low DSP mixture in theprimitive airbreathing fish, to intermediatecholesterol/intermediate DSP in the derivedlungfish and the amphibians, to low choles-terol/high DSP in the reptiles and mam-mals. We suggest that the cholesterol-en-riched surfactant may represent the primi-tive surfactant, or "protosurfactant" and theappearance of a pulmonary surfactant sys-tem was necessary for the evolution of air-breathing. Surfactant function also appearsto have evolved from the primitive role,which may have been to act as an antiglue,to the highly derived function of providingalveolar stability in the broncho-alveolarlung of mammals. Surfactant retains itsmalleable form within individuals by alter-ing its composition in response to changesin temperature, lung structure and maturity.Temperature and/or the Autonomic Ner-vous System controls the release of surfac-tant in the lungs of noneutherian verte-brates.

ACKNOWLEDGMENTS

This work has been primarily supportedby an Australian Research Council (ARC)grant to CBD and an ARC Fellowship toSO. We acknowledge the support and co-operation of the South Australian NationalParks and Wildlife Service.

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