Daniels, Orgeig - 2003 - Pulmonary Surfactant the Key to the Evolution of Air Breathing

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(Fig. 1A). The DPPC molecules can be compressed tightlytogether by virtue of their two fully saturated fatty acid chains.In so doing, they exclude water molecules from the air-liquidinterface, thereby eliminating surface tension (19).

Lipids can exist in either a fluid liquid-crystalline state or ina solid gel state. The transition between these two phasesoccurs at the phase transition temperature (Tm) of that lipid.For the surfactant lipids to spread over the alveolar surfaceupon inspiration, the surfactant film must exist in the liquid-crystalline state. Because DPPC has a Tm of 41C, a pureDPPC film will exist in the gel form at mammalian body tem-

peratures and hence adsorb extremely slowly to the air-liquidinterface (19). The addition of other lipids, e.g., Chol or USP,

into the surface film upon inspiration lowers the Tm of the lipidmixture, enabling it to exist in the fluid state at the same bodytemperature. In this state, the lipids are able to disperse to coathe surface of the expanding fluid layer.

Temperature therefore has a profound influence on thestructure and function of surfactant lipids. Given that themajority of animals have much lower body temperatures thanhomeothermic mammals, how can animals regulate the fluidity of their surfactant film at low and/or fluctuating body tem-peratures? Furthermore, the low metabolic rates of nonmammals also have profound implications for the rates of synthesis

of new components. Hence, for example, the need for addi-tional quantities of USP in surfactant requires PL synthesis

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FIGURE 1.A: schematic diagram of the life cycle of pulmonary surfactant (moving from bottom to top). Pulmonary surfactant components are synthesized inthe endoplasmic reticulum (ER), transported to the Golgi aparatus, and packaged into lamellar bodies (LB). LB are secreted into the liquid lining the alveoli(hypophase) via exocytosis across the type II cell plasma membrane. Here the lamellar bodies swell and unravel, forming a cross-hatched structure termed tubular myelin (TM), which consists of lipids and proteins. This structure supplies lipids to the surface film as well as the surface-associated phase (SAP). As the mixedmolecular film is compressed, lipids are squeezed out of the film into the SAP to produce a dipalmitoylphosphatidylcholine (DPPC)-enriched film that is capa-ble of reducing surface tension (ST) to near 0 mN/m. It is possible that some lipids from the SAP reenter the surface film (dashed arrow). Lipids from the surfacefilm and the SAP are eventually recycled and taken back up by the type II cell via endocytosis. The role of some of the surfactant proteins (SP-A, -B, and -C) inregulating these processes is indicated with (stimulation) or (inhibition). Reproduced with permission from Elsevier from Ref. 17. B: structure of the lungepithelium of the lizard Ctenophorus nuchalis, demonstrating the location of type II cells (t) in between the capillaries (c), which bulge into the airspace. Thecapillaries contain macrophages (m) and erythrocytes (r). Surfactant material (s) has been extruded into the airspace. C: electron micrograph demonstrating thesurfactant structures: lamellar bodies (lb) and tubular myelin (tm) in the airspace of a lizard lung. B and Care reprinted with permission from Ref. 14.


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which may take some time. Hence USPs do not appear to beappropriate for controlling fluidity of surfactant in the face ofthe very rapid or step changes in body temperature, whichcommonly occur in most nonmammals and in torpid or hiber-nating mammals.

Chol is able to affect the fluidity of PLs directly. Chol isthought to increase the separation between PL molecules, thusdisrupting the intermolecular interactions between their head

groups and allowing greater rotational movement. At 10% byweight, or 20 mol%, Chol is the second most abundant lipidcomponent of pulmonary surfactant. Although the source ofsurfactant Chol is not known, the ready availability of Chol inthe alveolar compartment and in the circulating plasma makesthis molecule a good candidate for the rapid regulation of sur-factant fluidity (16). Hence, titrating the Chol/DSP ratio in theface of rapid body temperature changes is an excellentmethod for maintaining fluidity and a functional surfactant atlow body temperatures.

The protein component of pulmonary surfactant represents~10% by weight, and four surfactant proteins have been

described. These are SP-A, SP-B, SP-C, and SP-D, which aresynthesized in alveolar type II cells and are all associated withpurified surfactant (11). Both the secretion and the reuptake ofsurfactant PLs into type II cells appear to be regulated by SP-A. Both SP-A and SP-B are essential for the formation andstructural integrity of surfactant components (Fig. 1A). Thehydrophobic surfactant proteins, SP-B and SP-C, stronglyinteract with the lipids and promote the formation and adsorp-tion of the surface film to the air-liquid interface. However, thehydrophilic surfactant proteins, SP-A and SP-D, are predomi-nantly involved in the innate host-defense system of the lung(11).

Using surfactant protein analysis, we determined that sur-factant had a single evolutionary origin that predated the evo-lution of the vertebrates. We demonstrated that an SP-A-likeprotein is present in surfactant from all vertebrate classes,including in goldfish swimbladders (20) (Fig. 2A). Further-more, the ultrastructure of the surfactant system is highly con-served. Lamellar bodies and tubular myelin-like structureshave been observed in the lungs of reptiles (Fig. 1), birds, andamphibians. Lamellar bodies have been observed in the threeextant species of lungfish, in the lungs of primitive air-breath-ing fish, and in the swimbladder of the rainbow trout. Thussurfactant from nonmammalian vertebrates appears to be pro-

duced, stored, and released in a similar manner to mam-malian surfactant. Moreover, the system predates the evolu-tion of lungs (reviewed in Ref. 4).

Lungs developed as out-pouchings of the gut, and the pri-mary selection pressure for the evolution of lungs was proba-bly aquatic hypoxia (18). The ancestral bony vertebrate wasmost likely lunged, inhabiting warm stagnant pools and gulp-ing air to gain sufficient oxygen. The cells that produce sur-factant, and contain SP-A, have been located in the gut ofmammals (9). In the gut, surfactant may be important in con-trolling fluid-fluid interactions between liquids of different vis-cosities (e.g., the mucus and serous fluid layers). Alternatively,

or perhaps additionally, gut surfactant may be involved ininnate immunity (11). Hence it makes sense to propose that

the surfactant-secreting cells migrated with the air-filled out-pouchings of the gut before the lung surfactant took on its cur-rent surface tension-controlling functions. Hence the surfac-tant system predated the evolution of lungs and was crucial forthe evolution of air breathing.

Surfactant and the evolution of air breathing

Here we examine the evolution of the surfactant system inassociation with three of the major evolutionary steps for thevertebrates: 1) the separation of the Actinopterygiian (bony)fish from the Sarcopterygiia (lungfish) and the Tetrapods (land-dwelling vertebrates); 2) the Land-Water Transition; and 3)changes in body temperature, particularly the general patternof increasing from cold ectotherms to warm heliotherms andendotherms. An evolutionary analysis of surfactant composi-tion of the vertebrates ranging from air-breathing fish, teleosts,and lungfish to amphibians, reptiles, birds, and mammals hasrevealed that fish lungs and the lungs of the primitive Aus-tralian lungfish, Neoceratodus forsteri, have surfactant with

approximately threefold greater amounts of Chol relative to PLthan any of the other vertebrate groups (5, 15) (Fig. 2B). How-ever, DSP as a percentage of PL demonstrates the oppositetrend, with only the mammals and some reptiles having highlevels of 4050% (5, 6) (Fig. 2C). Teleost swimbladders andthe lungs of the air-breathing fish and N. forstericontain PLsthat are two- to fourfold less saturated than those of thederived dipnoans, the African and South American lungfish,and the majority of the amphibians and fivefold less thanthose of reptiles and mammals (5, 6). These opposite trends inChol/PL and DSP/PL ratios result in a very dramatic pattern forthe Chol/DSP ratio (5, 16). The fish and N. forsteriwith their

relatively simple baglike lungs have a Chol/DSP ratio up to anorder of magnitude greater than the reptiles and mammals.The amphibians and the derived Dipnoans have intermediatelevels of Chol relative to DSP, i.e., the ratio is approximatelydouble that of the reptiles and mammals (6, 16).

Differences among terrestrial groups in the composition ofsurfactant probably reflect the temperature-dependent fluidityof surfactant PL. Because DPPC undergoes a phase transition

from a gel to a liquid-crystalline state at 41C, and realisticallyonly homeotherms and the most heliothermic of the reptilesare likely to have body temperatures that approach this value,it follows that only these groups are likely to be capable of tol-erating high DSP/PL ratios (5) (Fig. 2C). In contrast, advancedDipnoans and amphibians generally have much lower bodytemperatures and have incorporated less DSP in their surfac-tant, with DSP/PL ratios of only 1530% (5). Chol is not as

effective as DSP at reducing surface tension but is able tolower the normal Tm of DSP, thereby maintaining the mixture

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...surfactant had a single evolutionary origin

that predated the evolution

of the vertebrates.


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in a fluid, easily adsorbable state over a much broader rangeof temperatures (reviewed in Ref. 16). Similarly, USP have amuch lower Tm than their saturated counterparts. The surfac-tant of fish and amphibians, having greater Chol/DSP ratios(20130%) compared with those of the warmer reptiles andmammals (1015%), appear to benefit from this increased flu-idity (5, 6). Bats have the lowest amount of surfactant Cholever recorded. The two species we examined had 6 and 15times less surfactant Chol than all other mammals studied todate (1) (Fig. 2B). The extraordinarily complex structure of batlungs, with the tiny alveoli and high ventilatory capacity, cor-relates with the most sophisticated surfactant so far recorded.

It also appears that the combination of extremely high sur-factant Chol and very low DSP may be a universal function of

simple, largely avascular lungs, which are either not used oronly infrequently used for respiratory purposes (16). This issupported by the fact that the smooth baglike or saccular lungused for gas storage in the rattlesnake has a threefold greaterChol/DSP ratio than the septated faveolar lung containing therespiratory tissue (8). Furthermore, the goldfish swimbladderwhich is an internal gas-holding structure that is used pre-dominantly for buoyancy, had one of the highest Chol/DSPratios of any surfactant system (5). It is not clear why lungs andswimbladders have such high relative proportions of Chol. Isit a primitive feature, or does it represent an adaptive requirement? Possibly it is related to an increased need for spread-

ability in organs where the surfactant secretory cells are rela-tively few and far between. Alternatively, Chol may represent


FIGURE 2.A: Western blot analysis of lavage proteinprobed with a rabbit anti-human SP-A polyclonal anti-body. The antibody cross-reacted with all species

examined, with predominant cross-reactivity atmolecular weights of 5565 kDa (dimer) and less at2835 kDa (monomer) and 120 kDa (tetramer).Species are: mouse (Mus musculus), dunnart(Sminthopsis crassicaudata), crocodile (Crocodylus

porosus), sea turtle (Caretta caretta), freshwater turtle(Emydura kreffti), salamander (Amphiuma tridacty-lum), African lungfish (Protopterus annectens), Aus-tralian lungfish (Neoceratodus forsteri), bichir(Polypterus senegalensis), gar (Lepisosteus osseus), andgoldfish (Carassius auratus). Reprinted with permis-sion from Ref. 20. B and C: Cholesterol/phospholipidratio (Chol/PL; g/g; B) and disaturated phospho-lipid/phospholipid ratio (% DSP/PL; C) expressed as apercentage of total phospholipid of lavage materialobtained for a range of air-breathing vertebrates.Species are: teleost fish, the goldfish C. auratus (C.aur)(5); the air-breathing Actinopterygiian fishes P. sene-

galensis (P.sen), Calamoicthys calabaricus (C.cal), andL. osseus (L.oss) (5); the Australian and African lungfishN. forsteri(N.for) and P. annectens (P.ann) (15); thetiger salamanderAmbystoma tigrinum (A.tig) (5); theamphibians A. tridactylum (A.tri), Siren intermedia(S.int), Bufo marinus (B.mar), and Xenopus laevis(X.lae) (5); the rattlesnake Crotalus atrox (C.atr) (8); thelizard Ctenophorus nuchalis (C.nuc) (2); the chickenGallus gallus (G.gal) (12); the rat Rattus norvegicus(R.nor) (5); human (H.sap) (5); the fat-tailed dunnartSminthopsis crassicaudata (S.crass) (13); and the

microchiropteran bats Nyctophilus geoffroyi(N.geoff)and Chalinolobus gouldii (C.goul) (1). The lizard, thedunnart, and the bats were at their warm-active bodytemperature (3337C). Data are expressed as means SE; n = 49.


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a primitive antioxidant, protecting the inner lining from oxida-tive damage. However, because fish surfactant is so simple,poorly surface active, dominated by Chol, and located withinlungs only occasionally used for respiration (i.e., these speciesare facultative air breathers), we have termed the surfactant offish and the Australian lungfish the protosurfactant (16).

In addition to the large-scale evolutionary changes attribut-able to temperature as the primary selection pressure, temper-

ature is also the major acute controller for the lipid composi-tion of surfactant. The first documentation that alveolar surfac-tant Chol can change dynamically in response to a physiolog-ical change occurred in the lizard, Ctenophorus nuchalis (2).This central Australian lizard withstands variations in bodytemperature from 13 to 44C, with a mean preferred temper-ature of 36.4C (2). A step decrease in temperature from 37 to19 or 14C for 4 h increased the Chol/PL ratio from 8% at37C to 15% at 19C and 18% at 14C (2). The increase in theratio was due solely to an increase in the amount of Chol.Changes in the ratio were evident after as little as 2 h and weremaintained for up to 48 h. Hence it appears that at low body

temperatures the animals respond by selectively increasingthe level of Chol in their surfactant, presumably to maintain

fluidity (2). Similar changes in surfactant Chol occur inresponse to torpor in the fat-tailed dunnart, Sminthopsis cras-sicaudata, a desert-dwelling marsupial capable of reducing itsbody temperature to as little as 13C for an average of 8 h anda maximum of 19 h. The absolute amounts of Chol and PLincreased by up to 50% after as little as 1 h of torpor, and theChol/PL and Chol/DSP ratios increased after 8 h in torpor (13).Similarly, an increase in Chol relative to total PL and DSP

occurred in the microchiropteran bat, Chalinolobus gouldii, inresponse to a torpor bout at a body temperature of 25C.However, another microchiropteran bat, Nyctophilus geof-froyi, which has the lowest Chol level ever recorded in a mam-mal, did not demonstrate this classic pattern (1).

What is the evolutionary significance in surfactantfunction?

We have proposed that the main function of surfactant innonmammals is to act as an antiadhesive that prevents adhe-sion of adjacent epithelial surfaces at low lung volumes (5)

(Fig. 3,A and B). If the fluid lining the tissue-tissue interfacehas a high surface tension, then the work of initially separat-


FIGURE 3. Scanning electron micrograph of thefaveoli of the lizard C. nuchalis during the breathingcycle. A: lung is inflated and the faveolar walls arenot closely opposed. B: lung is deflated and oppos-

ing sheets of epithelial tissue are in close contact,particularly in the corners. o, Outer wall of lung; t,trabecular network; e, epithelial surface of faveoli.Reprinted with permission from Ref. 3. C: transmis-sion electron micrograph showing surfactant mater-ial preventing the adherence of adjacent epithelialsurfaces in the lung of C. nuchalis. Reprinted withpermission from Ref. 14. D: mechanism of airflow(thick arrows) into and out of the faveoli (f) duringbreathing in the lizard. At expiration (thin arrows) theribs move the outer wall (o) of the lung toward thetrabecular network (t), folding the faveoli like a bel-lows, decreasing volume and pushing air into thecentral air space. Upon inspiration, the outwardmovement of the ribs pulls the outer wall of the lungaway from the inner trabecular network, increasingthe volume of the faveoli. Air enters the faveoli fromthe central air space as the faveolar volumeincreases. Note that the trabeculae and the mouthof the faveoli do not change shape or location duringbreathing. Reprinted with permission from Elsevierfrom Ref. 4.


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ing the tissues will be high. As the surfaces are further sepa-rated and the fluid retreats to the corners, surface tensionshould play a smaller and smaller role in the work of breath-ing. Without an agent to lower the surface tension of the fluidintervening between the contacting epithelial surfaces (Fig.3C), inspiration after lung collapse might be impossible, or atleast extremely costly. The antiadhesive property of surfactantis therefore a biological manifestation of the ability of the

lipids to lower surface tension.Using scanning electron microscopy and computerizedtomography scanning, a model was developed for the breath-ing dynamics of the lizard, Ctenophorus nuchalis (3). Duringlung deflation, the epithelial tissues, which are strung betweenthe outer lung wall and the inner trabecular network, fold inon each other like a concertina (Fig. 3D). This causes largeportions of epithelial tissue to come into contact, a situation inwhich the antiadhesive function of surfactant may be critical.Such a function can be demonstrated in nonmammalian ver-tebrates (reptiles, Actinopterygiian fish, and salamanders) bydemonstrating an increase in opening pressure following the

removal of surfactant by rinsing with isotonic saline, a processtermed lavage (5). Opening pressure is defined as the pressurerequired to inflate a completely collapsed isolated lung (Fig. 4,

A and B). Surfactant also performed an antiadhesive functionin the goldfish swimbladder (7). Furthermore, in almost allcases, filling pressure (i.e., the pressure required to continue toinflate the lung after initial lung opening) was extremely low(14 cmH2O) and remained unchanged before and afterlavage (5). Therefore, the surfactant lipids appear to be impor-tant only during the initial phase of inflating a collapsed lungand not during further inflation.

The pattern and mode of breathing of nonmammalian ver-

tebrates also indicate that an antiadhesive function might beessential for these animals, particularly when they are verycold. The aquatic amphibians Amphiuma and Siren collapsetheir lungs completely upon expiration, as do dipnoan lung-fish and Polypterus. Complete collapse of the lung may alsooccur at end-expiration in some small frogs, and sea snakescycle air by emptying and collapsing the saccular lung(reviewed in Ref. 5). Furthermore, at low body temperatures,the lizard C. nuchalis exhibits periods of apnea during whichthe lungs collapse and the epithelial surfaces may come intocontact (5). The low metabolic rate of cold ectothermsdecreases the need for frequent ventilations, and the lungs

collapse for prolonged periods. Here surfactant is critical todecrease the work of separating the contacting epithelial sur-faces when the occasional breath is required.

Conclusion

One of the great steps in vertebrate evolution has been thedevelopment of an internal lung. However, despite the mor-phological diversity of lungs, they are all essentially internal,fluid-lined structures that cycle air by changing volume. More-over, lungs all face similar physical challenges. Organismsmust maximize the surface for gas exchange while maximiz-

ing lung compliance to minimize the work of breathing. Theymust minimize the threat of infection, prevent the accumula-

tion of irritants, and also prevent the lung from collapsing orfilling with fluid. There are some constants in the evolution oaerial breathing, particularly the presence of a surfactant system. It appears that a surfactant system evolved before theevolution of lungs in vertebrates. It also appears that there aretwo different types of surfactant: one in the Actinopterygiianfishes, which is high in Chol and USP, and one in the Sar-copterygiian fishes and tetrapods, which is relatively low in

Chol and USP and higher in DSP. The fish surfactant is highlyspreadable but not very surface active. The tetrapod surfactanis much more surface active and may have enabled the devel-opment of more complex lungs with smaller respiratory unitsand a greater total respiratory surface area, paving the way forthe occupation of land. It is possible that fish surfactant is aprotosurfactant that evolved into tetrapod surfactants buwas retained as a protective lipid lining for the gas bladders inthe modern fish and in gas-holding structures that are not usedfor respiration.

Within the tetrapods, the concentration of DSP increaseswith the evolution from cold, wet ectotherms (amphibians) to


FIGURE 4. Opening pressure of the lungs of nonmammalian vertebrates.Aschematic representation of the pressure required to open and then fill a completely collapsed lung with air infused at a constant rate (usually 1 ml/min)Note that initially the pressure increases before the lung begins to inflate, thenonce open the lung inflates with little change in pressure. B: the opening pressure before and after surfactant removal by lavage of collapsed lungs from arange of vertebrates. T.ord, Thamnophis ordinoides (garter snake). Othe

abbreviations are as for Fig. 2. Data are expressed as means SE; n = 48Reprinted with permission from Ref. 6.


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warm heliotherms and endotherms (some reptiles, birds, andmammals). It appears that the DSP/PL ratio is set by the ther-mal programming of the animal and does not represent ashort-term method for maintaining fluidity. Chol declines inimportance with the evolution of the vertebrates but retains animportant function as a rapid-response method of maintain-ing fluidity of the surfactant lipids, particularly for ectothermsor heterothermic mammals faced with major diurnal fluctua-

tions in body temperature.The majority of nonmammalian lungs are essentiallybaglike, without a bronchial tree, and with only a few excep-tions (e.g., tracheal lungs of snakes or parabronchial lungs ofbirds) regularly collapse completely. An important function ofsurfactant in these lung types is to prevent the epithelial sur-faces from adhering to each other. But nonmammalian surfac-tant does not influence inflation compliance. Because there islittle doubt that lungs of this type, and breathing patterns ofthis nature, represent the primitive form, we hypothesize thatacting as an antiadhesive may be the original function of sur-factant, but one which led naturally to the alveolar stability

and compliance roles that dominate mammalian surfactantfunction.

This work was funded by the Australian Research Council.

References

1. Codd JR, Daniels CB, and Orgeig S. Thermal cycling of the pulmonarysurfactant system in small heterothermic mammals. In: Life in the Cold.Eleventh International Hibernation Symposium, edited by Heldmeier Gand Klingenspor M. Berlin: Springer-Verlag, 2000, p. 187197.

2. Daniels CB, Barr HA, Power JHT, and Nicholas TE. Body temperaturealters the lipid composition of pulmonary surfactant in the lizardCtenophorus nuchalis. Exp Lung Res 16: 435449, 1990.

3. Daniels CB, McGregor LK, and Nicholas TE. The dragons breath: a modelfor the dynamics of breathing and faveolar ventilation in agamid lizards.Herpetologica 50: 251261, 1994.

4. Daniels CB and Orgeig S. The comparative biology of pulmonary surfac-tant: past, present and future. Comp Biochem Physiol A 129: 936, 2001.

5. Daniels CB, Orgeig S, and Smits AW. Invited Perspective: The evolution of

the vertebrate pulmonary surfactant system. Physiol Zool 68: 539566,1995.

6. Daniels CB, Orgeig S, Wood PG, Sullivan LC, Lopatko OV, and Smits AW.The changing state of surfactant lipids: new insights from ancient animals.

Am Zool 38: 305320, 1998.7. Daniels CB and Skinner CH. The composition and function of surface

active lipids in the goldfish swim bladder. Physiol Zool 67: 12301256,1994.

8. Daniels CB, Smits AW, and Orgeig S. Pulmonary surfactant lipids in thefaveolar and saccular lung regions of snakes. Physiol Zool 68: 812830,

1995.9. Engle MJ and Alpers DH. Surfactant-like particles mediate tissue-specific

functions in epithelial cells. Comp Biochem Physiol A 129: 163171,2001.

10. Goerke J. Pulmonary surfactant: functions and molecular composition.Biochim Biophys Acta 1408: 7989, 1998.

11. Haagsman HP and Diemel RV. Surfactant-associated proteins: functionsand structural variation. Comp Biochem Physiol A 129: 91108, 2001.

12. Johnston SD, Orgeig S, Lopatko OV, and Daniels CB. Development of thepulmonary surfactant system in two oviparous vertebrates.Am J PhysiolRegul Integr Comp Physiol 278: R486R493, 2000.

13. Langman C, Orgeig S, and Daniels CB. Alterations in composition andfunction of surfactant associated with torpor in Sminthopsis crassicau-data.Am J Physiol Regul Integr Comp Physiol 271: R437R445, 1996.

14. McGregor LK, Daniels CB, and Nicholas TE. Lung ultrastructure and thesurfactant-like system of the central netted dragon, Ctenophorus nuchalis.Copeia 1993: 326333, 1993.

15. Orgeig S and Daniels CB. The evolutionary significance of pulmonary sur-factant in lungfish (Dipnoi).Am J Respir Cell Mol Biol 13: 161166, 1995.

16. Orgeig S and Daniels CB. The roles of cholesterol in pulmonary surfac-tant: insights from comparative and evolutionary studies. Comp BiochemPhysiol A 129: 7589, 2001.

17. Orgeig S, Daniels CB, and Sullivan LC. The development of the pul-monary surfactant system. In: The Lung: Development, Aging and theEnvironment, edited by Harding R, Pinkerton K, and Plopper C. London:Academic, 2003.

18. Perry SF. Structure and function of the reptilian respiratory system. In:Comparative Pulmonary Physiology.Current Concepts (1st ed.), edited byWood SC. New York: Marcel Dekker, 1989, p. 193236.

19. Possmayer F. Physicochemical aspects of pulmonary surfactant. In: Fetaland Neonatal Physiology, edited by Polin RA and Fox WW. Philadelphia:W. B. Saunders, 1997, p. 12591275.

20. Sullivan LC, Daniels CB, Phillips ID, Orgeig S, and Whitsett JA. Conser-vation of surfactant protein A: evidence for a single origin for vertebratepulmonary surfactant.J Mol Evol 46: 131138, 1998.


Daniels, Orgeig - 2003 - Pulmonary Surfactant the Key to the Evolution of Air Breathing

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