Respiration Physiology, 95 (1994) 249-258 1994 Elsevier Science B.V. All rights reserved. 0034-5687/94/$07.00 SSDI: 0034-5687(93)E0096-4

RESP 02105

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Pulmonary-type surfactants in the lungs of terrestrial and aquatic amphibians

Christopher B. Daniels, Sandra Orgeig, J. Wilsen and Terence E. Nicholas Department of Human Physiology, School of Medicine, Flinders University, Bedford Park, Adelaide, South

Australia 5042, Australia

(Accepted 4 October 1993)

Abstract. We examined the composition and function of pulmonary surfactants in amphibians inhabiting aquatic and terrestrial habitats with particular regard to the influences of( l) variations in body temperature, (2) external hydrostatic pressure and (3) breathing pattern. Two fully aquatic salamanders, and the com- pletely terrestrial cane toad Bufo marinus (all maintained at 21-23 C) were selected. Whereas one of the salamanders (Siren intermedia) possessed gills and lungs, Amphiuma tridactylum only possessed lungs. We determined the amounts of cholesterol (Chol), disaturated phospholipids (DSP) and total phospholipid (PL) in lavage of all three species, and also determined the types of phospholipids orB. marinus and A. tridactylum. DSP lowers surface tension at the air-water interface in the lung, while Chol and unsaturated phospho- lipids assist spreading and maintain the DSP in its disordered, liquid-crystalline state at high lung volumes. All three species had significant amounts of pulmonary-type surfactant. The two aquatic salamanders had identical ratios of both Chol/PL and D SP/PL both of which in turn were nearly twice those of B. marinus. All three species had similar Chol/DSP ratios. Aquatic salamanders sustain high external hydrostatic pressures exerted by the aquatic environment and tend to collapse their lungs during expiration. We hypothesize that these salamanders might require a DSP-rich surfactant to prevent the epithelial surfaces from adhering and large amounts of Chol to keep the DSP fluid. The terrestrial B. marinus has less DSP, suggesting a surfaetant which is fluid over a large range of temperatures. Possibly, cane toads do not require a DSP rich surfactant as they neither collapse their lungs on deflation, nor experience external hydrostatic pressures promoting lung collapse. The PL profile orB. magnus lavage was similar to that of other frogs and mammals, containing phosphatidylcholine (PC) as the predominant phospholipid together with substantial amounts of phosphatidylglycerol (PG). On the other hand, although A. tridactylum exhibited high levels of

PC, it contained phosphatidylinositol (PI) in place of PG, a pattern typical of reptiles and birds.

Amphibians, salamanders (Siren intermedia, Amphiuma tridactylum), toad (Bufo marinus); Pressure, hydro- static; Surfactant, composition, temperature, pressure; Temperature

Pulmonary surfactant is a complex mixture of phospholipids, neutral lipids and pro- teins which lines the inner surface of the lung. In all the vertebrates so far examined, these lipids apparently act to lower the surface tension of the thin fluid layer located between the tissue and air, thereby reducing the work of inspiration and the risks of both actelectasis on expiration and alveolar oedema (Farrell, 1982). In mammals, the

Correspondence to." Christopher B. Daniels, Department of Human Physiology, School of Medicine, Flinders University, Bedford Park, Adelaide, South Australia 5042, Australia Tel. (618)2044100; Fax (618)204 5768.

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active component of pulmonary surfactant is a disaturated phospholipid (DSP) which by virtue of its highly hydrophobic, saturated acyl tails, can dramatically reduce sur- face tension by packing extremely closely together as a monomolecular layer (Farrell, 1982). However, D S P exhibit relatively high phase transition temperatures and only the addition of either cholesterol or unsaturated phospholipids, or both, allow them to alternate between the gel and liquid-crystalline states during breathing (Farrell, 1982; Hadley, 1985). Virtually all our knowledge of the inter-relationships between the chemi- cal components of pulmonary surfactants is derived from mammalian studies (Farrell, 1982). However, it is established that reptiles respond to either acute or chronic changes in body temperature by altering either the amount of cholesterol or the degree of unsaturation of the phospholipids (Daniels et al., 1990; Lau and Keough, 1981).

The presence of surfactant in the lungs of amphibians is well documented. In gen- eral, amphibian surfactant appears to be secreted as lamellated osmiophilic bodies (LOB's) from specific secretory pneumocytes into the airspaces (Bell and Stark-Vancs, 1983; Goniakowska-Witalinska, 1978, 1980; Pattle et al., 1977; Stark-Vancs et al., 1984). Amphibian pulmonary-type surfactant may (or may not) be present as the tubular myelin structural stage in the air space (Bell and Stark-Vancs, 1983; Goniakowska-Witalinska, 1978, 1980). The surfactant is composed primarily of phos- phatidylcholine (PC) (Baxter et al., 1969; Hallman and Gluck, 1976; Vergara and Hughes, 1981), is present in comparatively large amounts (Clements et al., 1970), and is not very surface active (Pattle and Hopkinson, 1963; Pattle et al., 1977). However, the function of amphibian pulmonary surfactant is unknown. Possibly it assists in maintaining lung patency in aquatic amphibians breathing underwater and exposed to significant hydrostatic pressure from the surrounding medium. Finally, how surfactant systems cope with the frequently low and variable body temperatures observed during the activity period of these animals also remains contentious.

In this study we examined the composition and function of pulmonary surfactants in amphibians inhabiting aquatic and terrestrial habitats with particular regard to the influences of (1) variations in body temperature, (2) external hydrostatic pressure and (3) breathing pattern. The two species of salamanders were selected because they have the most well developed lung of the fully aquatic amphibians (Bell and Stark-Vancs, 1983; Stark-Vancs et al., 1984). Moreover, one species, the siren (Siren intermedia) also has small but functional gills and is capable of varying the gill and lung contributions to overall gas exchange. Amphiuma tridactylum does not possess gills. Both species inhabit warm stagnant marshes and still, weed-filled ponds. The body temperatures of these two salamanders are usually around 20-24 C during their activity period (Brattstrom, 1963; Ultsch, 1973). A. tridactylum inhabits channels and rivers as well as ponds, while S. intermedia may inhabit temporary ponds and eastivate through the summer months when the pond dries up (Ultsch, 1973). Finally, we have compared the composition and function of their pulmonary-type surfactant with that of the tropical terrestrial anuran, the cane toad (Bufo marinus). Although the body tempera- ture of the cane toad may undergo a greater daily variation than the aquatic species, it also prefers body temperatures between 20-24 C (Brattstrom, 1963).

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Materials and methods

Animals. Amphiuma tridactylum (mass range: 89.9-822.3g; mean_+SE: 317.5__+ 133.0 g; n = 6) and Siren intermedia (mass range: 37.1-62.8 g; mean _+ SE: 47.8 _+ 4.2 g; n = 5) were purchased from commercial suppliers (Amphibians of North America; Nashville TN, USA), held in the lab in water at 23 C and fed diced catfish flesh twice per week. Bufo marinus (mass range 56.7-70.4 g: mean + SE: 62.7_+ 2.55 g; n= 8) were purchased from Australian commercial suppliers and maintained at an air tem- perature of 22 C on moist vermiculite and fed catfood. A. tridactylum and S. inter- media were killed by immersion in a solution of 0 .53 MS222 (pH 7.40) while B. marinus were injected intraperitoneally with 0.2 ml/kgBW (body weight) of pento- barbitone sodium.

Lavage procedure. Salamander lungs were lavaged with three volumes (0.1 ml/gBW) of ice-cold 0.15 M NaC1. Each volume was flushed through the lung three times. This procedure recovered 75-80 3 of the phospholipids recoverable in a serial lavage re- peated six times. The highly septated, compartmentalized nature of the B. marinus lung, coupled with the extremely delicate nature of the pharynx, required a different lavage procedure. The lung was filled with ice cold saline, then gently massaged before the saline was released through a small incision in the base. Although the saline contained lung lipids with little blood contamination, this method was not as efficient as the la- vage procedures applied to the salamanders: Thus, for comparative purposes, only the lipid ratios are presented for this species.

Biochemical analysis. The lavage fluid was centrifuged at 150 g for 5 rain to remove cell debris and then lyophilized. Lipids were extracted with chloroform:methanol (2:1) (Bligh and Dyer, 1959). Phosphorous content was measured by the method of Bartlett (1959), and total phospholipid calculated by multiplying the phosphorous content by 25. Disaturated phospholipids were complexed with osmium tetroxide on aluminium oxide columns, eluted separately from the neutral lipids, and a total phosphorous analysis performed on the eluate (Mason et aL, 1976). Cholesterol was quantified by gas chromatography using a flame ionisation detector and cholesteryl isopropyl ether as the internal standard (Whiting et aL, 1981).

Thin layer chromatography. Bufo marinus and Amphiuma tridactylum lung lavage phos- pholipids were separated by the method of Touchstone et al. (1980) on Whatman LK5 thin layer chromatography (TLC) plates. Samples containing 1.5 #g of phosphorus were placed on a TLC plate and developed. The !ipids were charred by dipping in a solution containing 10~o cupric sulphate 8~ phosphoric acid, heating first at 120 C for 5 rain and then at 185 C for 3 rain. The chromatograms were scanned using a Quick Scan Jr. Densitometer (Helena Laboratories, Beaumont Texas, USA). Start-

. ' L dards were included on each plate.

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Results

Lung structure. The two aquatic salamanders, Amphiuma tridactylum and Siren inter- media, both possessed a two chambered, tubular lung which stretched along the entire length of the body. Whereas the left lung was approximately 70 ~o of the length of the right in A. tridactylum, they were both of equal size in S. intermedia. There were no apparent histological differences between the two lungs. Complete but fine rings of supporting tissue were located at regular intervals along the length of each tubular lung. The lungs were comparatively uncompliant; they were well vascularized, pale to dark red in colour, septated and possessed a large respiratory surface area. In A. tridactylum lung mass represented 0.83 + 0.09~o of body mass (wet) (n = 6) while S. intermedia lungs comprised 0.56 +_ 0.05~ of body mass (n = 5). Bufo marinus had a two cham- bered, highly septated lung, which was richly vascularized and very compliant. The lung was relatively densely packed with respiratory tissue and was multi-c0mpartmental. Unlike the aquatic salamander lungs, the B. marinus lung lacked a central air space.

Phospholipid composition of amphibian lavage. When expressed as a percentage of the total phospholipid in the lavage material, phosphatidylcholine (PC) was the dominant phospholipid in both B. marinus and A. tridactylum (Table 1). Consistent but small amounts ofphosphatidylethanolamine (PE), and a combination ofphosphatidylinositol (PI) and phosphatidylserine (PS) were found in both the salamander and the anuran (Table i). Whereas B. marinus lavage material had a phospholipid profile similar to that of mammals, with substantial amounts of phosphatidylglycerol (PG), this phospholipid was absent in A. tridactylum. The presence of PG in B. marinus was confirmed by phosphorous analysis of the PG band on the TLC plate. Sphingomyelin (S) was also present in both species but only in trace amounts despite being a dominant blood phospholipid. This established that the surfactant phospholipids were not contami- nated by blood (Table 1).

TABLE 1

The phospholipid profile from Bufo marinus and A. tridactylum lung lavage material

PC (%) PE (%) PG (%) PS/PI (%) S (%)

Bufo marinus Mean 62.63 8.97 13.10 8.57 6.73 SE 4.48 1.53 1.14 1.99 0.67

Amphiuma tridactylum Mean 77.50 1.17 0 17.48 3,90 SE 1.93 1.16 0 2,79 0.12

Data are expressed as % of total phospholipids. PC = phosphatidylcholine; PE = phosphatidylethanolamine; PG = phosphatidylglycerol; PS = phosphatidylserine; PI = phosphatidylinositol; S = Sphingomyelin. PS and PI run together and cannot be distinguished from each other using one-dimensional thin layer chromatog- raphy. N= 3 for both species.

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TABLE 2

The composition of pulmonary-type surfactants extracted from the lungs of aquatic and terrestrial amphibians

Lipid B. marinus S. intermedia A. tridactylum

PL (/~g/gWL) - 227.2 + 33.0 (5) 273.2 + 72.8 (6) DSP (~g/gWL) - 60.2 + 9,2 (4) 75.3 _+ 25.2 (6) Chol (#g/gWL) - 16.8 + 2.2 (5) 23.0 + 9.0 (6) Chol/PL (%) 3,5 + 0.5 (4) 7.8 + 1.1 (5) 7.6 _+ 0.8 (6) DSP/PL (~o) 13.2_+2.2(3) 27.7_+3.8 (4) 25.8_+2.1 (6) Chol/DSP (%) 29.7 _+ 7.6 (8) 28.5 _+ 5.2 (4) 29.8 3.1 (6)

Data are expressed as mean + SE. Number of animals in parentheses, gWL = gram wet lung, PL = phospholipid, DSP = disaturated phospholipid, Chol = cholesterol.

Cholesterol and DSP. The two aquatic salamanders contained similar amounts of phospholipid/gram wet lung (gWL) (Table 2). Both species also contained similar amounts of cholesterol and DSP/gWL (Table 2). As it was not possible to lavage the B. marinus lung as rigorously, the absolute amounts of lipid harvested are not pre- sented. However, the relationship between the lipids differed greatly between the aquatic and the terrestrial species. The surfactant of both aquatic species contained a very high DSP/PL ratio, which was double that of B. marinus (Table 2). However, the surfac- rant of S. intermedia and A. tridactylum contained twice the cholesterol (as a fraction of PL) than did that of B. marinus (Table 2). All three species had a very similar Chol/DSP ratio.

Discussion

Phospholipidsfrom amphibian lavage. The lavage phospholipid profiles of the amphib- ians A. tridaetylum and B. marinus were similar to those observed for other vertebrates, in that PC was the predominant phospholipid. In terrestrial vertebrate surfactants, particularly reptiles and mammals, PC comprises over 70 ~o of the total phospholipid, and it is the disaturated PC which is responsible for lowering surface tension. PE represents only a smaU proportion of lavagable phospholipid in the cane toad, and is less than 6~o of PL in other frogs (Hallman and Gluck, 1976; Vergara and Hughes, 1981), but it is a dominant lipid in cell membranes, and represents a major phospho- lipid in whole lung homogenates (Baxter et al., 1969). Although PE is a characteristic trace lipid in mammalian surfactant, it is absent in lizard lavage (Daniels et al., 1989). Toad lung lavage phospholipids contained significant amounts of PG which is thought to be important in the optimal functioning of mammalian surfactant. Our B. marinus data closely match previous anuran PG determinations which range between 9-11 ~o of PL (Hallman and Gluck, 1976; Vergara and Hughes, 1981). However, PG is absent in lizard, snake, turtle, salamander and bird lavage material (Daniels etal., 1989;

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Farrell, 1982; Lau and Keough, 1981). In fact, PG has only ever been observed in the lavage of anurans, one snake (Daniels and Orgeig, unpublished) and mammals. The fraction of PG in toads is similar to that in humans (Hallman and Gluck, 1976). Lau and Keough (1981) suggested that as both PG and PI have negatively charged head groups and are formed by the same synthetic pathway, they may be functionally interchangeable; In conjunction with the relatively high cholesterol content and low level of saturation, these phospholipid profiles suggest that both anuran and urodele amphibians have a lipid mixture which is relatively fluid, with a low phase transition temperature.

Cholesterol and DSP in lavage. Both the aquatic salamanders, S. intermedia and A. tridactylum, had a surfactant rich in DSP and cholesterol. DSP is the only mate- rial capable of virtually eliminating surface tension at the air-water interface under relatively high dynamic compression forces. In the laboratory tanks, the aquatic am- phibians often rest on the bottom and stretch vertically, often over 10-15 cm, to the surface to breathe. The compression forces exerted by the environment on the lung are therefore not inconsiderable, and oppose inspiration and promote lung collapse on expiration. A. tridaetylum and S. intermedia collapse their lungs almost completely on expiration (Guimond and Hutchison, 1974, 1976; Martin and Hutchison, 1979; Stark- Vancs et al., 1984), and Stark-Vancs et al. (1984) suggested that the release of surfac- tant by the pneumocytes prevents the collapsed surfaces from sticking together by reducing the surface tension of the aqueous film between them. Similarly in lizards, the surfactant may act as an 'anti-glue' at very low lung volumes preventing apposing epithelial surfaces from adhering when the faveoli pleat and fold (Daniels et al., 1989, 1990). Such a function appears critical for all species which lack a conducting airways system, a diaphragm or a sealed pleuro-peritoneal chamber where the lungs also re- ceive structural support from a rib system. Such species may regularly collapse their lungs and may also have pleats and folds within the respiratory units (McGregor et al., 1993). Moreover, whereas a surfactant acting as an anti-glue would be required to lower surface tension, it would not necessarily have to exhibit the classical ability to vary surface tension on expanding spherical surfaces (Sanderson et aL, 1976). Hence a more 'detergent-like' surfactant may be more than adequate for an anti-glue func- tion. This may explain the relatively poor surface activity displayed by amphibian surfactants when compared with that of mammals (Pattle et al., 1977). However, until definitive surface tension measurements can be performed using either a bubble sur- factometer, Wilhelmy balance or the new captive bubble technique, and an accurate measurement technique for anti-glue properties is developed, this hypothesis remains untested.

A DSP-rich surfactant may allow aquatic species to decrease the work of inflation, and also reduce the risk of permanent atelectasis. However, the high phase transition temperature of DSP and the relatively low water temperatures (23 C) could result in the saturated lipids always existing in the non-spreadable, rigid-chain, gel form. Such a state is only advantageous at very low lung volumes when very low surface tensions

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are required. At high lung volumes (e.g. during inspiration) a fluid surfactant is required to coat the unfolding and expanding respiratory surface. In the absence of dynamic compression forces, the relatively high cholesterol content (approximately 30~o of DSP and 8 ~o of total PL by mass or approximately 50 and 15 mol~o, respectively) and the presence of unsaturated phospholipids will lower the phase transition temperature of the DSP located at the air-water interface, converting it to the rapidly adsorbed, liquid-crystalline form. Cholesterol, in particular, is capable of enhancing the fluidity of a DSP mixture at temperatures below phase transition, by inhibiting the van der Waals forces between apposed acyl chains. Below the transition temperature, choles- terol molecules align so that the 3-fi-OH group favours the oxygen on the 2-acyl and thus disrupts the acyl-acyl interactions, allowing increased movement of the tail and enhancing fluidity (Hadley, 1985). Hence a surfactant lipid mixture high in both DSPs and cholesterol appears ideal for assisting in the ventilation process.

Both the phase transition of DSP and the interactions between Chol and DSP are profoundly influenced by temperature. In particular, at very low body temperatures (especially below 10 C) DSP is always in the gel state, regardless of the cholesterol content and therefore potentially compromising lung function. However, a fall in body temperature may not interfere with respiration in the two aquatic salamanders for two reasons. First, water, particularly a large body, is relatively thermally inert, requiring large or prolonged changes in air temperature to institute relatively small changes in water temperature. Moreover, large bodies of water usually establish a thermocline separating the warmer, less dense upper layer from the cooler depths. Many aquatic amphibians can control their body temperatures within precise and narrow ranges by ascending and descending within the water column or moving to shallower or deeper water (Brattstrom, 1963; Feder etal., 1982). It is therefore possible for aquatic am- phibians to exhibit body temperatures at values over 20 C during their activity peri- ods (Brattstrom, 1963). DeWitt and Friedman (1979) and Feder et al. (1982) thor- oughly documented the skewed distribution of body temperatures of aquatic salamanders towards warmer temperatures and also found most species showed mean values between 20 and 30 C. Brattstrom (1963) observed body temperatures of 24 C for Amphiuma means. Therefore, it is possible that neither S. intermedia nor A. tridactylum usually experiences large rapid or short term fluctuations in body tem- perature which could compromise lung function by interfering with the surfactant biomechanics.

Second, some species of aquatic amphibians are occasionaliy active at body tem- peratures below 10 C (Brattstrom, 1963; Feder etal., 1982). For example, Ultsch (1973) observed two species of Siren to have body temperatures between 8 and 26 C. However, sirens, like most aquatic salamanders, can also respire either via gills or transdermally. Lungs are most well developed in aquatic amphibians inhabiting warm, hypoxic and/or hypercapnic waters (Bell and Stark-Vancs, 1983; Stark-Vancs et aI., 1984). Oxygen is more soluble in cold water, while amphibian metabolic rates are also lower at lower body temperatures. It is possible that many aquatic amphibians respire primarily via the gills or through the skin at low water temperatures and do not use

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their lungs. Hence an inactive lung surfactant may not impede the gas exchange process. These amphibians may utilize the lung only at higher temperatures when the surfac- tant can enter the liquid-crystalline form.

The terrestrial cane toad Bufo marinus appears to face slightly different respiratory problems and solves them using a different mixture of the three types of lipids. The phospholipids are dominated by unsaturated phospholipids rather than DSP and there is relatively little cholesterol. We suggest two advantages for such a lipid profile. First, a surfactant low in DSP will remain fluid at very low body temperatures. Compared with many other terrestrial anurans, the cane toad appears to be relatively intolerant of cold, rarely surviving protracted periods below 7 C (Brattstrom, 1963). However, because amphibians lose water through their skins, they will act as wet bulb thermom- eters. Therefore, hydrated individuals at thermal equilibrium will assume a body tem- perature equivalent to the wet bulb temperature at a particular relative humidity (RH). Should the RH fall below 50~o, the individuals may have body temperatures at least 2 C below their environment (Orgeig, Daniels and Smits unpublished). This charac- teristic may assist cooling at high body temperatures but will be disadvantageous at low body temperatures and may also result in a large diurnal fluctuation in body tempera- ture. Daniels et al. (1990) observed that lizards greatly increase the surfactant Chol/PL ratio with a step decrease in body temperature, and hypothesized that altering the cholesterol content may be an acute and short term response to maintain fluidity of the lipids. Lau and Keough (1981) observed an increased in the unsaturation of the sur- factant phospholipids in cold hibernating turtles, suggesting a synthetic change in re- sponse to chronic exposure to cold. It is possible that a lower level of saturation in the phospholipids of the cane toad may be a response to the risk of decreases or large and protracted variations in body temperature. Unlike aquatic amphibians with gills and skin as alternative gas exchange systems, terrestrial, nonburrowing amphibians must rely primarily on their lungs for gas exchange and maintaining a fluid surfactant capable of functioning over a very broad range of temperatures may be critical for survival.

Second, a surfactant rich in unsaturated phospholipids is less capable of reducing surface tension than a DSP rich film. However, unlike Amphiurna (Martin and Hutchi- son, 1979) anurans do not collapse their lungs completely during a respiratory cycle (Guimond and Hutchison, 1974), and terrestrial species are not exposed to high en- vironmental pressures promoting lung collapse and inhibiting inspiration. Therefore, B. marinus may not require a surfactant as rich in DSP as its aquatic counterparts.

We conclude that at the same body temperature (23 C) terrestrial cane toads main- tain the same Chol/DSP relationship in their lung lining as aquatic salamanders, al- beit by exhibiting half the Chol/PL and half the DSP/PL. This confirms the very strong relationship between Chol and DSP, most likely related to body temperature. More- over, pulmonary surfactants are clearly malleable, with composition and presumably also function, varying with lung structure and function, body temperature and a range of other physiological-ecological factors. Although surfactant composition is far more species specific than previously believed, the basic physico-chemical relationships are maintained despite the plasticity of approaches.

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Acknowledgements. The research was funded by a Australian Research Council grant to C.B.D. and T.E.N., and a Flinders University Research Scholarship and Overseas Postgraduate Research Scholarship to S.O. We thank A.W. Smits for providing the facilities in Texas and advice and support throughout this and re- lated projects. We also thank J.A. Christie for technical assistance.

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