Chapter 4. The surface activity of pulmonary surfactant from diving. mammals

Transcription

1 Chapter 4 The surface activity of pulmonary surfactant from diving mammals 4.1. Introduction Surface tension (γ) arises from interactions between the molecules within a liquid. A substance, which can interfere with the interaction of the surface water molecules and therefore vary or reduce the γ is termed a surface-active molecule and its behaviour is referred to as surface activity. Pulmonary surfactant lines the air-liquid interface on the inner surface of the lung to reduce and vary γ. Physiological variables which affect γ of surfactant include changing body temperature (Lempert and Macklem, 1971; Inoue et al., 1981; Schürch et al., 1985; Langman et al., 1996; Lopatko et al., 1998; Codd et al., 2002; Codd et al., 2003) and ph (Meban, 1978; Amirkhanian and Merritt, 1995), ventilation (Veldhuizen et al., 2002), disease (Hallman et al., 1982a; Gregory et al., 1991; Kazzi et al., 2000), lung injury (Nachtman et al., 1986; Paterson et al., 1992; Putman et al., 1997; Currie et al., 1998) and development (Stevens et al., 1987; Farrell et al., 1990). Pressure is another relatively unstudied variable that may affect the surfactant system. Pressure is especially important in diving mammals, where the external pressure can reach up to 100 atmospheres (see section for the effects of pressure on the lungs and surfactant system). Currently, almost nothing is known about the function of the surfactant system in diving mammals. In particular it is likely that surfactant will have functions peculiar to diving mammals because of the extended periods of lung collapse under high external pressure. Surfactant from northern elephant seals appears to have poor surface activity, and is unable to reduce γ to the low values required during breathing (Wood et al., 2000b; Spragg et al., 2004). This poor ability to lower γ is a common characteristic of reptilian surfactants, and relates to the anti-adhesive function these surfactants exhibit when cold 168

2 reptiles collapse their lungs. Here I hypothesise that an anti-adhesive function may be an important role for pinniped surfactant. Interestingly, reptiles have very large faveoli (the functional respiratory units analogous to alveoli) and Daniels et al. (1994a; 1995b) have suggested that the very large (absolute) size of the reptile respiratory units might influence the properties and function of surfactant. Since the size of alveoli is related to body size (Siegwart et al., 1971), we compared the properties of the surfactant of diving mammals with those of the surfactant from large terrestrial mammals. Spragg et al. (2004) found that surfactant from both California sea lions and Harbour seals was able to reduce γ adequately, i.e. to similar levels as other terrestrial mammals. The reasons for the apparent differences in surfactant surface activity between pinniped species are explored here. I have investigated the functional properties of surfactant from diving mammals by using the captive bubble surfactometer (CBS). The CBS is currently the best available method for assessing the surface-active properties of surfactant (Schürch et al., 2001). There are many advantages to this technique over others (Schürch et al., 2001; Possmayer, 2004), and therefore should most accurately determine the surface activity of pulmonary surfactant isolated from diving mammals Methods Animals Lungs from one adult California sea lion (Zalophus californianus), one northern elephant seal (Mirounga angustirostris), one northern fur seal (Callorhinus ursinus), six ringed seals (Phoca hispida), five sheep (Ovis aries) and four cows (Bos taurus) were obtained as in section Approximate age and body mass of animals were obtained for all pinniped species. For California sea lion, one adult (108 kg) was available for use. The northern elephant seal was weaned (approximately 3-8 months old) and weighed 45 kg. Northern fur seal was an adult weighing 26 kg. All ringed seal samples were from adults of body mass 24.7 ± 3.1 kg. Since all animals were wild-caught, exact age could not be determined. Cow and 169

3 sheep were all under 1 year and body mass was approximately 300 kg and 40 kg, respectively Lavage protocol and surfactant extraction Surfactant was removed from the lungs and purified following the methods of section Purified surfactant was dissolved in a solution containing 154 mm NaCl and 1.5 mm CaCl 2, aliquoted into smaller volumes and either frozen or lyophilised for shipping. One aliquot was used to extract total lipids in chloroform: methanol in a ratio of 1:2 (Bligh and Dyer, 1959) before being analysed for phospholipid (PL) or cholesterol composition (chapter 3) Captive bubble surfactometry Surface activity was measured using a CBS, according to the method of Codd et al. (2002). The frozen pellet samples were freeze-dried and shipped to Prof. Samuel Schürch and co-workers at the University of Calgary in Alberta, Canada to perform the analyses. Upon arrival, the samples were reconstituted in Millipore water and centrifuged at 30,000 g for 30 min at 4 C to obtain a pellet containing large aggregate material. The supernatant was then removed and the pellet reconstituted in 3 µl of Millipore water, which was then injected into the sample chamber, close to the agarose ceiling. The sample chamber was filled with a buffered salt solution (10 mm HEPES, 2 mm CaCl2, ph 6.9) containing 10% sucrose. The sucrose was added to increase the density of the salt solution above that of surfactant, so that the surfactant will remain close to the agarose ceiling. A bubble of approximately 3 mm in diameter was then injected into the chamber Bubble measurements Adsorption Once the bubble had come to rest at the ceiling, adsorption of surfactant to the airliquid interface was measured for 5 min. The bubble was then rapidly (within 0.05 s) 170

4 expanded to its maximum size of approximately 8 mm, and again adsorption was measured for up to 5 min after the expansion of the bubble. Throughout the experiment, the bubble was recorded continuously on Hi-8 video using a Sony EV-S5000 NTSC video recorder and Pulnix TM CN camera. Bubble volume, area and γ were calculated from digitised bubble images using height and diameter (Schoel et al., 1994) Quasi-static and dynamic compression After adsorption had occurred, four quasi-static compressions (QS) were performed as described by Schürch et al. (1992). Briefly, after equilibrium surface tension (γeq) was reached (approximately mn/m), the first quasi-static cycle was performed by slowly increasing the pressure within the chamber until the first bubble click. This took approximately 1-2 min. The bubble was then slowly expanded back to the original bubble size in approximately 1-2 min. This was repeated for four cycles, with a 5 min delay in between each cycle. After a 5 min waiting period at completion of the quasi-static cycles, 20 consecutive dynamic cycles (D) were performed at 20 cycles per minute Compressibility Film compressibility curves were produced by plotting γ against the area of the bubble during a dynamic cycle. This varied between samples, but was generally found within QS3 to D9. The curves were then fitted with a 4 th degree polynomial curve. Compressibility was then calculated using the following formula: da C = 1 A dγ where A is the film area at a particular γ and da/dγ is the inverse of the derivative of the curve at point (A, γ) (Lee et al., 2004). Compressibility was calculated at 25 and 15 mn/m. In some cases, γ did not reach 25 or 15 mn/m, and so compressibility was measured at maximum and minimum surface tension (γ max and γ min ). 171

5 Statistical analysis Single mean comparisons were made using unpaired t-tests assuming equal variances. Multiple mean comparisons were made using a one-way analysis of variance (ANOVA) followed by a Bonferroni multiple comparisons test. Significance was set at p<0.05, and all data are presented as mean ± S.E. where applicable Results Adsorption γ eq obtained five minutes following expansion of the bubble is shown in Fig Samples from cow and sheep were able to reach γ values expected of a functional surfactant, as indicated by the dotted line (21.65 ± 0.29 and ± 1.48 mn/m, respectively). Samples from ringed seal, northern elephant seal and northern fur seal were unable to reach γ of this level, giving values of ± 1.68, 61.17, and mn/m respectively. The adult California sea lion was able to reach the γ eq levels of the cow and sheep (23.43 mn/m). The otariid species (California sea lion and northern fur seal) obtained a much lower γ eq than those obtained by either phocid species (ringed seal and northern elephant seal). 172

6 70 Surface tension (mn/m) Cow Sheep Ringed seal Northern elephant seal California sea lion Northern fur seal Fig Equilibrium surface tension of surfactant films after five minutes of adsorption. Dotted line indicates approximate equilibrium surface tension reached by highly surfaceactive material and pure DPPC films. indicates significantly different from sheep, and indicates significantly different from cow (p<0.05) using a one-way ANOVA with a Bonferroni post-hoc test Minimum surface tension Both sheep and cow surfactant were able to reach very low surface tensions, with values of 1.49 ± 0.41 and 1.41 ± 0.13 mn/m for γ min respectively (Fig. 4.2). γ min did not achieve values lower than 14 mn/m in any of the pinniped species studied, with ringed seal measuring ± 0.27 mn/m, the northern fur seal mn/m and the California sea lion mn/m. The northern elephant seal had the poorest surfactant, with γ min being mn/m after D

7 25 Surface tension (mn/m) Cow Sheep Ringed seal Northern elephant seal California sea lion Northern fur seal Fig The minimum surface tension of surfactant films. The minimum surface tension obtained over the entire cycling period was used, and this varied between animals, with some obtaining minimum surface tension during quasi-static cycling and others during dynamic cycling. indicates significantly different from sheep, indicates significantly different from cow (p<0.05) using a one-way ANOVA with a Bonferroni post-hoc test Film surface area reduction Fig. 4.3 shows the change in surface area when the film was compressed from γ max to γ min. Cow and sheep were both able to reduce γ dramatically, with only a minimal reduction in surface area (22.51 ± 4.69% and ± 3.58% respectively). All pinniped species needed a large decrease in surface area in order to reduce γ (ringed seal ± 0.71%, northern elephant seal 71.17%, northern fur seal 79.06%, and California sea lion 77.12%). 174

8 Area change (%) Cow Sheep Ringed seal Northern elephant seal California sea lion Northern fur seal Fig The percent area change upon compression during quasi-static or dynamic cycling. The change in area was measured during the cycle that obtained the minimum surface tension, which varied between animals. indicates significantly different from sheep, indicates significantly different from cow (p<0.05) using a one-way ANOVA with a Bonferroni post-hoc test Film compressibility Table 4.1 shows the film compressibility for all species at 25 mn/m and at 15 mn/m, as well as bovine lipid extract surfactant (BLES), which is used in surfactant therapy of premature infants and people with adult respiratory distress syndrome (Possmayer, 2004). Both cow and sheep showed compressibility values close to that of pure DPPC films (0.005 m/mn) (see also Lee et al. (2004)). Compressibility of cow surfactant did not change significantly as γ decreased, and only a minor change was seen in the sheep, although this was statistically significant (p=0009). Compressibility of the pinniped samples however, was always greater than that of the terrestrial mammal samples. There 175

9 Table 4.1. Compressibility of surfactant from different species of diving and terrestrial mammals. Species C 25 (m/mn) 1 C 15 (m/mn) 2 BLES ± ± Cow ± a ± Sheep ± a ± Ringed seal ± * ± Northern elephant seal b Northern fur seal California sea lion Compressibility was measured at 25 mn/m, or at the maximum surface tension if 25 mn/m was not reached (indicated by a ). 2 Compressibility was measured at 15 mn/m, or at the minimum surface tension if 15 mn/m was not reached (indicated by b ). 3 Data from Lee et al. (2004). * indicates significantly different from cow sample using a one-way ANOVA followed by a Bonferroni multiple comparisons test. indicates significantly different from C 25 using a paired t-test. Data are expressed as mean ± SE (where applicable), and significance is set at p<0.05. was also a large increase in compressibility as γ decreased in the ringed seal (0.026 ± m/mn to ± m/mn at 25 and 15 mn/m respectively), and large increases in compressibility were seen in the other pinniped species, although due to low sample size, this could not be determined statistically Discussion Validation of Methods As demonstrated in section , the NaBr gradient technique was successful in extracting surfactant-like material. TEM demonstrated the presence of lamellar bodies and other multilamellated structures and vesicles in the pelleted extracted material. These 176

10 structures are typically present in large aggregate material (Veldhuizen et al., 1996) and are essential for achieving low surface tensions on surface balances and captive bubble surfactometers (Veldhuizen et al., 1996; Schürch et al., 2001; Veldhuizen et al., 2002). Hence, the isolation procedure should not have prevented the material from functioning optimally in this study. Further evidence that NaBr purification did not affect the quality of the surfactant, comes from the fact that the cow and sheep surfactant material yielded similar values for equilibrium and minimum surface tension as previously recorded (Egberts et al., 1981; Danlois et al., 2003a) and as expected according to the classical theory for typical mammals. The northern elephant seal used in this study is a juvenile which was probably still undergoing the post-wean fast that all young northern elephant seals experience before they enter the water for the first time. This means that this individual probably had not experienced the effects of diving. It is suggested in Chapter 5 that the surfactant of California sea lion pups is more terrestrial in nature until they start diving, where the surfactant becomes more like the adult diving mammal. This does not appear to be the case in northern elephant seals, with the surface activity of surfactant from the pup used in this study being diving in nature even before the pup has entered the water. This was why this animal was included in the current chapter Surface properties of pinniped surfactant The surface activity of surfactant was measured using a CBS. Under static conditions, surfactant components adsorb spontaneously to the air-liquid interface, forming a surfactant film which lowers γ. The point at which no further change in γ occurs, within a defined time period, is called the γ eq. Under dynamic lateral compression of the surfactant film, γ is further reduced as the lipids pack more closely together and exclude water from the interface. The point at which continued compression does not result in further decreases in γ is known as γ min. While these are the most important measures of γ, there 177

11 are a number of others, including surface area reduction and film compressibility. These are discussed below Equilibrium surface tension (γ eq ) γ eq was significantly higher in ringed seals than cow and sheep, and neither the northern elephant seal nor the northern fur seal reached the expected γ eq for mammals (dotted line in Fig. 4.1). Interestingly, the California sea lion was able to reach this level, and the potential reasons for this will be discussed in chapter 5. The γ eq is dependent on the PL concentration of the sample (Schürch et al., 1992), as well as cholesterol and protein concentrations (Yu and Possmayer, 1998; Possmayer et al., 2001). Since all samples were of the same initial PL concentration (20 µg/µl), this should not have affected γ eq. However, the relative contribution of cholesterol to the surfactant in these species was highly variable. When expressed as a percentage of total PL, ringed seal had significantly higher levels of cholesterol than cow and sheep, California sea lion was not significantly different from either cow or sheep, and northern elephant seal was much lower than cow and sheep (see chapter 3). Northern fur seal also had slightly elevated levels of cholesterol (9.69 µg/µmol PL). How cholesterol functions in surfactant is unclear, with numerous conflicting reports (Orgeig and Daniels, 2001). Cholesterol has been shown to have deleterious effects on surface activity, such as increasing γ min during compression or diminishing respreading during expansion (Suzuki, 1982; Yu and Possmayer, 1994; Taneva and Keough, 1997). The interference with the ability of surfactant films to reach near zero γ min upon film compression also appears to be due to the ability of cholesterol to resist squeeze-out upon film compression (Daniels et al., 1996; Yu and Possmayer, 1998). However, in pure lipid mixtures, cholesterol has also been shown to enhance adsorption and respreading of the monolayer after collapse (Notter et al., 1980b). Cholesterol has also been demonstrated to enhance the ability of SP-A to promote the adsorption of DPPC to the air-liquid interface (Yu and Possmayer, 1996). Another study 178

12 on lipid mixtures containing cholesterol found that cholesterol, in the presence of both SP- B and SP-C, has no effect on the surface activity under dynamic conditions (Diemel et al., 2002). The variability in cholesterol levels between pinniped species may contribute to some of the variability seen in surface-activity. For example, the deleterious effects of cholesterol may explain why the γ eq of ringed seal and northern fur seal were higher than expected. However, cholesterol does not explain all the variation in surface activity, as the level of cholesterol was very low in the northern elephant seal (2.35 µg/µmol PL), yet this species had the highest γ eq. A number of other different surfactant components are known to enhance the adsorption, and therefore the spreadability, of surfactant. The addition of USPs and short chain PLs to surfactant promotes rapid adsorption (as discussed in chapter 3), as does the addition of mono-, di-, and tri-acylglycerol (Veldhuizen et al., 1998). However, altering the proportion of these molecules may also alter the surface activity of the surfactant (Codd et al., 2003). SP-B and SP-C are also highly important for the surface-active properties of surfactant. SP-B is able to induce the formation of a monolayer from vesicles (Oosterlaken-Dijksterhuis et al., 1991), facilitate respreading of the monolayer from the collapsed phase (Taneva and Keough, 1994), and also induces film refinement by selective enrichment of the monolayer with DPPC during dynamic cycling (Veldhuizen et al., 2000a). The SP-B levels in all species of pinniped are significantly lower compared with both sheep and cow (see chapter 3). This is in contrast to the findings of Spragg et al. (2004), who found that SP-B was elevated in northern elephant seal surfactant, compared with human surfactant, whereas SP-B in California sea lion and Harbour seal surfactant did not differ significantly from that of humans. The lower levels of SP-B in pinnipeds (chapter 3) are consistent with a decrease in the rate of adsorption of lipids to the monolayer, which may explain the relatively high γ eq values of diving mammals compared with terrestrial mammals. 179

13 Minimum surface tension (γ min ) According to the classical theory, in order for a surfactant to function effectively in the mammalian lung, it must be able to achieve a γ min of close to 0 mn/m (Zasadzinski et al., 2001). This is necessary for the optimisation of alveolar area for efficient gas exchange at low lung volumes (Schürch and Bachofen, 1995), and for alveolar stability (Daniels et al., 1998a). The surfactant extracted from pinnipeds was unable to reduce γ to the level regularly observed in terrestrial mammals. This may also be due to low levels of SP-B, since this protein is involved in film refinement (Veldhuizen et al., 2000a). In chapter 3, I suggested that the decreased levels of SP-B may be compensated for by increased levels of SP-C. As SP-C is mainly involved in the retention of fluid lipids in the monolayer via the surface-associated reservoir (Amrein et al., 1997; Takamoto et al., 2001), an increase in SP-C would result in a decrease in surface activity, since these lipids are unable to form a tightly packed monolayer at the air-liquid interface. However, without measuring SP-C this is completely speculative. This poor surface activity was also found by Spragg et al. (2004) in northern elephant seals whose surfactant only achieved a γ min range of 9.9 to 14.6 mn/m. Whilst this value obtained by Spragg et al. (2004) is lower than that achieved in this study (Fig. 4.2), the difference is slight and may be explained by a difference in techniques used to isolate surfactant (see section on different surfactant extraction techniques) and also to measure surface activity (see section on different techniques for measuring surface activity). The reduction in surface area of the bubble in the CBS required to achieve γ min indicates how efficiently surfactant reduces γ during compression (Daniels et al., 1998a). A highly surface-active material will reduce γ to a very low value with minimal change in surface area. Cow and sheep were both able to reduce γ with a very small change in surface area, indicating that these surfactants have a very high surface activity (as defined by Daniels et al. (1998a)). All pinniped species required a large change in surface area to 180

14 reach γ min (Fig. 4.3), and this coupled with high values for γ min, implies that surface activity in these animals is very low. The values for γ min and change in surface area in these animals are comparable to values obtained for amphibians (Daniels et al., 1998a), reptiles (Phleger et al., 1978; Meban, 1980) and birds (Fujiwara et al., 1970; Bernhard et al., 2004), where surfactant functions more as an anti-adhesive to prevent the adherence of respiratory surfaces during lung collapse (see section ) Film compressibility The compressibility of a film can be an indication of its composition. A film comprised of pure DPPC will have a low compressibility due to its rigid structure, however a film with USP or neutral lipids will have a high compressibility due to its more fluid and flexible nature (Lee et al., 2004). There is no great difference in DSP concentrations in pinniped and terrestrial mammal surfactant (chapter 3), although a few species exhibited an increased cholesterol concentration. As mentioned above, SP-B and SP-C can prevent the loss of fluidising lipids from the monolayer during compression by creating a surfaceassociated reservoir, which enables rapid respreading during expansion (Takamoto et al., 2001). If there is an increase in the SP-C concentration (as suggested in chapter 3), this would result in a functional increase in USP in the monolayer, which may account for the high compressibility. It is clear from this study, and that of Spragg et al. (2004), that the surfactant of pinnipeds has poor surface activity. Moreover, as will be discussed in chapter 5, the quality of the surfactant alters with age, as the animals move from a terrestrial environment to an aquatic one. The functions of pinniped surfactant may therefore relate to their respiratory patterns, lung structure, and surfactant functions. 181

15 Functions of Pinniped Surfactant Alveolar stability In the mammalian lung, a γ min of near zero at the end of expiration is necessary for alveolar stability. The alveoli are lined with fluid of an average thickness of 0.2 µm (Bastacky et al., 1995). The surfactant film on top of the fluid hypophase generates the surface tension-related elastic retractile force. According to the law of Young and LaPlace, P = where γ is surface tension measured in mn/m and r is the radius of curvature measured in metres, a very small radius of curvature would result in very large collapse pressures (P), unless γ was extremely low. Surfactant reduces γ to near zero at very low lung volumes such that the retractile forces are equivalent both within separate regions of an alveolus as well as between the respiratory units (Bachofen et al., 1970; Schürch, 1982). This, together with the structural interdependence of the alveoli, provides the lung with alveolar stability and maintains a relatively large alveolar surface area necessary for efficient gas 2γ r exchange at low lung volumes (Bachofen et al., 1987). However, the respiratory units of large mammals are much larger than alveoli of small mammals, since alveolar size is related to body size (Siegwart et al., 1971). This large size may itself confer alveolar stability by substantially reducing the collapse pressures and therefore a highly surface-active surfactant may not be necessary. On the other hand, cows and sheep have alveoli of similar size to those of pinnipeds and still exhibit excellent surface activity by their surfactant. Hence it is not likely that the compositional differences observed are related to alveolar size, and the differences in surface activity of the surfactant are not related to an alveolar stability function in pinnipeds Static lung compliance In the mammalian lung, surfactant increases lung compliance, and thereby reduces the work of breathing. Static compliance describes the retractile nature of the lung at a set 182

16 volume and characterises the pressure due to the recoil forces of the lung (Daniels et al., 1998a). In mammals, static lung compliance is determined by the surface tension of the fluid lining the lung (66%) and the physical structure of the lung (33%) (von Neergaard, 1929). We did not measure static compliance of the pinnipeds in this study, nor have I discovered any direct study on marine mammal lung compliance. However, it has been suggested that lung compliance in aquatic mammals is not different from that of large terrestrial mammals (Denison et al., 1971; Kerem et al., 1975; Schroter, 1980). Moreover, during diving the lungs are not inflated, and the external pressure exerted by the surrounding water would maintain the lung in a compressed state. Given the episodic nature of breathing in these animals, it seems unlikely that the compositional and functional differences observed between pinnipeds and terrestrial mammals relate to static lung compliance Acting as an anti-adhesive Before a dive, pinnipeds expel the majority of air from their lungs to enable lung collapse, and to prevent the deleterious effects of the build-up of high pressure gases (see section 1.2). During this time, there is no gas exchange across the respiratory surface. Upon resurfacing, the lung needs to reinflate rapidly. Moreover, during their time on land, pinnipeds experience a breathing pattern consisting of bouts of breaths followed by extended periods of breath-hold. This type of breathing pattern is unusual for mammals, but similar to that of reptiles. In many studies, Daniels, Orgeig and others have related this type of breathing pattern to the requirement for a surfactant to act as an anti-adhesive (see below). Surfactant can act as an anti-adhesive by preventing the adhesion of adjacent epithelial surfaces at low lung volumes (Wilson and Farber, 1933) or, in the case of diving mammals, during the lung collapse associated with deep diving. Without an anti-adhesive to lower the γ of the fluid between the contacting epithelial surfaces, inspiration after lung 183

17 collapse would be more difficult (Daniels et al., 1998a), and may result in damage to the epithelial lining (Hallman et al., 2001). The anti-adhesive function may be important in all mammalian lungs. Mammalian alveoli are usually very small and may inflate in a very complex manner involving the unpleating and unfolding of the alveolar walls (Daniels et al., 1998a). If the surface tension of the hypophase increases, then the respiratory tissue will fold in upon itself more extensively on expiration, resulting in an increase in the work required to unfold the alveoli on subsequent inspiration (Daniels et al., 1998a). An elevated γ therefore increases the elastic recoil of the lung, which results in a decrease in lung compliance. However, pulmonary surfactant is present in the hypophase. As the air is forced in and the two apposing surfaces peel apart, the lipids rise to the expanding air-water interface to lower the surface tension, and thereby decrease the work required to separate the two surfaces. For a lipid mixture to act as an anti-adhesive it must be capable of reducing the surface tension of the hypophase to relatively low levels, although near 0 mn/m is not required (Daniels et al., 1998a). And, more importantly, it must be able to rapidly adsorb to the airliquid interface. Thus an anti-adhesive may often demonstrate a moderate ability to reduce surface tension without exhibiting pronounced surface activity (similar to a detergent). Lung collapse occurs frequently in a range of vertebrates. Radiographs of the breathing cycle of Lepidosiren paradoxa demonstrated almost complete lung collapse on expiration (Bishop and Foxon, 1968). Aquatic salamanders also collapse their lungs almost completely on expiration possibly to assist in generating a negative lung pressure upon inspiration (Guimond and Hutchison, 1976; Martin and Hutchison, 1979; Stark- Vancs et al., 1984). Low body temperatures have been shown to induce periods of apnoea in the lizard Ctenophorus nuchalis, during which the lungs collapse (Frappell and Daniels, 1991a, b; McGregor et al., 1993). Diving mammals, reptiles and amphibians also collapse their alveoli at depth when external compression forces are elevated (Daniels et 184

18 al., 1998a). In all these situations, a surfactant with an anti-adhesive function appears essential to assist the reinflation of deflated lungs (Daniels et al., 1995a) Controlling fluid balance Lungs are highly susceptible to fluid disturbances because they have a large surface area, high blood flow and relatively leaky capillaries (Daniels et al., 1998a). However, the amount of fluid in the lung is tightly regulated. The negative pressure within the fluid lining the alveolar corners, crevices and creases will be large (because the radius of curvature here is very small), but small along the sides of the alveolus. Theoretically, fluid flow occurs from the interstitial space into the alveolar hypophase via alveolar seams, crevices and corners, and may flow back into the interstitium via either the side regions or at corners where type II cells are located. Type II cells are involved in controlling fluid balance by pumping Na + through the basolateral membranes (Goodman et al., 1984; Daniels et al., 1998a; Matalon et al., 2002). By lowering surface tension at the air-liquid interface, surfactant reduces the negative pressure in the hypophase and therefore reduces the tendency of fluid to enter the alveoli. The ability of surfactant to reduce the tendency of fluid to pass into the hypophase, and the rate of removal of fluid by type II cells, ensure that the depth of the hypophase remains constant and small (Guyton et al., 1984). Acting as an anti-oedema agent is therefore likely to be a crucial role of surfactant in pinniped lungs due to the reduction of the radius of curvature during compression, and therefore the potential increase in fluid flowing in through these regions. Unfortunately there are no experimental tests for this function of surfactant Summary In conclusion, it appears that pinniped surfactant has a greatly different function to surfactant from other closely related, similar-sized mammals: Pinniped surfactant is unable to achieve the γ eq of terrestrial mammals. This may be due to different levels of cholesterol or to the very low levels of SP-B. The only exception 185

19 in this study is the California sea lion, which is able to achieve the γ eq of terrestrial mammals, but it is unlike terrestrial mammals in all other respects. It is unclear why this would occur. Pinniped surfactant cannot reach the low γ min that can be obtained by terrestrial mammal surfactant. This may also be caused by the decreased levels of SP-B, perhaps in conjunction with an increase in levels of SP-C. Compressibility is high in pinnipeds and it takes a greater reduction in surface area to reduce surface activity than in terrestrial mammals. This may be due to the increase in USP and short chain phospholipids (chapter 3), which could also be brought about by the decrease in SP-B concentration and the potential increase in SP-C concentration. In terrestrial mammals, the highly surface-active material is crucial in preventing atelectasis of the alveoli and increasing static compliance. It is suggested that diving mammals require a less surface-active material to act as an anti-adhesive. Diving mammal species regularly exhibit collapsed lungs and/or have pleats and folds within the respiratory units (Denison et al., 1971). This function might also be very important during periods of apnoea when residual volume is greatly reduced and epithelial surfaces within the lung come into contact. As with all other vertebrates, surfactant may be crucial in the prevention of pulmonary oedema. If acting as an anti-adhesive and/or as an anti-oedema agent are the functions of surface-active material in lungs of diving mammals, then a more detergent-like surfactant is sufficient to fulfil these roles. Promoting alveolar stability and increasing lung compliance appear to be functions restricted to terrestrial animals or perhaps in pinnipeds when they come onto land Future directions Pinniped samples are incredibly difficult to obtain due to the behaviour of the animals (most of the time is spent at sea), and also because many pinniped species are classified as vulnerable or endangered. This is why for the California sea lion, northern elephant seal and northern fur seal only one sample from each species was collected. Having a 186

20 number of ringed seal samples enabled educated estimates of the functions of these species even though sample size was small, but future studies may wish to repeat the current study with a greater sample size. From this chapter and the previous one, it has been suggested that pinniped surfactant may function more as an anti-adhesive rather than a surface tension reducer. Sanderson et al. (1976) developed a device that could measure the reduction in the force required to separate two surfaces when surfactant was applied to them. This device could be applied to pinniped surfactant to determine how effective it truly is as an anti-adhesive. Several studies from our laboratory have used opening pressure as a measure of the anti-adhesive property in isolated deflated lungs before and after lavage (Orgeig et al., 1994; Wood and Daniels, 1996; Wood et al., 1997). The pressure in a completely deflated lung or swim bladder chamber increases as air is infused into the structure (at a constant rate) until the chamber opens. This pressure spike is the opening pressure, after which the pressure decreases. If acting as an anti-adhesive is important, then the opening pressure will increase after the removal of surfactant, when γ increases to that of water. Most non-mammals that collapse their lungs have demonstrated an increase in opening pressure after lavage and hence an anti-adhesive function of surfactant. This technique could also be applied to diving mammal lungs to determine the anti-adhesive nature of their surfactant. 187

21 Chapter 5 The development of the pulmonary surfactant system in the California sea lion, Zalophus californianus 5.1. Introduction The pulmonary surfactant system varies greatly between species and appears to be influenced by evolutionary selection pressures such as body temperature, lung structure and function, as well as metabolic strategies (see chapters 3 and 4; (Bernhard et al., 2001a; Postle et al., 2001; Ormond, 2004). The surfactant system has also been shown to vary within a species when an animal is undergoing a physiological stress such as torpor (Langman et al., 1996; Codd et al., 2002; Codd et al., 2003). One of the most commonly studied changes in the surfactant system is during development. The composition (Benson et al., 1983; Burdge et al., 1993; Johnston et al., 2000; Bernhard et al., 2001b; Rau et al., 2004), function (Gluck et al., 1970; Stevens et al., 1987; Farrell et al., 1990) and control (Miller et al., 2001b; Sullivan et al., 2001; Sullivan et al., 2002b) of pulmonary surfactant have all been shown to change during development in many different types of vertebrates. Chapters 3 and 4 demonstrated that pinniped surfactant is different to terrestrial mammal surfactant due to the differences in lung structure and physiology of the animals. Adult pinnipeds spend a large amount of their lives under water, and hence both lung and surfactant function reflect the different requirements for oxygen delivery. However, unlike cetaceans, which are born in the water and spend their whole lives there, pinnipeds are born on land, and spend the majority of their development out of the water. California sea lion pups do not leave the rookery until after approximately 6 months of age (Melin et al., 2000). Before this time, they are nursed by their mothers, and there is no evidence that they follow their mothers on their foraging trips (Melin et al., 2000). Therefore, pups under 6 months of age probably do not dive. Given this terrestrial nature in the first part of life, 188

22 followed by an aquatic lifestyle after 6 months of age, I hypothesise that surfactant development (in terms of composition and function) will reflect this shift in life style, due to the different stresses imposed by the aquatic environment and the diving behaviour of the adult sea lions. It is therefore hypothesised that California sea lion pups will have a similar surfactant system to terrestrial mammals until they first start to forage for themselves. Once they enter the water, it is hypothesised that they will develop the adult California sea lion surfactant, which will facilitate their aquatic behaviour Methods Animals Lungs from California sea lions (Zalophus californianus) were obtained from wildcaught animals held at the Diving Mammal Centre, Sausalito, California. One male newborn, mass 4.5 kg, and one female adult, mass 104 kg, were obtained. Both adult and pup died from non-respiratory related illness (demoic acid poisoning), which was not considered to affect the quality of the data obtained. Demoic acid is passed to the pup from the mother via the milk, resulting in the death of the pup Experimental protocols Phospholipid classes and molecular species, cholesterol, SP-A and SP-B were measured in lavage extracts using the same protocols as in section 3.2. Surface activity was measured using the protocols as in section Results Phospholipids Table 5.1 shows the composition of phospholipid classes in the newborn and adult California sea lion. Phosphatidylcholine (PC) is by far the dominant phospholipid class and is very similar in both the adult and newborn. Phosphatidylglycerol (PG) is 4-fold higher in the adult compared with the newborn. PI and PS are both negligible in the adult 189

23 Table 5.1. Total phospholipid classes of lavage extracts from a newborn and adult California sea lion. PC PG PI PS LPC Newborn Adult PC = phosphatidylcholine, PG = phosphatidylglycerol, PI = phosphatidylinositol, PS = phosphatidylserine, LPC = lysophosphatidylcholine. (<1% each), whereas PS and PI combined make up >8% in the newborn. LPC is similar in the newborn and the adult. The levels of PI and especially PG in the newborn are significantly below average for most mammals (see Table 5.1 and Table 1.2 in chapter 1). Fig. 5.1 shows the saturation levels of lavage extracts from a newborn and an adult California sea lion. The adult has higher levels of disaturated PC and PG than the newborn. Levels of disaturated PI are negligible in both the newborn and the adult. The newborn has greater levels of polyunsaturated PC than the adult, which corresponds to the the lower levels of disaturated PC. The newborn is also higher in polyunsaturated PG resulting from the lower levels of both disaturated and monounsaturated PG. The newborn has lower levels of monounsaturated PI than the adult, resulting in greater levels of polyunsaturated PI. The individual molecular species of PC, PG and PI from the newborn and adult California sea lion are shown in Fig There are several differences between newborn and adult molecular species for all three phospholipid classes, but only those molecular species that account for more than 5 mol% are considered to be functionally important. The adult contains approximately twice the amount of PC16:0/14:0 than the newborn (11.05 and 5.59 mol%, respectively; Fig. 5.2a). However, the newborn contains a 190

24 significantly higher amount of long chain molecular species (10.06 and 2.27 mol% for the newborn and adult, respectively). The newborn also contains slightly more PC16:0/18:1 (12.97 and 9.41 mol% for newborn and adult, respectively), but both PC16:0/16:0 and PC16:0/16:1 are relatively similar between the adult and the newborn. The PG and PI molecular species are vastly different between ages. The adult surfactant contains relatively few species of PG, with PG16:0/16:0 and PG16:0/18:1 being the highest contributors. There are many more newborn PG molecular species, with PG16:0/18:1 and PG16:2a/16:1 being the highest. The newborn appears to be higher in the longer chain PG molecular species, whereas the adult is high in the shorter chain species. Adult PI also contains fewer molecular species than newborn PI, with the polyunsaturated PI16:0/20:4, PI18:1/18:2, PI16:0/22:6 and PI18:0/22:6 being unique to newborns. Both the adult and newborn have PI16:0/18:1 as the highest contributor to the PI molecular species, but the adult has an approximately 50% greater amount. The adult is also greater in PI18:1/18:1, PI18:0/18:1 and PI18:0/22:5. 191

25 A 100% B 100% C 100% 90% Phosphatidylcholine (%) 80% 60% 40% 20% Phosphatidylglycerol (%) 80% 70% 60% 50% 40% 30% 20% Phosphatidylinositol (%) 80% 60% 40% 20% Poly-USP Mono-USP DSP 10% 0% Newborn Adult 0% Newborn Adult 0% Newborn Adult Fig Saturation of phospholipid classes in lavage extracts of a newborn and an adult California sea lion. A. Phosphatidylcholine. B. Phosphatidylglycerol. C. Phosphatidylinositol. DSP = disaturated phospholipid, Mono-USP = monounsaturated phospholipid, Poly-USP = polyunsaturated phospholipid. Mono-USP consist of molecular species that have one fatty acid with one double bond (e.g. 16:0/16:1). Poly-USP consist of molecular species that have more than one double bond in total (e.g. 16:0/16:2 or 18:1/18:1). 192

26 Fig Molecular species composition of lavage extracts from a newborn and an adult California sea lion. A. Phosphatidylcholine. Short chain consists of molecular species not indicated here which have one fatty acid of the 14:x moiety. Long chain consists of molecular species not indicated here that have one fatty acid containing more than 20 carbons in its chain. B. Phosphatidylglycerol. C. Phosphatidylinositol. Concentrations are measured as a percentage of the total phospholipid class. 193

27 A 50 Phosphatidylcholine (mol%) short chain 16:0/14:0 16:0a/16:0 16:0/16:1 16:0/16:0 16:0a/18:1 16:0a/18:0 16:0/18:2 16:0/18:1 16:0/18:0 18:0a/18:1 18:0/18:2 18:0/18:1 16:0/22:6 long chain Newborn Adult

28 :0/16:0 16:2a/16:1 16:2/16:2 16:1/16:0 16:0/16:0 16:2/18:1 16:0/18:2 16:0/18:1 16:0/18:0 18:2a/18:2 18:1a/18:0 16:0/20:4 18:1/18:2 18:0/18:2 18:0/18:1 18:0a/20:1 16:0/22:6 Phosphatidylglycerol (mol%) Newborn Adult B :0/16:1 16:0/18:2 16:0/18:1 16:0/20:4 18:1/18:2 18:1/18:1 18:0/18:1 16:0/22:6 18:1/20:4 18:0/20:4 18:0/20:3 18:0/20:2 18:0/22:6 18:0/22:5 Phosphatidylinositol (mol%) Newborn Adult C

29 Cholesterol The cholesterol content of surfactant from the newborn and adult California sea lion is shown in Fig When expressed as a percentage of total phospholipid, there appears to be no difference between the levels of cholesterol in the newborn (7.15%) and those of the adult (7.55%). However, when expressed as a percentage of DPPC, the adult is slightly higher in cholesterol than the newborn (19.11% and 16.67%, respectively) Newborn Adult Cholesterol (%) Chol/PL Chol/DPPC Fig Content of cholesterol in lavage extracts from a newborn and an adult California sea lion, expressed as a ratio of total phospholipid (PL) or as a ratio of dipalmitoylphosphatidylcholine (DPPC) Proteins Fig. 5.4a and b show the SP-A and SP-B contents, respectively of surfactant from the newborn and adult California sea lions. When expressed as a ratio of total protein, total 193

30 A Newborn Adult 6.0 SP-A ratios SP-A/total protein SP-A/PL SP-A/DPPC B Newborn Adult SP-B ratios SP-B/total protein SP-B/PL SP-B/DPPC Fig Ratios of surfactant proteins in lavage extracts of a newborn and an adult California sea lion. A. SP-A expressed as a ratio of total protein (ng SP-A/µg total protein), total phospholipid (PL) (ng SP-A/µmol PL) and dipalmitoylphosphatidylcholine (DPPC) (ng SP-A/µmol DPPC). B. SP-B expressed as a ratio of total protein (ng SP-B/µg total protein), PL (ng SP-B/µmol total phospholipid) and DPPC (ng SP-B/µmol DPPC). 194

31 phospholipid or DPPC, there appears to be no difference in the levels of SP-A between the newborn and the adult. However, all SP-B ratios are approximately three times higher in the adult than in the newborn Surface activity Several different variables associated with the surface-active function of surfactant were measured in newborn and adult California sea lions. Fig. 5.5a shows the equilibrium surface tension after 5 min adsorption. The adult is able to reach a lower γ eq than the newborn (23.4 and 37.2 mn/m, respectively), with the adult value being similar to that of a typical highly surface-active surfactant, as indicated in Fig. 5.5a. Fig. 5.5b shows the minimum surface tension achieved by newborn and adult California sea lions during dynamic cycling of 20 cycles per minute. The newborn is able to reach a much lower γ min than the adult (5.9 and 16.3 mn/m, respectively), but is still unable to reach the almost 0 mn/m that can be achieved by other mammalian surfactants (see chapter 4). The surface tension to area (compressibility) curve of newborn and adult California sea lion surfactant is shown in Fig. 5.5c. Both animals have a similar compressibility curve, although due to the lower γ min achieved by the newborn, the curve is angled downwards in the lower area range. Both animals need a large reduction in surface area to reduce surface tension, compared with a typical mammalian surfactant. 195

32 A 40 B Surface tension (mn/m) Surface tension (mn/m) Newborn Adult 0 Newborn Adult C Surface tension (mn/m) Newborn Adult Area (cm 2 ) Fig Surface activity variables of lavage extracts from a newborn and an adult California sea lion. A. Equilibrium surface tension achieved after 5 min adsorption. Dotted line indicates approximate equilibrium surface tension of a highly surface-active surfactant. B. Minimum surface tension achieved during dynamic cycling. C. Surface tension to area curves demonstrating compressibility of surface films. 196

33 5.4. Discussion Previous studies on the biochemical characterisation of pulmonary surfactant have always focussed on PC16:0/16:0 as the major surface-active component (Adams et al., 1970; Ikegami and Jobe, 1983), with PG and the apoproteins (particularly SP-B) being important for their contribution to lipid adsorption and surfactant homeostasis (Ingenito et al., 2000; Veldhuizen et al., 2000a). However, it is now apparent that surfactant composition, function and capability for functional adaptation are much more complex than previously thought. For example, there is now increasing evidence that fluidic surfactant components other than PG contribute to the dynamic properties of surfactant (Walters et al., 2000; Piknova et al., 2001). Initial adsorption of surfactant to the air-liquid interface depends on the charge of the anionic head groups rather than their fatty acid chains (Walters et al., 2000). Surfactant from the adult California sea lion is higher in total anionic phospholipids than that from the newborn, which may explain its increased adsorbance on the CBS (as indicated by the lower γ eq values, Fig. 5.5a). Newborn pigs have a surfactant which demonstrates improved function compared to adolescent pigs, associated with a decrease in PC16:0/16:0 and an increase in PC16:0/14:0, PC16:0/16:1, SP-B and SP-C (Rau et al., 2004). Conversely, levels of PC16:0/14:0 and SP-B were higher in the adult California sea lion than the newborn, but PC16:0/16:1 and PC16:0/16:0 were at similar levels in both the adult and the newborn. Hence, adult sea lion surfactant had better adsorptive properties, whereas the newborn surfactant had better surface tension lowering properties. This discrepancy may be due to changes in the lung physiology of the animals during development. PC16:0/14:0 is usually regarded as being unable to lower surface tension, as collapse of the monolayer would occur at temperatures far blow body temperature (Goerke and Gonzales, 1981). However, diving adults collapse their lungs, while pups on the beach do not. The higher levels of PC16:0/14:0 in the adult California sea lion may result in a more collapsible monolayer, which may be important for a lung that frequently collapses. The increased levels of SP-B in the adult also support this, with SP-B (and SP-C) enabling 197