Lopatko, Olga V., Sandra Orgeig, Christopher B. Daniels, and David Palmer. Alterations in the surface properties of lung surfactant in the torpid marsupial Sminthopsis crassicaudata. J. Appl. Physiol. 84(1): 146–156, 1998.—Torpor changes the composition of pulmonary surfactant (PS) in the dunnartSminthopsis crassicaudata [C. Langman, S. Orgeig, and C. B. Daniels. Am. J. Physiol. 271 (Regulatory Integrative Comp. Physiol. 40): R437–R445, 1996]. Here we investigated the surface activity of PS in vitro. Five micrograms of phospholipid per centimeter squared surface area of whole lavage (from mice or from warm-active, 4-, or 8-h torpid dunnarts) were applied dropwise onto the subphase of a Wilhelmy-Langmuir balance at 20°C and stabilized for 20 min. After 4 h of torpor, the adsorption rate increased, and equilibrium surface tension (STeq), minimal surface tension (STmin), and the %area compression required to achieve STmin decreased, compared with the warm-active group. After 8 h of torpor, STmin decreased [from 5.2 ± 0.3 to 4.1 ± 0.3 (SE) mN/m]; %area compression required to achieve STmindecreased (from 43.4 ± 1.0 to 27.4 ± 0.8); the rate of adsorption decreased; and STeqincreased (from 26.3 ± 0.5 to 38.6 ± 1.3 mN/m). ST-area isotherms of warm-active dunnarts and mice at 20°C had a shoulder on compression and a plateau on expansion. These disappeared on the isotherms of torpid dunnarts. Samples of whole lavage (from warm-active and 8-h torpor groups) containing 100 μg phospholipid/ml were studied by using a captive-bubble surfactometer at 37°C. After 8 h of torpor, STmin increased (from 6.4 ± 0.3 to 9.1 ± 0.3 mN/m) and %area compression decreased in the 2nd (from 88.6 ± 1.7 to 82.1 ± 2.0) and 3rd (from 89.1 ± 0.8 to 84.9 ± 1.8) compression-expansion cycles, compared with warm-active dunnarts. ST-area isotherms of warm-active dunnarts at 37°C did not have a shoulder on compression. This shoulder appeared on the isotherms of torpid dunnarts. In conclusion, there is a strong correlation between in vitro changes in surface activity and in vivo changes in lipid composition of PS during torpor, although static lung compliance remained unchanged (see Langman et al. cited above). Surfactant from torpid animals is more active at 20°C and less active at 37°C than that of warm-active animals, which may represent a respiratory adaptation to low body temperatures of torpid dunnarts.
- body temperature
- surface tension
- lipid composition
pulmonary surfactant is a complex mixture of lipids [primarily phospholipids (PLs)] and proteins that lines the air-liquid interface of the lungs. By reducing the surface tension of the fluid lining the inner lung, pulmonary surfactant reduces the work of breathing on inspiration and stabilizes the gas-exchange area of the lung on expiration (28, 36). The ability to lower and vary surface tension during a cycle of alveolar expansion and compression is termed “surface activity,” a property of surfactant that is strongly dependent on its lipid and protein composition. Dipalmitoylphosphatidylcholine (DPPC), a disaturated PL (DSP), is the major surfactant PL and is primarily responsible for the potent surface tension-lowering properties of surfactant. A pure film of DPPC on a surface balance is capable of lowering the surface tension to nearly 0 mN/m under dynamic compression (25). Unsaturated PLs and the neutral lipid cholesterol (Chol) are thought both to promote the adsorption of lipids to form a monolayer at the air-liquid interface and to aid in the respreading of the molecules after compression (10,23). The specific surfactant-associated proteins are also capable of modifying the biophysical properties of surfactant as they significantly influence the rate of adsorption and dominate monolayer formation (24).
The effect of temperature on the surface activity of pulmonary surfactant appears to be controversial. Avery and Mead (1) using an unprimed Wilhelmy-Langmuir balance (WLB) reported that a temperature change from 21 to 38°C did not influence the surface tension-area relationship of human lung extracts. Similarly, King and Clements (13,14) using a highly purified fraction of surfactant on an unprimed WLB did not find a difference in minimum surface tension (STmin) between 20 and 37°C. Lempert and Macklem (16) and Wildeboer-Venema (37), all using an unprimed WLB and fresh mammalian surfactant, reported that an increase in temperature (from 21 and 29 to 37 and 40°C, respectively) decreased the maximum surface tension and increased the STmin (the latter from <10 to >14 mN/m). However, Schürch et al. (30), using the in situ microdroplet spreading procedure, demonstrated very low surface tensions (<10 mN/m) at 37°C. Furthermore, using an improved microdroplet technique and measuring surface tension over the entire pressure-volume curve at 22 and 37°C, Schürch et al. (29) determined that the alveolar surface tension and the surface tension-to-volume relationship are both essentially identical at the two temperatures. Recently, Miles et al. (19) reported that the minimal surface tension of fresh whole surfactant, isolated from excised rat lungs ventilated at 37°C and measured at 22°C with a primed WLB, did not differ between surfactant extracted from lungs ventilated at 22 and 37°C (remaining <10 mN/m) but did increase significantly at 42°C (19 mN/m) (19). The differences in the effect of temperature on the behavior of surfactant films in these studies are, in all likelihood, a result of differences in methodologies employed. The apparent increases in STmin with increasing temperature, seen in some of the earlier studies, are most probably due to surface film leakage in the apparatus used. It appears likely that the results obtained with the more recent techniques such as the primed WLB, captive-bubble surfactometer (CBS), or the in situ microdroplet technique (29, 30) are the more reliable.
Although from these studies it appears that the in vitro temperature does not greatly affect the surface properties of isolated pulmonary surfactant, to our knowledge, no study has addressed the effect of an in vivo change in body temperature on the surface activity of pulmonary surfactant. We have a model with which we can test the effect of in vivo changes in body temperature on surfactant composition, lung function, and surface properties of surfactant. The fat-tailed dunnart,Sminthopsis crassicaudata, is a heterothermic nocturnal marsupial commonly occurring in southern Australia. It regularly enters periods of torpor, both in the wild and in captivity (9), during which its body temperature decreases from 35°C to as low as 13°C (9, 15). Torpor is associated with a short-term (maximally 19 h in S. crassicaudata) reduction in metabolic rate and body temperature, which enables the conservation of energy during periods of low food availability and reduced ambient temperature (9, 17). As these animals continue to breathe during torpor and are capable of arousing completely from torpor within a few minutes (9), pulmonary surfactant must be capable of performing its functions both at very low and at relatively high body temperatures. Our previous research (15) demonstrated that both the composition and the amount of pulmonary surfactant changed markedly throughout the first 8 h of torpor inS. crassicaudata. The amount of surfactant PL increased significantly during torpor [from 21.8 ± 2.9 mg PL/g dry lung wt (DL) in warm-active dunnarts to 36.1 ± 4.0 mg PL/g DL after 4 h of torpor and to 38.4 ± 1.8 mg PL/g DL after 8 h of torpor]. There was an increase in the ratio of Chol/PL from 0.068 ± 0.006 in the warm-active group to 0.079 ± 0.005 after 4 h and to 0.107 ± 0.01 after 8 h of torpor. There was also an increase in the Chol/DSP ratio (from 0.176 ± 0.024 to 0.218 ± 0.022), %DSP/PL (from 40.2 ± 2.6 to 49.3 ± 1.6%), and percentage of phosphatidylinositol (PI) (from 13.5 ± 2.2 to 21.8 ± 2.1%), with a corresponding decrease in percentage of phosphatidylcholine (from 70.7 ± 2.1 to 58.7 ± 2.6%) after 8 h of torpor, when compared with the warm-active group (15). On the other hand, when we measured static compliance before and after lavage and with saline inflation, we found that after 8 h of torpor there was no difference in these parameters compared with warm-active animals, despite the major changes in surfactant composition at this time. Because changes in pressure-volume curves can be used to indicate changes in surface tension at the air-liquid interface of the lung (38), our results would suggest that after 8 h of torpor there was no difference in surface tension compared with warm-active animals. However, since changes in lipid composition greatly influence the surface tension of monolayers (11, 23), we decided to investigate more directly whether the in vivo compositional changes also correlated with changes in the surface properties of pulmonary surfactant when measured in vitro. We used the surface balance WLB primed with DPPC and lanthanum to measure a range of surface properties of whole surfactant at 20°C and the adsorption rate only at 37°C. The entire range of surface properties at 37°C was measured with a CBS. These two temperatures are similar to the body temperatures of torpid and warm-active animals, respectively (9, 15).
MATERIALS AND METHODS
Animals and Experimental Groups
Male S. crassicaudata (mass range: 8–13 g, n = 24) were purchased from a colony at the University of Adelaide, Australia. Animals were housed individually in cages of 15 × 28 cm and were subjected to a 16:8-h light-dark cycle, which commenced at 10:30 PM. Ambient temperature was maintained at 24°C. The dunnarts were fed ad libitum a mixture of dried and canned commercial pet food. The animals were randomly assorted to three experimental groups (see below). In addition, six Swiss White mice of either sex were housed as above on a 12:12-h light-dark cycle commencing at 8:00 AM.
To induce torpor, food was removed from the dunnart cages at 5:00 PM on the day before experimentation, and cages were placed into a constant-temperature cabinet (modified Kelvinator) held at 24°C. The following day at 9:00 AM, initial rectal temperature was measured with a thermocouple probe (9V FLUKE digital thermometer, Everett, WA, Australia) to confirm a body temperature >34°C. In this condition, S. crassicaudata are active and alert and were termed “warm-active.” The cabinet temperature was then reduced to 10°C. Animals usually took 30–90 min to enter torpor. Animals that did not enter torpor were removed from the cabinet and fed, to be used on another day. Torpid animals were identified by a characteristic body posture, which included either lying flat with splayed legs or curled tightly into a ball. Once a torpid posture was adopted, rectal temperature was again recorded to confirm torpor. Animals with a body temperature <20°C were defined as torpid. Three experimental groups (eachn = 4–6 animals) included a warm-active group of dunnarts, killed before ambient temperature was lowered (at 9:00 AM), and two groups of torpid dunnarts that were killed 4 and 8 h after the onset of torpor. Torpid animals were checked frequently to ascertain that they did not rouse from torpor during this time (15).
Animals were killed quickly with an intraperitoneal overdose of pentobarbital sodium (∼600 mg/kg) (Boehringer Ingelheim, Sydney, NSW, Australia). The trachea was exposed and cannulated. The cannula was ligated tightly to prevent leakage and connected to a syringe. The thorax was opened, but the lungs remained in situ. Lungs were ventilated manually several times with a tidal volume of ∼0.8 ml for dunnarts (total lung capacity = 1.0 ml) and ∼1.5 ml for mice (total lung capacity = 2.5 ml). Lungs were lavaged with 0.15 M NaCl. Three consecutive volumes (0.85 ± 0.05 ml for dunnarts and 1.5 ± 0.1 ml for mice) were infused and withdrawn three times from a syringe filled with 2 ml of saline (15). All lavage material was briefly stored on ice and then centrifuged at 150 gfor 5 min (Beckman model TJ-6 centrifuge) to remove macrophages and cellular debris. Of the total lavage volume (6 ml) obtained from each animal, 1 ml was used for lipid extraction and the determination of total PL. The remaining supernatant was lyophilized and stored at −13°C before the surface activity measurements.
Lipid Extraction and Phosphorus Analysis
Lipids were extracted in chloroform-methanol (1:2) mixture by using the method of Bligh and Dyer (4). The lipid extract was analyzed for total phosphorus. PL content was ascertained by multiplying the phosphorus content by 25 (6). PL amounts were expressed in milligrams per milliliter of lavage fluid.
Surface Activity Measurements
Surface WLB. A modified WLB was used for surface activity measurements at 20°C of surfactant from mice and from warm-active, 4-h, and 8-h torpid dunnarts and for adsorption studies at 37°C in warm-active and 8-h torpor groups (n = 4–6 animals). The surface balance trough, milled from a solid block of Teflon, had inside dimensions of 10 × 4 × 2 cm (parameters are similar to those of the Kimray Greenfield surfactometer; Kimray Medical Associates, Oklahoma City, OK). To prevent deformation of the Teflon over long time periods and at high temperatures, the trough was fitted to an aluminum frame (35). A tight-fitting, motor-driven Teflon barrier was used to vary the surface area (SA) from 100% (36 cm2) to 20% (7.2 cm2) at a constant speed of 1 mm/s (0.4 cm2/s), which is within the standard range of speeds for the WLB (36). Surface tension was continuously measured with a Wilhelmy dipping plate, which consisted of a 22 × 22 mm glass microscope coverslip (33), rigidly suspended from a force-displacement transducer (model FT 03 C, Grass Instruments, Quincy, MA), and the resulting force was recorded continuously for each compression-expansion cycle on a polygraph chart recorder (model 7C, Grass Instruments). The force transducer was calibrated with standard weights. Surface tension was calculated as a force per wetted perimeter of the coverslip. Measurements had the units of millinewtons per meter.
Measurements were performed at a relative humidity of 98–100%, which was controlled by means of two shallow metal baths filled with water, attached to either side of the balance, above the aluminum frame. The entire structure was housed within a Plexiglas chamber, with a sloping ceiling and with overall dimensions of 12 × 15 × 12 cm. For the adsorption studies in the WLB at 37°C, the environmental temperature within the chamber was maintained at 37°C by means of small heaters, attached to the aluminum frame and metal waterbaths. Both temperature and humidity were monitored continuously during all measurements. The Teflon trough and barrier were cleaned before each measurement by washing in hot tap water with detergent and subsequently rinsed with cold tap water, highly purified water, methanol, and then with chloroform. The last two steps were repeated three times, and the chloroform was allowed to evaporate for at least 10 min.
The trough was filled with 50 ml of highly purified water, and a glass coverslip (cleaned as above) was suspended from the force transducer and submerged 2 mm into the water for 1 min. The coverslip was then raised by using the transducer-mounting bracket until only 1 mm was submerged. This position was maintained for all surface tension measurements (20). The trough and subphase were considered free of contamination if the surface tension of water remained constant at 72.7 and 70.5 mN/m at 20 and 37°C, respectively, and if the surface tension of the subphase solution (see below) remained constant at 73.2 and 71.1 mN/m at 20 and 37°C, respectively, throughout a complete compression-expansion cycle.
Before surfactant samples were measured, the Teflon trough and the leading side of the barrier were primed with DPPC by using the method of Hildebran et al. (11). The trough was then filled with Ringer solution containing 0.15 M NaCl, 0.003 M MgCl2, 0.003 M CaCl2 and was buffered to pH 7.35 with 0.005 M Trizma buffer (at 20 and 37°C) (13). Each sample of freeze-dried lavage fluid was dissolved in the original volume of highly purified water immediately before surface activity measurements. Samples containing 200 μg of PL were carefully applied dropwise onto the surface of the subphase with a 500-μl Hamilton microsyringe through a 3-mm-diameter injection hole in the Plexiglas chamber. This amount of PL represented the equivalent of 5.5 μg/cm2 trough area, which forms a surface-excess film (23). As soon as sample application was completed, we measured surface tension at the air-liquid interface (0 time point, indicating an initial adsorption) and then after 5, 10, 15, and 20 min. All samples were allowed to equilibrate for 20 min because the surface tension of all experimental groups reached its lowest value after 15 min and remained constant thereafter. In pilot experiments, we allowed samples from all experimental groups to stabilize for up to 45 min. We observed no changes in surface tension after 15–20 min. The surface tension measurement at 20 min represented the equilibrium surface tension (STeq). Thereafter, for the samples studied at 20°C, dynamic compression-expansion cycling began, and surface tension was measured continuously. All films were compressed past the collapse point, which we define as the point at which STmin is achieved (22, 36).
For each sample, we recorded three to five successive compression-expansion cycles. To standardize data collection, we used the 1st, 2nd, and 3rd cycles for statistical analyses. Values for STeq and surface tension at minimal SA, STmin, were obtained, and the percent SA compression required to achieve STmin from the theoretical equilibrium point of 25 mN/m was calculated. For each sample, we generated an individual surface tension-area isotherm (data not shown) by using surface tension values at every 10% change in relative area and also at 25% on both compression and expansion limbs of the 2nd cycle. We then generated a characteristic surface tension-area isotherm for each experimental group by using the averaged data (4–6 animals) (see Figs. 2 and 3).
CBS. A CBS was used for surface activity measurements at 37°C for surfactant obtained from warm-active and 8-h torpid dunnarts (3 or 4 animals in each group). The design of the CBS has been previously described (26, 27). Before the surface activity measurements, freeze-dried surfactant samples were reconstitiuted to a final lipid concentration of 100 μg/ml in 0.15 M NaCl containing 1.5 mM CaCl2 (pH 6.9). Samples were placed into the small sample chamber of the CBS (200 μl), warmed to 37°C, and continually stirred for 20–25 min. After 5 min of incubation at 37°C, an air bubble with a diameter of 2–3 mm was introduced into the chamber. The moment the bubble assumed its resting shape was considered time 0 and indicated the initial adsorption. The bubble remained undisturbed for 20 min, and surface tension was calculated at 5, 10, 15, and 20 min of adsorption. Surface tension after 20 min was regarded as STeq. Thereafter, the bubble was compressed stepwise until it reached STmin (that point where further compression does not result in a further reduction in ST) and then expanded stepwise to the original volume. Each hemicycle lasted ∼1–2 min, depending on the STmin that could be reached, and the surfactant film was allowed to stabilize for ∼8 s after each step. We performed four to five compression-expansion cycles on each bubble with 5 min of equilibration time between cycles. The bubble was monitored continuously using a video system so the surface tension, area, and volume of the bubble were calculated from digitized images (26). Where possible, samples were measured in duplicate. Bubble SA corresponding to the surface tension of 25 mN/m was regarded as 100% area, and surface tension values at each 10% change of area during compression and expansion were used to generate a surface tension-area isotherm for the 1st, 2nd, and 3rd cycles (data not shown). The average surface tension values were then used to generate a characteristic curve for each experimental group (see Fig. 5). We also calculated the %SA compression required to achieve STmin from 25 mN/m of the 1st, 2nd, and 3rd compression-expansion cycles.
Statistical analyses of data (surface tension at 0, 5, 10, and 15 min of adsorption, STeq, STmin, and %SA compression from the 1st, 2nd, and 3rd compression-expansion cycles) between the different experimental groups were performed by using a one-way analysis of variance followed by post hoct-tests.
Surface Properties of Mouse Surfactant and Warm-Active Dunnart Surfactant (Measured in WLB at 20°C)
All the measured surface properties, i.e., the adsorption rate and STeq, STmin, and %SA compression required to achieve STmin were identical in mice and warm-active dunnarts (Table1, Fig. 1). The shapes of the surface tension-area isotherms of the surfactant from both species appeared relatively similar (Fig.2).
Effect of Torpor on the Surface Properties of Dunnart Surfactant
Surface properties measured in the WLB at 20°C.
ADSORPTION RATE AND STEQ.
The behavior of surfactant films changed during torpor. Surfactant samples from the 4-h torpor group adsorbed significantly faster onto an air-liquid interface than those of warm-active dunnarts at all time points studied (0 min: t = 3.888, df = 8,P = 0.002; 5 min:t = 2.862, df = 8,P = 0.01; 10 min:t = 1.851, df = 10,P = 0.047; 15 min:t = 2.011, df = 10,P = 0.036) (Fig. 1). STeq (i.e., adsorption rate after 20 min) was also less after 4 h of torpor (t = 2.071, df = 10,P = 0.032) when compared with warm-active dunnarts, although it remained within the normal range and did not differ from STeq of mouse surfactant (t = 0.545, df = 8, P = 0.300) (Fig. 1). After 8 h of torpor, surfactant adsorption was significantly slower at all time points when compared with either the warm-active, 4-h torpor, or mouse groups (Fig. 1). This resulted in a significantly higher STeq compared with the warm-active group (t = 8.824, df = 10,P = 2 × 10−6) as well as the 4-h torpor group (t = −8.605, df = 10, P = 3 × 10−6) (Fig. 1).
STMIN AND %SA COMPRESSION.
STmin decreased after both 4 h of torpor (t = 3.13, df = 8,P = 0.007) and 8 h of torpor (t = 2.887, df = 8,P = 0.01) to the same extent (Table1). As the three surface tension-area isotherms of the three experimental groups had a different initial surface tension (i.e., STeq), compression began at a different point. Hence, the %SA compression required to achieve STmin was measured from 25 mN/m (theoretical equilibrium point) rather than from STmax at 100% relative area. The %SA compression required to achieve STmin calculated from the 1st, 2nd, and 3rd cycles decreased after both 4 and 8 h of torpor when compared with the warm-active group (1st cycle after 4 h:t = 2.578, df = 8,P = 0.002; after 8 h:t = 4.747, df = 8,P = 0.0007; 2nd cycle after 4 h:t = 5.211, df = 8,P = 0.0004; after 8 h:t = 12.20, df = 8,P = 9.5 × 10−7; 3rd cycle after 4 h:t = 2.264, df = 8,P = 0.027; after 8 h:t = 4.382, df = 8,P = 0.001) (Table 1). The %SA compression was significantly greater in the 1st cycle than in the 2nd or 3rd cycles in three of the experimental groups (1st vs. 2nd cycle, mice: t = 7.765, df = 8,P = 3 × 10−5; warm-active:t = 5.495, df = 8,P = 0.0003; 4 h:t = 3.926, df = 8,P = 0.0022; 1st vs. 3rd cycle, mice:t = 4.45, df = 5,P = 0.003; warm-active:t = 2.954, df = 8,P = 0.009; 4 h:t = 2.739, df = 8,P = 0.013) (Table 1). In the 8-h torpor group, this parameter was equally low for each of the three cycles (1st vs. 2nd cycle: t = 1.498, df = 8, P = 0.086; 1st vs. 3rd cycle:t = 1.553, df = 8,P = 0.079) (Table 1).
SURFACE TENSION-AREA ISOTHERMS.
The shape of the compression limb of the surface tension-area isotherms differed between surfactant films from warm-active and torpid dunnarts (Fig. 3). A prominent shoulder (indicated by the relatively horizontal region of the curve) was seen on the compression limb (at ∼70% relative area) (Fig. 3), and a long plateau was seen on the expansion limb of isotherms obtained from warm-active dunnarts and mice (Fig. 2). Both the shoulder and the plateau disappeared in the isotherms recorded after 4 and 8 h of torpor (Fig. 3). The surface tension-area isotherms from warm-active and torpid dunnarts all demonstrated a final collapse plateau at a relative area of 25–20% (Fig. 3).
Surface properties measured in the WLB and CBS at 37°C.
ADSORPTION RATE AND STEQ.
At 37°C in the WLB, adsorption of surfactant form the 8-h torpor group was slower than that of the warm-active group at all time points (0 min: t = −3.22, df = 5,P = 0.012; 5 min:t = −2.57, df = 5,P = 0.025; 10 min:t = −2.17, df = 5,P = 0.041; 15 min:t = −2.178, df = 5,P = 0.041; 20 min:t = −2.178, df = 5,P = 0.041) (Fig.4). However, in the CBS, there was no statistically significant difference between these two groups. There was no statistically significant difference in STeq between surfactants measured at 37°C in the WLB and the CBS (warm-active, WLB vs. CBS:t = −0.396, df = 5,P = 0.354; 8-h torpor, WLB vs. CBS:t = −0.890; df = 5,P = 0.207) (Fig. 4).
Surface properties measured in the CBS at 37°C.
STMIN AND %SA COMPRESSION.
Surfactant from the 8-h torpor group demonstrated a final collapse plateau after reaching a surface tension of ∼9 mN/m. The STmin was significantly higher than that of surfactant from warm-active animals (t = −5.77, df = 27,P = 1 × 10−6) (Fig.5). The %SA compression required to achieve STmin was larger in the 1st cycle than in the 2nd and 3rd cycles in the 8-h torpor group (1st vs. 2nd cycle: t = 4.273, df = 7,P = 0.002; 1st vs. 3rd cycle:t = 2.513, df = 8,P = 0.018) (Table 1). This parameter did not differ between cycles in the warm-active dunnart group (Table 1).
SURFACE TENSION-AREA ISOTHERMS.
A prominent shoulder (indicated by the relatively horizontal region of the curve) was seen on the compression limb (at ∼70% relative area) of the isotherm obtained for surfactant from torpid animals. Such a shoulder was absent on the surface tension-area isotherm representing the behavior of surfactant from warm-active dunnarts at 37°C (Fig.5).
A comparison of surface properties measured at 20°C (with WLB) and 37°C (with CBS). Adsorption of surfactant from both warm-active and 8-h torpor groups measured at 20°C was slower than when measured at 37°C (Figs. 1 and 4). STmin was lower and %SA compression was smaller at 20°C than at 37°C in both the 8-h torpor group and the warm-active group. Surfactant from the 8-h torpor group at 20°C reached a lower STmin at the end of compression (t = 3.939, df = 16,P = 0.0006) and required a smaller change in SA to achieve STmin than surfactant from the warm-active group at 37°C (1st cycle:t = 10.146, df = 8,P = 4 × 10−6; 2nd cycle:t = 32.482, df = 7,P = 3 × 10−9; 3rd cycle:t = 23.919, df = 7,P = 3 × 10−8). There was no significant difference in %SA compression between the 1st, 2nd, and 3rd cycles in either the 8-h torpor group at 20°C or the warm-active group at 37°C. In all the other groups studied at both 20 and 37°C, this parameter was significantly higher in the 1st cycle compared with the 2nd and 3rd cycles (Table 1). Surface tension-area isotherms for the warm-active group measured at 37°C (Fig. 5) and for both 4- and 8-h torpor groups measured at 20°C (Fig. 3) lacked a horizontal plateau on their compression limbs. This plateau was present on the surface tension-area isotherms for the 8-h torpor group measured at 37°C (Fig. 5) and for warm-active dunnart and mouse groups measured at 20°C (Fig. 3).
Validation of Methods
Despite a number of criticisms, the surface WLB remains one of the most popular methods for the determination of surface activity of pulmonary surfactant because it is relatively inexpensive and easy to make. There are a number of different modifications to the standard surface WLB, which together with their various advantages and disadvantages have been widely discussed (3, 10, 21, 25, 27, 35, 36). We chose the instrument with the tight-fitting “dam”- type barrier (as opposed to the “ribbon”-type barrier) because the former yields SA cycling, which is linear with barrier position. The ribbon-type barrier is more cumbersome to work with, as it requires a complex takeup mechanism and does not necessarily give SA changes that are linear with barrier position (35). Furthermore, the dam-type barrier lends itself to easier and more efficient cleaning. The major disadvantage of the surface WLB is the tendency for the surface film to leak between the trough walls and the barrier and to creep up the trough walls and the leading side of the barrier, particularly during compression. We primed the trough and leading side of the barrier with lanthanum and DPPC, as has previously been described (11), and checked the quality of priming by measuring the surface tension of the subphase behind the barrier after compressing either DPPC or surfactant films to the minimum SA (35). The surface tension of the subphase behind the barrier remained at 70–72 mN/m after six to seven cycles.
Values obtained by using the surface balance method are also dependent on the contact angle between the Wilhelmy plate and the surface film (3, 25, 27). Whereas the contact angle is taken to be zero during compression, during film expansion the meniscus is forced to advance over an unclean film-coated plate, and the contact angle often increases, resulting in a reverse hysteresis (where the surface tension on expansion is lower than that on compression). Thorough cleaning of the Wilhelmy plate minimizes changes in the contact angle. Moreover, we periodically checked for the presence of the effects caused by changes in the contact angle by slightly raising the glass coverslip during the course of surface cycling (23). We never observed a reverse hysteresis or unusually low surface tension on barrier withdrawal.
The composition of the aqueous subphase used in the WLB (particularly the ionic composition) can also influence the adsorption, spreading, and other surface properties of surfactant (14). King and Clements (14) found that a surface-active material spreads more effectively over a subphase of Ringer solution than over a distilled water subphase. The calcium content of the subphase will dramatically influence the adsorption rate of surfactant and also mediate the actions of the surfactant proteins (31, 39). However, the composition of the aqueous hypophase in the lung is poorly understood and characterized, and, therefore, the subphase composition varies tremendously between studies. For simplicity and convenience, and because it contains 3 mM calcium, we chose Ringer solution buffered with Trizma buffer to a physiological pH of 7.35.
As the surface-active properties cannot be measured precisely at 37°C in the WLB, we used the CBS to determine the behavior of pulmonary surfactant from warm-active and torpid animals at 37°C. We used a relatively low concentration of PL in surfactant samples in this study (100 μg/ml), because S. crassicaudata is a very small animal (8–13 g) with a correspondingly low amount of total PL in the lavage fluid. However, surfactants with similar and lower concentrations of PL have been studied previously by using the CBS (27). If we assume that samples with a similar STeq had similar amounts of PL at the air-liquid interface before compression-expansion cycling, then we can compare the surface activity of surfactant with the two types of apparatus. In this study, surfactant from the warm-active group and/or the 8-h torpor group measured in the WLB had a similar surface activity as surfactant from those groups measured in the CBS.
To view the surface properties of dunnart surfactant in the context of classical mammalian surfactant properties, we also measured the surface activity of mouse surfactant at 20°C with the WLB. Of all the mammalian species studied to date, mice are the most closely matched to dunnarts in body mass. Under identical experimental conditions, surfactant from mice and warm-active dunnarts exhibited virtually identical surface properties, and our mice values agree closely with values of other mammals in the literature (16, 19, 20, 37).
Effect of Torpor on the Surface Properties of Dunnart Surfactant
Adsorption rate and STeq.
STeq is either a result of spontaneous adsorption of PL from a suspension to an air-liquid interface or a result of the spontaneous spreading of surfactant when applied dropwise to an air-liquid interface (10, 25). The rate of adsorption and spreading is strongly dependent on the chemical composition of pulmonary surfactant and the concentration of PL (10,27). The STeq resulting from an excess of saturated and unsaturated PL spread at the air-liquid interface is ∼25 mN/m (10, 25). In this study, the same amount of PL was always applied to the subphase in the WLB, generating a surface-excess film. STeq of mouse surfactant measured in the WLB or warm-active dunnart surfactant measured in both the WLB and the CBS was similar to the literature values for mammals (25.6, 26.3, and 26.2 mN/m, respectively) (10, 25).
After 4 h of torpor, the rate of adsorption at 20°C was significantly higher and STeq was lower (23 mN/m) than that of the warm-active dunnart group, although it remained within the mammalian range (22–26 mN/m) (36). In contrast, after 8 h of torpor, the adsorption in the WLB at both 20 and 37°C was slower and STeq was significantly higher than in any other group. However, when measured at 37°C in the CBS, the rate of adsorption of surfactant from the 8-h torpor group was similar to that of surfactant from warm-active animals. The difference in the results may be due to the differences in the two methodologies used. For example, in the WLB, surfactant was applied dropwise onto the surface, whereas in the CBS adsorption occured from the subphase. The rate of film formation when surface-active material is applied dropwise onto the surface (as in the WLB) characterizes both the spreadability and the ability of surfactant to adsorb onto the air-liquid interface. Possibly, surfactant from the 8-h torpor group was less spreadable at both 20 and 37°C than surfactant from warm-active animals while its ability to adsorb from the subphase at 37°C was not significantly altered. STeq of surfactant from warm-active and 8-h torpid dunnarts at the end of adsorption at 37°C was not significantly different between WLB and CBS studies.
The changes in surfactant adsorption in the WLB after 4 and 8 h of torpor may be the result of the changes in the lipid components of surfactant that we have demonstrated previously (i.e., an increase in Chol/PL and DSP/PL) (15) and is possibly related to the relative tendencies of the different surfactant components to adsorb to the air-liquid interface. After 4 h of torpor, we found a small increase in Chol/PL (15). The addition of Chol to a PL film will increase its fluidity (23) and, therefore, enhance the adsorption rate (39), which, in turn, may lead to a reduction in the observed STeq. However, after 8 h of torpor, there was a further increase in the Chol/PL ratio together with an increase in the PL saturation (15). The latter change in composition would lead to a reduction in fluidity that could counteract the fluidizing effect of Chol, possibly even resulting in an overall decrease in fluidity and, therefore, in adsorption and spreadability and an increase in STeq of this surfactant, particularly at 20°C. This may explain why at the lower temperature of 20°C the surfactant from 8-h torpid dunnarts, which is enriched in DSP, does not reduce the surface tension very effectively on initial adsorption but is able to reach very low STmin after compression (see below). However, the high Chol content of the surfactant from 8-h torpid dunnarts would increase the rate of adsorption at the higher temperature of 37°C in the CBS and reduce the surface tension-lowering ability, compared with the same surfactant tested at 20°C.
The protein composition of surfactant may also change with torpor, thereby influencing the surface active properties. The three surfactant-associated proteins, SP-A, SP-B, and SP-C, have all been shown to influence the adsorption of surfactant films to different extents (24, 25, 27, 31, 39). Furthermore, the proteins also act synergistically. For example, SP-A alone contributes little to lipid adsorption, but in the presence of both SP-B and SP-C it promotes adsorption considerably (24). Moreover, Yu and Possmayer (39) demonstrated that SP-A increased the STeq of bovine lipid extract surfactant containing an excess of Chol. The appparently conflicting changes in adsorption rate and STeq during torpor (i.e., a decrease after 4 h and an increase after 8 h) could be at least in part due to alterations in the proportions of the surfactant proteins. It would be interesting to determine the effect of torpor on the composition of the surfactant proteins to ascertain this possibility.
The STeq may also be affected by plasma proteins that adsorb more rapidly to the air-liquid interface and have a higher STeq than surfactant PL (12). However, the proteins also reduce the surface tension-lowering properties of the surfactant (8). Considering that the surfactant after 8 h of torpor attained a STmin of 4 mN/m at 20°C, it is unlikely that the high STeq values are a result of contamination with plasma proteins.
STmin and %SA compression.
The ability to lower surface tension under dynamic compression is the most well-known and important property of lung surfactant. A low STmin is required to provide the optimal alveolar area available for gas exchange at low lung volumes (28). In the WLB, it is only possible to recognize “true” STmin when the surfactant film is compressed past the point of film collapse, i.e., where the film has reached its closest packed monolayer structure (22). In the present study, we have defined STmin as the surface tension achieved after compression past the collapse point. Recently, Schürch (27) suggested that, when the surface tension-lowering properties of surfactant films are measured, one of the most sensitive parameters to consider is the change in area compression required to achieve minimum surface tension. The sensitivity of this parameter is demonstrated by the example that small concentrations of serum proteins added to surfactant did not affect STmin, but the area compression required to achieve minimum surface tension was significantly increased (32).
When measured at 20°C, surfactant films of torpid dunnarts achieved a lower STmin than surfactant from warm-active dunnarts either at 20°C in the WLB or at 37°C in the CBS and also demonstrated a substantially smaller film %SA compression from 25 mN/m to STmin.The %SA compression was significantly greater in the 1st cycle than in the 2nd or 3rd cycles in all experimental groups, with the exception of the 8-h torpor group, in which this parameter was low in each of the three cycles. Possibly, surfactant from the 8-h torpor group did not purify further during compression at 20°C.
At 37°C, surfactant from the 8-h torpor group did not achieve STmin below 5 mN/m, and a final collapse plateau appeared at ∼8–9 mN/m. Therefore, the ability of this surfactant to lower surface tension under compression at 37°C was reduced compared with surfactant from warm-active animals. The %SA compression, required to achieve STmin from 25 mN/m at 37°C for surfactant from both experimental groups studied in the CBS, was outside the physiological range, most likely because of the relatively low concentration of PL in surfactant samples (100 μg/ml). However, there was a statistically significant difference in this parameter between warm-active and 8-h torpor groups. The %SA compression required to achieve STmin in the 2nd and 3rd compression-expansion cycles was smaller in the 8-h torpor group compared with the warm-active dunnart group. In the latter group, this parameter remained unchanged in the 1st, 2nd, and 3rd cycles. Therefore, at this concentration, surfactant from warm-active animals did not appear to undergo further purification during compression to achieve low STmin, whereas surfactant from the 8-h torpor group was probably purified further under compression at 37°C.
The changes in chemical composition of surfactant during torpor may cause changes in its surface tension-lowering properties. For example, the increased amount of DSP in 8-h torpid dunnarts, compared with warm-active animals (15), could explain the greater ability of the former to lower surface tension at 20°C. However, the surfactant from 4-h torpid dunnarts demonstrated a reduced STmin at 20°C, without any changes in DSP (15). Furthermore, surfactant from 4- and 8-h torpid animals also contained greater absolute amounts of Chol (15). In vitro, the addition of Chol to a surface-active film reduces the ability of the lipid mixture to lower surface tension (5, 34, 39), although DPPC surface-excess films containing as much as 10 mol% Chol are still capable of generating STmin of <1 mN/m at 23°C (23). Furthermore, whereas the addition of Chol to reconstituted lipid-protein complexes significantly reduces surface activity, it does not influence the surface activity of fresh surfactant measured at room temperature (34). Yu and Possmayer (39) also demonstrated that the addition of 10% by weight of Chol only partially impaired the surface activity of bovine lipid extract surfactant. It is possible that SP-C or some minor PL components may contribute to the regulation of adsorption of Chol (39). Hence, it is often difficult to relate the behavior of a film of natural surfactant to that of an artificial lipid mixture with a similar molar ratio of Chol and DSP. The three-dimensional arrangement of the lipid molecules in a surfactant film will almost certainly differ from that of an artificial lipid mixture, which may influence the accessibility of different lipids to the interface. A further possibility for the observed decrease in STmin (and the %SA compression required to achieve it) at 20°C is that the change in the DSP/PI ratio with torpor (15) may alter the surface activity. The preferential “squeeze-out” of the unsaturated PL is especially effective in both 9:1 and 7:3 DPPC/PI mixtures, forming an almost pure, highly stable DPPC monolayer at the end of compression (7).
At 37°C, surfactant from 8-h torpid dunnarts was less surface active than surfactant from warm-active animals at 37°C, possibly because of the difference in the phase-transition temperatures of these two surfactants. It is likely that surfactant from 8-h torpid dunnarts has a lower phase-transition temperature than that from warm-active animals as it contains more Chol and PI and, therefore, may be too fluid to be equally active at 37°C.
In addition, it is possible that changes in surfactant-associated proteins during torpor may alter the surface tension-reducing ability of the different surfactants. For example, SP-B has been shown to increase the ability of surfactant to reduce surface tension, possibly by enhancing the squeeze-out of unsaturated PLs from the monolayer, resulting in an enrichment in DPPC (24, 25). This function of SP-B would also have the effect of reducing the area compression required to achieve STmin. It would be interesting to investigate whether the function of surfactant proteins changes with decreasing temperature in vitro.
Surface tension-area isotherms. The overall shapes of the surface tension-area isotherms, obtained at both 20 and 37°C, differed between the experimental groups. At 20°C, however, it appears that the differences between the expansion limbs are predominantly due to the differences in the ability of the three surfactants to adsorb (STeq) and respread from the collapse phase. This means that each of the three surface tension-area isotherms has a different initial surface tension that precedes the following compression. Consequently, the differences between the compression limbs could be due to the different starting points. However, once film compression has reached the theoretical STeq (∼25 mN/m), differences between the compression limbs are unlikely to be solely influenced by the initial surface tension. Hence, a comparison between the curves below ∼25 mN/m may be more meaningful. It is at this point that the compression limb of mouse and warm-active dunnart surface tension-area isotherms, measured at 20°C, demonstrated a prominent shoulder, which corresponded to a relative area of ∼70%. Neither the 4-h nor the 8-h torpor isotherms demonstrated such a shoulder at 20°C. In contrast, at 37°C, a similar shoulder was visible on the 8-h torpor isotherm and was absent on the isotherm from warm-active animals.
In surface tension-area isotherms of pure DPPC, a shoulder on the compression limb corresponds to a change from the liquid-expanded state where the acyl chains are highly mobile to a liquid-condensed state where they are more restricted. Further compression eventually results in a solid-gel form at STmin, which is represented on the compression limb as the final collapse plateau (25). However, in compressed films of whole lung surfactant, the shoulder probably represents a process of squeeze-out of other lipids such as Chol and unsaturated PL (10, 11). In surfactant from torpid dunnarts at 20°C and in surfactant from warm-active animals at 37°C, which both lacked a shoulder, the squeeze-out of other lipids may occur continuously throughout compression with a smooth transition from the liquid-expanded to the liquid-condensed state, resulting ultimately in a DSP-enriched film, which may be in the solid-gel state. Hildebran et al. (11) demonstrated that the shoulder visible on pure DPPC films was attenuated by the addition of Chol and unsaturated PLs. Schürch et al. (32) observed a smooth compression limb (i.e., lacking a squeeze-out plateau) when surfactant films were compressed only to STmin without reaching the collapse phase. It is possible that the lack of a squeeze-out plateau indicates that the film is already enriched in DSP (32). If the surfactant film from torpid dunnarts at 20°C or warm-active dunnarts at 37°C is already highly enriched in DSP at equilibrium, then further purification may not occur during compression. This supports our previous observations (see above) that %SA compression in the 1st, 2nd, and 3rd cycles of 8-h torpid dunnarts at 20°C remained unchanged, which may also indicate that no further film purification occurs. Similarly, the %SA compression of surfactant from warm-active dunnarts measured at 37°C did not differ between the 1st, 2nd, and 3rd cycles, again supporting the finding that there was no shoulder on the surface tension-area isotherm, possibly indicating that no further film purification occurs on compression.
It appears that the increased Chol content of surfactant from 8-h torpid dunnarts is not contributing to the overall adsorption of the surfactant. However, since both DSP and Chol by themselves spread poorly, it is possible that the two lipids are acting synergistically to aid in their spreading. For example, Yu and Possmayer (39) demonstrated that Chol by itself could not adsorb onto the air-water interface but could be transported to the interface by PL, most probably in association with DPPC. Similarly, suspensions of pure DPPC adsorb very poorly (10). Because the addition of Chol and/or unsaturated PLs to monolayers of DPPC enhances the respreading after compression past monolayer collapse (23), it is highly likely that they may also enhance the initial adsorption of DPPC monolayers. Hence, it is possible that the predominant role of Chol in film adsorption is to enhance the adsorption of DSP. Therefore, the changes in the proportions of Chol, unsaturated PL, and DSP (and, possibly, the surfactant proteins) in surfactant after 8 h of torpor may result in a selective adsorption of DSP to the air-liquid interface in preference to other lipids at 20°C but not at 37°C.
Functional Significance of Changes in Surfactant
The duration of torpor significantly affected the lipid composition of pulmonary surfactant (15). After 1 h of torpor, only the absolute amounts of alveolar surfactant increased. After 4 h of torpor, there were minor changes in surfactant lipid composition. The most significant compositional modifications occurred after 8 h of torpor. In the present study, we determined that changes in surface properties correlated with those that occurred in the lipid composition of surfactant during the period of torpor, with the most significant changes occurring after 8 h. Surface tension-lowering ability of dunnart surfactant after 8 h of torpor and measured at 20°C was greater than that of surfactant from warm-active dunnarts at both 20 and 37°C. Langman et al. (15) found a significant decrease in the static compliance of the tissue after 1 and 4 h into torpor, which was abolished by 8 h of torpor. However, this change was not reflected in the overall static lung compliance (measured in the presence of surfactant) (15). It appears, therefore, that the changes in the composition and the surface properties of surfactant, particularly by 8 h of torpor, do not relate to the traditional function of surfactant, which is to increase lung compliance. It is possible that the physiological significance of the changes observed in surfactant composition and surface properties is related to other aspects of the respiratory system that may change during periods of torpor. For example, in some hibernators, the breathing pattern is characterized by low tidal volumes (18).
The present study demonstrated that the surfactant of torpid dunnarts can attain lower surface tensions at the end of expiration when measured at 20°C. Using morphometric analysis and surface tension measurements, Bachofen et al. (2) demonstrated that at a surface tension of 6 mN/m the alveolar SA was 16% smaller than at a surface tension of 1 mN/m. Hence, a small decrease in surface tension can result in a relatively larger increase in alveolar SA available for gas exchange. Therefore, the lower STmin of surfactant from torpid dunnarts may provide a relatively greater alveolar SA available for gas exchange at low lung volumes. Moreover, the surfactant of torpid dunnarts required a smaller change in area compression to attain STmin, compared with surfactant from warm-active dunnarts at either 20 or 37°C. This property of surfactant may also contribute to the stabilization of the optimal alveolar SA available for gas exchange at low tidal volumes, when the extent of area compression is reduced.
Because the surfactant from 8-h torpid dunnarts did not appear to require further film purification on compression when measured at 20°C (unlike the measurements at 37°C), this may imply that the surfactant isolated from torpid dunnarts is more suited to function at relatively low temperatures. Conversely, the surfactant isolated from warm-active dunnarts appeared to be more suited to function at the higher temperature of 37°C.
In conclusion, the changes in surface activity of pulmonary surfactant in the heterothermic mammal S. crassicaudata correlate with changes in surfactant lipid composition that occur during torpor. We found that surfactant from torpid dunnarts had a greater surface tension-lowering ability when measured at 20 than at 37°C. Moreover, we found that the surfactant from 8-h torpid dunnarts measured at 37°C had a reduced surface activity when compared with surfactant from warm-active dunnarts measured at the same temperature. These findings would imply that the alterations in surfactant composition and activity during torpor represent an adaptation to the greatly reduced body temperatures of torpid dunnarts. Furthermore, the compositional alterations during torpor appear to render the surfactant less functional at 37°C. Therefore, further studies are needed to clarify the following: How do cold dunnarts respond to extremely rapid (15–20 min) arousals from torpor? How rapidly and by which mechanisms does the surfactant from arousing dunnarts assume the warm-active profile?
The authors are grateful to Carly Langman and Phil Wood for assisting with different aspects of the project. We also thank Brian Purdell, Dennis Pfeiffer, and Barry Andrew of the Medical School Workshop (University of Adelaide, Australia) for building the Wilhelmy-Langmuir surface balance. We are particularly grateful to Prof. Samuel Schürch for providing the captive-bubble surfactometer as well as expertise and laboratory facilities for the present study.
Address for reprint requests: S. Orgeig, Dept. of Physiology, University of Adelaide, Adelaide, South Australia 5005, Australia.
This research was funded by an Australian Research Council (ARC) Postdoctoral Research Fellowship to S. Orgeig and by ARC and Faculty of Medicine (University of Adelaide) grants to S. Orgeig and C. B. Daniels.
- Copyright © 1998 the American Physiological Society