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Alterations in surface activity of pulmonary surfactant in Gould’s wattled bat during rapid arousal from torpor
BBRC Biochemical and Biophysical Research Communications 308 (2003) 463–468 www.elsevier.com/locate/ybbrc
Alterations in surface activity of pulmonary surfactant in Gould’s wattled bat during rapid arousal from torpor Jonathan R. Codd,a,1 Sandra Orgeig,a,* Christopher B. Daniels,a and Samuel Sch€ urchb b
a Department of Environmental Biology, Adelaide University, Adelaide, SA 5005, Australia Respiratory Research Group, Department of Physiology and Biophysics, University of Calgary, Health Sciences Centre, Calgary, Canada, Alb. T2N 4N1
Received 7 July 2003
Abstract The small microchiropteran bat, Chalinolobus gouldii, undergoes large daily fluctuations in metabolic rate, body temperature, and breathing pattern. These alterations are accompanied by changes in surfactant composition, predominantly an increase in cholesterol relative to phospholipid during torpor. Furthermore, the surface activity changes, such that the surfactant functions most effectively at that temperature which matches the animal’s activity state. Here, we examine the surface activity of surfactant from bats during arousal from torpor. Bats were housed at 24 °C on an 8:16 h light:dark cycle and their surfactant was collected during arousal (28 < Tb < 32 °C). Surface activity was examined with a Captive Bubble Surfactometer at 24 and 37 °C. Surfactant from arousing bats was more active at 37 °C than at 24 °C, indicated by a lower STmin and reduced film area compression required to reach STmin . It appears that the arousal-induced changes in surfactant composition, i.e., lower levels of cholesterol, inhibit adsorption of surfactant at 24 °C. Furthermore, the alterations in surfactant composition during arousal are very rapid, such that the mixture behaves more like surfactant from warm-active bats, and therefore, functions more effectively at 37 °C. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Surfactant; Arousal; Torpor; Bats; Cholesterol
Many small mammals and birds regularly enter periods of torpor, which are characterised by a reduction in body temperature and metabolic rate. Torpor is thought to have evolved primarily as an energy saving strategy. The entry into and maintenance of torpor are accompanied by numerous biochemical and physiological changes. Alterations in the pulmonary surfactant system in response to torpor have previously been demonstrated in a marsupial, the fat-tailed dunnart, Sminthopsis crassicaudata [1]. Pulmonary surfactant lines the inner surface of the lung where it regulates the surface tension at the air–liquid interface with changing lung volume. This system consists of lipids and proteins and is sensitive to changes *
Corresponding author. Fax: +61-8-8303-4364. E-mail address: [email protected] (S. Orgeig). 1 Present address: Apt. Morphologie und Systematik, Universit€at Bonn, Institut f€ ur Zoologie, Poppelsdorfer Schloss, Bonn 53115, Germany. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01427-X
in body temperature [2]. Temperature-associated changes in surfactant cholesterol have previously been demonstrated in the Central Australian lizard, Ctenophorous nuchalis [3], and cold acclimation of map turtles has also been demonstrated to increase the level of unsaturated phospholipids [4]. Adding cholesterol (Chol) and unsaturated phospholipids (USP) to pulmonary surfactant is thought to reduce the phase transition temperature of the surfactant mixture, which consists of up to 80% of the disaturated phospholipid (DSP), dipalmitoylphosphatidylcholine (DPPC). Vesicles of pure DPPC, below the gel-to-liquid crystalline phase transition of 41 °C, form a surfactant monolayer extremely slowly. In addition, DPPC aggregates at the air–liquid interface, which may be formed upon film compression and remain associated with the monolayer, are unable to spread across the respiratory surface as the alveolus expands during inflation [5,6]. However, DPPC is primarily responsible for the surface tension lowering ability of surfactant. DPPC molecules produce near zero
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surface tensions upon film compression by excluding water and by achieving a tightly packed highly ordered state at the air–liquid interface. Therefore, DPPC has to be transported to, and accumulate at, the interface. The reduction in phase transition temperature by the addition of cholesterol and USPs improves the spreadability of the surfactant mixture [6]. Hence, the surface activity of a surfactant is essentially its ability to lower surface tension upon film compression, which can be quantified by three different parameters: the rate of film formation by adsorption from the subphase, the minimum surface tension (STmin ) achieved upon film compression, and the amount of film area compression (% SA compression) required to achieve the minimum surface tension from equilibrium surface tension (STeq ) at the end of adsorption [7]. In the fat-tailed dunnart, changes in surfactant composition during torpor correlate with changes in surface activity. After 4 h of torpor, when measured at 20 °C, the surfactant demonstrates an increased rate of adsorption and decreases in STeq , STmin , and the % SA compression, compared with the surfactant isolated from warm-active dunnarts [8]. After 8 h of torpor there is a further decrease in STmin , rate of adsorption, and STeq . Hence, surfactant from torpid animals is more active at 20 °C than it is at 37 °C when compared with warm-active surfactant. Therefore, in vitro changes in surface activity correlate with changes in lipid composition, in S. crassicaudata, and may be required to optimise the alveolar surface area available for gas exchange at very low lung volumes [8,9]. Recently, we have performed similar experiments on the small microchiropteran bat, Chalinolobus gouldii. Unlike the dunnart which periodically enters torpor under conditions of cold-stress or food-shortage, the bat regularly enters into daily torpor at a range of ambient temperatures even when food is plentiful [10]. During the natural daily activity cycle of these bats at a constant ambient temperature of 24 °C, alterations in the pulmonary surfactant system occur. However, the amounts of phospholipid (PL) and DSP per gram dry lung do not change during torpor [10]. Upon arousal from torpor the amount of PL increases transiently, which is thought to represent an adrenergic surge related to behavioural changes such as arousal, grooming, jostling for roost sites, and preparing to fly and feed [10]. Relative to both PL and DSP, the amount of cholesterol increases during torpor in C. gouldii [10], resulting in an increase in the Chol/PL and Chol/DSP ratios. Furthermore, we demonstrated that surfactant isolated from bats was more surface-active at the temperature that matched the body temperature and activity status of the animal [10]. These alterations in the surface-active properties of surfactant appeared to correlate with fluctuations in surfactant composition, most notably surfactant cholesterol [10].
Periodic arousal from torpor is typical of all animals that enter into periods of torpor or hibernation. Arousal from torpor involves a rapid increase in metabolic rate and breathing rate, with temperature increases of up to 0.8 °C/min recorded for the little brown bat Eptesicus fuscus [11]. In the fat-tailed dunnart, arousal from torpor, induced by physically handling the torpid animals [9], resulted in temperature increases of 15–30 °C at the rate of 1 °C/min. Arousal was accompanied by rapid decreases in cholesterol within 5 min of reaching 30 °C and this change in composition correlated with a rapid decrease in STmin [9]. Hence, rapid arousal was accompanied by both changes in surfactant composition and changes in surface activity. Here, we examine the surface activity of surfactant from the microchiropteran bat, C. gouldii, during spontaneous arousal from torpor. We have previously published the corresponding changes in surfactant composition, which occur during warm-active and torpid states from the same samples of this species of bat [10]. We hypothesise that changes in surfactant activity occur so rapidly that surfactant from arousing bats will be more surface-active at warm-active body temperatures (37 °C) than at torpid body temperatures (24 °C).
Materials and methods Animals. C. gouldii are widespread and common across much of Australia. The bats used in this study were trapped, using harp traps, from February to May 1999 in the Flinders Ranges, South Australia. Bats were kept at a fixed ambient temperature of 24 °C and fed mealworms (Tenebrio molitor) and crickets (Acheta domestica) ad libitum. Water was supplied ad libitum. During a two week acclimatisation period, before experimentation began, body mass did not alter significantly. A total of 15 adult male C. gouldii were used. Body mass, forearm, and foot measurements were recorded just prior to experimentation. For C. gouldii, body mass ranged from 8.17 to 9.65 g (means SE; 9.3 0.25 g); mean forearm length was 43.65 0.18 mm; and mean foot length was 7.65 0.09 mm. Five bats were sampled during arousal from torpor and were assigned to this group on the basis of body temperature and activity status. Bats that were arousing from torpor had a body temperature of 28–32 °C [10]. Lavage procedure. Animals were killed with an intraperitoneal overdose of sodium pentobarbitone (0.1 ml, 325 mg/ml). A thermocouple probe was used to measure rectal Tb immediately. The trachea was exposed and cannulated and lungs were then rinsed (lavaged) with three 1.5-ml volumes of ice-cold saline (0.15 M NaCl), each volume instilled and withdrawn three times. Over 90% of the surfactant in the lung is removed using this method [1]. Sample preparation. The lavage was centrifuged for 5 min at 150g to pellet and remove any macrophages (Beckman model TJ-6). The supernatant was then centrifuged at 40,000g (J2 series Beckman Highspeed centrifuge) for 30 min and the pellet reconstituted to a concentration of 10 mg/ml phospholipid in a buffered salt solution (140 mM NaCl, 10 mM Hepes, and 2.5 mM CaCl2 , pH 6.9). Samples were then freeze-dried before being finally reconstituted to a concentration of 20 mg/ml phospholipid with distilled water for experiments on the Captive Bubble Surfactometer (CBS). Surface activity. The surface tension, area, and volume of a bubble, with a surfactant film at the air–water interface, were determined using
J.R. Codd et al. / Biochemical and Biophysical Research Communications 308 (2003) 463–468 45
40
* *
ST (mN/m)
a CBS. The volume of the bubble is controlled, by varying the pressure in the chamber. As the chamber volume is reduced, by moving the chamber base and the chamber up, relative to the stationary piston, the surface area reduces and the surface tension of the surfactant film at the bubble surface falls. Alterations in the surface tension at the air– liquid interface in the CBS are visualised by a change in the bubble shape from spherical to more oval as the surface tension falls [12]. We used a new spreading technique [13] to examine the surface activity of high concentration/low volume samples. A buffered salt solution (140 mM NaCl, 10 mM Hepes, and 2.5 mM CaCl2 , pH 6.9) containing 10% (by mass) sucrose, to increase the density of the salt solution to approximately 1.04 g/ml above that of the surfactant (1.01 g/ml), was used to fill the sample chamber on the CBS. Then 2 ll of 20 mg/ml phospholipid was injected into the chamber. A small bubble, 2–3 mm in diameter, was created, by winding the chamber base down, to draw in air. Adsorption was measured after the bubble had come to rest and assumed an equilibrium shape [14]. A Sony EV-S5000 NTSC video recorder and Pulnix TM 7 CN camera were used to continuously record the bubble. From digitised images, bubble volume, area, and surface tension were calculated using height and diameter [15]. Adsorption. The new spreading technique results in rapid adsorption [13]. The bubble comes to rest at the chamber ceiling, makes contact with the surfactant, assumes an equilibrium shape, and adsorption starts immediately (time zero). Adsorption was measured from time 0 to 1 min (data are presented for the first 10 s). Quasi-static and dynamic compressions. The original bubble, 2–3 mm in diameter, was expanded to 7–8 mm in diameter in approximately 0.1 s. After this rapid expansion the surfactant films were always reproducible, in that the same equilibrium surface tension was always achieved [13]. Below surface tensions of 20 mN/m, film compressibilities and the minimum surface tensions upon the first quasi-static compression were equal ( 1 mN/m). Quasi-static cycling commenced after a 5 min delay at maximum surface tension. The bubble volume was reduced by 5% at each step, by always taking the volume of the previous step as 100%. To avoid overcompression, bubble compression ceased as soon as the first ‘bubble click’ was observed [13,16,17]. There was a 5 min inter-cycle delay between each of the four quasi-static cycles. Dynamic cycling was conducted after a further 5 min delay at maximum bubble volume. Samples were subjected to 25 dynamic cycles at 24 and 37 °C. Two to three independent experiments were conducted with each sample on the CBS at each temperature setting. The starting temperature was alternated for successive measurements, i.e., for samples started at 37 °C, the following measurements were performed at 24 °C using the same chamber filling. For samples started at 24 °C, the following measurements were performed at 37 °C and so on. A combination of water and methanol was used to clean out the chamber between each filling. Statistical analysis. Data are presented as means SEM where applicable. Within group comparisons were made using a one-way ANOVA followed by a Student–Newman–Keuls test for post hoc between group comparisons.
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35
* *
30 * *
*
25
20 0
1
2
3
4
5
6
7
8
9
10
Time (seconds) Fig. 1. Time course for adsorption of surfactant collected from bats arousing from torpor, at a Tb between 28 and 32 °C, examined at both 37 and 24 °C on the CBS. Symbols indicate points that are significantly different at p < 0:05, n ¼ 4. Solid line ¼ 37 °C, Dashed line ¼ 24 °C.
The adsorption of surfactant from arousing bats was significantly faster at 37 °C than at 24 °C, indicated by a significantly lower STeq (37 °C: 26.79 0.80 mN/m, 24 °C: 37.19 0.55 mN/m, p ¼ 4:06 105 , n ¼ 4, Fig. 1). At 37 °C, surfactant from arousing bats adsorbed
A
B
Results Adsorption Upon film formation by spreading and adsorption, an equilibrium surface tension (STeq ) of 23–25 mN/m was reached in less than 10 s. Adsorption of surfactant from bats that were arousing from torpor was examined at both 24 and 37 °C. Temperature had a significant effect on the adsorption rate of bat surfactant.
Fig. 2. Quasi-static surface tension–area relations for surfactant collected from arousing bats, at 28 6 Tb 6 32 °C, examined at (A) 37 °C and (B) 24 °C. Sample concentration was 20 mg/ml in both cases, n ¼ 11, Solid line, quasi-static (QS-1) and dashed line, QS-4.
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significantly faster at all time points than the same sample examined at 24 °C (p < 0:05, n ¼ 4, for all time points examined, Fig. 1). Quasi-static cycles The film area compression required to reach minimum surface tension (STmin ) from equilibrium values was determined for surfactant collected from bats that were arousing from torpor at both 24 and 37 °C. Temperature had a significant effect on both the STmin achieved and the area compression required to reach STmin . Surfactant isolated from arousing bats required significantly less area compression on the first quasi-static cycle (Q1) to reach an STmin of 1 mN/m when examined at 37 °C than when the same preparation was examined at 24 °C (37 °C: 17.9 2.8%, 24 °C: 25.4 2.9%, p ¼ 0:05, n ¼ 8, Figs. 2A and B). The STmax reached on
A
35 30
Q1 was also significantly higher at 24 °C than at 37 °C (37 °C: 24.52 0.81 mN/m, 24 °C: 32.67 0.70 mN/m, p ¼ 1:3 107 , n ¼ 11, Figs. 2A and B). The STmin reached on Q1 was significantly lower at 37 °C than at 24 °C (37 °C: 1.67 0.11 mN/m, 24 °C: 2.97 0.14 mN/ m, p ¼ 2:1 107 , n ¼ 11, Figs. 2A and B). There was no significant difference in the area compression required to reach STmin on the fourth quasi-static cycle (Q4). However, the minimum ST was still significantly lower on Q4 at 37 °C than at 24 °C (37 °C: 1.57 0.099 mN/m, 24 °C: 2.99 0.080 mN/m, p ¼ 2:65 1010 , n ¼ 11, Figs. 2A and B). Dynamic cycles Surfactant collected from arousing bats and dynamically cycled at 37 °C reached a significantly lower STmin than the same sample examined at 24 °C (37 °C: 1.77 0.11 mN/m, 24 °C: 2.16 0.12 mN/m, p ¼ 0.02, n ¼ 6, Figs. 3A and B). The maximum ST reached on dynamic cycling was significantly higher at 24 °C than at 37 °C (24 °C: 28.56 0.21 mN/m, 37 °C: 26.51 0.19 mN/m, p ¼ 1:57 105 , n ¼ 6, Figs. 3A and B). However, there was no significant difference in the area compression required to reach STmin at either temperature.
ST (mN/m)
25
Discussion
20 15 10 5 0 0.86
0.88
0.90
0.92
0.94
0.96
0.98
1.00
1.02
Relative Area
B
35 30
ST (mN/m)
25 20 15 10 5 0 0.86
0.88
0.90
0.92
0.94
0.96
0.98
1.00
1.02
Relative Area Fig. 3. Surface tension–area relation for surfactant isolated from arousing bats examined at (A) 37 °C and (B) 24 °C, and dynamically cycled at 25 cycles/min. Typical results are presented for 1 bat and three consecutive cycles (9, 10, and 11) centred around the 10th cycle.
Surfactant isolated from bats that were arousing from torpor, when examined at 37 °C, demonstrated equilibrium surface tensions of 25 mN/m and minimum surface tensions of 1 mN/m. Similarly, surfactant from arousing bats demonstrated significantly faster adsorption at 37 °C than the same surfactant examined at 24 °C. Surfactant from arousing bats functions optimally at 37 °C, as indicated by a lower STmin and reduced area compression required to reach STmin . It appears that the transition in the surfactant biochemistry that occurs during arousal is very rapid, and has a dynamic effect on surface activity, such that the mixture functions more effectively at the rapidly increasing body temperature. We have previously demonstrated that although absolute amounts of cholesterol increased initially and transiently upon arousal in C. gouldii [10], absolute amounts of phospholipid also increased at the same time points, such that the relative amount of cholesterol (i.e., Chol/PL) was decreasing throughout the arousal period towards warm-active levels, such that the lipid composition was significantly different at the end of the arousal period. Hence, when compared with animals that were in torpor there was significantly less cholesterol relative to PL (i.e., lower Chol/PL) present in surfactant from warm-active bats [10,13]. As the surfactant from arousing animals was less active at 24 °C than 37 °C, it
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appears that the arousal-induced changes in surfactant composition, i.e., significantly lower levels of cholesterol are inhibiting adsorption of surfactant at the lower temperature. This suggests that above a certain temperature, high levels of cholesterol are no longer required as fluidising components. In the case of C. gouldii, this critical temperature appears to be approximately 28 °C, which was the lowest body temperature of this group of arousing bats. Hence, the surfactant isolated from bats at a Tb > 28 °C appears to function equally well at 37 °C, as surfactant isolated from fully active bats. It appears that throughout their daily torpor–arousal–activity cycles, bats are capable of precisely regulating the concentration of surfactant cholesterol, such that the isolated surfactant functions optimally only at the temperature, which matches the body temperature of the animal. Upon arousal from torpor there appears to be a crucial transition, in terms of cholesterol composition, back to the warm-active state—which, once achieved, reproduces performance at warm-active levels. The increase in cholesterol during torpor in the fattailed dunnart, S. crassicaudata [1], is thought to influence the adsorption and surface activity during torpor [8]. Hence, in the fat-tailed dunnart, changes in surfactant composition correlate with alterations in surface activity, such that surfactant from torpid animals is more active at 20 °C than at 37 °C. Furthermore, upon arousal from torpor in the fat-tailed dunnart, cholesterol levels are immediately reduced to warm-active levels and are significantly lower than the level of cholesterol in torpid animals [9]. These changes are reflected in the Chol/PL and Chol/DSP ratios, which both decrease to warm-active levels as rapidly as 5 min after arousal [9]. These rapid alterations in surfactant cholesterol composition correlate with an immediate improvement in the surface activity of surfactant from the fat-tailed dunnart, when examined at 37 °C [9]. Surfactant isolated from arousing bats behaved more like surfactant isolated from warm-active bats [13], consistent with the return of cholesterol levels to warmactive levels. Hence, surfactant function is maintained despite large fluctuations in body temperature, as alterations in lipid composition appear to maximise surface activity. However, it is not possible to attribute all the observed changes in surface activity to the observed changes in cholesterol. The levels of surfactant cholesterol found in C. gouldii are around six times lower than those in other mammals [10]. These low levels of cholesterol may reflect important differences in the regulation and/or function of surfactant relative to other mammals. Other surfactant components may possibly be involved in regulating surfactant fluidity and surface activity. A number of different surfactant components are known to enhance the adsorption rate, and therefore the spreadability, of surfactant. The addition of fluid
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acidic phospholipids to surfactant enhances its adsorption rate, as does the addition of mono-/di-, and tri-acylglycerol [6]. However, altering the proportion of these molecules may also alter the surface activity of the surfactant. The surfactant proteins, on the other hand, do not affect surface activity per se, but they do enhance the adsorption rate by interacting with the phospholipids [6]. Reconstitution studies have also demonstrated that the most surface-active preparations do not contain cholesterol, but rather contain relatively large amounts of surfactant proteins [6]. The two small surfactant proteins SP-B and SP-C play important roles in maintaining the surface-active film and in fluidising the mixture [18]. Future studies examining any alterations in the amounts of the four surfactant-associated proteins may shed further light on the control of surfactant fluidity during torpor. In summary, C. gouldii cycle daily through periods of torpor, arousal, and activity. The large decreases in body temperature, which occur during daily torpor have a marked effect on surfactant composition and function. In C. gouldii, an increase in alveolar PL and DSP during arousal may serve to counteract the stiffness of the lung tissues due to the reduced temperatures of torpor. The most notable alteration in surfactant composition is the increased level of cholesterol associated with torpor, which is thought to enhance the fluidity of the mixture. The alterations in lipid composition with changes in body temperature correlate with changes in the surfaceactive properties and may serve to maximise the surface tension lowering ability of surfactant. The results from this study indicate that bats, which are arousing from torpor, experience a rapid rise in body temperature with a concomitant decrease in the Chol/PL ratio towards warm-active levels. These changes, at least in part, result in a surfactant, which is more surface-active at 37 °C than at 24 °C.
Acknowledgments This work was supported by the Australian Research Council, the Alberta Heritage Foundation for Medical Research, the Canadian Institute of Health Research, and the Silva Casa Foundation. The authors thank Dr. Michael Schoel, Stanley Cheng, and Ian Douglas for assistance in the laboratory and in preparing the graphs, Dr. Phil Wood for assistance with the surgery, and Terry Reardon for assistance in the field. Animals were collected under S.A.N.P.W.S. permit No: W24091-3 and surgery was performed under University of Adelaide Ethics Committee approval No: M/55/98.
References [1] C. Langman, S. Orgeig, C.B. Daniels, Alterations in composition and function of surfactant associated with torpor in Sminthopsis crassicaudata, Am. J. Physiol. 271 (1996) R437–R445.
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[2] C.B. Daniels, S. Orgeig, P.G. Wood, L.C. Sullivan, O.V. Lopatko, A.W. Smits, The changing state of surfactant lipids: new insights from ancient animals, Am. Zool. 38 (1998) 305–320. [3] C.B. Daniels, H.A. Barr, J.H.T. Power, T.E. Nicholas, Body temperature alters the lipid composition of pulmonary surfactant in the lizard Ctenophorus nuchalis, Exp. Lung Res. 16 (1990) 435– 449. [4] M.J. Lau, K.M.W. Keough, Lipid composition of lung and lung lavage fluid from map turtles (Malaclemys geographica) maintained at different environmental temperatures, Can. J. Biochem. 59 (1981) 208–219. [5] F. Possmayer, Biophysical activities of pulmonary surfactant, in: R.A. Polin, W.W. Fox (Eds.), Fetal and Neonatal Physiology, WB Saunders Co, Philadelphia, 1991, pp. 459–962. [6] R.A.W. Veldhuizen, K. Nag, S. Orgeig, F. Possmayer, The role of lipids in pulmonary surfactant, Biochim. Biophys. Acta 1408 (1998) 90–108. [7] S. Sch€ urch, Surface tension properties of surfactant, Clin. Perinatol. 20 (1993) 669–682. [8] O.V. Lopatko, S. Orgeig, C.B. Daniels, D. Palmer, Alterations in the surface properties of lung surfactant in the torpid marsupial Sminthopsis crassicaudata, J. Appl. Physiol. 84 (1998) 146–156. [9] O.V. Lopatko, S. Orgeig, D. Palmer, S. Sch€ urch, C.B. Daniels, Alterations in pulmonary surfactant after rapid arousal from torpor in the marsupial Sminthopsis crassicaudata, J. Appl. Physiol. 86 (1999) 1959–1970. [10] J.R. Codd, N.C. Slocombe, C.B. Daniels, P.G. Wood, S. Orgeig, Periodic fluctuations in the pulmonary surfactant system in
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Alterations in surface activity of pulmonary surfactant in Gould’s wattled bat during rapid arousal from torpor Jonathan R. Codd,a,1 Sandra Orgeig,a,* Christopher B. Daniels,a and Samuel Sch€ urchb b
a Department of Environmental Biology, Adelaide University, Adelaide, SA 5005, Australia Respiratory Research Group, Department of Physiology and Biophysics, University of Calgary, Health Sciences Centre, Calgary, Canada, Alb. T2N 4N1
Received 7 July 2003
Abstract The small microchiropteran bat, Chalinolobus gouldii, undergoes large daily fluctuations in metabolic rate, body temperature, and breathing pattern. These alterations are accompanied by changes in surfactant composition, predominantly an increase in cholesterol relative to phospholipid during torpor. Furthermore, the surface activity changes, such that the surfactant functions most effectively at that temperature which matches the animal’s activity state. Here, we examine the surface activity of surfactant from bats during arousal from torpor. Bats were housed at 24 °C on an 8:16 h light:dark cycle and their surfactant was collected during arousal (28 < Tb < 32 °C). Surface activity was examined with a Captive Bubble Surfactometer at 24 and 37 °C. Surfactant from arousing bats was more active at 37 °C than at 24 °C, indicated by a lower STmin and reduced film area compression required to reach STmin . It appears that the arousal-induced changes in surfactant composition, i.e., lower levels of cholesterol, inhibit adsorption of surfactant at 24 °C. Furthermore, the alterations in surfactant composition during arousal are very rapid, such that the mixture behaves more like surfactant from warm-active bats, and therefore, functions more effectively at 37 °C. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Surfactant; Arousal; Torpor; Bats; Cholesterol
Many small mammals and birds regularly enter periods of torpor, which are characterised by a reduction in body temperature and metabolic rate. Torpor is thought to have evolved primarily as an energy saving strategy. The entry into and maintenance of torpor are accompanied by numerous biochemical and physiological changes. Alterations in the pulmonary surfactant system in response to torpor have previously been demonstrated in a marsupial, the fat-tailed dunnart, Sminthopsis crassicaudata [1]. Pulmonary surfactant lines the inner surface of the lung where it regulates the surface tension at the air–liquid interface with changing lung volume. This system consists of lipids and proteins and is sensitive to changes *
Corresponding author. Fax: +61-8-8303-4364. E-mail address: [email protected] (S. Orgeig). 1 Present address: Apt. Morphologie und Systematik, Universit€at Bonn, Institut f€ ur Zoologie, Poppelsdorfer Schloss, Bonn 53115, Germany. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01427-X
in body temperature [2]. Temperature-associated changes in surfactant cholesterol have previously been demonstrated in the Central Australian lizard, Ctenophorous nuchalis [3], and cold acclimation of map turtles has also been demonstrated to increase the level of unsaturated phospholipids [4]. Adding cholesterol (Chol) and unsaturated phospholipids (USP) to pulmonary surfactant is thought to reduce the phase transition temperature of the surfactant mixture, which consists of up to 80% of the disaturated phospholipid (DSP), dipalmitoylphosphatidylcholine (DPPC). Vesicles of pure DPPC, below the gel-to-liquid crystalline phase transition of 41 °C, form a surfactant monolayer extremely slowly. In addition, DPPC aggregates at the air–liquid interface, which may be formed upon film compression and remain associated with the monolayer, are unable to spread across the respiratory surface as the alveolus expands during inflation [5,6]. However, DPPC is primarily responsible for the surface tension lowering ability of surfactant. DPPC molecules produce near zero
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surface tensions upon film compression by excluding water and by achieving a tightly packed highly ordered state at the air–liquid interface. Therefore, DPPC has to be transported to, and accumulate at, the interface. The reduction in phase transition temperature by the addition of cholesterol and USPs improves the spreadability of the surfactant mixture [6]. Hence, the surface activity of a surfactant is essentially its ability to lower surface tension upon film compression, which can be quantified by three different parameters: the rate of film formation by adsorption from the subphase, the minimum surface tension (STmin ) achieved upon film compression, and the amount of film area compression (% SA compression) required to achieve the minimum surface tension from equilibrium surface tension (STeq ) at the end of adsorption [7]. In the fat-tailed dunnart, changes in surfactant composition during torpor correlate with changes in surface activity. After 4 h of torpor, when measured at 20 °C, the surfactant demonstrates an increased rate of adsorption and decreases in STeq , STmin , and the % SA compression, compared with the surfactant isolated from warm-active dunnarts [8]. After 8 h of torpor there is a further decrease in STmin , rate of adsorption, and STeq . Hence, surfactant from torpid animals is more active at 20 °C than it is at 37 °C when compared with warm-active surfactant. Therefore, in vitro changes in surface activity correlate with changes in lipid composition, in S. crassicaudata, and may be required to optimise the alveolar surface area available for gas exchange at very low lung volumes [8,9]. Recently, we have performed similar experiments on the small microchiropteran bat, Chalinolobus gouldii. Unlike the dunnart which periodically enters torpor under conditions of cold-stress or food-shortage, the bat regularly enters into daily torpor at a range of ambient temperatures even when food is plentiful [10]. During the natural daily activity cycle of these bats at a constant ambient temperature of 24 °C, alterations in the pulmonary surfactant system occur. However, the amounts of phospholipid (PL) and DSP per gram dry lung do not change during torpor [10]. Upon arousal from torpor the amount of PL increases transiently, which is thought to represent an adrenergic surge related to behavioural changes such as arousal, grooming, jostling for roost sites, and preparing to fly and feed [10]. Relative to both PL and DSP, the amount of cholesterol increases during torpor in C. gouldii [10], resulting in an increase in the Chol/PL and Chol/DSP ratios. Furthermore, we demonstrated that surfactant isolated from bats was more surface-active at the temperature that matched the body temperature and activity status of the animal [10]. These alterations in the surface-active properties of surfactant appeared to correlate with fluctuations in surfactant composition, most notably surfactant cholesterol [10].
Periodic arousal from torpor is typical of all animals that enter into periods of torpor or hibernation. Arousal from torpor involves a rapid increase in metabolic rate and breathing rate, with temperature increases of up to 0.8 °C/min recorded for the little brown bat Eptesicus fuscus [11]. In the fat-tailed dunnart, arousal from torpor, induced by physically handling the torpid animals [9], resulted in temperature increases of 15–30 °C at the rate of 1 °C/min. Arousal was accompanied by rapid decreases in cholesterol within 5 min of reaching 30 °C and this change in composition correlated with a rapid decrease in STmin [9]. Hence, rapid arousal was accompanied by both changes in surfactant composition and changes in surface activity. Here, we examine the surface activity of surfactant from the microchiropteran bat, C. gouldii, during spontaneous arousal from torpor. We have previously published the corresponding changes in surfactant composition, which occur during warm-active and torpid states from the same samples of this species of bat [10]. We hypothesise that changes in surfactant activity occur so rapidly that surfactant from arousing bats will be more surface-active at warm-active body temperatures (37 °C) than at torpid body temperatures (24 °C).
Materials and methods Animals. C. gouldii are widespread and common across much of Australia. The bats used in this study were trapped, using harp traps, from February to May 1999 in the Flinders Ranges, South Australia. Bats were kept at a fixed ambient temperature of 24 °C and fed mealworms (Tenebrio molitor) and crickets (Acheta domestica) ad libitum. Water was supplied ad libitum. During a two week acclimatisation period, before experimentation began, body mass did not alter significantly. A total of 15 adult male C. gouldii were used. Body mass, forearm, and foot measurements were recorded just prior to experimentation. For C. gouldii, body mass ranged from 8.17 to 9.65 g (means SE; 9.3 0.25 g); mean forearm length was 43.65 0.18 mm; and mean foot length was 7.65 0.09 mm. Five bats were sampled during arousal from torpor and were assigned to this group on the basis of body temperature and activity status. Bats that were arousing from torpor had a body temperature of 28–32 °C [10]. Lavage procedure. Animals were killed with an intraperitoneal overdose of sodium pentobarbitone (0.1 ml, 325 mg/ml). A thermocouple probe was used to measure rectal Tb immediately. The trachea was exposed and cannulated and lungs were then rinsed (lavaged) with three 1.5-ml volumes of ice-cold saline (0.15 M NaCl), each volume instilled and withdrawn three times. Over 90% of the surfactant in the lung is removed using this method [1]. Sample preparation. The lavage was centrifuged for 5 min at 150g to pellet and remove any macrophages (Beckman model TJ-6). The supernatant was then centrifuged at 40,000g (J2 series Beckman Highspeed centrifuge) for 30 min and the pellet reconstituted to a concentration of 10 mg/ml phospholipid in a buffered salt solution (140 mM NaCl, 10 mM Hepes, and 2.5 mM CaCl2 , pH 6.9). Samples were then freeze-dried before being finally reconstituted to a concentration of 20 mg/ml phospholipid with distilled water for experiments on the Captive Bubble Surfactometer (CBS). Surface activity. The surface tension, area, and volume of a bubble, with a surfactant film at the air–water interface, were determined using
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40
* *
ST (mN/m)
a CBS. The volume of the bubble is controlled, by varying the pressure in the chamber. As the chamber volume is reduced, by moving the chamber base and the chamber up, relative to the stationary piston, the surface area reduces and the surface tension of the surfactant film at the bubble surface falls. Alterations in the surface tension at the air– liquid interface in the CBS are visualised by a change in the bubble shape from spherical to more oval as the surface tension falls [12]. We used a new spreading technique [13] to examine the surface activity of high concentration/low volume samples. A buffered salt solution (140 mM NaCl, 10 mM Hepes, and 2.5 mM CaCl2 , pH 6.9) containing 10% (by mass) sucrose, to increase the density of the salt solution to approximately 1.04 g/ml above that of the surfactant (1.01 g/ml), was used to fill the sample chamber on the CBS. Then 2 ll of 20 mg/ml phospholipid was injected into the chamber. A small bubble, 2–3 mm in diameter, was created, by winding the chamber base down, to draw in air. Adsorption was measured after the bubble had come to rest and assumed an equilibrium shape [14]. A Sony EV-S5000 NTSC video recorder and Pulnix TM 7 CN camera were used to continuously record the bubble. From digitised images, bubble volume, area, and surface tension were calculated using height and diameter [15]. Adsorption. The new spreading technique results in rapid adsorption [13]. The bubble comes to rest at the chamber ceiling, makes contact with the surfactant, assumes an equilibrium shape, and adsorption starts immediately (time zero). Adsorption was measured from time 0 to 1 min (data are presented for the first 10 s). Quasi-static and dynamic compressions. The original bubble, 2–3 mm in diameter, was expanded to 7–8 mm in diameter in approximately 0.1 s. After this rapid expansion the surfactant films were always reproducible, in that the same equilibrium surface tension was always achieved [13]. Below surface tensions of 20 mN/m, film compressibilities and the minimum surface tensions upon the first quasi-static compression were equal ( 1 mN/m). Quasi-static cycling commenced after a 5 min delay at maximum surface tension. The bubble volume was reduced by 5% at each step, by always taking the volume of the previous step as 100%. To avoid overcompression, bubble compression ceased as soon as the first ‘bubble click’ was observed [13,16,17]. There was a 5 min inter-cycle delay between each of the four quasi-static cycles. Dynamic cycling was conducted after a further 5 min delay at maximum bubble volume. Samples were subjected to 25 dynamic cycles at 24 and 37 °C. Two to three independent experiments were conducted with each sample on the CBS at each temperature setting. The starting temperature was alternated for successive measurements, i.e., for samples started at 37 °C, the following measurements were performed at 24 °C using the same chamber filling. For samples started at 24 °C, the following measurements were performed at 37 °C and so on. A combination of water and methanol was used to clean out the chamber between each filling. Statistical analysis. Data are presented as means SEM where applicable. Within group comparisons were made using a one-way ANOVA followed by a Student–Newman–Keuls test for post hoc between group comparisons.
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35
* *
30 * *
*
25
20 0
1
2
3
4
5
6
7
8
9
10
Time (seconds) Fig. 1. Time course for adsorption of surfactant collected from bats arousing from torpor, at a Tb between 28 and 32 °C, examined at both 37 and 24 °C on the CBS. Symbols indicate points that are significantly different at p < 0:05, n ¼ 4. Solid line ¼ 37 °C, Dashed line ¼ 24 °C.
The adsorption of surfactant from arousing bats was significantly faster at 37 °C than at 24 °C, indicated by a significantly lower STeq (37 °C: 26.79 0.80 mN/m, 24 °C: 37.19 0.55 mN/m, p ¼ 4:06 105 , n ¼ 4, Fig. 1). At 37 °C, surfactant from arousing bats adsorbed
A
B
Results Adsorption Upon film formation by spreading and adsorption, an equilibrium surface tension (STeq ) of 23–25 mN/m was reached in less than 10 s. Adsorption of surfactant from bats that were arousing from torpor was examined at both 24 and 37 °C. Temperature had a significant effect on the adsorption rate of bat surfactant.
Fig. 2. Quasi-static surface tension–area relations for surfactant collected from arousing bats, at 28 6 Tb 6 32 °C, examined at (A) 37 °C and (B) 24 °C. Sample concentration was 20 mg/ml in both cases, n ¼ 11, Solid line, quasi-static (QS-1) and dashed line, QS-4.
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significantly faster at all time points than the same sample examined at 24 °C (p < 0:05, n ¼ 4, for all time points examined, Fig. 1). Quasi-static cycles The film area compression required to reach minimum surface tension (STmin ) from equilibrium values was determined for surfactant collected from bats that were arousing from torpor at both 24 and 37 °C. Temperature had a significant effect on both the STmin achieved and the area compression required to reach STmin . Surfactant isolated from arousing bats required significantly less area compression on the first quasi-static cycle (Q1) to reach an STmin of 1 mN/m when examined at 37 °C than when the same preparation was examined at 24 °C (37 °C: 17.9 2.8%, 24 °C: 25.4 2.9%, p ¼ 0:05, n ¼ 8, Figs. 2A and B). The STmax reached on
A
35 30
Q1 was also significantly higher at 24 °C than at 37 °C (37 °C: 24.52 0.81 mN/m, 24 °C: 32.67 0.70 mN/m, p ¼ 1:3 107 , n ¼ 11, Figs. 2A and B). The STmin reached on Q1 was significantly lower at 37 °C than at 24 °C (37 °C: 1.67 0.11 mN/m, 24 °C: 2.97 0.14 mN/ m, p ¼ 2:1 107 , n ¼ 11, Figs. 2A and B). There was no significant difference in the area compression required to reach STmin on the fourth quasi-static cycle (Q4). However, the minimum ST was still significantly lower on Q4 at 37 °C than at 24 °C (37 °C: 1.57 0.099 mN/m, 24 °C: 2.99 0.080 mN/m, p ¼ 2:65 1010 , n ¼ 11, Figs. 2A and B). Dynamic cycles Surfactant collected from arousing bats and dynamically cycled at 37 °C reached a significantly lower STmin than the same sample examined at 24 °C (37 °C: 1.77 0.11 mN/m, 24 °C: 2.16 0.12 mN/m, p ¼ 0.02, n ¼ 6, Figs. 3A and B). The maximum ST reached on dynamic cycling was significantly higher at 24 °C than at 37 °C (24 °C: 28.56 0.21 mN/m, 37 °C: 26.51 0.19 mN/m, p ¼ 1:57 105 , n ¼ 6, Figs. 3A and B). However, there was no significant difference in the area compression required to reach STmin at either temperature.
ST (mN/m)
25
Discussion
20 15 10 5 0 0.86
0.88
0.90
0.92
0.94
0.96
0.98
1.00
1.02
Relative Area
B
35 30
ST (mN/m)
25 20 15 10 5 0 0.86
0.88
0.90
0.92
0.94
0.96
0.98
1.00
1.02
Relative Area Fig. 3. Surface tension–area relation for surfactant isolated from arousing bats examined at (A) 37 °C and (B) 24 °C, and dynamically cycled at 25 cycles/min. Typical results are presented for 1 bat and three consecutive cycles (9, 10, and 11) centred around the 10th cycle.
Surfactant isolated from bats that were arousing from torpor, when examined at 37 °C, demonstrated equilibrium surface tensions of 25 mN/m and minimum surface tensions of 1 mN/m. Similarly, surfactant from arousing bats demonstrated significantly faster adsorption at 37 °C than the same surfactant examined at 24 °C. Surfactant from arousing bats functions optimally at 37 °C, as indicated by a lower STmin and reduced area compression required to reach STmin . It appears that the transition in the surfactant biochemistry that occurs during arousal is very rapid, and has a dynamic effect on surface activity, such that the mixture functions more effectively at the rapidly increasing body temperature. We have previously demonstrated that although absolute amounts of cholesterol increased initially and transiently upon arousal in C. gouldii [10], absolute amounts of phospholipid also increased at the same time points, such that the relative amount of cholesterol (i.e., Chol/PL) was decreasing throughout the arousal period towards warm-active levels, such that the lipid composition was significantly different at the end of the arousal period. Hence, when compared with animals that were in torpor there was significantly less cholesterol relative to PL (i.e., lower Chol/PL) present in surfactant from warm-active bats [10,13]. As the surfactant from arousing animals was less active at 24 °C than 37 °C, it
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appears that the arousal-induced changes in surfactant composition, i.e., significantly lower levels of cholesterol are inhibiting adsorption of surfactant at the lower temperature. This suggests that above a certain temperature, high levels of cholesterol are no longer required as fluidising components. In the case of C. gouldii, this critical temperature appears to be approximately 28 °C, which was the lowest body temperature of this group of arousing bats. Hence, the surfactant isolated from bats at a Tb > 28 °C appears to function equally well at 37 °C, as surfactant isolated from fully active bats. It appears that throughout their daily torpor–arousal–activity cycles, bats are capable of precisely regulating the concentration of surfactant cholesterol, such that the isolated surfactant functions optimally only at the temperature, which matches the body temperature of the animal. Upon arousal from torpor there appears to be a crucial transition, in terms of cholesterol composition, back to the warm-active state—which, once achieved, reproduces performance at warm-active levels. The increase in cholesterol during torpor in the fattailed dunnart, S. crassicaudata [1], is thought to influence the adsorption and surface activity during torpor [8]. Hence, in the fat-tailed dunnart, changes in surfactant composition correlate with alterations in surface activity, such that surfactant from torpid animals is more active at 20 °C than at 37 °C. Furthermore, upon arousal from torpor in the fat-tailed dunnart, cholesterol levels are immediately reduced to warm-active levels and are significantly lower than the level of cholesterol in torpid animals [9]. These changes are reflected in the Chol/PL and Chol/DSP ratios, which both decrease to warm-active levels as rapidly as 5 min after arousal [9]. These rapid alterations in surfactant cholesterol composition correlate with an immediate improvement in the surface activity of surfactant from the fat-tailed dunnart, when examined at 37 °C [9]. Surfactant isolated from arousing bats behaved more like surfactant isolated from warm-active bats [13], consistent with the return of cholesterol levels to warmactive levels. Hence, surfactant function is maintained despite large fluctuations in body temperature, as alterations in lipid composition appear to maximise surface activity. However, it is not possible to attribute all the observed changes in surface activity to the observed changes in cholesterol. The levels of surfactant cholesterol found in C. gouldii are around six times lower than those in other mammals [10]. These low levels of cholesterol may reflect important differences in the regulation and/or function of surfactant relative to other mammals. Other surfactant components may possibly be involved in regulating surfactant fluidity and surface activity. A number of different surfactant components are known to enhance the adsorption rate, and therefore the spreadability, of surfactant. The addition of fluid
467
acidic phospholipids to surfactant enhances its adsorption rate, as does the addition of mono-/di-, and tri-acylglycerol [6]. However, altering the proportion of these molecules may also alter the surface activity of the surfactant. The surfactant proteins, on the other hand, do not affect surface activity per se, but they do enhance the adsorption rate by interacting with the phospholipids [6]. Reconstitution studies have also demonstrated that the most surface-active preparations do not contain cholesterol, but rather contain relatively large amounts of surfactant proteins [6]. The two small surfactant proteins SP-B and SP-C play important roles in maintaining the surface-active film and in fluidising the mixture [18]. Future studies examining any alterations in the amounts of the four surfactant-associated proteins may shed further light on the control of surfactant fluidity during torpor. In summary, C. gouldii cycle daily through periods of torpor, arousal, and activity. The large decreases in body temperature, which occur during daily torpor have a marked effect on surfactant composition and function. In C. gouldii, an increase in alveolar PL and DSP during arousal may serve to counteract the stiffness of the lung tissues due to the reduced temperatures of torpor. The most notable alteration in surfactant composition is the increased level of cholesterol associated with torpor, which is thought to enhance the fluidity of the mixture. The alterations in lipid composition with changes in body temperature correlate with changes in the surfaceactive properties and may serve to maximise the surface tension lowering ability of surfactant. The results from this study indicate that bats, which are arousing from torpor, experience a rapid rise in body temperature with a concomitant decrease in the Chol/PL ratio towards warm-active levels. These changes, at least in part, result in a surfactant, which is more surface-active at 37 °C than at 24 °C.
Acknowledgments This work was supported by the Australian Research Council, the Alberta Heritage Foundation for Medical Research, the Canadian Institute of Health Research, and the Silva Casa Foundation. The authors thank Dr. Michael Schoel, Stanley Cheng, and Ian Douglas for assistance in the laboratory and in preparing the graphs, Dr. Phil Wood for assistance with the surgery, and Terry Reardon for assistance in the field. Animals were collected under S.A.N.P.W.S. permit No: W24091-3 and surgery was performed under University of Adelaide Ethics Committee approval No: M/55/98.
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