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Torpor-associated fluctuations in surfactant activity in Gould’s wattled bat
Biochimica et Biophysica Acta 1580 (2002) 57^66 www.bba-direct.com
Torpor-associated £uctuations in surfactant activity in Gould's wattled bat Jonathan R. Codd a , Samuel Schu«rch b , Christopher B. Daniels a , Sandra Orgeig
a;
*
a
b
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, AB, Canada T2N 4N1 Received 25 July 2001; received in revised form 5 October 2001; accepted 19 October 2001
Abstract The primary function of pulmonary surfactant is to reduce the surface tension (ST) created at the air^liquid interface in the lung. Surfactant is a complex mixture of lipids and proteins and its function is influenced by physiological parameters such as metabolic rate, body temperature and breathing. In the microchiropteran bat Chalinolobus gouldii these parameters fluctuate throughout a 24 h period. Here we examine the surface activity of surfactant from warm^active and torpid bats at both 24³C and 37³C to establish whether alterations in surfactant composition correlate with changes in surface activity. Bats were housed in a specially constructed bat room at Adelaide University, at 24³C and on a 8:16 h light:dark cycle. Surfactant was collected from bats sampled during torpor (25 6 Tb 6 28³C), and while active (Tb s 35³C). Alterations in the lipid composition of surfactant occur with changes in the activity cycle. Most notable is an increase in surfactant cholesterol (Chol) with decreases in body temperature [Codd et al., Physiol. Biochem. Zool. 73 (2000) 605^612]. Surfactant from active bats was more surface active at higher temperatures, indicated by lower STmin and less film area compression required to reach STmin at 37³C than at 24³C. Conversely, surfactant from torpid bats was more active at lower temperatures, indicated by lower STmin and less area compression required to reach STmin at 24³C than at 37³C. Alterations in the Chol content of bat surfactant appear to be crucial to allow it to achieve low STs during torpor. ß 2002 Elsevier Science B.V. All rights reserved. Keywords: Surfactant; Surface activity; Torpor; Bat; Cholesterol
1. Introduction The entire alveolar surface area is lined with a Abbreviations: CBS, captive bubble surfactometer; Chol, cholesterol; DPPC, dipalmitoylphosphatidylcholine ; DSP, disaturated phospholipid; PL, phospholipid; Q, quasi-static cycle; ST, surface tension; STmax , surface tension maximum ; STmin , surface tension minimum; SP-B, surfactant protein B; SP-C, surfactant protein C; USP, unsaturated phospholipid * Corresponding author. Fax: +61-8-8303-4364. E-mail address: [email protected] (S. Orgeig).
continuous thin aqueous £uid. This air^liquid interface in the lung has the potential to generate high surface tensions (STs). Molecules on the surface experience unequal forces of attraction, being more strongly attracted to molecules within the bulk phase of the liquid than to the much fewer molecules in the vapor phase. High ST has the potential to compromise lung function. Pulmonary surfactant lines the air^liquid interface and lowers the ST to near zero values upon alveolar compression. Any material which lowers ST is referred to as surface active.
1388-1981 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 1 ) 0 0 1 8 5 - 8
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The surface activity of surfactant can be measured using a captive bubble surfactometer (CBS) [1]. Under static conditions the surfactant forms a ¢lm by adsorbing the surfactant components to the air^ liquid interface and lowering ST. The point at which no further change in ST occurs, within a certain time period is called the ST equilibrium (STeq ). Under dynamic compression of the surfactant ¢lm, the ST is further reduced as the lipids pack more closely together and exclude water from the interface. The point at which continued compression does not result in further decreases in ST is known as the minimum ST (STmin ). In reducing ST, the surfactant ¢lm greatly reduces the work of breathing and stabilizes the terminal air spaces. The disaturated phospholipid (DSP) dipalmitoylphosphatidylcholine (DPPC) is the most abundant phospholipid (PL) in surfactant and is primarily responsible for the ST lowering ability of surfactant. At 37³C, in the alveolar mesophase, DPPC forms aggregates, excluding water, in which the lipid is in a semi-solid state. However, upon in£ation of the lung, these aggregates are unable to spread at the air^liquid interface as the alveolus expands [2]. The addition of unsaturated PLs (USPs) and cholesterol (Chol) is thought to £uidize the aggregates, facilitating lipid adsorption and spreading on in£ation over the respiratory surface [2]. This function of Chol and USPs appears to be particularly important during periods of low body temperature. For example, the Central Australian lizard, Ctenophorous nuchalis, undergoes large changes in body temperature, which are associated with an increase in the Chol to PL ratio (Chol/PL), which may promote £uidity in surfactant aggregates [3]. Cold acclimation of map turtles increases the amounts of surfactant PLs and signi¢cantly increases levels of USPs. The increase in USPs may £uidize surfactant at low temperatures [4]. Torpor, which occurs widely in small mammals and birds, is associated with reduced body temperatures and metabolic rates and largely functions as a means of conserving energy. After 1 h of torpor in the fat-tailed dunnart, Sminthopsis crassicaudata, total PL increases when compared to warm^active animals. These levels remain elevated throughout 4 and 8 h of torpor. The amount of DSP per gram dry lung increases after 8 h of torpor, as does the %DSP/PL
[5]. The amount of surfactant Chol also increases after 1 h of torpor and these levels remain elevated through 8 h of torpor [3,5]. The increase in Chol is greatest, such that there is an overall increase in the Chol/PL and Chol/DSP ratios. This increase is thought to lead to an increase in surfactant £uidity, which may promote ¢lm formation by adsorption and spreading of surfactant at low temperatures. Although Chol is thought to be an important £uidizing component of surfactant, it does not appear to be stored in the lamellar bodies. Its origin and processing pathway remain unknown [6]. Recently we have shown that Gould's wattled bat, Chalinolobus gouldii, readily enters torpor at an ambient temperature of 24³C, where it reduces its body temperature by V10³C [7]. We have demonstrated that the amounts of PL and DSP per gram dry lung do not change during torpor [7]. Upon arousal from torpor the amount of PL increases transiently, which is thought to represent an adrenergic surge related to behavioral changes such as arousal, grooming, jostling for roost sites and preparing to £y and feed [7]. Relative to both PL and DSP, the amount of Chol increases during torpor in C. gouldii [7], resulting in an increase in the Chol/PL and Chol/DSP ratios. This increase in Chol may act to increase the £uidity and aid in the respreading of surfactant as body temperature falls. Here we examine the surface activity of surfactant collected from the same warm^active and torpid bats used in the above experiments [7], at 24³C and 37³C. Surfactant isolated from bats was found to be more surface active at the temperature which matched the body temperature and activity status of the animal. These alterations in the surface active properties of surfactant appeared to correlate with £uctuations in surfactant composition, most notably surfactant Chol. 2. Materials and methods 2.1. Animals C. gouldii were trapped using harp traps in the Flinders Ranges of northern South Australia, over the period from February to May 1999 and housed at Adelaide University. The `bat room' walls were
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partially covered with hessian to facilitate roosting and the room was maintained at an ambient temperature of 24³C. Bats were fed mealworms (Tenebrio molitor) and crickets (Acheta domestica) ad libitum. Water was supplied ad libitum. In this study, 15 adult male C. gouldii were used. For 2 weeks before experimental work began the bats were acclimatized to the room, during which time body mass did not alter signi¢cantly. Body mass, forearm and foot measurements were recorded just prior to experimentation. For C. gouldii, body mass ranged form 7.18 to 12.72 g (mean þ S.E.M.; 10.01 þ 0.229); mean forearm length was 43.65 þ 0.18 mm; mean foot length was 7.65 þ 0.09 mm. 2.2. Experimental protocol C. gouldii, as part of their natural daily cycle, go through periods of activity and torpor [7]. Bats (5^6 animals) were sampled whilst torpid and warm^active and were assigned to each group on the basis of body temperature and activity status [7]. Torpid bats had a body temperature of 25^28³C, whereas active bats had a body temperature of s 35³C [7]. 2.3. Lavage procedure Animals were killed with an intraperitoneal overdose of sodium pentobarbitone (0.1 ml, 325 mg/ml). Rectal Tb was measured, with a thermocouple probe, just prior to lavage. In order to harvest surfactant the trachea was exposed and cannulated. 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. This lavage protocol removes s 90% of the surfactant [5]. 2.4. Sample preparation Surfactant was ¢rst spun at 150Ug, for 5 min, to pellet and remove any macrophages. The supernatant was then centrifuged at 40 000Ug (J2 series Beckman Ultracentrifuge) for 30 min and the pellet reconstituted in a bu¡ered salt solution (140 mM NaCl/10 mM HEPES/2.5 mM CaCl2 , pH 6.9) to a concentration of 10 mg/ml of PL. Samples were then freeze-dried before being ¢nally reconstituted to a concentration of 20 mg/ml of PL with distilled water.
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2.5. Surface activity We used a CBS, a leak proof system, which enables the determination of ST, area and volume of a bubble with a surfactant ¢lm at the air^water interface. The volume of the bubble is controlled by varying the pressure in the chamber. As volume is reduced, the surface area is reduced and the ST of the surfactant ¢lm at the bubble surface falls. The shape of the bubble changes, depending on the ST, from spherical to more oval as the ST falls. The CBS consists of a sample chamber with a tight ¢tting piston sealed with an O ring. The chamber ceiling, formed by the concave surface of a 1% agarose gel plug attached to the piston, makes a completely hydrophilic surface for the bubble to contact. We used a new spreading technique (Schu«rch, S. and Codd, J.R., unpublished), which allowed us to examine the surface activity of high concentration/low volume samples. Brie£y, the chamber is ¢lled with a bu¡ered salt solution (140 mM NaCl, 10 mM HEPES, 2.5 mM CaCl2 , pH 6.9), which contained 10% (by mass) sucrose. The sucrose is added to increase the density of the salt solution to approximately 1.04 g/ml above that of the surfactant (V1.01 g/ml). Two Wl of 20 mg/ml from each sample is then injected into the chamber near its agarose ceiling. The injected surfactant rests against the agarose plug at the top of the chamber. A bubble, 2^3 mm in diameter, was then created, by moving the chamber down, and drawing air in by means of a small hole at the chamber base. Adsorption was measured after the creation of a bubble by drawing air into the chamber once the bubble had come to rest and assumed a Laplacian shape [8]. 2.6. ST, area and volume calculations Throughout the experiment the bubble was recorded continuously on Hi-8 video using a Sony EV-S5000 NTSC video recorder and Pulnix TM 7 CN camera. Bubble volume, area and ST were calculated from digitized bubble images using height and diameter [9]. 2.7. Adsorption The new spreading technique results in rapid ad-
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sorption (Fig. 1A,B). Almost simultaneously, the bubble comes to rest at the chamber ceiling, makes contact with the surfactant, assumes a Laplacian shape and adsorption starts. Time zero for the adsorption was taken at the moment when the bubble assumed a Laplacian shape. Adsorption was measured from time zero to 1 min (data are presented for the ¢rst 10 s).
were done at 37³C and so on. The chamber was washed out between each sample ¢lling using a combination of water and methanol. 2.9. Statistical analysis
2.8. Quasi-static and dynamic compressions
Data are presented as means þ S.E.M. where applicable. Multiple mean comparisons were made using a one-way analysis of variance followed by a Newman^Keuls test for post hoc comparisons.
The original bubble, 2^3 mm in diameter, was expanded to V7^8 mm in diameter in approximately 0.1 s. Previous control experiments with bovine lipid extract surfactant (BLES) have shown that the surfactant ¢lms, after this rapid expansion of the bubble, were reproducible, in that the same STeq of 23^ 25 mN/m was always achieved. Film compressibilities below STs of V20 mN/m were less than 0.01 m/mN and the minimum STs upon the ¢rst quasistatic compression were below 2 mN/m and equal within the error range of 1 mN/m. After a 5 min delay, quasi-static cycling commenced with each step representing a 5% compression of the new initial volume. The bubble compressions continued until the ¢rst click was seen. At this point, there was no further compression to avoid overcompression. `Bubble clicks' essentially refer to the spontaneous and rapid increase in ST accompanied by a reduction in surface area, visualized as a sudden rounding of the bubble [10,11]. There was a 5 min inter-cycle delay between each of the four quasi-static cycles. Finally, after a 5 min waiting period at maximum bubble volume 25 dynamic cycles were conducted with each sample. Measurements for each sample from both warm^active and torpid bat groups were performed at two temperatures (24 and 37³C) matching the activity status of both groups: warm^active (37³C) and torpid bats (24³C). This enabled us to determine whether changes in surfactant composition were speci¢c to temperature. Two to three independent experiments were conducted with each sample on the CBS at each temperature setting. For successive measurements, the starting temperature was alternated, i.e. for samples started at 37³C the following measurements were done at 24³C using the same chamber ¢lling. For samples started at 24³C, the following measurements
Fig. 1. Time course for adsorption of surfactant collected from (A) warm^active bats sampled at 37³C (solid line) and 24³C (dashed line), and (B) torpid bats sampled at 24³C (dashed line) and 37³C (solid line). Symbols indicate points that are signi¢cantly di¡erent at (A) P90.0075, (B) P90.0041, n = 6.
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3. Results 3.1. Adsorption Upon ¢lm formation by spreading and adsorption, as described in Section 2, an STeq of 23^25 mN/m was reached in less than 10 s. Adsorption of surfactant from warm^active and torpid bats was examined at both 24³C and 37³C. Temperature had a signi¢cant e¡ect on the adsorption rate of bat surfactant. The adsorption of surfactant, from warm^active bats, examined at 37³C was signi¢cantly faster than adsorption of the same sample at 24³C (P 9 0.0075, n = 6, at all time points, Fig. 1A). STeq was also signi¢cantly higher for warm^active samples at 24³C than at 37³C (24³C: 43 þ 1.2 mN/m, 37³C: 28 þ 0.8 mN/m, P = 2U1035 , n = 6). Conversely, surfactant from torpid bats (Tb 6 28³C), when examined at 24³C, had a signi¢cantly lower STeq than at 37³C (24³C: 28 þ 0.9 mN/m, 37³C: 36 þ 2.1 mN/m, P = 0.0175, n = 6). At 24³C surfactant from torpid bats had signi¢cantly faster adsorption than the same sample examined at 37³C (P 9 0.041, n = 6, at all time points, Fig. 1B). 3.2. Quasi-static cycles The ¢lm area compression required to reach minimum STs from equilibrium values was determined for surfactant collected from warm^active and torpid bats at both 24³C and 37³C. Temperature had a signi¢cant e¡ect on both minimum STs achieved and the area compression to reach STmin . For `warm^active' surfactant examined at 37³C the area compression required to reach an STmin of 6 1 mN/m on the ¢rst quasi-static cycle, quasi-static 1 (Q1), at 37³C was signi¢cantly lower than for the same preparation examined at 24³C (37³C: 19.9 þ 2.2%, 24³C: 28.3 þ 2.4%, P = 0.01, n = 13, Fig. 2A,B). The STmin reached on Q1 was signi¢cantly lower at 37³C than at 24³C (37³C: 1.65 þ 0.04 mN/ m, 24³C: 3.62 þ 0.12 mN/m, P = 8.66U1039 , n = 13, Fig. 2A,B). The maximum ST (STmax ) reached on Q1 was signi¢cantly higher at 24³C than at 37³C (24³C: 34.4 þ 0.9 mN/m, 37³C: 24.9 þ 0.3 mN/m, 39 P = 1.23U10 , n = 13, Fig. 2A,B). There was no signi¢cant di¡erence in area compression for Q4 at either 24³C or 37³C with both requiring approximately
Fig. 2. Quasi-static ST^area relations for surfactant collected from warm^active bats at Tb V37³C examined at (A) 37³C and (B) 24³C. Q1 (solid line) and Q4 (dashed line) are shown here for comparison, there was no signi¢cant di¡erence between Q4 and Q2 or Q3. Sample concentration was 20 mg/ml in both cases, n = 13.
16% changes in area to reach STmin . STmin , however, was still signi¢cantly lower on Q4 (37³C: 1.36 þ 0.06 mN/m, 24³C: 3.42 þ 0.09 mN/m, P = 9.6U10316 , n = 13, Fig. 2A,B). For surfactant collected from torpid bats, the area compression required to reach STmin on Q1 was signi¢cantly lower at 24³C than at 37³C (24³C: 19.4 þ 3.4%, 37³C: 26.3 þ 3.7%, P = 0.05, n = 10, Fig. 3A,B). The STmin reached on Q1 was signi¢cantly lower at 24³C than at 37³C (24³C: 1.48 þ 0.2 mN/ m, 37³C: 2.92 þ 0.1 mN/m, P = 3.21U1039 , n = 10, Fig. 3A,B). The STmax reached on Q1 was also signi¢cantly lower at 24³C than at 37³C (24³C: 24.27 þ 0.6 mN/m, 37³C: 29.37 þ 1.6 mN/m, P = 0.005, n = 10, Fig. 3A,B). There was no signi¢-
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around 1 mN/m upon continuous cycling at 25 cycles per minute. Results presented here are from three consecutive cycles centered around the 10th cycle. Surfactant collected from warm^active bats and dynamically cycled at 37³C reached a signi¢cantly lower STmin when compared with the same sample examined at 24³C (37³C: 1.2 þ 0.008 mN/m, 24³C: 2.5 þ 0.09 mN/m, P = 2.08U1035 , n = 6, Fig. 4A,B). The area compression required to reach STmin at 37³C was signi¢cantly lower than that required for the same samples examined at 24³C (37³C: 11.8 þ 0.1%, 24³C: 17.2 þ 0.5%, P = 6.7U1037 , n = 6, Fig. 4A,B). For surfactant from torpid bats, dynamically cycled at 24³C, a signi¢cantly lower STmin was reached when compared with the same samples ana-
Fig. 3. Quasi-static ST^area relations for surfactant collected from torpid bats at Tb V25³C examined at (A) 37³C and (B) 24³C. Q1 (solid line) and Q4 (dashed line) are shown here for comparison, there was no signi¢cant di¡erence between Q4 and Q2 or Q3. Sample concentration was 20 mg/ml in both cases, n = 10.
cant di¡erence in area compression for Q4 at either 24³C or 37³C with both requiring approximately 20% compression to reach STmin . STmin was still signi¢cantly lower on Q4 (24³C: 1.15 þ 0.09 mN/m, 37³C: 3.3 þ 0.15 mN/m, P = 5.15U10311 , n = 10, Fig. 3A,B). There was no di¡erence in any surface activity parameters measured between surfactant isolated from warm^active and torpid bats, when they were examined at the temperature matching body temperature. 3.3. Dynamic cycles When examined under temperature conditions matching the body temperature of the warm^active and torpid bats, samples exhibited an STmin of
Fig. 4. ST^area relation for surfactant isolated from warm^active bats (Tb V37³C) examined at (A) 37³C and (B) 24³C, and dynamically cycled at 25 cycles per minute. Typical results are presented for one bat and three consecutive cycles centered around the 10th cycle are illustrated.
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Fig. 5. ST^area relation for surfactant isolated from torpid bats (Tb V25³C) examined at (A) 37³C and (B) 24³C, and dynamically cycled at 25 cycles per minute. Typical results are presented for one bat and three consecutive cycles centered around the 10th cycle are illustrated.
lyzed at 37³C (24³C: 1.2 þ 0.2 mN/m, 37³C: 3.76 þ 0.04 mN/m, P = 2.63U1037 , n = 6, Fig. 5A,B). The area compression to reach STmin was also signi¢cantly smaller at 24³C than 37³C (24³C: 8.75 þ 0.2%, 37³C: 10.1 þ 0.2%, P = 0.0006, n = 6, Fig. 5A,B). 4. Discussion For surfactant to function e¡ectively in the lung, it must be able to achieve an STmin of V1 mN/m. A low STmin is required to provide the optimal alveolar area for respiratory gas exchange at low lung volumes [12]. However, other aspects of the biophysical behavior of surfactant on a CBS also provide impor-
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tant information. While the adsorption rate of surfactant, for the present experiments, provides information about the spreading of surfactant at an air^ liquid interface, quasi-static cycles demonstrate the quality of the ¢lms formed with regard to the puri¢cation process of surfactant. These are related to ¢lm compressibility and mechanical stability at low ST. The ¢lm compressibility below STs of 20 mN/m may decrease with successive cycling. This is also seen in the decreasing amount of ¢lm compression required to achieve near zero STmin from an initial near STeq of V25 mN/m. A measure of the mechanical stability of the ¢lm at STmin may be determined by the bubble clicks at STmin . Such clicks are demonstrated in Fig. 2A,B, as they contribute to the ST^ area hysteresis of the quasi-static cycles. This can clearly be seen in the ¢rst cycle in Fig. 2A. The point of the curve which follows that of the STmin of V3 mN/m and a relative area of 0.83 has an ST of 6 mN/ m at a relative bubble area of 0.78. This is the result of the mechanical instability at STmin , which causes a sudden jump in the ST of the bubble accompanied with a decrease in the surface area at constant bubble volume (bubble clicks). Surfactant from warm^active bats sampled at 37³C, and investigated at 37³C, forms ¢lms with lower STmin and higher ¢lm stability at minimum STs. There is also no substantial change in the amount of ¢lm area compression required to achieve STmin from the STeq of V25 mN/m (Fig. 2A). In contrast, in Fig. 2B, the ¢rst quasi-static cycle shows a large ¢lm area compression (V35%) needed to obtain the STmin . Upon successive cycling, the decrease in the area compression (V20%) indicates ¢lm puri¢cation toward a ¢lm enriched in DPPC. Dynamic cycling provides information on the surfactant ¢lm properties upon cycling rates closer to physiological conditions. After 3^4 consecutive cycles, the cycles become nearly stationary, as they are highly reproducible. The low minimum STs and a low hysteresis area indicate mechanically stable ¢lms, while the relatively low maximum STs of V30 mN/ m demonstrate fast respreading of surfactant material associated with the ¢lm resulting in a fast reformation of the ¢lm. The relatively low ¢lm area compressions (V13%) needed to achieve near zero STmin are also indicative of an e¤cient ¢lm reformation upon repeated dynamic cycling (Figs. 4A,B and 5A,B). Dynamic cycling provides information on
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¢lm quality by examining the area change required to achieve STmin . 4.1. Adsorption rates Adsorption is a¡ected substantially by the composition of surfactant and, in particular, the PL concentration. We applied the same volume and concentration of surfactant in the CBS during this study and when surfactant was analyzed at a temperature matching the body temperature of the bat, equilibrium STs of V25 mN/m and minimum STs of V1 mN/m were achieved, similar to literature values for other mammals [2,13]. Adsorption was signi¢cantly slower, when surfactant from warm^active bats was analyzed at 24³C compared with 37³C. Conversely, surfactant from torpid bats demonstrated much faster adsorption at 24³C than at 37³C. Hence, bat surfactant demonstrates signi¢cant thermal sensitivity. Chol has been shown to adversely a¡ect adsorption rates in bovine surfactant preparations at 37³C [2]. Hence, it is possible that the increased concentration of Chol associated with the decreased temperatures of torpor inhibits surfactant adsorption at warm^active temperatures [7,14]. Furthermore, at the lower temperature, the increased Chol appears to increase the spreadability of surfactant. 4.2. Surface properties of bat surfactant Quasi-static and dynamic cycling of surfactant from warm^active bats at 37³C yields lower STmin and %area compression than at 24³C. Hence, the ability of `warm^active' surfactant to reduce ST was impaired at the lower temperature. Conversely, surfactant from torpid bats reaches a lower STmin and requires less %area compression to reach low STmin at 24³C than at 37³C. Hence, bat surfactant appears to function optimally at the temperature at which it was isolated. 4.3. The e¡ect of lipid composition on surface activity The compositional change in Chol appears to be crucial to maintain surfactant function at reduced body temperatures. How Chol functions in surfactant is unclear. The reduction in ST is unlikely to be directly brought about by the observed changes
in Chol [15]. However, there is evidence that Chol can a¡ect the biophysical behavior of mixed lipid^ protein ¢lms indirectly by interactions with one or other of the surfactant proteins. Chol has been demonstrated to promote mixing of DPPC and surfactant protein C (SP-C) in ternary ¢lms over that observed in DPPC/SP-C binary ¢lms at 23³C [19]. Signi¢cantly greater concentrations of SP-C were retained within the monolayer in the presence of Chol, whereas in the absence of Chol, SP-C is squeezed out at an ST of 20 mN/m. Chol has also been demonstrated to enhance the ability of surfactant protein A to enhance the adsorption of DPPC to the air^liquid interface [17]. Biophysical studies of mixed lipid, or mixed lipid^ protein ¢lms have yielded contradictory results. On the Wilhelmy balance at room temperature, Chol enhances the adsorption of DPPC and improves ¢lm respreading after collapse [15,16]. However, some studies have shown that Chol may inhibit the reduction in ST upon compression [15]. The enhancement of respreading and adsorption is presumably due to the increase in the £uidity of the mixture in the aqueous phase. The interference with the capacity of surfactant ¢lms to reach near zero STmin upon ¢lm compression appears to be due to the ability of Chol to resist squeeze-out upon ¢lm compression [17,18]. In mixed DPPC/protein ¢lms in the Wilhelmy balance, Chol has a negative in£uence on the surface properties thought to be important for surfactant function [19]. For example, the addition of Chol to a spread monolayer of DPPC plus surfactant protein B (SP-B) did not improve the adsorption and spreading properties compared to a ¢lm of DPPC and SP-B alone [19]. Moreover, the addition of Chol to aqueous dispersions of DPPC with SP-B or SP-C caused a reduction in the rate of adsorption to the air^water interface at 35³C [19]. In investigations with the pulsating bubble surfactometer, the addition of Chol to BLES or DPPC/1-palmitoyl-2oleoyl-phosphatidylglycerol/SP-B mixtures at 37³C also caused instability and an impairment of surface activity [20]. However, in the CBS, Chol appeared to have minimal e¡ects on ST reduction of the arti¢cial surfactant preparations Curosurf and BLES [21]. The cause for these di¡ering results obtained in vitro is likely related to the methodology, especially to the characteristics of the various ST measuring device.
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For example, ¢lms in the Langmuir^Wilhelmy balance are formed by dropwise spreading of surfactant material onto the air^liquid interface, building up an equilibrium surfactant ¢lm (V25 mN/m) as drops are added to the surface. In addition, in the Langmuir^Wilhelmy surface balance surfactant components can creep along constraining walls and between the walls and the barrier, causing ¢lm leakage and preventing the ST to reach near zero ST values upon ¢lm compression [22]. Film leakage is also a problem in the pulsating bubble surfactometer, as surfactant may spread up at the plastic^air and plastic^£uid interfaces of the tube, causing considerably less surface compression than that (50%) given by the change in the bubble surface area. Films at STeq (V25 mN/m) containing Chol are more £uid than those without Chol and so are more likely to su¡er from leakage artifacts than those without Chol [23]. It is possible, therefore, that the results obtained in the Langmuir^Wilhelmy balance, especially at 35 or 37³C, are in£uenced by ¢lm leakage [24]. Therefore, it is extremely di¤cult to predict how Chol functions in vivo in the surfactant system. Natural surfactant contains substantial amounts of Chol [25] and it is clear that surfactant Chol has profound e¡ects on surfactant function. 4.4. The e¡ect of surfactant composition on surface activity The large decreases in body temperature, associated with torpor in C. gouldii, in£uence the surfactant system. Amounts of Chol were found to increase during torpor in these bats [7]. Chol is thought to be the primary £uidizing component of surfactant [6]. Chol has also been found to act as a £uidizer during torpor in the fat-tailed dunart, S. crassicaudata [23]. The surface activity of surfactant changes both during torpor and upon arousal from torpor. After 4 h of torpor, the adsorption rate increases and STeq , STmin and the area compression required to achieve STmin decrease, compared with the warm^active group [23]. After 8 h of torpor there is a further decrease in STmin , rate of adsorption and STeq . Hence, changes in surfactant composition correlate with alterations in surface activity such that surfactant from torpid animals is more active at 20³C than at 37³C.
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Increasing the amount of Chol during torpor appears to enable bat surfactant to achieve an STmin of around 1 mN/m. However, higher levels of Chol may not be required at warm^active temperatures. The absolute surfactant Chol levels found in C. gouldii are lower than those in other mammals [7]. The addition of small amounts of Chol (8^10% by weight) to either bovine lipid surfactant or isolated lipid protein mixtures has been shown to impair adsorption and surface activity [18,19]. Hence, although the changes we see are small (2^3%), they are still likely to have a signi¢cant e¡ect. The low levels of Chol, in bats, may re£ect important di¡erences in the regulation and/or function of Chol relative to other mammals. Low levels of Chol in C. gouldii may also re£ect the possibility that other surfactant components are involved in regulating surfactant £uidity. Future experiments adding small amounts of Chol to surfactant samples from warm^active bats examined at 24³C may help to further elucidate the role of Chol during torpor. A number of di¡erent surfactant components are known to enhance the adsorption rate, and therefore the spreadability, of surfactant. The addition of £uid acidic PLs to surfactant enhances its adsorption rate, as does the addition of mono-/di-, and tri-acylglycerol [24]. However, altering the proportion of these molecules may also alter the surface activity of the surfactant [26]. The surfactant proteins on the other hand do not a¡ect surface activity per se, but they do enhance the adsorption rate by interacting with the PLs [24]. Reconstitution studies have demonstrated that the most surface active preparations do not contain Chol, but rather contain relatively large amounts of surfactant proteins [24]. The two small surfactant proteins SP-B and SP-C play important roles in maintaining the surface active ¢lm and in £uidizing the mixture [27]. In conclusion, the pulmonary surfactant system of bats demonstrates remarkable temperature speci¢city and sensitivity. The biochemical changes in surfactant associated with torpor, most notably the relative enrichment in Chol, appeared to enable the surface activity parameters of STmin , STmax and area compression required to achieve STmin , to be equivalent to warm^active values, despite the reduced body temperature. Hence, surfactant function is maintained despite large £uctuations in body temperature.
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Throughout their daily torpor activity cycle, bats appear to be capable of precisely regulating the concentration of surfactant Chol, such that the isolated surfactant functions optimally only at the temperature, which matches the body temperature of the animal. Whether the observed alterations in biophysical activity are solely due to the alterations in lipid composition is unknown. Future work should examine the possibility that the concentration of surfactant proteins, other neutral lipids and negatively charged PLs may also alter during the daily activity cycle of bats and hence contribute to the observed alterations in biophysical activity. Acknowledgements 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 would like to thank Dr. Michael Schoel for assistance with captive bubble software and experimentation, Stanley Cheng and Ian Douglas for assistance in the lab and in preparing the graphs, Dr. Phil Wood for assistance with the surgery and helpful comments on the manuscript, Terry Reardon and the cooper family for assistance in the ¢eld. Animals were collected under S.A.-N.P.W.S. permit No. W24091-3 and under approval of the University of Adelaide Ethics Committee: approval No. M/55/98. References [1] S. Schu«rch, H. Bachofen, J. Goerke, F. Possmayer, J. Appl. Physiol. 67 (1989) 2389^2396. [2] F. Possmayer, in: R.A. Polin, W.W. Fox (Eds.), Fetal and Neonatal Physiology, 1st edn., WB Saunders Co., Philadelphia, PA, 1991, pp. 459^962. [3] C.B. Daniels, H.A. Barr, J.H.T. Power, T.E. Nicholas, Exp. Lung Res. 16 (1990) 435^449. [4] M.J. Lau, K.M.W. Keough, Can. J. Biochem. 59 (1981) 208^219.
[5] C. Langman, S. Orgeig, C.B. Daniels, Am. J. Physiol. 271 (1996) R437^R445. [6] S. Orgeig, C.B. Daniels, Comp. Biochem. Physiol. A 129 (2001) 75^89. [7] J.R. Codd, N.C. Slocombe, C.B. Daniels, P.G. Wood, S. Orgeig, Physiol. Biochem. Zool. 73 (2000) 605^612. [8] S. Schu«rch, F.H.Y. Green, H. Bachofen, Biochim. Biophys. Acta 1408 (1998) 180^202. [9] W.M. Schoel, S. Schu«rch, J. Goerke, Biochim. Biophys. Acta 1200 (1994) 281^290. [10] S. Schu«rch, H. Bachofen, J. Goerke, F. Green, Biochim. Biophys. Acta 1103 (1992) 127^136. [11] S. Schu«rch, H. Bachofen, F. Possmayer, Comp. Biochem. Physiol. A 129 (2001) 195^207. [12] S. Schu«rch, H. Bachofen, in: B. Robertson, H.W. Teausch (Eds.), Surfactant Therapy for Lung Disease, Marcel Dekker, 1995, pp. 3^32. [13] J. Goerke, J.A. Clements, in: P.T. Macklem, J. Mead (Eds.), Handbook of Physiology, Section 3: The Respiratory System. Vol III: Mechanics of Breathing, Part I, American Physiological Society, Washington, DC, 1985, pp. 247^260. [14] J.R. Codd, C.B. Daniels, S. Orgeig, in: G. Heldmeier, M. Klingenspor (Eds.), Life in the Cold, Eleventh International Hibernation Symposium, Springer, Berlin, 2000, pp. 187^ 197. [15] R.H. Notter, S.A. Tabak, R.D. Mavis, J. Lipid Res. 21 (1980) 10^22. [16] B.D. Fleming, K.M.W. Keough, Chem. Phys. Lipids 49 (1988) 81^86. [17] S.-H. Yu, F. Possmayer, J. Lipid Res. 37 (1996) 1278^1288. [18] S.-H. Yu, F. Possmayer, J. Lipid Res. 39 (1998) 555^568. [19] S. Taneva, K.M.W. Keough, Biochemistry 36 (1997) 912^ 922. [20] S. Yu, F. Possmayer, Biochim. Biophys. Acta 1211 (1994) 350^358. [21] D. Palmer, S. Cheng, F. Green, S. Schu«rch, Am. J. Respir. Crit. Care Med. 155 (1997) A214. [22] B. Robertson, S. Schu«rch, in: S. Uhlig, A.E. Taylor (Eds.), Methods in Pulmonary Research, Birkha«user, Basel, 1998, pp. 349^386. [23] O.V. Lopatko, S. Orgeig, C.B. Daniels, D. Palmer, J. Appl. Physiol. 84 (1998) 146^156. [24] R.A.W. Veldhuizen, K. Nag, S. Orgeig, F. Possmayer, Biochim. Biophys. Acta 1408 (1998) 90^108. [25] C.B. Daniels, S. Orgeig, P.G. Wood, L. Sullivan, O.V. Lopatko, A.W. Smits, Am. Zool. 38 (1998) 305^320. [26] C.B. Daniels, O.V. Lopatko, S. Orgeig, Clin. Exp. Pharmacol. Physiol. 25 (1998) 716^721. [27] T.E. Weaver, Biochim. Biophys. Acta 1408 (1998) 173^179.
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Torpor-associated £uctuations in surfactant activity in Gould's wattled bat Jonathan R. Codd a , Samuel Schu«rch b , Christopher B. Daniels a , Sandra Orgeig
a;
*
a
b
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, AB, Canada T2N 4N1 Received 25 July 2001; received in revised form 5 October 2001; accepted 19 October 2001
Abstract The primary function of pulmonary surfactant is to reduce the surface tension (ST) created at the air^liquid interface in the lung. Surfactant is a complex mixture of lipids and proteins and its function is influenced by physiological parameters such as metabolic rate, body temperature and breathing. In the microchiropteran bat Chalinolobus gouldii these parameters fluctuate throughout a 24 h period. Here we examine the surface activity of surfactant from warm^active and torpid bats at both 24³C and 37³C to establish whether alterations in surfactant composition correlate with changes in surface activity. Bats were housed in a specially constructed bat room at Adelaide University, at 24³C and on a 8:16 h light:dark cycle. Surfactant was collected from bats sampled during torpor (25 6 Tb 6 28³C), and while active (Tb s 35³C). Alterations in the lipid composition of surfactant occur with changes in the activity cycle. Most notable is an increase in surfactant cholesterol (Chol) with decreases in body temperature [Codd et al., Physiol. Biochem. Zool. 73 (2000) 605^612]. Surfactant from active bats was more surface active at higher temperatures, indicated by lower STmin and less film area compression required to reach STmin at 37³C than at 24³C. Conversely, surfactant from torpid bats was more active at lower temperatures, indicated by lower STmin and less area compression required to reach STmin at 24³C than at 37³C. Alterations in the Chol content of bat surfactant appear to be crucial to allow it to achieve low STs during torpor. ß 2002 Elsevier Science B.V. All rights reserved. Keywords: Surfactant; Surface activity; Torpor; Bat; Cholesterol
1. Introduction The entire alveolar surface area is lined with a Abbreviations: CBS, captive bubble surfactometer; Chol, cholesterol; DPPC, dipalmitoylphosphatidylcholine ; DSP, disaturated phospholipid; PL, phospholipid; Q, quasi-static cycle; ST, surface tension; STmax , surface tension maximum ; STmin , surface tension minimum; SP-B, surfactant protein B; SP-C, surfactant protein C; USP, unsaturated phospholipid * Corresponding author. Fax: +61-8-8303-4364. E-mail address: [email protected] (S. Orgeig).
continuous thin aqueous £uid. This air^liquid interface in the lung has the potential to generate high surface tensions (STs). Molecules on the surface experience unequal forces of attraction, being more strongly attracted to molecules within the bulk phase of the liquid than to the much fewer molecules in the vapor phase. High ST has the potential to compromise lung function. Pulmonary surfactant lines the air^liquid interface and lowers the ST to near zero values upon alveolar compression. Any material which lowers ST is referred to as surface active.
1388-1981 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 1 ) 0 0 1 8 5 - 8
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The surface activity of surfactant can be measured using a captive bubble surfactometer (CBS) [1]. Under static conditions the surfactant forms a ¢lm by adsorbing the surfactant components to the air^ liquid interface and lowering ST. The point at which no further change in ST occurs, within a certain time period is called the ST equilibrium (STeq ). Under dynamic compression of the surfactant ¢lm, the ST is further reduced as the lipids pack more closely together and exclude water from the interface. The point at which continued compression does not result in further decreases in ST is known as the minimum ST (STmin ). In reducing ST, the surfactant ¢lm greatly reduces the work of breathing and stabilizes the terminal air spaces. The disaturated phospholipid (DSP) dipalmitoylphosphatidylcholine (DPPC) is the most abundant phospholipid (PL) in surfactant and is primarily responsible for the ST lowering ability of surfactant. At 37³C, in the alveolar mesophase, DPPC forms aggregates, excluding water, in which the lipid is in a semi-solid state. However, upon in£ation of the lung, these aggregates are unable to spread at the air^liquid interface as the alveolus expands [2]. The addition of unsaturated PLs (USPs) and cholesterol (Chol) is thought to £uidize the aggregates, facilitating lipid adsorption and spreading on in£ation over the respiratory surface [2]. This function of Chol and USPs appears to be particularly important during periods of low body temperature. For example, the Central Australian lizard, Ctenophorous nuchalis, undergoes large changes in body temperature, which are associated with an increase in the Chol to PL ratio (Chol/PL), which may promote £uidity in surfactant aggregates [3]. Cold acclimation of map turtles increases the amounts of surfactant PLs and signi¢cantly increases levels of USPs. The increase in USPs may £uidize surfactant at low temperatures [4]. Torpor, which occurs widely in small mammals and birds, is associated with reduced body temperatures and metabolic rates and largely functions as a means of conserving energy. After 1 h of torpor in the fat-tailed dunnart, Sminthopsis crassicaudata, total PL increases when compared to warm^active animals. These levels remain elevated throughout 4 and 8 h of torpor. The amount of DSP per gram dry lung increases after 8 h of torpor, as does the %DSP/PL
[5]. The amount of surfactant Chol also increases after 1 h of torpor and these levels remain elevated through 8 h of torpor [3,5]. The increase in Chol is greatest, such that there is an overall increase in the Chol/PL and Chol/DSP ratios. This increase is thought to lead to an increase in surfactant £uidity, which may promote ¢lm formation by adsorption and spreading of surfactant at low temperatures. Although Chol is thought to be an important £uidizing component of surfactant, it does not appear to be stored in the lamellar bodies. Its origin and processing pathway remain unknown [6]. Recently we have shown that Gould's wattled bat, Chalinolobus gouldii, readily enters torpor at an ambient temperature of 24³C, where it reduces its body temperature by V10³C [7]. We have demonstrated that the amounts of PL and DSP per gram dry lung do not change during torpor [7]. Upon arousal from torpor the amount of PL increases transiently, which is thought to represent an adrenergic surge related to behavioral changes such as arousal, grooming, jostling for roost sites and preparing to £y and feed [7]. Relative to both PL and DSP, the amount of Chol increases during torpor in C. gouldii [7], resulting in an increase in the Chol/PL and Chol/DSP ratios. This increase in Chol may act to increase the £uidity and aid in the respreading of surfactant as body temperature falls. Here we examine the surface activity of surfactant collected from the same warm^active and torpid bats used in the above experiments [7], at 24³C and 37³C. Surfactant isolated from bats was found to be more surface active at the temperature which matched the body temperature and activity status of the animal. These alterations in the surface active properties of surfactant appeared to correlate with £uctuations in surfactant composition, most notably surfactant Chol. 2. Materials and methods 2.1. Animals C. gouldii were trapped using harp traps in the Flinders Ranges of northern South Australia, over the period from February to May 1999 and housed at Adelaide University. The `bat room' walls were
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partially covered with hessian to facilitate roosting and the room was maintained at an ambient temperature of 24³C. Bats were fed mealworms (Tenebrio molitor) and crickets (Acheta domestica) ad libitum. Water was supplied ad libitum. In this study, 15 adult male C. gouldii were used. For 2 weeks before experimental work began the bats were acclimatized to the room, during which time body mass did not alter signi¢cantly. Body mass, forearm and foot measurements were recorded just prior to experimentation. For C. gouldii, body mass ranged form 7.18 to 12.72 g (mean þ S.E.M.; 10.01 þ 0.229); mean forearm length was 43.65 þ 0.18 mm; mean foot length was 7.65 þ 0.09 mm. 2.2. Experimental protocol C. gouldii, as part of their natural daily cycle, go through periods of activity and torpor [7]. Bats (5^6 animals) were sampled whilst torpid and warm^active and were assigned to each group on the basis of body temperature and activity status [7]. Torpid bats had a body temperature of 25^28³C, whereas active bats had a body temperature of s 35³C [7]. 2.3. Lavage procedure Animals were killed with an intraperitoneal overdose of sodium pentobarbitone (0.1 ml, 325 mg/ml). Rectal Tb was measured, with a thermocouple probe, just prior to lavage. In order to harvest surfactant the trachea was exposed and cannulated. 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. This lavage protocol removes s 90% of the surfactant [5]. 2.4. Sample preparation Surfactant was ¢rst spun at 150Ug, for 5 min, to pellet and remove any macrophages. The supernatant was then centrifuged at 40 000Ug (J2 series Beckman Ultracentrifuge) for 30 min and the pellet reconstituted in a bu¡ered salt solution (140 mM NaCl/10 mM HEPES/2.5 mM CaCl2 , pH 6.9) to a concentration of 10 mg/ml of PL. Samples were then freeze-dried before being ¢nally reconstituted to a concentration of 20 mg/ml of PL with distilled water.
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2.5. Surface activity We used a CBS, a leak proof system, which enables the determination of ST, area and volume of a bubble with a surfactant ¢lm at the air^water interface. The volume of the bubble is controlled by varying the pressure in the chamber. As volume is reduced, the surface area is reduced and the ST of the surfactant ¢lm at the bubble surface falls. The shape of the bubble changes, depending on the ST, from spherical to more oval as the ST falls. The CBS consists of a sample chamber with a tight ¢tting piston sealed with an O ring. The chamber ceiling, formed by the concave surface of a 1% agarose gel plug attached to the piston, makes a completely hydrophilic surface for the bubble to contact. We used a new spreading technique (Schu«rch, S. and Codd, J.R., unpublished), which allowed us to examine the surface activity of high concentration/low volume samples. Brie£y, the chamber is ¢lled with a bu¡ered salt solution (140 mM NaCl, 10 mM HEPES, 2.5 mM CaCl2 , pH 6.9), which contained 10% (by mass) sucrose. The sucrose is added to increase the density of the salt solution to approximately 1.04 g/ml above that of the surfactant (V1.01 g/ml). Two Wl of 20 mg/ml from each sample is then injected into the chamber near its agarose ceiling. The injected surfactant rests against the agarose plug at the top of the chamber. A bubble, 2^3 mm in diameter, was then created, by moving the chamber down, and drawing air in by means of a small hole at the chamber base. Adsorption was measured after the creation of a bubble by drawing air into the chamber once the bubble had come to rest and assumed a Laplacian shape [8]. 2.6. ST, area and volume calculations Throughout the experiment the bubble was recorded continuously on Hi-8 video using a Sony EV-S5000 NTSC video recorder and Pulnix TM 7 CN camera. Bubble volume, area and ST were calculated from digitized bubble images using height and diameter [9]. 2.7. Adsorption The new spreading technique results in rapid ad-
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sorption (Fig. 1A,B). Almost simultaneously, the bubble comes to rest at the chamber ceiling, makes contact with the surfactant, assumes a Laplacian shape and adsorption starts. Time zero for the adsorption was taken at the moment when the bubble assumed a Laplacian shape. Adsorption was measured from time zero to 1 min (data are presented for the ¢rst 10 s).
were done at 37³C and so on. The chamber was washed out between each sample ¢lling using a combination of water and methanol. 2.9. Statistical analysis
2.8. Quasi-static and dynamic compressions
Data are presented as means þ S.E.M. where applicable. Multiple mean comparisons were made using a one-way analysis of variance followed by a Newman^Keuls test for post hoc comparisons.
The original bubble, 2^3 mm in diameter, was expanded to V7^8 mm in diameter in approximately 0.1 s. Previous control experiments with bovine lipid extract surfactant (BLES) have shown that the surfactant ¢lms, after this rapid expansion of the bubble, were reproducible, in that the same STeq of 23^ 25 mN/m was always achieved. Film compressibilities below STs of V20 mN/m were less than 0.01 m/mN and the minimum STs upon the ¢rst quasistatic compression were below 2 mN/m and equal within the error range of 1 mN/m. After a 5 min delay, quasi-static cycling commenced with each step representing a 5% compression of the new initial volume. The bubble compressions continued until the ¢rst click was seen. At this point, there was no further compression to avoid overcompression. `Bubble clicks' essentially refer to the spontaneous and rapid increase in ST accompanied by a reduction in surface area, visualized as a sudden rounding of the bubble [10,11]. There was a 5 min inter-cycle delay between each of the four quasi-static cycles. Finally, after a 5 min waiting period at maximum bubble volume 25 dynamic cycles were conducted with each sample. Measurements for each sample from both warm^active and torpid bat groups were performed at two temperatures (24 and 37³C) matching the activity status of both groups: warm^active (37³C) and torpid bats (24³C). This enabled us to determine whether changes in surfactant composition were speci¢c to temperature. Two to three independent experiments were conducted with each sample on the CBS at each temperature setting. For successive measurements, the starting temperature was alternated, i.e. for samples started at 37³C the following measurements were done at 24³C using the same chamber ¢lling. For samples started at 24³C, the following measurements
Fig. 1. Time course for adsorption of surfactant collected from (A) warm^active bats sampled at 37³C (solid line) and 24³C (dashed line), and (B) torpid bats sampled at 24³C (dashed line) and 37³C (solid line). Symbols indicate points that are signi¢cantly di¡erent at (A) P90.0075, (B) P90.0041, n = 6.
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3. Results 3.1. Adsorption Upon ¢lm formation by spreading and adsorption, as described in Section 2, an STeq of 23^25 mN/m was reached in less than 10 s. Adsorption of surfactant from warm^active and torpid bats was examined at both 24³C and 37³C. Temperature had a signi¢cant e¡ect on the adsorption rate of bat surfactant. The adsorption of surfactant, from warm^active bats, examined at 37³C was signi¢cantly faster than adsorption of the same sample at 24³C (P 9 0.0075, n = 6, at all time points, Fig. 1A). STeq was also signi¢cantly higher for warm^active samples at 24³C than at 37³C (24³C: 43 þ 1.2 mN/m, 37³C: 28 þ 0.8 mN/m, P = 2U1035 , n = 6). Conversely, surfactant from torpid bats (Tb 6 28³C), when examined at 24³C, had a signi¢cantly lower STeq than at 37³C (24³C: 28 þ 0.9 mN/m, 37³C: 36 þ 2.1 mN/m, P = 0.0175, n = 6). At 24³C surfactant from torpid bats had signi¢cantly faster adsorption than the same sample examined at 37³C (P 9 0.041, n = 6, at all time points, Fig. 1B). 3.2. Quasi-static cycles The ¢lm area compression required to reach minimum STs from equilibrium values was determined for surfactant collected from warm^active and torpid bats at both 24³C and 37³C. Temperature had a signi¢cant e¡ect on both minimum STs achieved and the area compression to reach STmin . For `warm^active' surfactant examined at 37³C the area compression required to reach an STmin of 6 1 mN/m on the ¢rst quasi-static cycle, quasi-static 1 (Q1), at 37³C was signi¢cantly lower than for the same preparation examined at 24³C (37³C: 19.9 þ 2.2%, 24³C: 28.3 þ 2.4%, P = 0.01, n = 13, Fig. 2A,B). The STmin reached on Q1 was signi¢cantly lower at 37³C than at 24³C (37³C: 1.65 þ 0.04 mN/ m, 24³C: 3.62 þ 0.12 mN/m, P = 8.66U1039 , n = 13, Fig. 2A,B). The maximum ST (STmax ) reached on Q1 was signi¢cantly higher at 24³C than at 37³C (24³C: 34.4 þ 0.9 mN/m, 37³C: 24.9 þ 0.3 mN/m, 39 P = 1.23U10 , n = 13, Fig. 2A,B). There was no signi¢cant di¡erence in area compression for Q4 at either 24³C or 37³C with both requiring approximately
Fig. 2. Quasi-static ST^area relations for surfactant collected from warm^active bats at Tb V37³C examined at (A) 37³C and (B) 24³C. Q1 (solid line) and Q4 (dashed line) are shown here for comparison, there was no signi¢cant di¡erence between Q4 and Q2 or Q3. Sample concentration was 20 mg/ml in both cases, n = 13.
16% changes in area to reach STmin . STmin , however, was still signi¢cantly lower on Q4 (37³C: 1.36 þ 0.06 mN/m, 24³C: 3.42 þ 0.09 mN/m, P = 9.6U10316 , n = 13, Fig. 2A,B). For surfactant collected from torpid bats, the area compression required to reach STmin on Q1 was signi¢cantly lower at 24³C than at 37³C (24³C: 19.4 þ 3.4%, 37³C: 26.3 þ 3.7%, P = 0.05, n = 10, Fig. 3A,B). The STmin reached on Q1 was signi¢cantly lower at 24³C than at 37³C (24³C: 1.48 þ 0.2 mN/ m, 37³C: 2.92 þ 0.1 mN/m, P = 3.21U1039 , n = 10, Fig. 3A,B). The STmax reached on Q1 was also signi¢cantly lower at 24³C than at 37³C (24³C: 24.27 þ 0.6 mN/m, 37³C: 29.37 þ 1.6 mN/m, P = 0.005, n = 10, Fig. 3A,B). There was no signi¢-
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around 1 mN/m upon continuous cycling at 25 cycles per minute. Results presented here are from three consecutive cycles centered around the 10th cycle. Surfactant collected from warm^active bats and dynamically cycled at 37³C reached a signi¢cantly lower STmin when compared with the same sample examined at 24³C (37³C: 1.2 þ 0.008 mN/m, 24³C: 2.5 þ 0.09 mN/m, P = 2.08U1035 , n = 6, Fig. 4A,B). The area compression required to reach STmin at 37³C was signi¢cantly lower than that required for the same samples examined at 24³C (37³C: 11.8 þ 0.1%, 24³C: 17.2 þ 0.5%, P = 6.7U1037 , n = 6, Fig. 4A,B). For surfactant from torpid bats, dynamically cycled at 24³C, a signi¢cantly lower STmin was reached when compared with the same samples ana-
Fig. 3. Quasi-static ST^area relations for surfactant collected from torpid bats at Tb V25³C examined at (A) 37³C and (B) 24³C. Q1 (solid line) and Q4 (dashed line) are shown here for comparison, there was no signi¢cant di¡erence between Q4 and Q2 or Q3. Sample concentration was 20 mg/ml in both cases, n = 10.
cant di¡erence in area compression for Q4 at either 24³C or 37³C with both requiring approximately 20% compression to reach STmin . STmin was still signi¢cantly lower on Q4 (24³C: 1.15 þ 0.09 mN/m, 37³C: 3.3 þ 0.15 mN/m, P = 5.15U10311 , n = 10, Fig. 3A,B). There was no di¡erence in any surface activity parameters measured between surfactant isolated from warm^active and torpid bats, when they were examined at the temperature matching body temperature. 3.3. Dynamic cycles When examined under temperature conditions matching the body temperature of the warm^active and torpid bats, samples exhibited an STmin of
Fig. 4. ST^area relation for surfactant isolated from warm^active bats (Tb V37³C) examined at (A) 37³C and (B) 24³C, and dynamically cycled at 25 cycles per minute. Typical results are presented for one bat and three consecutive cycles centered around the 10th cycle are illustrated.
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Fig. 5. ST^area relation for surfactant isolated from torpid bats (Tb V25³C) examined at (A) 37³C and (B) 24³C, and dynamically cycled at 25 cycles per minute. Typical results are presented for one bat and three consecutive cycles centered around the 10th cycle are illustrated.
lyzed at 37³C (24³C: 1.2 þ 0.2 mN/m, 37³C: 3.76 þ 0.04 mN/m, P = 2.63U1037 , n = 6, Fig. 5A,B). The area compression to reach STmin was also signi¢cantly smaller at 24³C than 37³C (24³C: 8.75 þ 0.2%, 37³C: 10.1 þ 0.2%, P = 0.0006, n = 6, Fig. 5A,B). 4. Discussion For surfactant to function e¡ectively in the lung, it must be able to achieve an STmin of V1 mN/m. A low STmin is required to provide the optimal alveolar area for respiratory gas exchange at low lung volumes [12]. However, other aspects of the biophysical behavior of surfactant on a CBS also provide impor-
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tant information. While the adsorption rate of surfactant, for the present experiments, provides information about the spreading of surfactant at an air^ liquid interface, quasi-static cycles demonstrate the quality of the ¢lms formed with regard to the puri¢cation process of surfactant. These are related to ¢lm compressibility and mechanical stability at low ST. The ¢lm compressibility below STs of 20 mN/m may decrease with successive cycling. This is also seen in the decreasing amount of ¢lm compression required to achieve near zero STmin from an initial near STeq of V25 mN/m. A measure of the mechanical stability of the ¢lm at STmin may be determined by the bubble clicks at STmin . Such clicks are demonstrated in Fig. 2A,B, as they contribute to the ST^ area hysteresis of the quasi-static cycles. This can clearly be seen in the ¢rst cycle in Fig. 2A. The point of the curve which follows that of the STmin of V3 mN/m and a relative area of 0.83 has an ST of 6 mN/ m at a relative bubble area of 0.78. This is the result of the mechanical instability at STmin , which causes a sudden jump in the ST of the bubble accompanied with a decrease in the surface area at constant bubble volume (bubble clicks). Surfactant from warm^active bats sampled at 37³C, and investigated at 37³C, forms ¢lms with lower STmin and higher ¢lm stability at minimum STs. There is also no substantial change in the amount of ¢lm area compression required to achieve STmin from the STeq of V25 mN/m (Fig. 2A). In contrast, in Fig. 2B, the ¢rst quasi-static cycle shows a large ¢lm area compression (V35%) needed to obtain the STmin . Upon successive cycling, the decrease in the area compression (V20%) indicates ¢lm puri¢cation toward a ¢lm enriched in DPPC. Dynamic cycling provides information on the surfactant ¢lm properties upon cycling rates closer to physiological conditions. After 3^4 consecutive cycles, the cycles become nearly stationary, as they are highly reproducible. The low minimum STs and a low hysteresis area indicate mechanically stable ¢lms, while the relatively low maximum STs of V30 mN/ m demonstrate fast respreading of surfactant material associated with the ¢lm resulting in a fast reformation of the ¢lm. The relatively low ¢lm area compressions (V13%) needed to achieve near zero STmin are also indicative of an e¤cient ¢lm reformation upon repeated dynamic cycling (Figs. 4A,B and 5A,B). Dynamic cycling provides information on
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¢lm quality by examining the area change required to achieve STmin . 4.1. Adsorption rates Adsorption is a¡ected substantially by the composition of surfactant and, in particular, the PL concentration. We applied the same volume and concentration of surfactant in the CBS during this study and when surfactant was analyzed at a temperature matching the body temperature of the bat, equilibrium STs of V25 mN/m and minimum STs of V1 mN/m were achieved, similar to literature values for other mammals [2,13]. Adsorption was signi¢cantly slower, when surfactant from warm^active bats was analyzed at 24³C compared with 37³C. Conversely, surfactant from torpid bats demonstrated much faster adsorption at 24³C than at 37³C. Hence, bat surfactant demonstrates signi¢cant thermal sensitivity. Chol has been shown to adversely a¡ect adsorption rates in bovine surfactant preparations at 37³C [2]. Hence, it is possible that the increased concentration of Chol associated with the decreased temperatures of torpor inhibits surfactant adsorption at warm^active temperatures [7,14]. Furthermore, at the lower temperature, the increased Chol appears to increase the spreadability of surfactant. 4.2. Surface properties of bat surfactant Quasi-static and dynamic cycling of surfactant from warm^active bats at 37³C yields lower STmin and %area compression than at 24³C. Hence, the ability of `warm^active' surfactant to reduce ST was impaired at the lower temperature. Conversely, surfactant from torpid bats reaches a lower STmin and requires less %area compression to reach low STmin at 24³C than at 37³C. Hence, bat surfactant appears to function optimally at the temperature at which it was isolated. 4.3. The e¡ect of lipid composition on surface activity The compositional change in Chol appears to be crucial to maintain surfactant function at reduced body temperatures. How Chol functions in surfactant is unclear. The reduction in ST is unlikely to be directly brought about by the observed changes
in Chol [15]. However, there is evidence that Chol can a¡ect the biophysical behavior of mixed lipid^ protein ¢lms indirectly by interactions with one or other of the surfactant proteins. Chol has been demonstrated to promote mixing of DPPC and surfactant protein C (SP-C) in ternary ¢lms over that observed in DPPC/SP-C binary ¢lms at 23³C [19]. Signi¢cantly greater concentrations of SP-C were retained within the monolayer in the presence of Chol, whereas in the absence of Chol, SP-C is squeezed out at an ST of 20 mN/m. Chol has also been demonstrated to enhance the ability of surfactant protein A to enhance the adsorption of DPPC to the air^liquid interface [17]. Biophysical studies of mixed lipid, or mixed lipid^ protein ¢lms have yielded contradictory results. On the Wilhelmy balance at room temperature, Chol enhances the adsorption of DPPC and improves ¢lm respreading after collapse [15,16]. However, some studies have shown that Chol may inhibit the reduction in ST upon compression [15]. The enhancement of respreading and adsorption is presumably due to the increase in the £uidity of the mixture in the aqueous phase. The interference with the capacity of surfactant ¢lms to reach near zero STmin upon ¢lm compression appears to be due to the ability of Chol to resist squeeze-out upon ¢lm compression [17,18]. In mixed DPPC/protein ¢lms in the Wilhelmy balance, Chol has a negative in£uence on the surface properties thought to be important for surfactant function [19]. For example, the addition of Chol to a spread monolayer of DPPC plus surfactant protein B (SP-B) did not improve the adsorption and spreading properties compared to a ¢lm of DPPC and SP-B alone [19]. Moreover, the addition of Chol to aqueous dispersions of DPPC with SP-B or SP-C caused a reduction in the rate of adsorption to the air^water interface at 35³C [19]. In investigations with the pulsating bubble surfactometer, the addition of Chol to BLES or DPPC/1-palmitoyl-2oleoyl-phosphatidylglycerol/SP-B mixtures at 37³C also caused instability and an impairment of surface activity [20]. However, in the CBS, Chol appeared to have minimal e¡ects on ST reduction of the arti¢cial surfactant preparations Curosurf and BLES [21]. The cause for these di¡ering results obtained in vitro is likely related to the methodology, especially to the characteristics of the various ST measuring device.
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For example, ¢lms in the Langmuir^Wilhelmy balance are formed by dropwise spreading of surfactant material onto the air^liquid interface, building up an equilibrium surfactant ¢lm (V25 mN/m) as drops are added to the surface. In addition, in the Langmuir^Wilhelmy surface balance surfactant components can creep along constraining walls and between the walls and the barrier, causing ¢lm leakage and preventing the ST to reach near zero ST values upon ¢lm compression [22]. Film leakage is also a problem in the pulsating bubble surfactometer, as surfactant may spread up at the plastic^air and plastic^£uid interfaces of the tube, causing considerably less surface compression than that (50%) given by the change in the bubble surface area. Films at STeq (V25 mN/m) containing Chol are more £uid than those without Chol and so are more likely to su¡er from leakage artifacts than those without Chol [23]. It is possible, therefore, that the results obtained in the Langmuir^Wilhelmy balance, especially at 35 or 37³C, are in£uenced by ¢lm leakage [24]. Therefore, it is extremely di¤cult to predict how Chol functions in vivo in the surfactant system. Natural surfactant contains substantial amounts of Chol [25] and it is clear that surfactant Chol has profound e¡ects on surfactant function. 4.4. The e¡ect of surfactant composition on surface activity The large decreases in body temperature, associated with torpor in C. gouldii, in£uence the surfactant system. Amounts of Chol were found to increase during torpor in these bats [7]. Chol is thought to be the primary £uidizing component of surfactant [6]. Chol has also been found to act as a £uidizer during torpor in the fat-tailed dunart, S. crassicaudata [23]. The surface activity of surfactant changes both during torpor and upon arousal from torpor. After 4 h of torpor, the adsorption rate increases and STeq , STmin and the area compression required to achieve STmin decrease, compared with the warm^active group [23]. After 8 h of torpor there is a further decrease in STmin , rate of adsorption and STeq . Hence, changes in surfactant composition correlate with alterations in surface activity such that surfactant from torpid animals is more active at 20³C than at 37³C.
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Increasing the amount of Chol during torpor appears to enable bat surfactant to achieve an STmin of around 1 mN/m. However, higher levels of Chol may not be required at warm^active temperatures. The absolute surfactant Chol levels found in C. gouldii are lower than those in other mammals [7]. The addition of small amounts of Chol (8^10% by weight) to either bovine lipid surfactant or isolated lipid protein mixtures has been shown to impair adsorption and surface activity [18,19]. Hence, although the changes we see are small (2^3%), they are still likely to have a signi¢cant e¡ect. The low levels of Chol, in bats, may re£ect important di¡erences in the regulation and/or function of Chol relative to other mammals. Low levels of Chol in C. gouldii may also re£ect the possibility that other surfactant components are involved in regulating surfactant £uidity. Future experiments adding small amounts of Chol to surfactant samples from warm^active bats examined at 24³C may help to further elucidate the role of Chol during torpor. A number of di¡erent surfactant components are known to enhance the adsorption rate, and therefore the spreadability, of surfactant. The addition of £uid acidic PLs to surfactant enhances its adsorption rate, as does the addition of mono-/di-, and tri-acylglycerol [24]. However, altering the proportion of these molecules may also alter the surface activity of the surfactant [26]. The surfactant proteins on the other hand do not a¡ect surface activity per se, but they do enhance the adsorption rate by interacting with the PLs [24]. Reconstitution studies have demonstrated that the most surface active preparations do not contain Chol, but rather contain relatively large amounts of surfactant proteins [24]. The two small surfactant proteins SP-B and SP-C play important roles in maintaining the surface active ¢lm and in £uidizing the mixture [27]. In conclusion, the pulmonary surfactant system of bats demonstrates remarkable temperature speci¢city and sensitivity. The biochemical changes in surfactant associated with torpor, most notably the relative enrichment in Chol, appeared to enable the surface activity parameters of STmin , STmax and area compression required to achieve STmin , to be equivalent to warm^active values, despite the reduced body temperature. Hence, surfactant function is maintained despite large £uctuations in body temperature.
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Throughout their daily torpor activity cycle, bats appear to be capable of precisely regulating the concentration of surfactant Chol, such that the isolated surfactant functions optimally only at the temperature, which matches the body temperature of the animal. Whether the observed alterations in biophysical activity are solely due to the alterations in lipid composition is unknown. Future work should examine the possibility that the concentration of surfactant proteins, other neutral lipids and negatively charged PLs may also alter during the daily activity cycle of bats and hence contribute to the observed alterations in biophysical activity. Acknowledgements 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 would like to thank Dr. Michael Schoel for assistance with captive bubble software and experimentation, Stanley Cheng and Ian Douglas for assistance in the lab and in preparing the graphs, Dr. Phil Wood for assistance with the surgery and helpful comments on the manuscript, Terry Reardon and the cooper family for assistance in the ¢eld. Animals were collected under S.A.-N.P.W.S. permit No. W24091-3 and under approval of the University of Adelaide Ethics Committee: approval No. M/55/98. References [1] S. Schu«rch, H. Bachofen, J. Goerke, F. Possmayer, J. Appl. Physiol. 67 (1989) 2389^2396. [2] F. Possmayer, in: R.A. Polin, W.W. Fox (Eds.), Fetal and Neonatal Physiology, 1st edn., WB Saunders Co., Philadelphia, PA, 1991, pp. 459^962. [3] C.B. Daniels, H.A. Barr, J.H.T. Power, T.E. Nicholas, Exp. Lung Res. 16 (1990) 435^449. [4] M.J. Lau, K.M.W. Keough, Can. J. Biochem. 59 (1981) 208^219.
[5] C. Langman, S. Orgeig, C.B. Daniels, Am. J. Physiol. 271 (1996) R437^R445. [6] S. Orgeig, C.B. Daniels, Comp. Biochem. Physiol. A 129 (2001) 75^89. [7] J.R. Codd, N.C. Slocombe, C.B. Daniels, P.G. Wood, S. Orgeig, Physiol. Biochem. Zool. 73 (2000) 605^612. [8] S. Schu«rch, F.H.Y. Green, H. Bachofen, Biochim. Biophys. Acta 1408 (1998) 180^202. [9] W.M. Schoel, S. Schu«rch, J. Goerke, Biochim. Biophys. Acta 1200 (1994) 281^290. [10] S. Schu«rch, H. Bachofen, J. Goerke, F. Green, Biochim. Biophys. Acta 1103 (1992) 127^136. [11] S. Schu«rch, H. Bachofen, F. Possmayer, Comp. Biochem. Physiol. A 129 (2001) 195^207. [12] S. Schu«rch, H. Bachofen, in: B. Robertson, H.W. Teausch (Eds.), Surfactant Therapy for Lung Disease, Marcel Dekker, 1995, pp. 3^32. [13] J. Goerke, J.A. Clements, in: P.T. Macklem, J. Mead (Eds.), Handbook of Physiology, Section 3: The Respiratory System. Vol III: Mechanics of Breathing, Part I, American Physiological Society, Washington, DC, 1985, pp. 247^260. [14] J.R. Codd, C.B. Daniels, S. Orgeig, in: G. Heldmeier, M. Klingenspor (Eds.), Life in the Cold, Eleventh International Hibernation Symposium, Springer, Berlin, 2000, pp. 187^ 197. [15] R.H. Notter, S.A. Tabak, R.D. Mavis, J. Lipid Res. 21 (1980) 10^22. [16] B.D. Fleming, K.M.W. Keough, Chem. Phys. Lipids 49 (1988) 81^86. [17] S.-H. Yu, F. Possmayer, J. Lipid Res. 37 (1996) 1278^1288. [18] S.-H. Yu, F. Possmayer, J. Lipid Res. 39 (1998) 555^568. [19] S. Taneva, K.M.W. Keough, Biochemistry 36 (1997) 912^ 922. [20] S. Yu, F. Possmayer, Biochim. Biophys. Acta 1211 (1994) 350^358. [21] D. Palmer, S. Cheng, F. Green, S. Schu«rch, Am. J. Respir. Crit. Care Med. 155 (1997) A214. [22] B. Robertson, S. Schu«rch, in: S. Uhlig, A.E. Taylor (Eds.), Methods in Pulmonary Research, Birkha«user, Basel, 1998, pp. 349^386. [23] O.V. Lopatko, S. Orgeig, C.B. Daniels, D. Palmer, J. Appl. Physiol. 84 (1998) 146^156. [24] R.A.W. Veldhuizen, K. Nag, S. Orgeig, F. Possmayer, Biochim. Biophys. Acta 1408 (1998) 90^108. [25] C.B. Daniels, S. Orgeig, P.G. Wood, L. Sullivan, O.V. Lopatko, A.W. Smits, Am. Zool. 38 (1998) 305^320. [26] C.B. Daniels, O.V. Lopatko, S. Orgeig, Clin. Exp. Pharmacol. Physiol. 25 (1998) 716^721. [27] T.E. Weaver, Biochim. Biophys. Acta 1408 (1998) 173^179.
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