INTRODUCTION

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A MIXTURE OF LIPIDS and proteins lines the lungs of all air-breathing vertebrates (8). This mixture, termed pulmonary surfactant (PS), functions to modulate the surface tension of the fluid lining the lung. The lipids in PS are comprised predominantly of phospholipids (PL), and, of these, disaturated PLs (DSP) are largely responsible for the reduction in surface tension (8). A reduction in the surface tension of the air-liquid interface reduces the work of breathing, prevents pulmonary edema, and reduces the adhesion of epithelial surfaces that may come into contact within the lung during the breathing cycle (8). Surfactant is synthesized and stored in ciliated, cuboidal cells known as alveolar type II cells (30). Within the type II cell, surfactant is stored in characteristic organelles termed lamellar bodies. In mammals, a great many factors appear to affect the secretion and composition of pulmonary surfactant. The principal stimuli for release appear to be changes in ventilatory pattern and input from the sympathetic nervous system (11, 24). Ventilatory changes are thought to directly distort the type II cell and trigger PL release (31), whereas epinephrine (Epi) is believed to act on ₂ receptors situated on the type II cell (3). Although ACh stimulates surfactant PL release in the isolated perfused rat lung, it is thought to act indirectly, causing a contraction of intrapulmonary smooth muscle, which in turn distorts the type II cell (22). Direct application of ACh onto type II cells isolated from rats does not stimulate PL release (22). The parasympathetic nervous system, therefore, is not believed to have any direct effects on the secretion of PS in mammals (22).

In contrast to mammals, changes in ventilatory pattern in the isolated lizard lung have no affect on PL release (33). This is perhaps not surprising; lizards, like most nonmammals, have a respiratory cycle characterized by long periods of breath-holding, interspersed with shorter periods of breathing (14). It is possible that such a discontinuous breathing pattern is too irregular to provide a reliable mechanism for continued secretion. Alternatively, increases in tidal volume in the large saccular lungs of lizards may involve increases in the volume of the saccular portions of the lungs without concomitant increases in the volume of the respiratory tissue. As such, increases in tidal volume may not distort the type II cells within the lizard lung and stimulate surfactant secretion.

Both Epi and ACh trigger surfactant PL release in the isolated lizard lung (32, 33). It is possible that both the sympathetic and parasympathetic nervous systems control surfactant secretion in nonmammals, although the sites of action for either Epi or ACh remain unknown. Whereas mammalian type II cells possess both cholinergic and ₂-adrenergic receptors (16), it is not known whether either of these receptor types are present on lizard type II cells. ACh appears to act on muscarinic receptors in the isolated perfused lizard lung, but the location of those muscarinic receptors is unknown (32). ACh may act on receptors located on the type II cells or may also act on muscarinic receptors located on pulmonary smooth muscle, causing a contraction, which in turn may distort the type II cells and stimulate secretion.

In lizards, cholesterol (Chol) is a major component of surfactant and increases rapidly in vivo in response to decreases in body temperature (7). Whereas both Epi and ACh trigger PL release in the isolated lizard lung, neither agonist appears to affect surfactant Chol secretion (32, 33).

To determine whether both adrenergic and cholinergic agonists act directly on the type II cell, we examined the effects of autonomic neurotransmitters and receptor antagonists on the secretion of total PL, DSP, and Chol from primary cultures of type II pneumocytes isolated from the lungs of the bearded dragon Pogona vitticeps. Furthermore, a decrease in the incubation temperature of mammalian type II cells inhibits basal and agonist-stimulated PL secretion, an effect that may result from a decrease in membrane fluidity (5). A similar inhibition of secretion in animals that regularly undergo large reductions in body temperature may have profound effects on lung function in these animals. Therefore, the effect of changes in temperature on agonist-stimulated surfactant lipid secretion was also examined.

	MATERIALS AND METHODS

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Animals. Bearded dragons [Pogona vitticeps, 139.06 ± 12.75 g (mean ± SE), snout-to-vent length = 161 ± 5.29 mm] were collected in the northern Flinders Ranges of South Australia in mid-February. They were housed at 26-28°C in groups of 5 or 6 in 100 × 45 × 50-cm plastic containers, and fed fruit, vegetables, mealworms (Tenebrio larvae), and dried dog food twice weekly. Water was provided ad libitum. Lighting was maintained on a 12:12-h light-dark cycle.

Isolation and culture of type II pneumocytes from Pogona vitticeps. Type II pneumocytes were isolated from lizards using modifications of the methods of Dobbs et al. (10). Animals were killed with an intraperitoneal injection of pentobarbitone (Nembutal, 150 mg/kg body wt, Boehringer Ingelheim, Australia). The lungs were perfused via the pulmonary artery with a Ca²⁺- and Mg²⁺-free salt solution (120 mM NaCl, 3.2 mM KCl, 6 mM glucose, 10 mM HEPES, pH 7.4, 37°C) until free of blood. They were then lavaged to total lung capacity (~50 ml) with this solution supplemented with 0.2 mM EGTA and 10 µg/ml amphotericin B. The lungs were then removed from the body cavity and minced with scissors in the presence of DNase type I (10 µg/ml, Worthington Biochemical, Freehold, NJ). The tissue pieces were incubated at 37°C in collagenase type IV (200 U/ml in Ca²⁺- and Mg²⁺-free salt solution, pH 7.4, 5% CO₂ in air; Worthington Biochemical, Freehold, NJ) for 12-15 h. Cellular clumps were dispersed by trituration, and the resulting cell suspension was filtered through two layers of mesh (pore size, 1 mm and 150 µm). The filtrate was centrifuged (150 g, 5 min, Beckman model TJ-6 centrifuge) and the supernatant removed. The cell pellet was resuspended in DMEM (phosphate deficient; Sigma Chemical, St Louis, MO), and the suspension was poured over 60-mm bacteriologic petri dishes that had been precoated with 500 µg/ml bovine IgG. These dishes were prepared by incubation with 500 µg/ml bovine IgG in Tris base (pH 9.5) for 3 h at 22°C. The dishes with the cell suspension were then incubated at 37°C for 90 min, during which time macrophages and other immune cells adhere to the IgG, allowing the nonimmune cells, predominantly type II cells, to be decanted. The cell suspension was centrifuged (150 g, 5 min), and the pellet was resuspended in phosphate-free DMEM. The cells were counted using a hemocytometer and plated in six-well tissue culture trays (Becton Dickinson, Franklin Lakes, NJ, 5 ml media/well, 5 × 10⁵ cells/well) in phosphate-free DMEM supplemented with 25 mM HEPES, 24 mM NaHCO₃, 10% fetal bovine serum (Commonwealth Serum Laboratories, Adelaide, Australia), 25 U/ml penicillin, 25 µg/ml streptomycin, 10 µg/ml amphotericin B and 50 µg/ml gentamicin (Sigma Chemical). The pH was adjusted to 7.4, and the cells were incubated at 37°C and 5% CO₂ in air. Cell viability was assessed by exclusion of the vital dye trypan blue. Differential cell counts were performed on air-dried smears using a modified Papanicolaou stain (10).

Effects of autonomic neurotransmitters on surfactant lipid secretion from isolated type II cells. After incubation of the cells for 15 h, aliquots of the media were taken from each well [at "time 0" (T₀)]. After a further 3 h, the media were again sampled ["time 1" (T₁)]. This first 3-h experimental period, which is prior to the addition of agonists ("predrug"), provides an indication of basal changes in media PL, DSP, and Chol content. Agonists (100 µM carbamylcholine hydrochloride or 100 µM Epi hydrochloride) were added to each well, and the cells were incubated for a further 3 h, after which time the media was again sampled ["time 2" (T₂)]. This second experimental period ("postdrug"), following the addition of the agonists (or agonists/antagonists) provides an indication of changes in media PL, DSP, and Chol content in the presence of these agents. Control groups, into which no agonists or antagonists had been added, were also sampled throughout the 6-h experimental period. Media aliquots from four wells at each time point were pooled and centrifuged (1,000 g, 10 min) to remove cellular debris, the supernatant was decanted, and the total lipids were extracted. For experiments using antagonists (propranolol hydrochloride, 1 mM; atropine sulfate, 100 µM), these were added 5 min before addition of the agonist.

Surfactant lipid (PL, DSP, and Chol) secretion/uptake was calculated from changes in total media content of each lipid and is expressed as a ratio of the media lipid content at a given time point divided by that of the previous time point. For example, changes in media PL in the 3 h between T₀ and T₁ (i.e., before agonists are added to the media) are calculated by dividing total media PL at T₁ by total media PL at T₀. Similarly, changes in media PL in the 3 h between T₁ and T₂ (i.e., after agonists and/or antagonists are added to the media) are calculated by dividing total media PL at T₂ by total media PL at T₁. These ratios are then expressed as a percentage. Values <100% reflect lipid uptake and those >100% reflect lipid release.

Effect of lowering incubation temperature on surfactant lipid secretion from isolated type II cells. To test the effect of lowering incubation temperature on agonist-stimulated secretion of PL and Chol, cells were isolated and cultured at 37°C (as above). Twelve hours prior to commencement of secretion experiments, the incubation temperature of the cells was reduced to 18°C (32, 33) and the cells were allowed to equilibrate for another 12 h. The media was then sampled, and sampling was repeated (as above) after a further 3 h. Agonists (100 µM carbamylcholine chloride or 100 µM Epi hydrochloride) were added to each well, and the cells were incubated for a further 3 h, after which time the media was again sampled (postdrug).

In all experiments in which Epi was used, sodium ascorbate (1 mM) was also added to the media to prevent oxidation and inactivation of the Epi.

Biochemical analyses of media. Lipids were extracted from media samples into chloroform and methanol (1:2) using the method of Bligh and Dyer (2). The chloroform layer was removed, dried down under nitrogen, and reconstituted in a standard volume of chloroform. Phosphorus content was determined by the method of Bartlett (1). Total PL was calculated by multiplying the phosphorus content by 25, as phosphorus comprises ~4% of PL (6). The DSPs were separated from the unsaturated PLs and the neutral lipids on aluminum oxide following reaction with osmium tetroxide (21). The amount of DSP was determined using a phosphorus assay. The neutral lipid fraction was retained, dried under nitrogen, and reconstituted in isopropanol at 2°C. Chol was quantified using a high-pressure liquid chromatography system (9), comprising a Waters pumping system (model M-45; Waters, Milford, MA) and an LKB 2157 autosampler (Pharmacia LKB Biotechnology, Uppsala, Sweden). Twenty microliters of either sample or standard were injected onto a Waters ¹⁸C Novopak guard and analytical column (150 mm long, 4.6 mm ID) packed with 4-µm silica spheres. Isocratic elution of Chol was completed within 32 min at room temperature using a mobile phase consisting of acetonitrile, isopropanol, and water (6:3:1 vol/vol/vol). A flow rate of 1 ml/min and an operating pressure of 1,600 psi were maintained throughout the elution, and ultraviolet absorbance was recorded at 210 nm. The detector output was digitized using a Delta Chromatography data system for acquisition of data and integration of peaks (Digital Solutions, Brisbane, Australia). Standards were assayed in duplicate and were included at the beginning of each run (5-50 µg/ml).

Fixation of cells for electron microscopy. Cell pellets were fixed in 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and postfixed in 1% osmium tetroxide overnight. They were then stained en bloc in 1.5% uranyl acetate, dehydrated in 70%, 80%, 90%, and 100% acetone, and embedded in resin.

Statistical analyses. Percentage changes were arcsin transformed and data analyzed using one-way analyses of variance followed by paired or unpaired t-tests where appropriate. Statistical significance was assumed as P  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Electron microscopical examination of type II cells. Figure 1 depicts a typical type II cell isolated from the lungs of the bearded dragon Pogona vitticeps. The cells have large, osmiophilic lamellar bodies characteristic of mammalian type II cells. Cell viability, as assessed by exclusion of the vital dye trypan blue, was found to be >90%. Differential cell counts indicated that the cell populations were >80% type II cells.

View larger version (171K): [in this window] [in a new window]   Fig. 1.   Electronmicrograph of a type II cell isolated from lungs of bearded dragon Pogona vitticeps. Cells have large lamellated inclusions characteristic of mammalian alveolar type II cells. Scale bar = 2 µm.

Effects of autonomic neurotransmitters on surfactant lipid secretion from isolated type II cells. At 37°C, no change in media PL content occurred in a control group into which no agonists or antagonists were added over the 6-h experimental period (T₁/T₀: 100.44 ± 5.23%; T₂/T₁: 99.20 ± 0.84%). Media PL increased on addition of Epi (Epi: t = 2.59, df = 5, P = 0.02; Fig. 2A) but not on addition of ACh (Fig. 2A). The response to Epi was abolished in the presence of propranolol (Fig. 2A).

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Effect of epinephrine (Epi), Epi/propranolol (Epi/Prop), ACh, and ACh/atropine (ACh/At), respectively, on total surfactant phospholipid (PL) secretion (A; Epi: n = 4, Epi/Prop: n = 5, ACh: n = 9, ACh/At: n = 8), disaturated PL (DSP) secretion (B; Epi: n = 6, ACh: n = 8, ACh/At: n = 8), and cholesterol (Chol) secretion (C; Epi: n = 5, ACh: n = 6) from type II cells isolated from Pogona vitticeps (37°C). Data are expressed as means ± SE and represent changes in secretion as %time 1/time 0 (predrug) and %time 2/time 1 (postdrug). Values <100% reflect lipid uptake, and those >100% reflect lipid release (see dotted line). * Significant difference from predrug (P = 0.02 in A and 0.003 in B).

No change in media DSP content occurred in a control group over the 6-h experimental period (T₁/T₀: 99.62 ± 4.05%; T₂/T₁: 96.07 ± 3.7%). Addition of Epi increased media DSP (Epi: t = 4.47, df = 5, P = 0.003; Fig. 2B). However, for unknown reasons, propranolol interferes with the assay used to determine DSP, and, as such, it was not possible to determine whether the Epi-stimulated DSP increase was blocked by propranolol. Addition of ACh did not affect DSP secretion (Fig. 2B). Chol release was unaffected by either Epi or ACh (Fig. 2C).

Effects of lowering incubation temperature on surfactant lipid secretion from isolated type II cells. At 18°C, no change in media PL content occurred in a control group over the 6-h experimental period (T₁/T₀: 98.52 ± 3.94%, T₂/T₁: 104.69 ± 1.96%). Addition of either Epi or ACh increased media PL content (Epi: t = 2.589, df = 5, P = 0.025; Fig. 3A; ACh: t = 3.282, df = 7, P = 0.007; Fig. 3A). No change in media DSP content occurred in the control group over the 6-h experimental period (T₁/T₀: 90.24 ± 10.37%, T₂/T₁: 96.98 ± 4.46%). Media DSP content also increased on addition of either Epi or ACh (Epi: t = 3.054, df = 7, P = 0.009; Fig. 3B; ACh: t = 2.489, df = 5, P = 0.028; Fig. 3B). Chol release was unaffected by either Epi or ACh (Fig. 3C).

View larger version (41K): [in this window] [in a new window]   Fig. 3.   Effect of Epi and ACh on total surfactant PL secretion (A; Epi: n = 6, ACh: n = 8), DSP secretion (B; Epi: n = 8, ACh: n = 6), and Chol secretion (C; Epi: n = 4, ACh: n = 5) from type II cells isolated from Pogona vitticeps (18°C). Data are expressed as means ± SE and represent changes in secretion as %time 1/time 0 (predrug) and %time 2/time 1 (postdrug). Values <100% reflect lipid uptake, and those >100% reflect lipid release (see dotted line). * Significant difference from predrug (Epi: P = 0.025, ACh: P = 0.007 in A; Epi: P = 0.009, ACh: P = 0.028 in B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Validation of methods. The current study presents a new methodology for the investigation of the factors that control surfactant secretion and composition in nonmammals, namely, the isolation and culture of type II pneumocytes from lizards. The earliest attempts to isolate mammalian type II cells predominantly used differential centrifugation, a technique that separates the cells based on size and density (17). Crude suspensions of lung cells are layered over gradients (usually Ficoll or Percoll), and the gradients are centrifuged. Mammalian type II cells separate to the boundary between the layers of density 1.04 and 1.08 (17). Whereas it is possible that this technique will work with the lung cells of nonmammals, it is first necessary to know the size of the type II cells, which appears to vary markedly from mammals to nonmammals and indeed even within the nonmammals. This technique also, by selecting for cells of a certain size, limits the size and possibly age of the cell population harvested, and, as such, the population obtained may not be truly representative of the type II cell population in the intact lung.

This is the first time that type II cells have been isolated and cultured from a nonmammalian species. Although the technique is founded on the method of Dobbs et al. (10), it varies considerably from the original technique, and, as such, the reasons for these variations require some explanation. The original technique of Dobbs et al. (10), designed for the isolation of type II cells from the lungs of rats, uses elastase, applied for 30 min, as the primary digestive enzyme used to dissociate the lung tissue. Whereas the mass of lung tissue obtained from the lizards used in this study is similar to that typically obtained from a rat, we have found elastase to be poorly effective in lizard lungs, probably due to the relatively high proportion of collagen in these lungs. Collagenase was therefore used, although a much longer exposure time was necessary for digestion of the lungs (12-15 h). To ensure maximum viability of the cells, a relatively low concentration of the enzyme was used. Exclusion of the vital dye trypan blue confirmed that this length of time in the enzyme solution did not adversely affect the health of the cells, and light and electron microscopic examination of the cells revealed that differentiation of the cells to type I cells had not commenced in this period. Preliminary experiments revealed that the cells also do not become adherent until after 5 days or more in culture. For this reason, cell suspensions and not adherent populations were used in all of these experiments. It was decided to commence the secretion experiments as soon as possible after cell isolation, after an equilibration period of ~12 h. This time allowed the cells to settle, while not being of sufficient duration to allow media infections to develop, as the animals used were wild caught and cells cultured from these animals appeared to be susceptible to infection.

The isolation technique of Dobbs et al. (10) involves the "panning" of crude lung cell suspensions over bacteriologic plates coated with IgG to remove immune cells, the predominant non-type II cells in the suspension. These researchers used homologous rat IgG for the isolation of rat type II cells. To isolate cells from the lungs of nonmammals, it was not possible to obtain homologous IgG. However, use of a nonhomologous antibody to isolate type II cells from the species used appears to work well, possibly because the F_c region (constant, non-antigen-binding segment) of the immunoglobulin molecule, to which immune cells attach, is highly conserved among the vertebrates (20). It appears that immune cells from the species used will bind to rat IgG.

The overwhelming majority of cellular secretion studies using mammalian type II pneumocytes preincubates the cells with radiolabeled precursors (particularly [³H]choline) of surfactant PLs (4, 11). These labels are taken up by the cells and incorporated into surfactant PLs. Subsequent secretion is quantified by liquid scintillation counting. Although this technique is undoubtedly easier and faster, it may be premature for these early studies using nonmammalian type II cells. Most radiolabeling studies are performed on adherent cell populations, and, as discussed earlier, adherence does not appear to occur with nonmammalian type II cells until at least several days after isolation. Without lengthy preliminary studies establishing rates of lipid uptake and incorporation by cells of the species examined, it is difficult to guarantee that radiolabeling will not lead to false-negative results. Prior to these studies it was also unknown which, if any, agonists stimulate surfactant secretion in nonmammals. However, with the use of our current methods, it was possible to reliably measure total PL, DSP, and Chol secretion from the cells.

Control of surfactant lipid secretion from isolated type II cells. A great many factors appear to affect the secretion of surfactant PL from type II cells. In mammals, the principals of these are -agonists, ATP and adenosine compounds, and ventilation-induced distortion of the type II cells. Although it does not appear that ventilation affects surfactant secretion in the lizard (33), -agonists do act directly on the type II cells to stimulate PL secretion. Cholinergic agonists also trigger PL secretion from lizard type II cells at 18°C, and it appears that they act directly on muscarinic receptors on the cell surface. In mammalian type II cells, a number of distinct signal transduction mechanisms are activated by different secretagogues prior to secretion. Adrenergic agonists trigger production of cAMP and subsequent activation of cAMP-dependent protein kinases (26). ATP and adenosine compounds trigger production of cAMP, diacylglycerol, and inositol triphosphate, with consequent increases in intracellular Ca²⁺. The subcellular responses to distortion of the type II cell are unclear, but may involve an increase in cytosolic Ca²⁺ (31). Many of the compounds produced after stimulation of the receptor are part of cascades that activate a range of enzymes that ultimately facilitate the movement of the lamellar bodies to the plasma membrane and subsequent exocytosis. For instance, an increase in cytosolic cAMP is thought to activate several cAMP-dependent protein kinases and mediate the polymerization of tubulin to microtubules (4). Similarly, increases in Ca²⁺ activate many Ca²⁺-dependent protein kinases (29) and promote the self-association of "docking" proteins, which assist in the fusing of the membrane of the cell with those of the lamellar bodies (27).

The subcellular mechanisms that are triggered prior to surfactant secretion from lizard type II cells are unknown. However, as -agonists trigger secretion from both lizard and mammal cells, it is possible that these secretagogues act via similar pathways in both groups. The signal transduction pathway through which ACh acts, however, is unclear. Application of cholinergic agonists to isolated rat type II cells elicits an increase in cytosolic Ca²⁺ of up to 20% (16). Mechanical distortion of rat type II cells increases cytosolic Ca²⁺ up to 3.5-fold (31). Whether the ACh-elicited increase in Ca²⁺ in mammalian type II cells is of significant magnitude to be physiologically relevant is unknown (16). It is possible that the ACh-mediated stimulation of PL release from lizard type II cells is also associated with an increase in cytosolic Ca²⁺.

Lowering the incubation temperature of mammalian type II cells to 5°C and of isolated lungs to 13°C virtually abolishes surfactant PL secretion (11, 18). Low temperature inhibits basal secretion and secretion elicited by respiratory alkalosis (28), hyperventilation (23), and the protein kinase-activating compound tetradecanoylphorbolacetate (11). The inhibition does not appear to result from a lowering in metabolic activity and may be related to a decrease in membrane fluidity (5). At 18°C, cells from P. vitticeps respond to stimulation by Epi and ACh and are capable of continued secretion. This continued secretion may be possible because the cellular membranes of lizard type II cells are sufficiently fluid or, alternatively, undergo some degree of homeoviscous adaptation as temperature decreases, maintaining fluidity via a modification of the lipid composition of the membranes.

Application of adrenergic agonists onto mammalian type II cells may increase PL secretion by over 100% (11). However, Epi increased the secretion of total PL and DSP from lizard type II cells by only 7% and 15%, respectively. Hence, although the reptilian lung typically contains 7-70 times more surfactant PL per centimeters squared of respiratory surface area (8), rates of secretion of PL from reptilian type II pneumocytes appear considerably lower than those of mammalian cells. Furthermore, it does not appear that lamellar bodies in lizards store less surfactant than those in mammals. Comparisons of the PL content of lamellar bodies from mice and central netted dragons (Ctenophorus nuchalis) reveal that, on a milligram of PL-per-gram wet lung basis, the lamellar bodies contain the same amounts of PL between the two species (6). The lower secretory (and probably surfactant turnover) rates of the lizard type II cells may be reflective of the substantially lower metabolic rates of reptilian cells, which, at 37°C, are three- to sixfold less than cells from equivalent mammalian tissues (12).

Cholinergic agents stimulate PL secretion in the isolated perfused rat lung at 23°C but not at 37°C (15). Similarly, cholinergic agents elicit surfactant PL secretion in the isolated perfused lizard lung at 22-23°C (32) and from primary cultures of lizard type II cells at 18°C, but carbamylcholine did not affect secretion from lizard cells at 37°C. The reasons for these differences are unclear, but it is possible that the cholinergic response is temperature sensitive. The temperature dependency of the response may result from a number of factors, including alterations in receptor sensitivity with temperature and an increase in the activity of the enzymes responsible for the breakdown of ACh, chiefly acetylcholinesterase. Increased enzyme activity may result in an accelerated degradation of applied ACh and, consequently, a reduction in the effective dose at the receptor.

The Chol component of surfactant is capable of rapid changes in vivo in response to changes in body temperature in lizards (7). A reduction in body temperature during torpor in the marsupial Sminthopsis crassicaudata is associated with the production of a surfactant enriched in Chol and DSP (19). In its pure form, DSP has a phase-transition temperature of 41°C, below which it exists in a gel state and cannot readily spread at the air-liquid interface (13). Addition of Chol to in vitro preparations of pure DSP decreases the phase transition point of the DSP, enabling the mixture to remain in a fluid, spreadable state well below 41°C (25). Chol is believed to interfere with the van der Waals intermolecular forces between the DSP molecules, preventing formation of the gel (13). Hence, in vivo increases in surfactant Chol with decreases in body temperature may result in a more surface-active surfactant that remains fluid and functional as body temperature decreases. Whereas it appears that Chol is a dynamic component of surfactant, titrated rapidly and independently of the PL component, its secretion was unaffected by any agonists used, and it appears that the secretion of Chol may not be under autonomic control.

Summary. As changes in ventilatory pattern in lizards may not be expected to stimulate regular surfactant secretion, the autonomic nervous system may predominantly control secretion. The effects of Epi and ACh on PL secretion in the isolated perfused lizard lung appear to be mediated, at least in part, through specific receptors for these agonists on the type II cells. At lower body temperatures, the type II cells in the lizard remain metabolically active and, unlike those of homeothermic mammals, are able to continue secretion as temperature falls. The Epi-stimulated secretion of lizard surfactant PL at 18°C suggests that adrenergic receptors on lizard type II cells may be insensitive to changes in temperature, and the cells remain sufficiently metabolically active to maintain some uptake and secretion of PL. Although Epi appears to stimulate PL secretion from lizard type II cells at low temperature, an in vivo increase in plasma Epi at low temperature will also increase metabolic rate at a time when the animal needs to conserve energy. ACh, on the other hand, may be a more appropriate stimulator of surfactant secretion at low temperatures. Furthermore, if the secretory response to ACh is indeed temperature dependent, parasympathetic effects on surfactant PL secretion may be inactive at high body temperatures. The relative dominance of the sympathetic and parasympathetic branches of the autonomic nervous system may therefore change with fluctuations in body temperature. Additionally, over a wide range of body temperatures, both the sympathetic and parasympathetic nervous systems may control secretion. However, the sympathetic nervous system may dominate at higher body temperatures, stimulating secretion via increases in circulating catecholamines and/or via an increased activity of intrapulmonary sympathetic nerves (32). In the isolated lizard lung, an increase in temperature also increases the reuptake of surfactant PL, suggesting that higher body temperatures in the lizard are associated with an increased turnover of surfactant PL (33). As body temperature decreases, the parasympathetic nervous system may become increasingly more influential, stimulating surfactant secretion without elevating metabolic rate.

Perspectives