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Am J Physiol Regul Integr Comp Physiol 281: R770-R777, 2001;
0363-6119/01 $5.00
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Vol. 281, Issue 3, R770-R777, September 2001

Dexamethasone and epinephrine stimulate surfactant secretion in type II cells of embryonic chickens

Lucy C. Sullivan and Sandra Orgeig

Department of Environmental Biology, University of Adelaide, Adelaide, South Australia 5005, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pulmonary surfactant (PS), a mixture of phospholipids and proteins secreted by alveolar type II cells, functions to reduce the surface tension in the lungs of all air-breathing vertebrates. Here we examine the control of PS during lung development in a homeothermic egg-laying vertebrate. In mammals, glucocorticoids and autonomic neurotransmitters contribute to the maturation of the surfactant system. We examined whether dexamethasone, epinephrine, and carbamylcholine hydrochloride (agonist for acetylcholine) increased the amount of PS secreted from cultured type II cells of the developing chicken lung. In particular, we wanted to establish whether dexamethasone would increase PS secretion through a process involving lung fibroblasts. We isolated and cocultured type II cells and lung fibroblasts from chickens after 16, 18, and 20 days of incubation and from hatchlings (day 21). Epinephrine stimulated phosphatidylcholine (PC) secretion at all stages, whereas dexamethasone stimulated secretion of PC at days 16 and 18. Carbamylcholine hydrochloride had no effect at any stage. This is the first study to establish the existence of similar cellular pathways regulating the development of surfactant in chickens and eutherian mammals, despite the vastly different birthing strategies and lung structure and function.

lung development; bird; phosphatidylcholine; type II cell culture; fibroblasts


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE EVOLUTION OF AIR BREATHING in the vertebrates has occurred independently many times since the Devonian period. Each vertebrate class has members that breathe through lungs, which can vary enormously in structure and complexity (14). Despite the tremendous structural diversity, all vertebrate lungs are internal structures lined with fluid. A high surface tension at the air-liquid interface in the lung would result in a number of ventilatory difficulties, including alveolar collapse and pulmonary edema. Pulmonary surfactant (PS), a mixture of lipids and proteins, functions to reduce surface tension at the air-liquid interface in the lung. PS has been discovered in the lungs of all air-breathing vertebrates examined, including in the parabronchial lung of birds (9).

The morphology and biochemistry of the surfactant system in all vertebrates is highly conserved. Disaturated phospholipids, unsaturated phospholipids, and cholesterol are the dominant lipids in surfactant (10), and phosphatidylcholine (PC) is the most abundant phospholipid class (49). Dipalmitoylphosphatidylcholine is the most abundant phospholipid in mammals and in birds (24, 39). Surfactant lipids and proteins are synthesized in specialized cuboidal alveolar cells, termed type II cells. The components are synthesized in the endoplasmic reticulum and stored in dense, multilayered structures called lamellar bodies (22). Type II cells and lamellar bodies occur in the lungs of reptiles (36, 37, 51), birds (29, 31, 48), and amphibians (16, 18). A surfactant specific protein, SP-A, isolated from mammals, is also present in surfactant isolated from members of each vertebrate group (41, 45). Furthermore, SP-A and SP-B have been detected in epithelial cells of the developing chick lung from the 15th day of incubation (57).

In all vertebrates, the surfactant system is probably most critical at the time when the animal draws its first breath. The removal of lung fluid, a decreased resistance to inflation, and a decrease in pulmonary arteriolar resistance must occur simultaneously with the first breath. The PS system plays a crucial role in the first two of these actions. However, there are many different birthing strategies among the vertebrates. Mammals and birds represent two dramatically different birthing strategies. The transfer from the uterus to air breathing occurs relatively quickly in eutherian mammals. In birds, hatching may take several days, allowing time for lung clearance before pulmonary ventilation. Birds commence breathing by "pipping" into an internal air cell while continuing respiration through the chorioallantoic membrane. Pipping in chickens normally occurs on day 19 of a 21-day incubation period, and pulmonary ventilation commences on day 20 (50).

There are also differences in the developmental processes between birds and mammals. During gestation, mammals are under the influence of maternal hormones. Although the developing chick is under the influence of some hormones in the yolk (30, 40), the transfer of these hormones is likely to differ in terms of the regulation and relative timing throughout incubation. Therefore, these differences in birthing strategy could lead to differences in the mechanisms and timing of control of development of the PS system. However, the surfactant system is crucial to the successful completion of the first breath. Furthermore, it has had only one origin and is highly conserved throughout the radiation of the vertebrates. Therefore, the mechanisms controlling the development of the surfactant system may also be conserved, suggesting that the system as a whole, including its control, has been subject to stabilizing selection.

The control of development of the PS system has never been described in a non-mammalian vertebrate. We know, however, that in chickens, the first lamellar bodies appear in epithelial cells of the parabronchi on day 16 of incubation and then rapidly increase in number (8). The lamellar bodies undergo accelerated differentiation after 18 days of incubation (32). By day 20 of incubation, the lung is fully differentiated to allow for normal breathing, and it contains numerous, large lamellar bodies (33). The total phospholipid content of the lung increases most rapidly after 18 days of incubation (24, 26). The major lung phospholipid throughout the entire incubation period is PC (24), and lamellar bodies isolated from the embryonic chick lung are particularly enriched in PC (32). The proportion of disaturated phospholipids increases significantly toward the end of incubation (26); however, the level of PC as a percentage of total phospholipid remains the same (24). The lung phospholipids in lavage from the adult bird are almost identical in composition to that of mammals (15). Therefore, there are many similarities between the development of the PS system in birds and mammals.

In mammals, maturation of the surfactant system is induced by a complex interaction of factors of both maternal and fetal origin. Glucocorticoids and autonomic neurotransmitters are among the most important factors controlling the development of the surfactant system in mammals. Glucocorticoids have been found to increase surfactant phospholipid synthesis, secretion, and lipid saturation (20) and are believed to mediate their effects on type II cells through interstitial fibroblasts (42). Although there is some evidence to suggest that glucocorticoids contribute to avian surfactant maturation (25, 46, 53), the mechanism remains unclear, in particular whether glucocorticoids will promote secretion of PS from type II cells via a pathway involving fibroblasts.

Adrenergic agonists act directly on mammalian fetal and adult type II cells (4, 19) through beta 2-receptors, stimulating a cAMP-dependent pathway (34). Adrenergic agonists also promote PS secretion in type II cells isolated from adult lizards, frogs, and dunnarts (a small heterothermic marsupial) (54, 55). In eutherian mammals, cholinergic agonists are believed to act indirectly to stimulate surfactant secretion. These agents may act on the adrenal glands to release epinephrine and norepinephrine, which in turn stimulates surfactant release (38). Alternatively, cholinergic agonists may cause contraction of smooth muscle cells in the intrapulmonary tissue, distorting the type II cells and stimulating surfactant secretion (35). However, acetylcholine elicits surfactant phospholipid release by acting directly on muscarinic receptors on type II cells in lizards, dunnarts, frogs, and lungfish (54, 55). It has been proposed that direct cholinergic stimulation may predominate at low body temperatures in heterothermic animals to trigger surfactant release without affecting metabolic rate (55).

Conservation of the pathways controlling development of the surfactant system in two such widely divergent groups of animals as birds and mammals would provide evidence that the surfactant system was crucial to the evolution of air breathing. Furthermore, by comparing two groups of homeotherms, we remove the compounding effects of variations in body temperature. In addition, the development of the chick lung morphology is well described. Therefore, in the present study we examine the effect of dexamethasone, epinephrine, and carbachol on lung cells isolated from the embryonic chicken Gallus gallus.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Fertilized chicken eggs (Gallus gallus) were purchased from a commercial supplier (Globe Derby Poultry, Bolivar, South Australia). The eggs were incubated under normoxic conditions at 39°C in a commercially purchased incubator equipped with an automatic turner (Bellsouth, Victoria, Australia). The embryos were sampled after 16, 18 (prepip), and 20 days (postpip) of incubation. Animals were also sampled within 24 h after hatching. Between five and nine individuals were used for each age group, and cells were isolated and cultured from individual animals without pooling cells.

Isolation and coculture of type II cells and fibroblasts. As glucocorticoids are believed to mediate their effects through interstitial fibroblasts, type II cells and fibroblasts were isolated and cocultured. The method used was based on that of Dobbs et al. (12) and Scott et al. (43). The animals were killed with an overdose of pentobarbital sodium (Nembutal; 150 mg/kg body wt, Abbott Laboratories). The lungs were perfused with heparinized PBS solution via the right ventricle until free of blood (2-3 min). The lungs were then excised and minced with scissors in 1 ml of a solution of DNase type I (1 mg/ml, Worthington Biochemicals, Freehold, NJ) in PBS. About 10 ml of a solution of trypsin (0.1%), EDTA (0.02%), and fungicide (10 µg/ml) was added to the minced lung mixture and incubated in a water bath at 37°C for 40 min to dissociate the tissue. The enzyme reaction was stopped by the addition of 10% fetal bovine serum (FBS; Commonwealth Serum Laboratories, Adelaide, Australia). Clumps of tissue were dispersed by trituration and filtered through two layers of gauze followed by filtering through a mesh screen (74 µm pore size). The cell suspension was then centrifuged at 150 g for 8 min, and the resulting cell pellet was gently resuspended in DMEM containing 24 mM NaHCO3 and 10 mM HEPES (Sigma Chemicals). To select type II cells and fibroblasts specifically, 60-mm bacteriologic plastic dishes were coated with a solution of bovine IgG (Sigma Chemicals) and allowed to incubate for 3 h at room temperature. The plates were then washed with 2× PBS and 1× DMEM before the cell suspension was poured onto the plates. The cells were incubated for 1 h 15 min at 37°C in a 10% CO2-90% air incubator. During this time, macrophages and other immune cells bind to the IgG molecules and adhere to the plates, allowing the remainder of the cells (the majority of which are type II cells and fibroblasts) to be poured off. The unattached cells were then centrifuged at 150 g for 8 min, and the pellet was resuspended and incubated in DMEM containing 10% FBS, 10 mM HEPES, 24 mM NaHCO3, 25 U/ml penicillin, 25 µg/ml streptomycin, and 10 µg/ml amphotericin B (Sigma Chemicals). Viability was assessed by exclusion of the vital dye trypan blue, and cell counts were performed using a hemocytometer. Cells were diluted to 2.5 × 106 cells/ml, and 1 µCi/ml [methyl-3H]choline chloride (Amersham) was added to the medium. The cells were then plated at a density of 500,000 cells/cm2 in 96-well tissue culture plates on a layer of human fibronectin (5 µg/cm2, Boehringer Mannheim) and allowed to adhere overnight at 37°C in 10% CO2.

Isolation of type II cells. Type II cells alone were isolated and cultured from seven 18-day embryos to determine if glucocorticoids act through a pathway involving fibroblasts and to confirm that epinephrine acts directly on type II cells. Before the incubation on IgG, the cell suspension was centrifuged at 150 g for 8 min. The cell pellet was resuspended in DMEM containing 10% FBS, poured over 10-cm tissue culture plates, and incubated for 1 h at 37°C in 10% CO2. The cell suspension was removed, and the process was repeated for another 1 h on a fresh plate. During this time, the fibroblasts adhered to the culture dishes, and the remaining cell suspension was centrifuged at 150 g for 8 min. The resulting pellet was resuspended in DMEM (without FBS), applied to the bacteriologic plates coated with IgG, and then isolated and cultured as above.

Agonist stimulation. After the overnight incubation, the cells were rinsed twice in fresh DMEM to remove unincorporated choline chloride and unattached cells. The cells were then incubated in the presence of an agonist dissolved in DMEM (without FBS) or control in DMEM alone for 4 h. The control and agonist groups were from the same preparation of cells (250,000 cells/well in a 96-well tissue culture plate), and each assay was done in triplicate. The agonists were epinephrine hydrochloride (100 µM), carbamylcholine hydrochloride (100 µM), and water-soluble dexamethasone (100 µM) (Sigma Chemicals). To prevent oxidation and inactivation of the agonists, 1 mM sodium ascorbate (Sigma Chemicals) was added to all the wells. At day 18, we included additional experiments to establish if the action of dexamethasone was mediated through the glucocorticoid receptor and dependent on protein synthesis. For this, we added 1 µM RU-486 (glucocorticoid receptor antagonist, Sigma) or 50 µM cycloheximide (protein synthesis inhibitor, Calbiochem) to wells containing 100 µM dexamethasone. The media and cell fractions were isolated separately from the wells. A mixture of chloroform and methanol (1:2) was used to extract lipids from the media and cellular fractions separately (3). Unlabeled egg yolk PC (70 µg/ml) (Sigma Chemicals) was added to the media samples as a carrier to improve the recovery of labeled PC. The chloroform layer was removed, and the samples were dried under nitrogen and resuspended in Ready Organic Scintillant (Beckman). The amount of radioactive PC was measured by liquid scintillation counting (Tricarb 2100TR, Canberra Packard), and the percentage of total PC secreted was determined by the following equation (44):
%Total PC secretion<IT>=</IT><FR><NU>DPM of media</NU><DE>DPM of media<IT>+</IT>cells</DE></FR><IT>×</IT>100
We confirmed that sphingomyelin (S) and lysophosphatidylcholine (LPC) did not contribute significantly to the amount of labeled PC in the medium by thin-layer chromatography using the method of Touchstone et al. (47). After extraction of the lipids, the samples were dried under nitrogen, resuspended in 25 µl of chloroform, and spotted on Whatman LK5 plates. The plates were developed in 15 ml chloroform-17 ml ethanol-17.5 ml triethylamine-4.25 ml water. PL standards were included on each plate. The lipids were visualized using iodine, and the bands corresponding to PC, S, and LPC were scraped into scintillation vials, resuspended in Ready Organic Scintillant (Beckman), and counted by liquid scintillation counting.

Cell viability. After the 4-h incubation period, viability of the cells in some wells containing the secretatgogues was assessed by exclusion of the vital dye trypan blue. In addition, a lactate dehydrogenase (LDH) cytotoxicity kit (Roche) was used as a sensitive measure of cell damage. Some test substances interfere with the measurement of LDH by this technique. Therefore, a number of controls were also tested for LDH activity, such as media alone and media containing the agonists. The LDH assay was performed according to the manufacturer's instructions.

Statistical analyses. Percent changes were arcsin transformed, and the data were analyzed using one-way analysis of variance followed by paired or unpaired t-tests. Statistical significance was assumed as P <=  0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell purity and viability. Cell yield varied between 2 and 6 million cells/animal, depending on the age of the animal and the degree of lung perfusion. In initial experiments, we found that only ~30% of the cells would attach to the culture dish without the presence of an attachment factor. Plating the cells overnight on a layer of human fibronectin increased the number of attached cells to >85%. After the overnight incubation and rinsing the cells, ~90% of the remaining cells were type II cells and fibroblasts, in a ratio of ~1:1. The few contaminating cell types were macrophages and red blood cells. Thin-layer chromatography indicated that ~90% of radiolabeled choline released into the media was PC. This percentage did not change over the different ages. LDH release was minimal and indicated that the high levels of secretion at day 18 were not due to cell injury. There was no increase in LDH release after the 4-h incubation period in the presence of any of the agonists.

Electron microscopy. The type II cells isolated from days 16, 18, 20, and hatchling chickens show microvilli, large nuclei, and lamellar bodies characteristic of type II cells. However, the number of lamellar bodies in the cells appears to differ between the various ages. The lamellar bodies at day 16 are small and few in number (Fig. 1A). At day 16, there are also a number of large, electron-dense bodies, believed to represent the precursor of lamellar bodies (17, 33). The number of lamellar bodies rapidly increases after 18 days of incubation, with the electron micrograph showing numerous, large lamellar bodies (Fig. 1B), which are maintained throughout development (Fig. 1, C and D).


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  		Fig. 1.   Appearance of type II cells isolated from embryos of the chicken after 16 days (A), 18 days (B), and 20 days (C) incubation and after hatching (D). All the cells have lamellar bodies, microvilli, and a large nucleus, characteristic of type II cells (scale bar = 2 µm).

Basal PC secretion. Cocultured type II cells and fibroblasts had the highest level of basal PC secretion at embryonic day 18. There was a significant increase in basal PC secretion from cocultured cells isolated from day 16 and day 18 embryos (day 16, n = 9; day 18, n = 7; P < 0.001, Fig. 2A). At day 18, the level of basal PC secretion was significantly higher in cocultured type II cells and fibroblasts than in type II cells alone (n = 7, P = 0.046, Fig. 2B).


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  		Fig. 2.   Basal phosphatidylcholine (PC) secretion from type II cells in coculture with fibroblasts (A: embryonic day 16, n = 9; day 18, n = 7; day 20, n = 8; hatch, n = 12) and comparison with type II cells alone (B: embryonic day 18 coculture, n = 7; type II, n = 7). Data are expressed as means ± SE. Pairs of #, significant difference in percentage PC secretion; P <=  0.05. Pip, onset of pulmonary ventilation within egg.

Agonist stimulation. Dose-response data for epinephrine and dexamethasone are provided in Table 1. The increase in PC secretion was significant with both agonists only at a concentration of 100 µM. This concentration was used in all other experiments. Epinephrine stimulated the secretion of PC in cocultured cells isolated after 16 (n = 8, P = 0.034), 18 (n = 7, P = 0.023), and 20 (n = 7, P = 0.037) days of incubation and also from hatchling chickens (n = 5, P = 0.044, Fig. 3A). Dexamethasone stimulated PC secretion in cocultured cells isolated from day 16 (n = 7, P = 0.048) and day 18 (n = 7, P = 0.05) embryos (Fig. 4A), but not in day 20 embryos or hatchling chickens. Dexamethasone had no effect on isolated type II cells in the day 18 chicken (n = 7, P = 0.313, Fig. 4B), whereas epinephrine stimulated PC secretion on type II cells alone at the same time point (n = 7, P = 0.05, Fig. 3B). Dexamethasone had no effect when in the presence of RU-486 (n = 6, P = 0.327, Table 2) or cycloheximide (n = 6, P = 0.325, Table 1) on cocultured cells at day 18. Carbachol failed to stimulate PC secretion at any embryonic age tested (Table 2).

                              
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  		Table 1.   Effect of concentrations of agonists and inhibitors on %PC secretion from cocultured type II cells and fibroblasts isolated from day 18 embryonic chickens



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  		Fig. 3.   Effect of epinephrine on PC secretion from cocultured type II cells and fibroblasts (A: embryonic day 16, n = 8; day 18, n = 7; day 20, n = 7; hatch, n = 5) and on type II cells alone (B: embryonic day 18, n = 7). Data are expressed as means ± SE. star Significant difference from control PC secretion; P <=  0.05.



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  		Fig. 4.   Effect of dexamethasone on PC secretion from cocultured type II cells and fibroblasts (A: embryonic day 16, n = 7; day 18, n = 7; day 20, n = 6; hatch, n = 7) and on type II cells alone (B: embryonic day 18, n = 7). Data are expressed as means ± SE. star Significant difference from control PC secretion; P <=  0.05.


                              
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  		Table 2.   Effect of carbamylcholine hydrochloride on %PC secretion from cocultured type II cells and fibroblasts


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Validation of methods. This is the first time that embryonic type II cells have been isolated from a non-mammalian species. It is also the first time that non-mammalian type II cells have been cocultured with fibroblasts. Earlier techniques used to isolate type II cells from mammals used differential centrifugation to separate cells on the basis of size and density (28). This technique is not applicable to embryonic type II cells because of the variation in cell size during development, which would result in an enrichment of "mature" type II cells and may not be a true indication of the cell population (7). More recently, elastase and differential adherence on IgG have been used to isolate type II cells (12). Successful isolation of embryonic type II cells has been achieved with the use of IgG adherence (21). However, the enzyme elastase preferentially digests the epithelial layer and hence would not have released fibroblasts from the surrounding tissue.

Our laboratory has previously used collagenase to isolate type II cells from lizard lungs (54). However, to maintain cell viability, a very low concentration was used, which necessarily increased the dissociation time to 12-15 h. This is not appropriate for embryonic type II cells, because they differentiate very rapidly in culture such that they may be no longer representative of the age of the animal from which they were originally isolated (11). Therefore, trypsin was used to release both type II cells and fibroblasts. The use of trypsin as a digestive enzyme has been criticized because it may damage cell membranes and receptors (4). Hence, trypsin was used at a low concentration (0.1%), and exposure to the enzyme was limited to 40 min. This procedure allowed for the rapid dissociation of fibroblasts and type II cells from the surrounding tissue. Minimal LDH release indicated that the cells remained healthy during the isolation and experimental period. Electron microscopy confirmed that the type II cell morphology was identical to that of other species we have previously published (36, 54, 55) as well as to chicken type II cells in situ (31, 32).

As shown by both the dexamethasone and epinephrine dose-response data (Table 1), it was necessary to use what is regarded as very high doses (100 µM). Although the concentrations of agonists are well above mammalian physiological levels, cell viability was not affected. This dose of epinephrine has also been previously published and is optimal for use on type II cells isolated from a range of other non-mammalian vertebrates, including lizards, frogs, and lungfish (54, 55) as well as the heterothermic mammal, the fat-tailed dunnart (55). It is possible that non-mammalian cells in general require higher doses to elicit similar responses. It is unclear why this would be the case, but could possibly be related to differences in metabolic rate or receptor number.

The time course of 4 h in the presence of the agonists was chosen on the basis of preliminary experiments. It was found that 2 h was often not long enough to elicit a response to dexamethasone, although a response to epinephrine was observed within this period. By 6 h, the response to the agonists had reached a plateau. This is probably due to increased surfactant recycling.

Basal PC secretion. Although the levels of PC secretion in vitro are not likely to be the same as those in vivo, we can compare rates between different in vitro preparations isolated from different age animals. In addition, we can relate this to various cellular characteristics in the embryonic chick lung. Levels of basal PC secretion were <1% at day 16. As this is the first day that lamellar bodies are observed in type II cells of embryonic chickens (23, 33), it is not surprising that basal PC secretion is so low. Basal secretion was higher at day 18 than at any other time point. The amount of total phospholipid in tissue (24) and lavage (26) from embryonic chicken lungs is also highest at incubation day 18. This is the day immediately before pulmonary ventilation. Therefore, the higher basal secretion observed in vitro might function to increase surfactant in vivo, which would prepare the animal for air breathing. Fibroblasts appear to have a stimulatory effect on embryonic chicken type II cells in culture at day 18, demonstrated by higher basal PC secretion in coculture. It has been reported that the growth factors produced from lung fibroblasts in the fetal rat are the same at all stages, but their amounts can vary during development (2). During late gestation in the fetal rat, fibroblast populations act to stimulate differentiation of the type II cell phenotype to enable the adequate production of surfactant (5). Therefore, it appears that type II cell-fibroblast interactions are important in the maturation of the surfactant system in birds and in mammals.

Agonist stimulation. This study demonstrates that epinephrine is a potent stimulator of PC secretion from embryonic chicken cells. Epinephrine stimulated the secretion of PC from cells isolated from embryonic chickens of all ages tested. Adrenergic agonists are also effective in stimulating surfactant secretion in isolated type II cells from rats immediately before birth and at 1, 7, 14, and 30 days after parturition (19). In chickens, the maximal stimulation at day 18 coincides with an increase in plasma epinephrine in the chick embryo. This increase is induced by hypoxia, which occurs toward the end of the developmental period (52). The plasma epinephrine may act directly on the type II cells to release PS, which in turn may prepare the animal for air breathing on day 19. The adrenergic pathway also appears to be effective posthatch, indicating that this pathway is important in maintaining surfactant secretion beyond the developmental period. Adrenergic agonists also stimulate surfactant secretion in type II cells isolated from adult mammalian (4, 6) and non-mammalian vertebrates (54, 55). It is believed that adrenergic agonists bind directly to beta -receptors located on the type II cells and activate a cAMP-dependent signaling pathway to increase surfactant secretion (4, 6). Our data from day 18 embryos also indicate that epinephrine acts directly on type II cells in the chicken. Although this interaction is not dependent on fibroblasts, the presence of fibroblasts in coculture does enhance the response. Although the specific adrenergic receptors on type II cells have not yet been characterized, it is likely that adrenergic agonists act via the same mechanism.

Dexamethasone is important in the maturation of PS in the chicken. The release of PC from lung cells isolated at embryonic days 16 and 18 was stimulated by dexamethasone. In vivo, the hypoxia in the chicken embryo induces a surge in plasma glucocorticoids, which peaks on day 17 (27). The administration of corticosterone to intact embryos stimulates disaturated PC synthesis in the lungs of day 18 chickens (25). In addition, the treatment of chicken eggs with dexamethasone on day 17 of incubation results in a stimulation of PS synthesis in the embryo at days 18 and 19 (53). In this study, PC secretion from type II cells was enhanced by glucocorticoids only when the cells were cocultured with fibroblasts. Furthermore, the effect of dexamethasone was blocked when in the presence of a glucocorticoid receptor antagonist and protein synthesis inhibitor. Taken together, this provides evidence that glucocorticoids act through a cellular, receptor-mediated pathway involving fibroblasts in chickens.

In vivo, fibroblasts and type II cells are only in direct communication during development and in cases of lung injury. This may explain why glucocorticoids have only a mild effect on surfactant synthesis and secretion in the adult (56). However, in cell cultures of day 20 chick embryos and of hatchlings, dexamethasone had no effect on PC secretion, despite the fact that fibroblasts and type II cells were in direct contact. Furthermore, dexamethasone increases choline incorporation into PC at days 18 and 19, but not on day 20 of incubation (53). Mature rat type II cells secrete a factor that inhibits fibroblast function and therefore the glucocorticoid response (1). It is possible that a similar factor may be secreted from mature chicken type II cells, modulating the glucocorticoid-mediated PC secretion in day 20 and hatchling chicken lung cells.

Carbachol was found to be ineffective in stimulating PC secretion at any embryonic age. It is generally believed that cholinergic agonists do not stimulate surfactant secretion directly from isolated type II cells of rats (4, 13). However, recently it has been shown that acetylcholine stimulates surfactant secretion directly from isolated type II cells from adult dunnarts (a heterothermic marsupial), lizards, frogs, and lungfish (54, 55). In heterothermic animals, the role of acetylcholine may be to stimulate PC secretion from type II cells without raising the metabolic rate of the animal. This may be important when the animal is cold or during torpor, when it is trying to conserve energy (55). Although adrenergic agonists are able to increase PL secretion at low body temperatures, this is an inappropriate mechanism as it also leads to an increase in metabolic rate. As chickens are homeothermic, the lack of a direct cholinergic response on type II cells is not surprising. Nevertheless, cholinergic agonists may have indirect effects on PC secretion in the chicken lung, possibly by increasing the level of circulating epinephrine. This remains to be investigated.

Perspectives

In mammals, the development of the surfactant system is controlled by many complex and interrelated factors. Two of the most important factors contributing to the maturation of the surfactant system are glucocorticoids and adrenergic agonists. This is the first study to use a coculture system to investigate the control of PS development in a non-mammalian vertebrate. We demonstrated that both glucocorticoids and adrenergic agonists are involved in the development of the PS system in chickens. Furthermore, we showed that adrenergic agonists act directly on type II cells, whereas glucocorticoids require the presence of lung fibroblasts. Therefore, similar control mechanisms are present during lung development in the chicken and in eutherian mammals, despite the vastly different birthing strategies and lung structure and function between the two groups. This provides evidence that all facets of the surfactant system, including its morphology, biochemistry, and regulation, are highly conserved among the vertebrates. Although further research is required to determine the exact nature of the cellular interactions involved in the regulation, our findings may provide the impetus for the use of nontraditional animal models in surfactant research in the future.


    ACKNOWLEDGEMENTS

The authors thank P. Wood, N. Miller, and S. Chamberlain for technical assistance. We thank C. Daniels for critically reviewing the manuscript and S. Johnston for technical help with preparing the manuscript.


    FOOTNOTES

This research was funded by an Australian Research Council (ARC) grant to C. B. Daniels and an ARC Research Fellowship to S. Orgeig.

Address for reprint requests and other correspondence: S. Orgeig, Dept. of Environmental Biology, Univ. of Adelaide, Adelaide, SA 5005, Australia (E-mail: sandra.orgeig{at}adelaide.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 20 April 2000; accepted in final form 1 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adamson, IY, Young L, and King GM. Reciprocal epithelial: fibroblast interactions in the control of fetal and adult rat lung cells in culture. Exp Lung Res 17: 821-835, 1991[ISI][Medline].

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Am J Physiol Regul Integr Comp Physiol 281(3):R770-R777
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