Neurochemical and thermal control of surfactant secretion by alveolar type II cells isolated from the marsupial, Sminthopsis crassicaudata

Pulmonary surfactant is synthesised in alveolar type II cells and secreted into the lining of the lung in response to ventilation, temperature changes and autonomic neurotransmitters. Type II cells were isolated from the heterothermic marsupial, Sminthopsis crassicaudata. The neurotransmitters, isoproterenol and carbamylcholine chloride significantly increased phosphatidylcholine secretion at 37 degrees C (basal: 14.2%, isoproterenol: 20.1%, carbamylcholine: 17.0%). Temperature reduced the rate of secretion from dunnart type II cells (e.g. basal: 14.2% at 37 degrees C; 7.2% at 18 degrees C). However, the change in secretory rate between 37 degrees C and 18 degrees C was less than expected if due to temperature alone (Q10= 1.4). The surfactant secretory pathway is therefore modulated by factors other than and in addition to, temperature. The response of dunnart type II cells to the agonists remained the same at both temperatures. Basal secretion was higher in dunnart type II cells (14.2% in 4 h) than has been reported in rat type II cells (1.9% in 3 h) and consequently, the agonist-stimulated increases in secretion from dunnart type II cells (41% above basal in 4 h) were much lower than observed for rat type II cells (200% above basal in 1.5 h).     

Developmental regulation of phospholipid secretion by fetal type II pneumocytes

Surfactant sufficiency is dependent upon adequate synthesis and secretion of surfactant by the type II alveolar epithelium. Our laboratory has previously shown that basal secretion of surfactant phospholipid by differentiated fetal type II cells is lower than the basal secretion by adult cells. The purposes of this study were to determine if undifferentiated fetal type II cells can secrete phosphatidylcholine, to determine if terbutaline, a β-adrenergic agonist, stimulates secretion of surfactant phospholipids by undifferentiated fetal cells and to examine the effects of differentiation on secretion of surfactant phospholipids by fetal cells. Constitutive (basal) secretion of phosphatidylcholine increased linearly as a function of time in both undifferentiated and differentiated cells, but the rate of secretion was greater in differentiated cells than the rate of secretion in undifferentiated cells. Terbutaline caused a concentration-dependent increase in secretion in both undifferentiated and differentiated cells. Maximal effective concentration and EC₅₀ were similar for undifferentiated (10⁻⁶ M, 0.2 μM) and differentiated (10⁻⁵ M, 0.3 μM) cells. The relative stimulation of secretion above control values was greater for undifferentiated cells. The kinetics of terbutaline stimulation varied significantly with cellular differentiation. Terbutaline resulted in 230% stimulation of secretion in undifferentiated cells at 30 min followed by a decline in the response to terbutaline at 60 to 120 min. In contrast, terbutaline stimulated secretion by differentiated cells showed a sustained linear increase from 0 to 120 min. This regulation of stimulated secretion is not present in undifferentiated cells. We conclude that undifferentiated type II cells are capable of the secretion of phosphatidylcholine and that terbutaline stimulates secretion by undifferentiated cells. Furthermore, basal secretion increases as a function of differentiation of type II cells and the regulation of stimulated secretion seen in differentiated cells is not developed in undifferentiated cells. The developmental regulation of the secretion of surfactant is complex and probably involves both excitatory as well as inhibitory mechanisms which develop at different stages of differentiation of the type II cell.     

The role of calcium in the secretion of surfactant by rat alveolar type II cells

Beta adrenergic agonists, tetradecanoylphorbol acetate, and the ionophore A23187 all stimulate surfactant secretion in type II cells isolated from rats. We found that combinations of these agonists cause augmented secretion, suggesting that the agonists may effect different steps in the secretory process. Previous studies have shown that cAMP is likely to be an intracellular 'second messenger' in type II cells. A23187, which has been reported to increase cAMP in some cell systems, did not increase the cAMP content of type II cells. We investigated the possible role of Ca2+ as another 'second messenger' by studying cellular 45Ca fluxes and the effect of extracellular calcium depletion on secretion. Depletion of extracellular calcium for as long as 3 h did not alter stimulated secretion, although basal secretion was increased. Secretagogues did not stimulate 45Ca influx from extracellular sources. A23187 and, to a lesser extent, terbutaline caused an acceleration of 45Ca efflux from type II cells. The addition of terbutaline or tetradecanoylphorbol acetate to A23187 further accelerated 45Ca efflux, suggesting that these agonists may act on separate calcium pools or by different mechanisms on the same calcium pool. Although secretion from type II cells is not inhibited by extracellular calcium depletion, the studies on 45Ca efflux suggest that Ca2+ plays a role in the regulation of surfactant secretion from isolated type II cells.     

Glycerol as a substrate for phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol and glucose utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozotocin-diabetic rats. In cells from diabetic rats, incorporation of [1,3-14C]glycerol into total phosphatidylcholine (PC), disaturated phosphatidylcholine (DSPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) occurred to a greater degree by the glycerol 3-phosphate pathway as opposed to the dihydroxyacetone phosphate pathway. Total incorporation of glycerol into each of the major cellular phospholipids was increased up to 6-fold in cells from diabetic rats, while the total incorporation of glucose into the same lipids was decreased 2-fold. While the percentage of both glucose and glycerol carbons incorporated into the backbone of DSPC was increased in cells from diabetic rats, the percentage of carbons from both substrates incorporated into the fatty acid moieties was decreased. As a measure of DSPC synthesis, choline incorporation into DSPC was significantly decreased in type II cells from diabetic animals if the cells were incubated in the presence of glucose, palmitate and choline but not glycerol. Addition of 0.1 or 0.3 mM glycerol to the incubation medium restored choline incorporation to the control value in cells from diabetic rats, but did not affect the rate of choline incorporation into DSPC in cells from normal rats. These results suggest that exogenous glycerol can compensate for reduced glucose metabolism in type II cells of diabetic animals to maintain a constant rate of DSPC synthesis.     

Effect of colchicine and pilocarpine on the secretory activity of rat type II alveolar cells: an ultrastructural study

Colchicine in a total dose of 0.6 mg/100 g body weight per day was shown to reduce the level of apical surfactant secretion by type II alveolar cells in random-bred male albino rats, thereby demonstrating that the cytoplasmic microtubules participate in the release of surfactant into the alveolar lumen. In addition, basal secretion of surface-active material was found in 51% of all the cells. In a single dose of 8 mg/100 g b.w., pilocarpine stimulated apical surfactant secretion. If injected after colchicine, it slightly increased the number of type II alveolar cells ready to release surfactant, but actual secretion was not observed; the level of basal secretion did not increase. It has been suggested that microtubular function is not completely responsible for basal secretion and is only partly responsible for apical surfactant secretion.     

Synexin and GTP increase surfactant secretion in permeabilized alveolar type II cells

We have previously suggested that synexin (annexin VII), a Ca(2+)-dependent phospholipid binding protein, may have a role in surfactant secretion, since it promotes membrane fusion between isolated lamellar bodies (the surfactant-containing organelles) and plasma membranes. In this study, we investigated whether exogenous synexin can augment surfactant phosphatidylcholine (PC) secretion in synexin-deficient lung epithelial type II cells. Isolated rat type II cells were cultured for 20-22 h with [(3)H]choline to label cellular PC. The cells were then treated with beta-escin, which forms pores in the cell membrane and releases cytoplasmic proteins including synexin. These cells, however, retained lamellar bodies. The permeabilized type II cells were evaluated for PC secretion during a 30-min incubation. Compared with PC secretion under basal conditions, the presence of Ca(2+) (up to 10 microM) did not increase PC secretion. In the presence of 1 microM Ca(2+), synexin increased PC secretion in a concentration-dependent manner, which reached a maximum at approximately 5 microg/ml synexin. The secretagogue effect of synexin was abolished when synexin was inactivated by heat treatment (30 min at 65 degrees C) or by treatment with synexin antibodies. GTP or its nonhydrolyzable analog beta:gamma-imidoguanosine-5'-triphosphate also increased PC secretion in permeabilized type II cells. The PC secretion was further increased in an additive manner when a maximally effective concentration of synexin was added in the presence of 1 mM GTP, suggesting that GTP acts by a synexin-independent mechanism to increase membrane fusion. Thus our results support a direct role for synexin in surfactant secretion. Our study also suggests that membrane fusion during surfactant secretion may be mediated by two independent mechanisms.     

Glycerol metabolism in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozocinin-diabetic rats. With glucose in the incubation medium, incorporation of exogenous [1,3-14C]glycerol into disaturated phosphatidylcholine, total phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) was increased 4-fold in cells from diabetic rats. In the absence of glucose, glycerol incorporation was 5-fold greater than in its presence in cells from normal animals, but was further increased 2.2-fold in cells from diabetic rats. Insulin treatment of diabetic rats returned all incorporation rates to control values. The increased glycerol incorporation rates were not due to differences in either phospholipid turnover or the size of the glycerol 3-phosphate precursor pool. Kinetic analysis of glycerol entry into the acid-soluble cell fraction indicated that glycerol transport occurred largely by simple diffusion, and was not rate limiting for its entry into lipids. Glycerol entry into the total lipid fraction was saturable, reaching a Vmax of 48 pmol/micrograms DNA per h in normal cells and 120 pmol/micrograms DNA per h in cells from diabetic rats, with no change in the Km (0.31 mM). While glycerol oxidation was reduced 23% in cells from diabetic rats in the presence of glucose and by 44% in the absence of glucose, glycerol kinase activity in sonicates of cells from diabetic animals was increased 210% and was reversed by in vivo insulin treatment. These results suggest that glycerol utilization in type II pneumocytes is a hormonally regulated function of both glycerol oxidation and glycerol phosphorylation.     

Regulation of very-low-density-lipoprotein lipid secretion in hepatocyte cultures derived from diabetic animals.

Hepatocytes were derived from 2-3-day streptozotocin-diabetic rats and maintained in culture for up to 3 days. Compared with similar cultures from normal animals, these hepatocytes secreted less very-low-density-lipoprotein (VLDL) triacylglycerol, but the decrease in the secretion of VLDL non-esterified and esterified cholesterol was not so pronounced. This resulted in the secretion of relatively cholesterol-rich VLDL particles by the diabetic hepatocytes. Addition of insulin for a relatively short period (24 h) further decreased the low rates of VLDL triacylglycerol secretion from the diabetic hepatocytes. The secretion of VLDL esterified and non-esterified cholesterol also declined. These changes occurred irrespective of whether or not exogenous fatty acids were present in the culture medium. Little or no inhibitory effect of insulin was observed after longer-term (24-48 h) exposure to the hormone. Both dexamethasone and a mixture of lipogenic precursors (lactate plus pyruvate) stimulated VLDL triacylglycerol and cholesterol secretion, but not to the levels observed in hepatocytes from normal animals. The low rate of hepatic VLDL secretion in diabetes contrasts with the increase in whole-body VLDL production rate. This suggests that the intestine is a major source of plasma VLDL in insulin-deficient diabetes.     

The effect of temperature on adrenergic receptors of alveolar type II cells of a heterothermic marsupial

Fat-tailed dunnarts, Sminthopsis crassicaudata, survive dramatic changes in body temperature during torpor without experiencing surfactant dysfunction. Adrenergic factors regulate surfactant secretion through beta(2)-adrenergic receptors on alveolar type II cells. Temperature has no effect on the secretory response of dunnart type II cells to adrenergic stimulation. We hypothesise that during torpor, dunnart type II cells up-regulate the number of adrenergic receptors present on type II cells to enable stimulation at lower concentrations of agonist. Here, we isolated type II cells from warm-active (35 degrees C) and torpid (15 degrees C) dunnarts and examined the effects of an in vitro temperature change on the number and activity of adrenergic receptors. Torpor did not affect the beta-adrenergic receptor number. However, we observed a significant decrease in adrenergic receptor number when cells from warm-active animals were incubated at 15 degrees C and when cells from torpid animals were incubated at 37 degrees C. cAMP production was significantly higher in type II cells from torpid dunnarts than warm-active dunnarts and this may contribute, in part, to the temperature insensitivity we have previously observed in the adrenergic regulation of surfactant secretion.     

Altered phospholipid biosynthesis in type II pneumocytes isolated from streptozotocin-diabetic rats

To determine whether type II pneumocytes isolated from diabetic animals could serve as a useful model for the study of surfactant phospholipid biosynthesis and its regulation, type II pneumocytes were isolated from adult streptozotocin-diabetic rats and placed in short-term primary culture. On a DNA basis, total cellular disaturated phosphatidylcholine (disaturated PC) and phosphatidylglycerol (PG) were decreased 36 and 66%, respectively, in type II cells from diabetic animals. 7 days of insulin treatment of diabetic rats returned the cellular disaturated PC and PG content to control values and increased the total cellular phosphatidylethanolamine (PE) content by 51%. The rates of glucose and acetate incorporation into disaturated PC per unit DNA were reduced 32 and 38%, respectively, in cells isolated from diabetic rats, while glycerol incorporation was increased by 143%. Insulin treatment of diabetic rats returned the glucose and glycerol incorporation rates to control values and increased acetate incorporation into disaturated PC by 66%. These data suggest that the biosynthesis of surfactant is altered by both diabetes mellitus and in vivo insulin treatment.     

Arachidonic acid metabolites stimulate phosphatidylcholine secretion in primary cultures of type II pneumocytes

There is evidence from whole animal and intact lung studies that prostaglandins are involved in the regulation of surfactant secretion. To explore this further we examined the effect of arachidonic acid on secretion of phosphatidylcholine in primary cultures of adult rat type II pneumocytes. Arachidonic acid stimulated phosphatidylcholine secretion and this effect was dependent on concentration in the range 1-8 microM. Arachidonic acid (8 microM) stimulated secretion by 79% from a basal rate of 1.17% total cellular phosphatidylcholine secreted in 90 min to 2.09%. We examined the effects of inhibitors of arachidonic acid metabolism on the stimulatory effect. Nordihydroguairaretic acid (0.1 microM), a lipoxygenase inhibitor, reduced the stimulatory effect by 64%. The same concentration of cyclooxygenase inhibitors had no effect. We conclude that arachidonic acid metabolites stimulate surfactant secretion in type II cells. Whether this effect is mediated by leukotrienes or other products remains to be established.     

The ontogeny of pulmonary surfactant secretion in the embryonic green sea turtle (Chelonia mydas).

Pulmonary surfactant, consisting predominantly of phosphatidylcholine (PC), is secreted from Type II cells into the lungs of all air-breathing vertebrates, where it functions to reduce surface tension. In mammals, glucocorticoids and thyroid hormones contribute to the maturation of the surfactant system. It is possible that phylogeny, lung structure, and the environment may influence the development of the surfactant system. Here, we investigate the ontogeny of PC secretion from cocultured Type II cells and fibroblasts in the sea turtle, Chelonia mydas, following 58, 62, and 73 d of incubation and after hatching. The influence of glucocorticoids and thyroid hormones on PC secretion was also examined. Basal PC secretion was lowest at day 58 (3%) and reached a maximal secretion rate of 10% posthatch. Dexamethasone (Dex) alone stimulated PC secretion only at day 58. Triiodothyronine (T(3)) stimulated PC secretion in cells isolated from days 58 and 73 embryos and from hatchling turtles. A combination of Dex and T(3) stimulated PC secretion at all time points.     

Stimulation of surfactant secretion by vasopressin in primary cultures of adult rat type II pneumocytes

The current study examined the effect of vasopressin on the secretion of phosphatidylcholine, the principal component of pulmonary surfactant, from adult rat alveolar type II pneumocytes in primary culture. Vasopressin stimulated secretion in a time- and dose-dependent manner. At a concentration of 10 nM, vasopressin stimulated release by 6-fold over the basal secretory rate. The concentration producing half the maximal response for vasopressin-induced secretion was 0.4 nM. The stimulation of phosphatidylcholine release by vasopressin was duplicated by the vasopressin fragment, amino acids 4 through 9. [Lys8]vasopressin and the selective vasopressin-2 agonist [deamino-8-D-Arg]vasopressin did not stimulate surfactant secretion effectively. The vasopressin- and fragment-induced secretion was inhibited by the vasopressin-1 receptor antagonist d(CH2)5TDAVP and the protein kinase C inhibitor, tetracaine, but not by the beta-adrenergic antagonist alprenolol. Vasopressin did not activate adenylate cyclase, which suggests that stimulation by vasopressin was independent of cyclic AMP. When vasopressin and isoproterenol were added concomitantly, the effects on phosphatidylcholine secretion were additive. This suggests that these two secretagogues operate via separate mechanisms.     

Surfactant-associated protein inhibits phospholipid secretion from type II cells

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.     

Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats

Pulmonary surfactant conventionally is prepared from material obtained by endobronchial lavage. Although it has been assumed that the components of surfactant are secreted by alveolar type II cells, direct proof of this assumption has not been available. Furthermore, it is possible that the final material obtained by lavage has been modified after secretion or altered during the isolation procedure. It has been shown previously that type II cells, after 1 day in primary culture, secrete saturated phosphatidylcholine, one of the lipid components of surfactant. Because saturated phosphatidylcholine is not unique to surfactant and because type II cells in culture lose differentiated characteristics over the first several days in culture, it has not previously been established how closely the secretory products of cultures of type II cells resemble surfactant as obtained by endobronchial lavage. We therefore studied the morphologic, physical and chemical characteristics of the material that type II cells secrete under basal conditions and after stimulation with terbutaline or 12-O-tetradecanoyl-13-phorbol acetate. The secreted material resembled surfactant obtained by lavage; it was similar morphologically to the lamellar material and tubular myelin seen in the fluid-filled alveoli of fetal rats, it lowered surface tension to 5 mN per meter, and it contained the 72000 dalton apolipoprotein of surfactant (as measured by the 'rocket' immunoelectrophoresis technique). When cells were incubated for 22 h with [1-(14)C]acetate, the distribution of radioactivity in the secreted material was very similar to the phospholipid composition of rat surfactant. We conclude that the material secreted by alveolar type II cells after 1 day in primary culture is similar to surfactant obtained by endobronchial lavage.     

Proteomic analysis of lamellar bodies isolated from rat lungs

Background  

Lamellar bodies are lysosome-related secretory granules and store lung surfactant in alveolar type II cells. To better understand the mechanisms of surfactant secretion, we carried out proteomic analyses of lamellar bodies isolated from rat lungs.     

Reorganization of cytoskeleton during surfactant secretion in lung type II cells: a role of annexin II

The secretion of lung surfactant requires the movement of lamellar bodies to the plasma membrane through cytoskeletal barrier at the cell cortex. We hypothesized that the cortical cytoskeleton undergoes a transient disassembly/reassembly in the stimulated type II cells, therefore allowing lamellar bodies access to the plasma membrane. Stabilization of cytoskeleton with Jasplakinolinde (JAS), a cell permeable actin microfilament stabilizer, caused a dose-dependent inhibition of lung surfactant secretion stimulated by terbutaline. This inhibition was also observed in ATP-, phorbol 12-myristate 13-acetate (PMA)- or Ca(2+) ionophore A23187-stimulated surfactant secretion. Stimulation of type II cells with terbutaline exhibited a transient disassembly of filamentous actin (F-actin) as determined by staining with Oregon Green 488 Phalloidin. The protein kinase A inhibitor, H89, abolished the terbutaline-induced F-actin disassembly. Western blot analysis using anti-actin and anti-annexin II antibodies showed a transient increase of G-actin and annexin II in the Triton X-100 soluble fraction of terbutaline-stimulated type II cells. Furthermore, introduction of exogenous annexin II tetramer (AIIt) into permeabilized type II cells caused a disruption in the cortical actin. Treatment of type II cells with N-ethylmaleimide (NEM) resulted in a disruption of the cortical actin. NEM also inhibited annexin II's abilities to bundle F-actin. The results suggest that cytoskeleton undergoes reorganization in the stimulated type II cells, and annexin II tetramer plays a role in this process.     

Splenic macrophage function in early syphilitic infection is complex. Stimulation versus down-regulation

Macrophages are important regulatory cells that can both stimulate and down-regulate various immune functions. During syphilitic infection, these cells phagocytize, kill, and lyse Treponema pallidum. They also modulate early T cell activation by decreasing IL-2 production through secretion of PG. This report focuses on additional complexities of macrophage regulation. Non-adherent splenic cells were stimulated with Con A to induce IFN-gamma synthesis. High levels were detected in preparations from normal rabbits and much lower levels in preparations from infected rabbits. The organisms also readily stimulated IL-1 synthesis by adherent spleen preparations from normal but not from infected rabbits. When indomethacin was added to these latter preparations, this IL-1 defect was reversed, implicating PG in this down-regulation. Spleen cells were obtained from normal rabbits and from rabbits infected testicularly for 9 to 12 days. Infection elevated basal levels of class II Ia Ag on adherent cells. In addition, macrophage Ia expression was increased during 4 days of in vitro incubation with treponemes. Non-adherent spleen cells from infected animals inhibited two different macrophage functions. First, culture filtrates obtained after 48 h of incubation contained a soluble factor that subsequently decreased LPS-induced IL-1 synthesis. Second, when macrophages were co-incubated with non-adherent cells, treponemal stimulation of macrophage Ia expression was inhibited; this inhibition was reversed by indomethacin implicating prostaglandins in this down-regulation. In further experiments an exogenous source of IFN-gamma was incubated with adherent cells from infected rabbits. This stimulated macrophage function as shown by increased IL-1 synthesis and Ia expression and decreased PGE2 secretion. Results are discussed in terms of the complexities of immunoregulation by macrophages during syphilitic infection.     

Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca²⁺ channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.     

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

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.