INTRODUCTION

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Glucagon and related peptides constitute a family included in the proglucagon molecule, which is identical in sequence in the pancreas, intestine, and brain (1), although postranslational processing of the precursor yields different products in these organs (2). In gut L cells, the C-terminal portion of proglucagon is predominantly processed to glucagon-like peptide 1 (GLP-1) and GLP-2. Further processing of GLP-1 in these cells produces the truncated and amidated forms of the peptide: GLP-1(1-36) amide, GLP-1(7-36) amide, and GLP-1(7-37).

Although the truncated forms of GLP-1 have been described to have strong incretin activity, it is currently known that they are also important in the functioning of other peripheral tissues and of the central nervous system (CNS). Both forms of the peptide are indistinguishable in their ability to produce biological effects through GLP-1 receptors located in pancreatic endocrine cells (3), gastric glands (4), and in adipocyte (5), lung (6), and rat brain membranes (7). These peptides stimulate insulin secretion in a glucose-dependent manner (11) and have significant effects on gastrointestinal motility and secretion (12). Also, GLP-1(7-36) amide significantly increases arterial blood pressure and heart rate in rats (13, 14).

In addition, GLP-1(7-36) amide and its own receptors are synthesized in the same brain regions, strongly supporting the actions of this peptide on the CNS. Thus, the perfusion of several brain nuclei with GLP-1(7-36) amide produces a selective release of neurotransmitters (15, 16), and central administration of this peptide induces an inhibitory effect on food and drink intake (17). Coexpression of GLP-1 receptors, glucokinase, and glucose transporter protein 2 (GLUT-2) in neurons involved in the control of food intake suggests that these cells may play a role in glucose sensing in the brain (18, 20).

Besides the above-described actions of GLP-1(7-36) amide a high content of GLP-1 receptors has been determined in the rat (6), in the submucosal glands of the trachea, the smooth muscle of pulmonary arteries, and in cells considered to be Type II pneumocytes. The GLP-1 receptor expressed in rat lung has a molecular mass of 55 kD, in disagreement with the data obtained in brain and pancreatic B cells. These differences in molecular masses may be related to a distinctive posttranslational processing, because studies of isolated rat lung GLP-1 receptor cDNA have shown that receptors in the pancreas, lung, and brain have identical sequences (20). The binding of GLP-1(7-36) amide to its receptors generates the signals needed to produce increases in mucous secretion, pulmonary smooth muscle relaxation, and increased pulmonary surfactant secretion by rat Type II pneumocytes (23, 24). In contrast to our knowledge of these issues in the rat, little is known about the effect of glucagon and related peptides on the human lung. The only report addressing this matter is that of Wei and Mojsov (25), who described that the GLP-1 receptor is expressed in the human lung. However, in another report, the expression of GLP-1 receptor mRNA transcripts was detected only in the human pancreas (26). These observations reinforce the interest in studying the action of GLP-1(7-36) amide and related peptides in the human lung. Accordingly, in the present work, we describe the results obtained with an experimental design in which the action of agonists and antagonists of GLP-1 receptors on the secretion of pulmonary surfactant by human Type II pneumocytes was studied.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Materials

D-Glucose labeled with radioactive carbon (¹⁴C) and a cAMP radioimmunoassay kit were obtained from the Radiochemical Centre (Amersham, Bucks, UK). Elastase, 2',7'-dichlorofluoroscein, propranolol, 8-bromo-cAMP (8-Br-cAMP), trypsin, 3-isobutyl-1-methylxanthine, 12-O-tetradecanoylphorbol 13-acetate (TPA), calphostin C (CalphC), and standard lipids were from Sigma (St. Louis, MO). Deoxyribonuclease I and fetal bovine serum were purchased from Boehringer Mannhein (Mannheim, Germany). Rp-isomer (Rp-cAMPS), and A23187 ionophore were supplied by Calbiochem (La Jolla, CA). KN-62 inhibitor was obtained from ICN Pharmaceuticals (Costa Mesa, CA). Percoll was from Pharmacia Fine Chemicals (Uppsala, Sweden). GLP-1(1-37), GLP-1(7-36) amide, and GLP-2 were obtained from Peninsula Laboratories (St. Helens, UK). Exendin-4 and exendin(9-39) were gifts from J. Eng (Department of Internal Medicine, Veterans Administration Hospital, Bronx, NY). RPMI 1640 medium was from ICN Flow (Costa Mesa, CA). All other chemicals were of analytical grade from E. Merck (Darmstadt, Germany).

Patients and Lung Tissue Procurement

Human lung tissue was obtained from cadaveric multiple organ donors. Ages ranged from 25 to 60 yr. Donors with a recent history of tobacco smoking, more than 72 h of receiving mechanical ventilation, or any radiologically demonstrated pulmonary infiltrate were excluded from the study. We have used organs only when lungs were not used for transplants because of the reasons cited below. All the organs of our donors were routinely offered to the local transplant teams through the Spanish Transplant Organization, which is also in connection with Eurotransplant for inquiring outside transplant teams within the safe distance limit. Unfortunately, in practice, donor lungs are sometimes discarded for different reasons, including lack of a suitable recipient, lack of an available lung transplant team, donor age > 50 yr old, anthropometric mismatches between donor and recipient, or refusal of the lungs by the transplant team for unknown reasons.

Immediately after harvesting the organs to be used for transplantation, a right lower lobectomy was performed, and the lobe was excised and placed in a cold (4° C) saline solution. The cold ischemia period was less than 3 h in all cases.

This investigation was conducted according to the Declaration of Helsinki principles and had the approval of the Ethics Committee of our institution.

Type II Pneumocyte Isolation Procedure

All solutions were made with double glass-distilled water. Solution I contained 140 mM NaCl, 5 mM KCl, 2.5 mM Na₂HPO₄, 10 mM HEPES, 6 mM glucose, 0.2 mM EGTA, and DNase (10 µg/ml). Solution II contained solution I with 2 mM CaCl, 1.3 mM MgSO₄, elastase (27 "orcein-elastin" units/ml), trypsin (0.5 mg/ml), and collagenase (0.5 mg/ml).

The time period between lobectomy and the beginning of isolation was never longer than 3 h. The lung tissue was rinsed with solution I and minced into small pieces (1-3 mm), which were then washed extensively with the same solution to remove as many blood cells as possible. Subsequently, the portions were digested by two consecutive exposures, of 30 min each, with solution II in a shaking water bath at 37° C. The tissue cellular suspension was filtered through two nylon meshes (200 and 20 µm, respectively), and centrifuged at 250 × g for 10 min. After removing the supernatant, the pellets were then resuspended in RPMI 1640 medium, added to 75-cm² flasks, precoated with fetal calf serum, and cultured in a 95% O₂-5% CO₂ air incubator at 37° C for 90 min, during which most of the alveolar macrophages adhered to the plastic. After 90 min, the nonadherent cells were removed, centrifuged (250 × g for 10 min), and resuspended in solution I. For further purification, the suspension was applied to a Percoll gradient by centrifuging the combination of Percoll with phosphate-buffered saline (25 mM NaH₂PO₄, 300 mM NaCl, pH 7.4) in a 6:7 (v/v) ratio for 10 min at 20,000 × g (27). Cells were counted in a standard hemacytometer. The mean yield obtained was 6.5 (± 0.9) × 10⁶ cells/g tissue. The percentage of cells that excluded trypan blue was 93.9 ± 7.1%. To determine the purity of the Type II pneumocyte preparation (86.4 ± 6.9%), both modified Papanicolaou stain and tannic acid and polychrome stain (28) were used.

Chromium-51 Release Assays

To address the possibility of a nonspecific effect due to cytotoxicity, we measured cell lysis by a standard Chromium-51 (⁵¹Cr) release assay as previously described by us (29). Cells were labeled by incubation at 37° C in 150 µl of RPMI 1640 medium/well with 2 µCi of ⁵¹Cr-labeled sodium chromate at 37° C for 24 h and then washed four times. The additives were diluted in RPMI 1640 medium and added to the cell culture; the plates were incubated at 37° C for 24 h, after which an aliquot (100 µl) of the medium was collected and counted in a  counter. Specific cell lysis was calculated as 100 × [(test medium cpm  spontaneous cpm) (total cpm  spontaneous cpm)].

Synthesis of Phospholipid by Human Type II Pneumocytes

Synthesis of pulmonary surfactant was measured as the incorporation of 10 mM D-[U-¹⁴C]glucose into its most important phospholipid component, phosphatidylcholine (PC), as an index. To accomplish this, Type II pneumocytes (10⁶ cells/ml) were incubated (collagen A-precoated microwells) in RPMI 1640 medium in a 95% O₂-5% CO₂ air incubator at 37° C for 24 h. The media were then replaced with fresh media with or without the substances to be tested and cultured again for a further 24 h in the presence of D-[U-¹⁴C]glucose. Antagonists were added 10 min before the use of agonists. Optimal agonist and antagonist concentrations were selected after dose-response studies were performed. At the end of this incubation period, the media were removed and the cells were rapidly frozen in acetone chilled with dry ice. After the addition of acid methanol, cells were sonicated in an MSE ultrasonic disintegrator (Branson, Danbury, CT). Following this, the lipids were extracted with 1.3 ml of chloroform and 0.4 ml of salt solution for 1 h at room temperature. The organic phase was then washed three times with 1 ml of the aqueous phase of a system composed of chloroform-methanol-salt solution-concentrated HCl (266: 133:100:1, by volume) after the addition of 30 µl of carrier lipids in chloroform-methanol-concentrated HCl (200:100:1, by volume). The organic phase was dried and redissolved in chloroform-methanol (2:1, by volume). Samples of the redissolved organic phase were then applied to precoated plates of silica gel 60 (20 × 20 cm; E. Merck) previously activated for 1 h at 110° C. Lipid separation was performed by unidimensional chromatography with two solvent systems, as described previously in depth (30). To identify the lipids, the plates were sprayed with 2',7'-dichlorofluorescein. Each spot was scraped off into a scintillation vial, and its radioactivity was measured. To exclude any "carrythrough" of unincorporated label, experiments in which label was added just before freezing cells were used as controls. A frozen aliquot of the cellular suspension was stored for the determination of proteins by the Coomassie Brilliant Blue spectrophotometric method.

Phospholipid Secretion from Human Type II Pneumocytes

Cells were cultured overnight in the presence of 10 mM D-[U-¹⁴C]glucose. At the end of this period, the medium was removed, and the cells were rinsed three times with RPMI 1640 medium to remove labeled glucose. The media were then replaced by fresh media with or without the substances to be tested and cultured again for a further 24 h. Thereafter, lipids were extracted from the media and identified as described above. PC secretion is expressed as the percentage of [¹⁴C]PC in the medium relative to the total amount of this compound.

Cyclic AMP Production by Human Type II Pneumocytes

Human type II pneumocytes were cultured in RPMI 1640 medium at 37° C for 24 h. The medium was then replaced by fresh medium, and the cells were incubated with terbutaline (10 µM), propranolol (10 µM), GLP-1(7-36) amide (1-1,000 nM), or other glucagon-related peptides (10 nM). Exendin(9-39) was used at a concentration of 100 nM. 3-Isobutyl-1-methylxanthine (0.5 mM) was present during the incubation to prevent cAMP hydrolysis. After 24 h, the media were removed, and 1 ml of cold ethanol was added to lyse the cells. After 1 h of incubation at 4° C, the wells were scraped, and the suspension was centrifuged for 10 min at 10,000 × g. Supernatants were removed and evaporated under vacuum. Residues were dissolved in assay buffer and assayed for cAMP with a competition binding assay kit (Radiochemical Centre).

Statistical Analyses

N represents the number of separate pneumocyte preparations used (each from a different donor lung). The different assays were performed in duplicate, and the means were calculated. The results are presented as the means (± SEM), obtained by combining the results from each cell preparation. Mean comparison was done by the Friedman analysis of variance of ranks, followed by a two-tailed Wilcoxon rank sum test for paired data to identify the source of the found differences; a confidence level  95% (p < 0.05) was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of Glucagon-like Peptides on Phosphatidylcholine Content and Secretion by Human Type II Pneumocytes

Human Type II pneumocytes in culture are a good tool for testing the effects of different agents on the release of pulmonary surfactant. After isolation, the viability of the cells was 92.1 ± 6.9% as determined by the exclusion of trypan blue, while the purity of the Type II pneumocyte preparations was 85.8 ± 6.2% according to the modified Papanicolaou stain and the tannic acid and polychrome stain methods.

Spontaneous release of ⁵¹Cr by cells incubated in RPMI 1640 alone was less than 20% of total ⁵¹Cr release, which was measured by dissolving the cells in 4% Triton X-100 for 6 h. Cell lysis (percentage of ⁵¹Cr release above spontaneous release) was insignificant in the presence of the different additives, suggesting that their effects were not mediated by alterations in cell viability.

As previously reported by some of us (31), the incorporation of D-[U-¹⁴C]glucose into the different phospholipid fractions by isolated Type II pneumocytes was time dependent. The incorporation of labeled glucose increased progressively with time in all lipid fractions. Isotopic equilibrium was apparently reached between 30 and 60 min, except for the PC fraction, which showed a tendency to reach isotopic equilibrium between 120 and 180 min. A higher percentage of radioactivity was incorporated into the PC fraction (46.6%) as compared with the smaller amounts determined in the lysophosphatidylcholine (LPC, 1.9%), diacylglycerol (DAG, 4.7%), phosphatidylglycerol (PGL, 12.8%), triglyceride (TG, 7.7%), phosphatidylethanolamine (PE, 6.6%), phosphatidic acid (PA, 5.1%), polyphosphoimositides (PPI, 3.6%), phosphatidylinositol (PI, 3.2%), and sphingomyelin (SF, 2.8%) fractions. Because PC is the major phospholipid responsible for the surface tension-lowering properties of surfactant, henceforth, only the data concerning the PC fraction will be shown, although in each experiment all the other phospholipids were also determined.

GLP-1(7-36) amide stimulated [¹⁴C]phosphatidylcholine secretion by Type II pneumocytes as a function of the incubation time (Figure 1), as well as in a concentration-dependent manner in the 1 to 1,000 nM range (Figure 2), while the opposite effect was found with the PC content in these cells (Figures 1 and 2). Because this correlation occurred in all the studies performed, below only the data concerning PC secretion are shown. When we tested the effect of other glucagon-like peptides on PC secretion by Type II pneumocytes, exendin-4 was seen to produce an effect similar to that of GLP-1(7-36) amide. No changes were found when the peptides GLP-2 and exendin(9-39) were added to the medium, and a minimum stimulation by GLP-1(1-37) was observed (Figure 3). Also, exendin(9-39) reversed the stimulatory effect of GLP-1(7-36) amide on PC secretion.

View larger version (13K): [in this window] [in a new window]   Figure 1.   Effect of GLP-1(7-36) amide on phosphatidylcholine content (A) and secretion (B) as a function of the incubation time. Human Type II pneumocytes were labeled and treated with or without 10 nM GLP-1(7-36) amide. Each point represents the mean ± SE of duplicate samples from five different experiments. Statistical comparisons versus cells incubated without GLP-1(7-36) amide: *p < 0.05; **p < 0.01; ***p < 0.001.

View larger version (10K): [in this window] [in a new window]   Figure 2.   Effect of GLP-1(7-36) amide on phosphatidylcholine content and secretion as a function of concentration. Human Type II pneumocytes were labeled and treated with or without different concentrations of GLP-1(7-36) amide, as indicated. Each point represents the mean ± SE of duplicate samples from six different experiments. Statistical comparisons versus cells incubated without GLP-1(7-36) amide: *p < 0.05; **p < 0.01; ***p < 0.001.

View larger version (29K): [in this window] [in a new window]   Figure 3.   Effect of glucagon-related peptides on phosphatidylcholine secretion. Human Type II pneumocytes were labeled and treated in the absence or presence of 10 nM GLP-1(1-37), GLP-1(7-36) amide, GLP-2, exendin-4, and 100 nM exendin(9-39), as described in METHODS. Each column represents the mean ± SE of duplicate samples from six different experiments. Statistical comparisons versus cells incubated without peptides: *p < 0.05; **p < 0.001.

Effect of Glucagon-like Peptides on cAMP Production by Human Type II Pneumocytes

It has been previously reported that cAMP acts as a chemical mediator of the truncated forms of GLP-1 in several target tissues. To ascertain the involvement of cAMP in the effects of GLP-1(7-36) amide on human Type II pneumocytes, these cells were incubated with glucagon-like peptides and with terbutaline as a positive control for activation. We selected the time of incubation, after determining the effect of GLP-1(7-36) amide on cAMP concentrations by Type II pneumocytes as a function of time. Both with short and long periods of time, GLP-1(7-36) amide augmented cAMP concentrations. Both GLP-1(7-36) amide and exendin-4 increased cAMP concentration by 8- to 9-fold over basal values (Figure 4), and this effect was reversed when Type II pneumocytes were stimulated in the presence of the antagonist exendin(9-39). By contrast, exendin(9-39) alone and GLP-2 had a minimal stimulatory effect, and GLP-1(1-37) did not modify cAMP formation at all. As expected, terbutaline induced a strong increase in cAMP concentrations that was completely blocked with the -adrenergic antagonist propranolol (Figure 4). This compound only partially reduced the effect of GLP-1(7-36) amide on cAMP levels. As shown in Figure 5, the stimulatory effect of GLP-1(7-36) amide on Type II pneumocytes was exerted in a concentration-dependent manner in the 1 to 1,000 nM range.

View larger version (27K): [in this window] [in a new window]   Figure 4.   Effect of glucagon-related peptides on cAMP levels of human Type II pneumocytes. Cells were treated in the absence or presence of 10 nM GLP-1(1-37), GLP-1(7- 36) amide, GLP-2, exendin-4, and 100 nM of exendin(9-39), and of terbutaline with or without propranolol, as described in METHODS. Each column represents the mean ± SE of duplicate samples from six different experiments. Statistical comparisons versus cells incubated without peptide: *p < 0.01; **p < 0.001.

View larger version (11K): [in this window] [in a new window]   Figure 5.   Effect of GLP-1(7-36) amide on cAMP levels of human Type II pneumocytes as a function of concentration. Cells were treated with or without of different concentrations of GLP-1(7-36) amide, as indicated. Each point represents the mean ± SE of duplicate samples from six different experiments. Statistical comparisons versus cells incubated without GLP-1(7-36) amide: *p < 0.001.

Effect of Stimulants and Inhibitors of Phosphatidylcholine Secretion by Human Type II Pneumocytes

The mechanism by which GLP-1(7-36) amide exerts its effect on pulmonary surfactant secretion was investigated with different agents known to be modulators of PC secretion. In this sense, the cAMP analog 8-Br-cAMP mimicks the secretion of PC induced by GLP-1(7-36) amide, and the stimulatory effect of terbutaline on PC was blocked by propranolol, while this -adrenergic blocker only partially reduced the effect of GLP-1(7-36) amide. The cAMP antagonist adenosine-3',5'-cyclic monophosphothioate, Rp-isomer (Rp-cAMPS), significantly decreased the stimulatory effect of GLP-1(7-36) amide (Figure 6). TPA, a direct activator of protein kinase C (PKC), elicited a strong increase in PC secretion by Type II pneumocytes, this effect being additive to the action of GLP-1(7-36) amide (Figure 7). Strikingly, the addition of CalphC, an inhibitor of PKC, to the culture medium did not modify the basal level but blocked the stimulatory effect of GLP-1(7-36) amide on PC secretion.

View larger version (38K): [in this window] [in a new window]   Figure 6.   Effects of GLP-1(7-36) amide and other secretagogues on phosphatidylcholine secretion. Human Type II pneumocytes were labeled and treated with or without GLP-1(7-36) amide (10 nM), terbutaline (10 µM), propranolol (10 µM), Br-cAMP (50 µM), Rp-cAMP (50 µM), and H-89 (10 µM). Each column represents the mean ± SE of duplicate samples from six different experiments. Statistical comparisons versus cells incubated without secretagogues: *p < 0.05; **p < 0.01; ***p < 0.001.

View larger version (33K): [in this window] [in a new window]   Figure 7.   Effects of GLP-1(7-36) amide and other secretagogues, including agonists and antagonists of PKC, on phosphatidylcholine secretion. Human Type II pneumocytes were labeled and treated with or without GLP-1(7-36) amide (10 nM), TPA (10 µM), and Calph C (0.5 µM). Each column represents the mean ± SE of duplicate samples from six different experiments. Statistical comparisons versus cells incubated without secretagogues: *p < 0.001.

Also, an increase in calcium influx into Type II pneumocytes as mediated by the ionophore A23187 produced a stimulatory effect on PC secretion that was additive to the action of GLP-1(7-36) amide. Interestingly, KN-62, a specific inhibitor of Ca²⁺-calmodulin-dependent kinase (Ca-Cm-PK), did not affect the stimulatory effect of the calcium ionophore A23187 (Figure 8).

View larger version (32K): [in this window] [in a new window]   Figure 8.   Effects of GLP-1(7-36) amide and other secretagogues, including activator and inhibitor of Ca-CM-PK, on phosphatidylcholine secretion. Human Type II pneumocytes were labeled and treated with or without GLP-1(7-36) amide (10 nM), A23187 ionophore (1 µM), and KN-62 (30 µM), as described in METHODS. Each column represents the mean ± SE of duplicate samples from six different experiments. Statistical comparisons versus cells incubated without secretagogues: *p < 0.001.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Although earlier studies indicated that the truncated forms of GLP-1 are intestinal peptides with a potent effect on glucose-dependent insulin secretion, later on it was found that several biological effects of these peptides can be detected in extrapancreatic tissues. As well as in the pancreas, the expression of GLP-1 receptor mRNA has also been found in human organs such as lung, heart, kidney, and brain (25) although, in contrast to our knowledge obtained in experimental animals, little is known about the actions of the truncated forms of GLP-1 in human extrapancreatic tissues. Wei and Mojsov (25) reported the presence of GLP-1 receptor mRNA in human lung, using an RNase protection assay, although other authors, using Northern blot analysis (26), found GLP-1 receptor expression only in the pancreas.

We also report here that the agonists of the GLP-1 receptor, GLP-1(7-36) amide and exendin-4, increase the production of cyclic AMP by human Type II pneumocytes, as has been described in brain, pancreatic acini, and insulinoma-derived cells. The dose-response curves of GLP-1(7-36) amide in producing increases in cyclic AMP formation and in phosphatidylcholine secretion by Type II pneumocytes were identical, suggesting that cyclic AMP acts as a chemical mediator of the effects induced by the agonists on the GLP-1 receptor. Indeed, the GLP-1 receptor is a member of the seven-transmembrane family of G protein-linked receptors that mediate increases in cyclic AMP by adenylate cyclase activation (20). Both GLP-1(7-36) amide and exendin-4 stimulated cyclic AMP formation and PC secretion in cultured Type II pneumocytes in a time- and dose-dependent manner. These effects seem to be specific, because other glucagon-like peptides, such as GLP-1(1-37), GLP-2, and exendin(9-39), alone did not modify PC secretion or cyclic AMP formation. However, the stimulatory effects of GLP-1(7-36) amide and exendin-4 were reversed by the antagonist exendin(9-39).

Exendin-4 is a peptide isolated from Helodermatidae venoms that share 53% structural homology with GLP-1(7-36) amide. Both peptides specifically interact with the same GLP-1 receptor and produce the same chemical mediator and biological effects, while exendin(9-39) inhibits all the effects of the other two peptides (32). The properties of exendin-4 as an agonist and of exendin(9-39) as an antagonist of the GLP-1 receptor have been used as tools to define the role of GLP-1(7- 36) amide in insulin secretion (32), arterial blood pressure (14), and food intake (17, 18). In this study we took advantage of these properties to attempt to determine the action of glucagon-like peptides on pulmonary surfactant secretion by human Type II pneumocytes.

Isolated Type II pneumocytes in primary culture conserved their physiological properties, permitting study of the effects of different agents on surfactant secretion in vitro. Radioactive labeling of these cells with [¹⁴C]glucose gave rise to several labeled phospholipids with surfactant properties. Surfactants are considered to be a complex mixture of lipids and proteins that reduces the surface tension of the air-alveolar interface in the lung. Phosphatidylcholine is the most abundant of surfactant phospholipids, its disaturated species being largely responsible for the tension-lowering properties of surfactant (33). Accordingly, experimental models designed to elucidate the regulation of pulmonary surfactant secretion have focused on the lipid components; in particular phosphatidylcholine. Current knowledge suggests that the release of this phospholipid would be mediated by at least three signal transduction mechanisms involving the activation of Ca-CM-PK, protein kinase A (PKA), and PKC (34). Thus, to gain further insight into the stimulatory effect of GLP-1(7-36) amide on PC secretion by human Type II pneumocytes, we cultured the cells with several secretagogues and specific inhibitors of signal transduction pathways. Thus, we used terbutaline, a -adrenergic agonist that increases cellular cyclic AMP levels; TPA, which is a direct activator of PKC; and A23187, a calcium ionophore that promotes calcium influx into the cell, which in turn activates a Ca²⁺-calmodulin-dependent protein kinase (Ca-CM-PK). All three compounds strongly increased PC secretion. Several lines of experimental evidence suggest that, at least in part, the stimulatory effects of GLP-1(7-36) amide and exendin-4 on PC secretion by human Type II pneumocytes would be produced through a cAMP-dependent protein kinase (PKA) mechanism. Such evidence is based on the facts that PC secretion induced by GLP-1(7-36) amide was mimicked by the cAMP analog 8-Br-cAMP and by terbutaline; that both the cAMP antagonist adenosine-3',5'-cyclic monophosphothioate, Rp-isomer (Rp-cAMP), significantly decreased the stimulatory effect of GLP-1(7-36) amide, and that this peptide increased cAMP formation by Type II pneumocytes, this effect being reversed in the presence of the antagonist exendin(9-39). In addition, the inhibitors of PKA partially blocked the effect of GLP-1(7-36) amide.

The observations that GLP-1(7-36) amide and TPA have additive effects on the secretion of PC by human Type II pneumocytes and that CalphC, a potent inhibitor of PKC, blocks most of the effect of GLP-1(7-36) amide, suggest that protein kinase C is also involved in the mechanisms of signal transduction required for phosphatidylcholine release by these cells.

Despite the report of increased intracellular Ca²⁺ and activation of phospholipase C in COS-7 cells transfected with the cloned GLP-1 receptor (26), we did not detect any effect of GLP-1(7-36) amide on PC secretion presumably mediated through an increase in intracellular calcium. Because calcium concentrations were not measured, only indirect statements may be made. Thus, both the calcium ionophore A23187 and GLP-1(7-36) amide were seen to exert additive effects on PC secretion, but the specific inhibitor of Ca-CM-PK, KN-62, inhibited the effect of A23187 but did not alter the stimulatory effect of GLP-1(7-36) amide. In summary, our data seem to indicate that PKA and PKC are involved in the stimulatory effect of GLP-1(7-36) amide on PC secretion by human Type II pneumocytes, while this peptide does not produce activating effects through a Ca-CM-PK mechanism. These findings also suggest that GLP-1(7-36) amide could be included in the group of peptides that act as neurotransmitters or hormones in the respiratory tract. These peptides, such as neuropeptide Y, cholecystokinin, galanin, calcitonin gene-related peptide, vasoactive intestinal polypeptide, gastrin-releasing peptide, neurotensin, somatostatin, and enkephalins (35), help to regulate pulmonary functions at different levels: mucous secretion, effects on the smooth muscle of the airways, pulmonary surfactant formation, vascular effects, and neurogenic inflammation.

Also, the stimulatory effect of GLP-1(7-36) amide on PC secretion by human Type II pneumocytes opens new avenues in the pathophysiology of the lung, mainly as regards states of respiratory distress, and especially during the perinatal period or in premature newborns. Further efforts focused on studying the development of GLP-1 receptors in human Type II pneumocytes and on the effects of glucagon-like peptides on pulmonary surfactant secretion during the perinatal age should be made in order to gain a better understanding of the pathophysiology and potential treatment of such disorders.