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Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, and the Department of Biochemistry and Molecular Biology III, Faculty of Medicine (E.B.), Universidad Complutense, 28040 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Maria A. Bosch, Department of Biochemistry and Molecular Biology, Faculty of Chemistry, Universidad Complutense, 28040 Madrid, Spain.
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Abstract |
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Introduction |
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Cloning of the human (3, 4) and rat (5) GLP-1 receptor from pancreatic islets has significantly improved our understanding of the mechanism of action of this peptide. The GLP-1 receptor complementary DNA encodes a 463-amino acid protein identical to those from lung, heart, kidney, and brain (6, 7). Specific high affinity binding sites for GLP-1-(7–36)amide have been identified in rat insulinoma cells (8), pancreatic ß-cells (9), gastric glands (10), and adipocyte (11), lung (12), and brain (13, 14, 15, 16) membranes. Both truncated forms of GLP-1 are indistinguishable in their ability to produce biological effects through the described receptors. These peptides stimulate insulin secretion in a glucose-dependent manner (17, 18) and at the same time increase somatostatin release and reduce glucagon secretion (19). GLP-1-(7–36)amide also has significant effects on gastrointestinal motility and secretion (20, 21). This peptide inhibits gastric acid secretion (20, 21), gastric emptying (21), and meal-induced pancreatic exocrine secretion (22), although the latter phenomenon may be secondary to its effect on gastric emptying. These effects seem to be centrally mediated because they are not found in vagotomized subjects (23). Similarly, intracerebroventricular administration of GLP-1-(7–36)amide reduces both food intake (24, 25, 26, 27, 28) and body temperature (29). GLP-1-(7–36)amide and exendin-4 significantly increase arterial blood pressure as well as heart rate, but previous treatment with exendin-(9–39) blocks the effects of both peptides (30, 31). These findings suggest that the action of the agonist is mediated through its own receptors.
High levels of the GLP-1 receptor and of its own messenger RNA (mRNA) have been found in rat lung (12). These receptors have been detected in the submucosal glands of the trachea and the smooth muscle of the pulmonary arteries, where ligands produce increases in mucous secretion and pulmonary smooth muscle relaxation, respectively (32). In addition, in situ hybridization experiments have identified GLP-1 receptor mRNA in cells morphologically considered type II pneumocytes (33) that are involved in the synthesis and secretion of pulmonary surfactant in alveolar regions (34). Surfactant is a complex mixture of lipids and proteins that reduces the tension at the air-alveolar interface in the lung and provides for alveolar stability. Phosphatidylcholine (PC) accounts for over 80% of surfactant phospholipids (35); its disaturated species is largely responsible for the surface tension-lowering properties of surfactant. PC secretion is a regulated process and in isolated type II cells can be induced by physiological and other agents that act via at least three signal transduction mechanisms involving the activation of different protein kinases (36).
The purpose of the present study was to examine the effect of GLP-1 and other related peptides in the secretory response of type II pneumocytes and the signal transduction pathway involved in this effect. The potential effect of GLP-1-(7–36)amide on the regulation of surfactant secretion may open new insights into the control of surfactant secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
Elastase and deoxyribonuclease I were obtained from Boehringer
Mannheim (Mannheim, Germany). FBS was purchased from BioWhitaker
(Veviers, Belgium). Trypsin, 12-O-tetradecanoylphorbol
13-acetate (TPA), rabbit IgG, DMEM, Earle’s Balanced Salt Solution,
thapsigargin (TSG), dimethylsulfoxide (DMSO), staurosporine,
sphingosine, terbutaline, and 3-isobutyl-1-methylxanthine were
purchased from Sigma Chemical Co. (St. Louis, MO). The Ca2+
chelator 1,2-bis(O-aminophenoxy)ethane,
N,N,N’,N’-tetraacetic acid, tetraaacetoxymethyl ester
(BAPTA-AM) was purchased from Molecular Probes (Leiden, The
Netherlands). 8-Bromo-cAMP (8-Br-cAMP), adenosine-3’,5’-cyclic
monophosphothioate, Rp-isomer (Rp-cAMPS), Ro-31–8220, H-89,
4-Br-A23187 ionophore, and bisindolylmaleimide I were supplied by
Calbiochem (La Jolla, CA). H-7 and KN-62 inhibitors were obtained from
ICN Pharmaceuticals (Costa Mesa, CA).
[Methyl-14C]choline chloride was purchased
from Amersham International (Aylesbury, UK). Percoll was obtained from
Pharmacia Biotech (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 Dr. John Eng, Department
of Internal Medicine, Veterans Administration Hospital (Bronx, NY).
Isolation and culture of type II pneumocytes
Type II pneumocytes were isolated from rat lungs as described
previously (37) with some modifications: the addition of trypsin (75
µg/ml) and deoxyribonuclease I (100 µg/ml) to improve yield and
minimize cell clumping, and a further purification step by differential
adherence to plates coated with IgG, as described by Dobbs et
al. (38). Freshly isolated cells were plated at a density of
106 cells/well on a 12-well tissue-culture plate (Cultek)
and cultured in 1 ml DMEM containing 10% FBS, streptomycin (100
µg/ml), and penicillin (100 U/ml) for 20 h at 37 C in 5%
CO2 in an air/water-saturated atmosphere. At this stage at
least 90% of the attached cells were type II pneumocytes, as
determined by alkaline phosphatase stain (39), and their viability was
95%, as determined by the exclusion of trypan blue.
PC secretion
[Methyl-14C]choline chloride (2
µCi/ml) was included in the medium during overnight culture of the
cells. At the end of this period the medium was removed, and the cells
were rinsed three times with antibiotic-free DMEM to remove
[14C]choline and unattached cells. Fresh DMEM was added,
and the incubation was allowed to proceed for 30 min, after which the
medium was changed, and the test agents were added. The incubation was
continued for 90 min, except for time-course experiments, as indicated.
All inhibitors were added 10 min before the addition of activators.
Some agents were dissolved in DMSO before addition to DMEM. The final
concentration of DMSO in the culture medium was 0.1%, and this amount
was also added to the medium of the corresponding control dishes. At
the end of the incubation period, the medium was aspirated and the
attached cells were lysed with ice-cold water. The spent medium was
centrifuged at 200 x g for 10 min to remove any
floating cells. Lipids were extracted from both the cell extract and
the medium with a mixture of chloroform and methanol by the method of
Bligh and Dyer (40) and separated by two-dimensional TLC on silica gel
G plates. PC fractions were identified by exposing the plates to iodine
vapor, and the incorporated radioactivity was measured in a Beckman
LS-3801 scintillation counter (Beckman, Palo Alto, CA).
PC secretion is expressed as the percentage of [14C]PC in the medium relative to the total amount (cells plus medium).
Assay for cAMP
Type II pneumocytes in primary culture, prepared as described
above, were incubated in DMEM with terbutaline (10 µM),
GLP-1-(7–36)amide (10 nM), or other related peptides for
20 min at 37 C. 3-Isobutyl-1-methylxanthine (0.5 mM) was
present during incubation to prevent cAMP hydrolysis. At the end of the
incubation period, the medium was removed, and 1 ml 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 x g. The supernatant was removed and evaporated
under vacuum at room temperature. The residue was dissolved in assay
buffer and assayed for cAMP with a competition binding assay kit
(Amersham).
Lactate dehydrogenase assay
The rate of lactate dehydrogenase released into the medium was
determined to assess cellular integrity. After the secretion
experiments, lactate dehydrogenase activity in the cells and medium was
assayed by measuring the disappearance of NADH at 340 nm (41). The
lactate dehydrogenase activity released into the medium did not exceed
1% of the total cellular content in all experiments.
Statistics and data analysis
Type II pneumocytes isolated from three rats were pooled in each
experiment and distributed among the various control and treated
groups. In secretion experiments, three wells were used for each group.
The wells were processed separately, and the values were averaged to
yield a single data point per group per experiment. Data from at least
four experiments were averaged, and the groups were compared
statistically with Student’s t test for paired samples.
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Results |
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). The
concentration of GLP-1-(7–36)amide required to produce a half-maximal
response was 10 nM. At this concentration, enhanced PC
secretion was observed up to at least 120 min (Fig. 2).
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, secretion was stimulated up to
the same level by GLP-1-(7–36)amide (1.33-fold) and the agonist
exendin-4 (1.47-fold). No changes vs. control values were
observed when the peptides GLP-1-(1–37) and GLP-2 or the antagonist
exendin-(9–39) were added to the medium. The latter reverted the
stimulatory effect of GLP-1-(7–36)amide and exendin-4 (Fig. 3). The
mechanism by which GLP-1-(7–36)amide exerts its stimulatory effect was
studied using different agents that are well known stimulants of PC
secretion. Thus, TPA, which is a direct activator of protein kinase C
(PKC); terbutaline, a ß-adrenergic agonist that increases cellular
cAMP levels; TSG, an endoplasmic reticulum Ca2+-adenosine
triphosphatase inhibitor; and the calcium ionophore A23187, which
promotes calcium influx into the cell that, in turn, activates a
Ca2+-calmodulin-dependent protein kinase (Ca-CM-PK), were
employed. When either TPA or A23187 plus GLP-1-(7–36)amide were
included in the culture medium (Fig. 4),
PC secretion was stimulated 3.3-fold over the basal rate; this effect
was additive to that of TPA (2.2-fold) or A23187 (2.1-fold) and
GLP-1-(7–36)amide (1.3-fold). The same additive effect was observed
when TSG plus GLP-1-(7–36)amide were added to the culture medium (Fig. 5). GLP-1-(7–36)amide did not produce
any change in the stimulatory effect observed with terbutaline (Fig. 4), and the intracellular Ca2+ chelator BAPTA suppressed
the stimulatory effect of TSG and A23187, but had no effect on PC
secretion mediated by GLP-1-(7–36)amide (Fig. 5). On the other hand,
the secretion induced by GLP-1-(7–36)amide was mimicked by the cAMP
analog 8-Br-cAMP; this effect was also observed with terbutaline (Fig. 6). Taken together, these data suggest
the involvement of a cAMP-dependent protein kinase (PKA) mechanism in
the stimulatory effect of GLP-1-(7–36)amide on PC secretion. The
involvement of PKA was tested using a strategy involving specific
inhibitors for signal transduction pathways. As shown in Table 1, bisindolylmaleimide and sphingosine,
which are inhibitors of PKC, reversed the stimulatory effect of TPA on
PC secretion. The increase in PC secretion mediated by
GLP-1-(7–36)amide and exendin-4 was reverted by two inhibitors of PKC
and PKA, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazone (H-7) and
staurosporine (Table 1). The specific inhibitor of Ca-Cm-PK, KN-62, did
not affect the stimulatory effect of secretagogues, except for the
calcium ionophore A23187 and TSG, which lost their stimulatory capacity
only in the presence of KN-62 (Table 1). These results are supported by
those shown in Table 2. The secretion induced by GLP-1-(7–36)amide and
terbutaline was reversed by H-89, a potent inhibitor of PKA that is
commonly used to discriminate between the effects of PKC and PKA,
whereas the highly specific PKC inhibitor Ro-31–8220 had no effect on
GLP-1- and terbutaline-stimulated PC secretion. The cAMP antagonist
adenosine-3’,5’-cyclic monophosphothioate, Rp-isomer (Rp-cAMPS)
significantly decreased the stimulatory effect of GLP-1 and terbutaline
(Table 2).
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). GLP-1-(7–36)amide and exendin-4
increased cAMP cellular levels by 10- to 12-fold over basal values;
this effect was reversed when type II pneumocytes were stimulated in
the presence of the antagonist exendin-(9–39) (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we observed that GLP-1-(7–36)amide stimulated PC secretion from cultured type II cells in a time- and dose-dependent manner without causing any cell damage. However, other glucagon-like peptides, such as GLP-1-(1–37) and GLP-2, did not modify PC secretion, whereas exendin-4 had almost the same stimulatory effect on secretion as GLP-1-(7–36)amide. The antagonistic effect of exendin-(9–39) on this parameter was also tested by prior treatment of the cells with this peptide. Thus, treatment with exendin-(9–39) alone did not change PC secretion, but the stimulatory effects of both GLP-1-(7–36)amide and exendin-4 were reverted by the antagonist, indicating a specific effect of the truncated form of GLP-1.
Exendin-4 is a peptide purified from Helodermatidae venoms that competes with GLP-1-(7–36)amide for the same receptor from pancreatic acini (42), insulinoma-derived cells, and lung membranes (43) and also stimulates glucose-induced insulin secretion in isolated rat islets and proinsulin gene expression at the transcriptional level in mouse insulinoma ßTC-1 cells (43). By contrast, exendin-(9–39) reduces or inhibits all of the aforementioned effects of GLP-1-(7–36)amide and exendin-4, indicating that exendin-4 is an agonist and exendin-(9–39) is an antagonist of GLP-1-(7–36)amide. These findings open the possibility of using these peptides to define the role of GLP-1-(7–36)amide in PC secretion by type II pneumocytes, as it has been used to study the action of GLP-1-(7–36) on arterial blood pressure (30, 31), food intake (24, 25, 26, 27, 28), and insulin secretion (17, 18).
To investigate the mechanisms involved in the stimulatory effect of GLP-1-(7–36)amide on PC secretion, we used a combination of assays to assess secretory responses and intracellular signals using a strategy of different secretagogues and specific inhibitors of signal transduction pathways.
The stimulatory effect of GLP-1-(7–36)amide was additive to that observed with TPA, a direct activator of PKC; TSG, an endoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor; or the calcium ionophore A23187, which permits calcium influx into the cell that, in turn, activates a Ca-CM-PK. Otherwise, GLP-1-(7–36)amide did not alter the increase on PC secretion due to the cAMP analog 8-Br-cAMP or terbutaline and lost its stimulatory capacity in the presence of H-7, staurosporine, and H-89, inhibitors of PKA that also reversed the stimulation due to terbutaline. Terbutaline is a well known surfactant phospholipid secretagogue that binds to ß-receptors coupled to AC via the heterotrimeric G protein Gs. Activation of AC results in the generation of cAMP, which, in turn, activates cAMP-dependent protein kinase (44). Phosphorylation of actin and/or other proteins is believed to lead ultimately to PC secretion. In this respect, we observed a significant increase in cAMP intracellular levels after GLP-1-(7–36)amide or exendin-4 stimulation; this effect was reversed by the antagonist exendin-(9–39). These results are in agreement with those reported in pancreatic acini, insulinoma-derived cells, and rat lung membranes (34, 35).
At present we know that several neuropeptides contribute to the regulation of lung functions. Peptides considered to be involved in such regulation are cholecystokinin, enkephalins, galanin, neurotensin, somatostatin, vasoactive intestinal polypeptide, neuropeptide Y, calcitonin gene-related peptide, and gastrin-releasing peptide. The results present here as well as the effect of GLP-1-(7–36)amide on the stimulation of mucous secretion from isolated rat trachea and the relaxation of the preconstricted vessels in isolated rings or pulmonary arteries (32) argue for a similar contribution of GLP-1-(7–36)amide. GLP-1 receptors have been described in rat lung membranes with an apparent molecular mass of 55 kDa, which is significantly smaller than that of receptors found in pancreatic ß-cells (12). However, the isolation of a rat lung GLP-1 receptor complementary DNA revealed that receptors in ß-cells and lung possess identical sequences (45). Thus, it seems likely that the GLP-1 receptor undergoes different posttranscriptional processing in lung and endocrine pancreas.
The biological effects induced by GLP-1-(7–36)amide on type II pneumocytes may be the consequence of the high levels of GLP-1 receptors (12) and GLP-1 receptor mRNAs detected in rat lung (33). It has been reported that GLP-1-(7–36)amide binds to receptors on the smooth muscle of the pulmonary arteries and on submucosal glands of the trachea (32). Also, in situ hybridization studies have shown that GLP-1 receptor mRNA is present in cells within the lung alveoli, which seem to be type II pneumocytes (33). These findings support the stimulatory effect of GLP-1-(7–36)amide on surfactant secretion and offer new insight into the pathophysiology of the lung, especially during the perinatal period or in premature newborns. Accordingly, it is of great importance to elucidate the ontogenic development of the GLP-1 receptor. These receptors are present in significant amounts in the lung of 19-day-old mouse fetuses and increase significantly after birth until reaching maximal levels at 5 weeks of extrauterine life (46). Lower concentrations of GLP-1 receptors in the lung of fetal mice seem to be sufficient for the GLP-1-(7–36)amide-induced secretion of the surfactant, but smaller amounts of the receptor at earlier stages might well lead to pathological complications.
Despite the available information about the GLP-1 receptor and the effect of GLP-1-(7–36)amide on different cell lines and tissues, the mechanism through which this peptide may regulate surfactant secretion has not been elucidated. Our results suggest the involvement of PKA in the stimulatory effect of GLP-1-(7–36)amide on PC secretion in such a way that GLP-1-(7–36)amide acting through a putative G protein-coupled cell surface receptor would activate AC, resulting in the generation of cAMP.
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Footnotes |
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2 Recipient of a research fellowship from Complutense
University.
Received October 2, 1997.
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)
different concentrations of GLP-1-(7–36)amide for 90 min. Each
point represents the mean ± SE
(bars) of triplicate samples from four different
experiments. *, P < 0.05 vs.
control.
, P < 0.05 vs. BAPTA-nonloaded
cells.