1 Center for Gastrointestinal Research and 2 Department of Morphology and Imaging, University of Louvain, B-3000 Louvain, Belgium; and 3 Intestinal Diseases Research Programme, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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The ability of
neuropeptides to modulate enteric smooth muscle proliferation was
examined in primary explant cultures of rabbit gastric antrum and colon
smooth muscle. Cell proliferation was determined by
[3H]thymidine
incorporation measurements and cell counting. Subcultured rabbit antrum
and colon myocytes (passages
2-6) preserved a smooth muscle phenotype, as
verified by immunohistochemistry for -smooth muscle actin and
electron microscopy. Both vasoactive intestinal polypeptide (VIP) and
pituitary adenylate cyclase-activating peptide-(1
38) [PACAP-(1
38)] concentration dependently
(10
10 to
10
6 M) inhibited the
serum-induced
[3H]thymidine
incorporation [in colon, 48.2 ± 5.8 and 55.6 ± 9.3% of
control with 10
6 M VIP and
10
7 M PACAP-(1
38)]
and inhibited increase in cell numbers in cultures derived from the
colon but not in those from the antrum. Effects of VIP and
PACAP-(1
38) were mimicked by forskolin
(10
7 to
10
6 M) but not by
8-bromo-cGMP, whereas theophylline enhanced the effects of VIP.
Inhibition of nitric oxide synthase with
NG-nitro-L-arginine
methyl ester (10
3.5 M) did
not alter the effects of VIP. Substance P, motilin, calcitonin gene-related peptide, and somatostatin had no effect. A single class of
125I-labeled VIP binding sites was
found in antrum and colon myocyte cultures with an equal affinity for
VIP and PACAP-(1
38) [dissociation constant
(Kd) in antrum = 3.4 ± 0.8 nM for VIP and 2.0 ± 1.0 nM for PACAP-(1
38);
Kd in colon = 2.0 ± 1.0 nM for VIP and 2.8 ± 1.6 nM for PACAP-(1
38)].
Density of binding sites in the antrum was higher than in the colon. In
disease states such as inflammatory bowel disease, inhibition of
myocyte proliferation by VIP and PACAP may serve to control smooth
muscle hyperplasia in the colon but not in the antrum.
enteric neuropeptides; smooth muscle pathophysiology; signal transduction; neuropeptide receptors
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INTRODUCTION |
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IN THE NORMAL muscularis propria, enteric myocytes have a "contractile" phenotype and a low rate of proliferation and secretion. In contrast, smooth muscle hyperplasia and hypertrophy are prominent histological features of acute and chronic enteric inflammation and may reflect phenotypic transformations. For example, in Crohn's disease, smooth muscle cells of the muscularis mucosae show a striking proliferation and secrete copious amounts of collagen (9, 14). Enteric smooth muscle cells in primary culture also transform phenotypically and secrete a diversity of proteins such as collagen and cytokines (13, 17).
The factors controlling enteric smooth muscle proliferation remain
largely unknown. Cytokines, such as interleukin-1, tumor necrosis
factor-, and transforming growth factor-
, have mitogenic effects
on enteric smooth muscle in culture (28, 42), whereas transforming
growth factor-
inhibits smooth muscle proliferation (30).
It is becoming increasingly evident that interactions between enteric nerves and smooth muscle cells influence the course of the inflammatory reaction in the deeper layers of the gut wall (5). Recently, we have submitted evidence that enteric neuropeptides enhance interleukin-1-induced interleukin-6 secretion by enteric smooth muscle cells in primary cultures (39). Therefore, we hypothesized that neuropeptides may modulate the proliferation of smooth cells in the muscle layers. Indeed, neuropeptides have been shown to regulate cell growth and differentiation at other sites of the body. For instance, vasoactive intestinal polypeptide (VIP) has trophic and mitogenic effects on embryonic neural tissue (2), but at the same time the peptide inhibits the mitosis of pancreatic tumors (31), colon adenocarcinoma cells (38), and murine Peyer's patch lymphocytes (33). VIP and the related peptide, pituitary adenylate cyclase-activating peptide, inhibit the proliferation of vascular and bronchial smooth muscle cells in primary culture (16, 22, 26), and substance P and somatostatin modulate vascular smooth muscle proliferation (29, 15). The aim of the present study, therefore, was to explore the role of enteric neuropeptides in the control of smooth muscle proliferation using a model of mammalian enteric myocytes in primary culture.
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MATERIALS AND METHODS |
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Enteric smooth muscle culture.
New Zealand White rabbits of either sex (2.5-3.0 kg) were
euthanized, and the gastric antrum and 10 cm of the distal colon were
removed and rinsed. The mucosa and submucosa of the antrum were removed
by sharp dissection, and the serosa was removed by peeling under a
stereoscopic microscope. The colonic segment was cleared of mucosa by
scraping with a scalpel blade, and the submucosa and serosa were peeled
off. Samples of the dissected muscle layers were rapidly frozen and
hematoxylin-eosin-stained sections were examined for the extent of
dissection using light microscopy. Explant cultures were prepared as
described by Kahn et al. (17). Briefly, fragments of the muscle layers
were explanted in 60-mm culture dishes (Corning, NY) and grown in DMEM
(GIBCO BRL, Grand Island, NY) with 100 U/ml penicillin G, 100 µg/ml
streptomycin, and 0.25 µg/ml amphotericin B (GIBCO BRL) and
supplemented with 10% fetal bovine serum (FBS; GIBCO BRL). The primary
cultures were incubated in a 5%
CO2 incubator at 37°C, and the
medium was changed twice weekly. When cultures reached confluency,
cells were dissociated with 0.125% trypsin-EDTA (GIBCO BRL),
concentrated by centrifugation at 500 rpm for 5 min and replated at the
desired density. Cells were used between passages
2 and 5. For cell
counts, trypsinized cells in suspension were diluted 1:4 with trypan
blue (0.4%) and loaded into a Burker chamber. Viability, as judged by
trypan blue exclusion, was always preserved in more than 90% of
trypsinized cells. Cells for immunohistochemistry were seeded in
24-well plates (Corning) on Thermanox inserts (Nunc, Naperville, IL).
Inserts with confluent cells attached were removed from medium and soon
after were fixed in acetone. Immunohistochemistry was performed on cell
cultures and on cryostat sections from snap-frozen (80°C)
undissected tissue samples using a three-step indirect immunoperoxidase
method with monoclonal antibodies against
-smooth muscle actin
(1/400; Sigma, St. Louis, MO), desmin (1/10; Boehringer, Mannheim,
Germany), vimentin (1/20; Amersham, Little Chalfont, UK), and
neuron-specific enolase (NSE; 1/10) and glial fibrillary acidic protein
(1/10; both from Monosan, Uden, The Netherlands). Cells for
transmission electron microscopy were seeded in six-well plates,
mobilized with 0.125% trypsin, and pelleted by centrifugation at 2.000 g for 10 min. Pellets were fixed in
2% glutaraldehyde, postfixed in osmium tetroxide, embedded in epon,
and further processed for light (semithin sections) and electron
microscopy (ultrathin sections).
Proliferation studies.
Cells were seeded in 24-well plates at a density of 5 × 104 per well and cultured for 48 h
in DMEM containing 10% FBS. Subconfluent cells were then growth
arrested by exposure to serum-free DMEM for 24 h. The medium was
subsequently replaced by either 1 ml of DMEM containing 2% FBS or DMEM
alone (control). Neuropeptides at different concentrations were added
to the medium, and cells were incubated for another 24 h; 0.3 µCi
[3H]thymidine (sp act
15 Ci/mmol; Amersham) was added for the final 18 h of incubation. Cells
were subsequently dehydrated by adding 1 ml of ice-cold methanol for 1 min and washed twice with 10% TCA. Cells were lysed with 1% SDS in
0.3 M NaOH, lysates were aspirated, and the retained radioactivity was
determined in a liquid scintillation counter (Tri-Carb 1500; Packard,
Meriden, CT). The following peptides were tested in the proliferation
studies: porcine VIP [which is identical to rabbit VIP
(19)] (Biogenesis, Poole, UK), ovine pituitary adenylate cyclase
activating peptide-(138) [PACAP-(1
38); Novabiochem,
Laüfelfingen, Switzerland],
[Ac-Tyr1,D-Phe2]growth
hormone releasing factor-(1
29)-amide
{[Ac-Tyr1,D-Phe2]GRF-(1
29)-NH2;
Peninsula, Belmont, CA}, substance P (Biogenesis), somatostatin
(UCB Bioproducts, Braine-l'Alleud, Belgium), and [13-norleucine]porcine motilin
([nle13]po-motilin;
Eurogentec, Namur, Belgium). Forskolin, theophylline, 8-bromo-cGMP
(8-BrcGMP), NG-nitro-L-arginine
methyl ester (L-NAME), and
cytosine arabinoside were all from Sigma.
125I-labeled VIP binding studies.
Binding studies were performed as described by Chijiwa et al. (4) with
minor modifications. Confluent myocytes in 24-well plates were washed
with 1 ml of HEPES buffer [containing (in mM) 120 NaCl, 4 KCl,
2.6 KH2PO4,
2 CaCl2, 0.6 MgCl2, 14 D-glucose, and 25 HEPES]
with 0.1% bacitracin (Sigma) and 0.1% BSA (Serva, Heidelberg,
Germany). Incubation with
125I-labeled VIP (NEN, Boston, MA;
sp act = 2,000 Ci/mmol, final concentration = 10 pM) alone or in the
presence of increasing concentrations of VIP or PACAP-(138) was
performed at 20°C in 0.5 ml of HEPES buffer containing 0.2% BSA,
0.01% serum trypsin inhibitor (Serva), and 0.1% bacitracin. After 20 min, the incubation buffer was aspirated, and cells were washed twice
with ice-cold HEPES buffer. Cells were lysed by SDS (1%) in 0.3 M
NaOH, and lysates were counted in a gamma counter (Cobra-II, Packard).
125I-VIP binding was corrected for
nonspecific binding, determined in the presence of
10
6 M VIP. For
desensitization experiments, cells were incubated with
10
7 M VIP or PACAP in DMEM
with 2% FBS at 37°C in a 5%
CO2 environment for 24 h, and
cells were washed four times with HEPES buffer before the binding experiment.
VIP radioimmunoassay. The RIA was performed as described by Long and Bryant (20) using commercial reagents. VIP and rabbit VIP antibodies were from Biogenesis (Poole, UK), and 125I-VIP from NEN (sp act = 2,000 Ci/mmol). The detection limit of the assay was 20 pg/ml. Interassay variation was <10%.
Data analysis. The modulation of [3H]thymidine incorporation by neuropeptides and other compounds is expressed as a percentage of the response to 2% FBS. Radlig-Ligand software (Elsevier-Biosoft, Cambridge, UK) was used for the analysis of 125I-VIP binding data.
All data are expressed as means ± SE. Statistical analysis was performed using Student's t-test when comparing two groups and one-way ANOVA, followed by Student's t-test or Student-Newman-Keuls test, when comparing three or more groups. Statistical significance was accepted at a P value of <0.05. ![]() |
RESULTS |
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Characterization of enteric myocytes.
Cultures were inspected at least twice weekly for growth and
microscopic appearance. Cells in primary cultures were spindle shaped
and displayed a hill-and-valley growth pattern. Subcultured cells had a
heterogeneous appearance at low density, but confluent cells were
spindle shaped and also adopted a hill-and-valley appearance (Fig.
1A).
More than 95% of confluent cells from colon and antrum (passages 2-4) stained
intensely for -smooth muscle actin, but staining for vimentin,
desmin, NSE, and glial fibrillary acidic protein was not observed. In
control tissue sections from rabbit colon and antrum, the
-smooth
muscle actin and desmin immunoreactivity was restricted to the
muscularis mucosae, muscularis propria, and vascular tunica media. The
appearance of confluent colonic and antral cells
(passage 3) using electron
microscopy was characterized by the presence of a discontinuous
basement membrane around individual cells, by caveolae of the plasma
membrane, and by cytoplasmatic actin filaments (Fig.
1B).
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Effects of neuropeptides on
[3H]thymidine incorporation.
[3H]thymidine was
incorporated in cultured myocytes from rabbit antrum and colon to an
equal extent (1,152 ± 403 and 1,131 ± 235 dpm/g protein for
antrum and colon, respectively). About 80% was specifically
incorporated into DNA because addition of cytosine arabinoside
(106 M), to inhibit DNA
biosynthesis, reduced thymidine incorporation to 19.0 ± 3.1 and to
19.9 ± 5.5%, respectively, of the control values obtained in 2%
FBS. When serum-free DMEM was added to the growth-arrested myocytes,
[3H]thymidine
incorporation was only 16.8 ± 8.7% (antrum) and 8.2 ± 3.3%
(colon) of the incorporation obtained with 2% FBS-containing medium.
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Effects of VIP and PACAP on cell counts.
The antiproliferative effects of both VIP and PACAP-(138) on colon
myocytes were verified by determining the increase in cell number for
several days. Cells were seeded at a density of 105/well in DMEM-10% FBS. After
24 h, the FBS content was reduced to 2% and the cell counts were
monitored for 3 more days. VIP (10
7 M) and PACAP-(1
38)
(10
7 M) added along with
2% serum significantly inhibited the increase in cell numbers of colon
but not of antrum myocytes. This effect was preserved for 3 days (Fig.
3). In all conditions, more than 90% of
the trypsinized cells excluded trypan blue.
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Transduction pathway for the effect of VIP.
The adenylate cyclase-stimulating compound forskolin concentration
dependently (108 to
10
6 M) inhibited the
[3H]thymidine
incorporation of colon but not of antrum myocytes. On the contrary,
8-BrcGMP (10
4 M) was
without effect and did not interfere with the effects of VIP
(10
7 M). Also, inhibition
of nitric oxide synthase with
L-NAME
(10
3.5 M) did not change
the effects of VIP (Table 2).
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Degradation of VIP by smooth muscle membranes. Incubation of VIP (289.5 ± 21.5 ng/ml) in the cell culture medium for 6 or 24 h at 37°C did not result in VIP degradation (89.4 ± 5.1% of control at 24 h; not significant, n = 4). In the presence of either antral or colonic smooth muscle cells, no significant decrease was detected after 6 h (87.3 ± 5.9 and 92.7 ± 4.6% of control VIP levels for antrum and colon myocytes; n = 4), but after 24 h partial degradation was evident [70.5 ± 11.5 and 67.7 ± 8.9% of control in antrum (P < 0.05) and colon (P < 0.02), respectively; n = 4].
Binding of 125I-labeled VIP to smooth
muscle membranes.
Specific 125I-labeled VIP binding
was observed in intact myocytes of both antrum and distal colon.
Nonspecific binding, in the presence of
106 M VIP, was 36.0 ± 5.7 (antrum) and 34.2 ± 5.6% (colon) of the total binding. These
values compare with the previously reported nonspecific binding in
myocytes from rabbit colon and guinea pig antrum (4, 10). Analysis of
the displacement studies in colon and antrum myocytes demonstrated the
presence of a single binding site in both preparations, with a
dissociation constant of 3.4 ± 0.8 nM in the antrum and 2.0 ± 0.8 nM in the colon (Fig. 4). The density of VIP binding sites
(Bmax) in the antrum (170.1 ± 39.5 fmol/mg protein) was significantly
(P < 0.05) higher than in the colon
(82.4 ± 21.4 fmol/mg protein). This corresponds to 17.9 ± 7.8 (× 103) binding sites per
cell for colon and 64.1 ± 11.9 (× 103) binding sites per cell for
antrum (P < 0.01) (the data are
summarized in Table 3). PACAP-(1
38)
competed with 125I-VIP for the
binding site. The potency of PACAP-(1
38) at displacing 125I-VIP from antrum and colon
binding sites was identical to that observed with VIP (Table 3 and Fig.
4). The Bmax values obtained with
PACAP-(1
38) were also not different from VIP (76.5 ± 23.6 and 163.9 ± 53.9 fmol/mg for colon and antrum, respectively).
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DISCUSSION |
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The results of the present study provide the first evidence that the enteric neuropeptides VIP and PACAP inhibit the proliferation of colon but not of antrum myocytes in culture. Moreover, we have shown that specific VIP receptors are expressed in primary cultures of both rabbit antrum and colon myocytes.
Cultures of rabbit enteric myocytes have been described before (10,
42), but, to the best of our knowledge, this is the first report on
primary and secondary cultures of rabbit antrum and colon myocytes
using an explant technique. Therefore, extensive characterization of
the cultures was performed. The hill-and-valley growth pattern, shape,
and ultrastructural appearance of confluent cells were typical for
smooth muscle cells. The presence of -smooth muscle actin and the
absence of vimentin staining, even after being subcultured, indicated
that the cells preserved the phenotype of smooth muscle cells and not
of myofibroblasts, which have been described to contain smooth muscle
-actin but invariably coexpress vimentin (32). Also, the absence of
staining for glial fibrillary acidic protein and for NSE excluded the
contamination of the cultures by a significant amount of enteric glial
cells or enteric nerves. It is also worth mentioning that both rabbit
antrum and colon myocytes in culture responded to agonists, such as ACh
and motilin, with elevations of intracellular calcium (40).
Nevertheless, the cultured myocytes were phenotypically transformed, since desmin staining was not observed, whereas smooth muscle in control tissue sections stained intensely for this filament. Phenotypic transformation of smooth muscle cells in primary culture is a general observation (3). The myocytes transform from a strictly contractile cell type to cells with secretory capacities. This transformation is also observed in most pathological conditions characterized by smooth muscle hyperplasia. In strictures caused by inflammatory bowel disease, phenotypically transformed myocytes are the principal cell type of the thickened gut wall and they are responsible for collagen deposition (14). Therefore, primary cultures of enteric myocytes appear to be a suitable model to study the regulation of smooth muscle proliferation.
Inhibition of myocyte growth by VIP and PACAP has been previously demonstrated in cultured myocytes from rat aorta and guinea pig bronchi with a similar potency as observed in the present study (22, 26). However, the antiproliferative effect of neuropeptides in the gut is region specific, since VIP and PACAP had an effect on myocytes of colonic but not of antral origin. Differences in phenotype were not observed between antrum and colon myocytes in culture and cannot account for this observation.
VIP is rapidly degraded by homogenized antral smooth muscle membranes (19), although it is not clear whether this is caused by a membrane-bound enzyme or by cytosolic hydrolases. The absence of an effect of VIP on antral myocytes could therefore have been caused by the rapid degradation of VIP. However, we found that VIP is only slightly degraded by intact colon and antrum myocytes. The stability of VIP in the presence of colon myocytes is further confirmed by the fact that with these cells VIP and PACAP effects on [3H]thymidine incorporation were preserved for 24 h and the reduction in cell numbers was preserved for 3 days.
The effects of substance P, motilin, and somatostatin, which have been shown to modulate cell growth in vitro in other systems, were explored. Substance P had no effect in contrast to previous observations of mitogenic effects in vascular and bronchial cultured myocytes (15, 25). Similarly, motilin, which was reported to enhance lymphocyte proliferation, and somatostatin, which inhibited vascular smooth muscle proliferation in allografts (15, 35), were without effect. The expression of specific receptors for these peptides was not further investigated; therefore, their lack of effect may be due to the lack of receptor expression.
The second messenger pathway involved in the observed effect of VIP and PACAP was further investigated, since modulation of myocyte proliferation by cAMP and cGMP in vitro has been reported in rat and rabbit aortic myocytes. (6, 18, 32). In the present study, the effects of VIP and PACAP were mimicked by the adenylate cyclase activator, forskolin, and enhanced by the phosphodiesterase inhibitor, theophylline. These results support a role for the cAMP-protein kinase A-dependent pathway, whereas the negative results with 8-Br cGMP, in a concentration known to modulate vascular smooth muscle proliferation (11), exclude the cGMP-protein kinase G pathway. The latter transduction mechanism was also rejected on the basis of the negative results obtained with the nitric oxide synthase inhibitor L-NAME. Nitric oxide is believed to be the downstream intermediate of mitogenic cytokines, such as interleukin-1, responsible for the activation of guanylate cyclase in vascular smooth muscle (8). VIP and PACAP are known to activate membrane-bound nitric oxide synthase in rabbit gastric smooth muscle (23). However, the cAMP-protein kinase A pathway appears to be the sole second messenger system involved in the VIP modulation of myocyte proliferation.
Serum-starved smooth muscle cells in culture become nonproliferative and are blocked in the late G0 phase of the cell cycle (17). The antiproliferative effects of both VIP and PACAP, however, do not seem to be cell cycle specific, since inhibition of the [3H]thymidine incorporation by VIP was present in both growth-arrested cells and in cells that had already been exposed to the mitogenic action of FBS. VIP was most effective at inhibiting thymidine incorporation when added 6 h after FBS (2%), indicating that the myocytes were more sensitive after the cell cycle had been reactivated. Furthermore, VIP and PACAP were effective in reducing cell numbers when administered 24 h after passage in medium with 10% serum.
VIP and PACAP have a very similar profile of biological activity, and
their receptors are commonly classified as "classical" VIP
receptors, which have an equal affinity for VIP and PACAP receptors and
PACAP receptors, which bind PACAP preferentially (34). In colon
myocytes, we characterized a single binding site with equal affinity
for both peptides. Moreover, the concentration dependence for the
antiproliferative effect is similar for VIP and PACAP and correlates
well with the binding affinity. Therefore, the antiproliferative
effects of these neuropeptides appear to be mediated through a
classical VIP receptor. Its affinity correlates well with the
high-affinity VIP binding site described in rabbit colon myocytes (10).
Two subtypes of the VIP receptor,
VIP1 and
VIP2, have been reported in other
species, but in rabbits only limited information became recently
available. In freshly isolated rabbit gastric myocytes, VIP receptors
were demonstrated (24); with the use of Northern blot analysis, using
primers based on the rat VIP receptor subtypes, only
VIP2 receptors appear to be
present (37). It is tempting to speculate that the differences in
responses of antrum and colon are due to VIP receptor subtypes; however, more detailed studies of VIP receptors in the rabbit are
needed to clarify this point. The fact that the GRF analog, [Ac-Tyr1,D-Phe2]GRF-(129),
a VIP antagonist in the rat pancreas (41) and rat vascular smooth
muscle (26), was ineffective in our study in the proliferation as well
as in the binding experiments may indicate species differences between
the rat and rabbit VIP receptors. Interestingly, more VIP receptors
were found in the antrum than in the colon myocytes, despite the
absence of antiproliferative effects. Also, receptor downregulation by
VIP was similar in antrum and colon myocytes. Therefore, downregulation
cannot account for the absence of an effect in antrum myocytes. Because
forskolin did not alter the thymidine incorporation of antrum myocytes, we suggest that the growth of antrum myocytes is insensitive to increases in cAMP despite the presence of a functional VIP receptor.
Neuropeptide-mediated antiproliferative effects may be of importance in disease states. For example, hypertrophy of the muscle layers has been documented in Crohn's colitis (9) and is accompanied by an increase in the number and size of VIPnergic nerves and of the VIP and PACAP content of the gut wall (1, 27). Because our results indicate that VIP and PACAP are putative endogenous inhibitors of smooth muscle proliferation in the mammalian colon, we propose that both neuropeptides serve to control unlimited muscle hyperplasia. The antiproliferative effects of VIP and PACAP illustrate the importance of the interactions between enteric nerves and smooth muscle as active participants of the inflammatory reaction in the gut. Because the observed effects were region specific for the colon, further studies will be required to outline the role of enteric nerves in controlling smooth muscle hyperplasia at different levels of the gastrointestinal tract.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Belgian National Science Foundation (NFWO Grant 3.0187.96) and the Belgian Ministry of Science (Geconcerteerde Onderzoeksactie 92/96-04 and Interuniversitaire Attractiepool P4/16). G. Van Assche is a doctoral fellow and I. Depoortere is a postdoctoral research fellow of the Belgian National Research Foundation.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: T. L. Peeters, Gut Hormone Lab, Gasthuisberg O&N, Herestraat 49, B-3000 Louvain, Belgium.
Received 21 January 1998; accepted in final form 19 October 1998.
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