The endothelin system in normal human colon
Giorgia
Egidy1,
Lucienne
Juillerat-Jeanneret2,
Petra
Korth1,
Fred T.
Bosman2, and
Florence
Pinet1
1 Institut National de la Santé et de la Recherche
Médicale Unit 36, Collège de France, 75005 Paris, France;
and 2 Institute of Pathology, Centre Hospitalier Universitaire
Vaudois, CH 1011 Lausanne, Switzerland
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ABSTRACT |
Endothelin
(ET)-1 is a potent vasoconstrictor and mitogenic peptide that has a
variety of biological effects in noncardiovascular tissues. The precise
cellular distribution of the ET-1 system in the wall of the normal
human colon was studied to identify the physiological role of ET in the
gut. In situ hybridization revealed ET-converting enzyme-1 (ECE-1) mRNA
in all vessels, the colon epithelium, and macrophages. Prepro-ET-1
(PPET-1) mRNA had a similar distribution except for a scattered signal
in mucosal microvessels. ETA and ETB receptor
mRNAs were mainly in the lamina propria, pericryptal myofibroblasts,
microvessels, and mononuclear cells, with ETA mRNA more
abundant than ETB mRNA. 125I-ET-1 binding
showed ETB along the crypts and in nerve fibers descending
from the ganglionic plexus that contained PPET-1, ECE-1, and
ETB transcripts, whereas glia contained ETA
receptors. The finding of the entire ET system in the normal mucosa
suggests its implication in some characteristic functions of the colon and its secretion as both a neuroactive and a vasoactive peptide.
in situ hybridization; immunocytochemistry; endothelial cell; smooth muscle cell
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INTRODUCTION |
THE POTENT
VASOCONSTRICTOR peptide endothelin (ET)-1 was identified by
Yanagisawa et al. (47). It belongs to a recently discovered family of three 21-amino acid peptides, the endothelins (ETs), which regulate vascular tone (18). The ETs act on
two distinct high-affinity ET receptor subtypes, ETA
(13) and ETB (33), which are
seven-transmembrane G protein-coupled receptors. ETA
receptors bind ET-1 and ET-2, but not ET-3, at physiological concentrations, whereas ETB receptors bind all three ETs
with a similar affinity. ETs are initially synthesized as large
precursor polypeptides, prepro-ETs (PPETs), that are cleaved at two
pairs of basic amino acids to generate intermediate peptides, the Big ETs. The Big ETs are then cleaved by ET-converting enzyme (ECE) (35) to produce the mature ETs. ECE is a key enzyme in the
synthesis of the ETs because Big ETs have negligible biological
activities (17). Two ECE-encoding genes have been cloned,
ECE-1 (42) and ECE-2
(8). Their sequences are 59% identical, but only ECE-1, the most abundant, has been studied in detail (see
Ref. 41 for a recent review). Targeted inactivation studies on
ECE-1 in mice showed that although there was still ET-1 in
the plasma because of ECE-2, it did not rescue the mutant
developmental phenotype, indicating that mature ETs must be produced at
specific sites to influence development (46). Also, the
cleavage of other substrates, vasoactive intestinal peptide (VIP) and
neurotensin, by ECE-1 cannot be excluded, because ECE-1 was recently
shown to have a relatively broad specificity (16).
ET-1 was initially believed to be a vasoconstrictor peptide, but it has
a variety of other biological activities, such as stimulation of
hormone release and regulation of central nervous system activity
(26), in nonvascular tissues. ET-1 is also a potent
mitogen in many cell types, including vascular smooth muscle cells
(11), playing a fundamental role in the development of the
cardiovascular system (23). ET-1 and ET-3 peptides are
also present in the gastrointestinal tract; they have been found in the
rat gut mucosa by immunoassay (27), by in situ
hybridization (ISH) (25) and by ET binding
(38). ET-3 plays a key role in the development of
the mouse enteric nervous system (10). Both ETB and ET-3 are necessary to prevent the premature
differentiation of crest-derived cells, which leads to aganglionosis
(45). Shortly after the discovery of ET-1, Whittle and
Esplugues (44) reported that ET could be pro-ulcerogenic
in the rat, in the pathogenesis of gastric damage and ulceration, and a
nonselective ET receptor antagonist was found to reduce injury in a rat
model of colitis (12). The pharmacological effects of
exogenous ET-1 in the intestine have been studied; the peptide seems to
be a potent intestinal secretagogue that increases colon contraction by
direct stimulation of smooth muscle (37, 38)
and transient transepithelial Cl
secretion mediated by
the enteric nerves (2, 19, 28). However, it is now becoming evident that the tissue and cellular distribution of ET receptors is species specific.
There have been few reports on the distribution of ET in the human
colon. Inagaki et al. (14) found ET-like immunoreactivity and binding sites for ET-1 in the human colon, and competition experiments suggested that there are two populations of ET receptors (9). Mutations in the ETB receptors have been
found in some families with Hirschsprung's disease or aganglionosis
(32). Kuhn et al. (22) recently showed that
ET-1 was a secretagogue for human colon mucosa in vitro. In a previous
study, we (20) showed that ECE-1 mRNA and its protein are
present in the adult human colon. We used ISH and immunohistochemistry
to demonstrate large amounts of ECE-1 in the epithelium and enteric
ganglia of the normal human colon.
However, none of these studies determined the receptor subtype,
substrate, and enzyme of the ET system in the same tissue, and most of
the studies were carried out at low resolution so that the cellular
distribution was not obtained. The present study was therefore carried
out to determine the precise cellular locations of all the components
of the ET-1 system in the human normal colon and so gain insight into
the possible role of ET-1 in gastrointestinal physiology. The
distributions of PPET-1, ECE-1, ETA, and ETB
receptor mRNAs were studied by ISH. The cells containing the mRNAs were further examined by comparing the distribution of these mRNAs with
those markers of endothelial cells, smooth muscle cells, and
macrophages. We checked for the presence of specific, functional ET
receptors by measuring ET binding in frozen sections after ISH for
receptor mRNAs.
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MATERIALS AND METHODS |
Human tissues.
Normal human colon tissue was obtained from patients undergoing
colectomy for cancer, after routine diagnostic examination of the
surgical specimen at the Institute of Pathology (Lausanne, Switzerland). Samples from 18 patients (41-84 years, 8 women and 10 men) were examined. Nine samples were from the cecum, and nine were
from the sigmoid colon. We examined only tissue that was macroscopically and microscopically normal after routine histological staining. All tissues used had a normal submucosa and had not been in
contact with cancerous tissue. Tissues were either snap-frozen in
liquid nitrogen and stored at
80°C (8 samples) or fixed in 4%
buffered paraformaldehyde for at least 24 h, processed, and embedded in paraffin (10 samples).
Preparation of radiolabeled probes.
The human PPET-1 partial cDNA (15), corresponding to
nucleotide sequence 70-630, was subcloned into pBK-CMV
(Stratagene). The recombinant plasmid was linearized by digestion with
Sac I to obtain the antisense or Kpn I to obtain
the sense RNA probe. Probes for human ECE-1 (35) were
prepared as described in Korth et al. (20). Briefly, the
recombinant plasmid ECE-1 partial cDNA corresponding to nucleotides
304-1666 was linearized by digestion with Hind III to
obtain the antisense or Xba I to obtain the sense RNA.
Probes for human ETA (13) and ETB
(33) receptors, subcloned into pcDNA3, were prepared as
described by Brand et al. (1). Briefly, ETA
and ETB cDNA were linearized by digestion with
Xho I and Kpn I, respectively, to obtain the
antisense probes. ETA and ETB cDNA were
linearized by digestion with Xba I to obtain the sense
probe. In vitro transcription and labeling with 35S-UTP
(Amersham) were carried out with T7 or SP6 RNA polymerase (Boehringer
Mannheim). Probes were precipitated with ammonium acetate and ethanol,
dried by Speed-vac centrifugation, and dissolved in TE-dithiothreitol
(DTT) (in mM: 10 Tris, 1 EDTA, and 20 DTT).
In situ hybridization.
The ISH protocol used for paraffin sections involved microwave
pretreatment to enhance the hybridization signal (36).
Paraffin-embedded sections (5 µm) were cut, and two adjacent sections
were mounted on each silane-coated slide. Deparaffinized sections were
immersed in 0.01 M citric acid (pH 6.0) and heated in a microwave oven for 12 min. The sections were then incubated with proteinase K (2 µg/ml, Boehringer Mannheim) for 20 min and dehydrated. ISH on frozen
sections used 7-µm sections fixed in paraformaldehyde-PBS and
dehydrated without microwaving. The same protocol was subsequently used
for both frozen and paraffin-embedded sections. Sections were incubated
overnight at 50°C with the respective antisense and sense riboprobes
(3-4 × 105 cpm per section). The slides were
washed with increasingly stringent solutions and treated with RNase A
(20 µg/ml, Sigma). The sections were dehydrated and placed in contact
with Biomax film (Kodak) for 1-3 days. They were then dipped in
NTB2 liquid emulsion (Kodak) and exposed for 2 wk with ECE-1 or PPET-1
probes and for 4 wk with ETA and ETB probes.
Sections were counterstained with toluidine blue. Figures 1-7 are
from ISH performed in paraffin-embedded tissue sections unless
otherwise stated.
125I-ET-1 binding.
Sections were cut using a cryostat (7 µm), thaw-mounted on
silane-coated slides, and stored overnight under vacuum at 4°C. Consecutive sections were fixed for 10 min in 4% formaldehyde-PBS and
then preincubated for 15 min in 50 mM Tris · HCl buffer, pH 7.5, containing 120 mM NaCl, 5 mM MgCl2, and 40 mg/l bacitracin. Sections were then incubated with 100 pM of 125I-labeled
ET-1 (2,125 Ci/mmol) in the previous buffer containing 1% BSA
(fraction V, protease free) and 1 mM phosphoramidon for 90 min at room
temperature. Sections were given four successive 1-min washes
in ice-cold 50 mM Tris · HCl, pH 7.4, dipped in ice-cold distilled water, air dried, and placed in contact with Biomax MR films
(Kodak). Nonspecific binding was determined in consecutive sections
incubated as described above with 1 µM unlabeled ET-1 (Bachem). The
receptor subtypes were identified by incubating consecutive sections as
described above with 1 µM BQ-123 (ETA antagonist), 10 nM
ET-3 (natural ETB agonist), or 0.2 µM sarafotoxin 6c
(S6c, selective ETB agonist). The sections were then air
dried, fixed in paraformaldehyde at 80°C for 2 h, dipped in NTB2
photographic emulsion (Kodak), exposed for 4 days, and counterstained
with toluidine blue.
Immunohistochemistry.
Paraffin-embedded sections (5 µm) were incubated with xylene (to
remove paraffin) and rehydrated in a graded ethanol series, and their
endogenous peroxidase was inactivated by incubation with 3% hydrogen
peroxide in methanol for 10 min. They were then washed in water and
incubated with monoclonal antibodies to CD31, CD68 (both from Dako),
-smooth muscle actin (
-SMA, Sigma), or MIB-1 (Dianova) according
to the manufacturers' instructions. The antiserum 473-17-A
(21) was used to stain for ECE-1. The bound anti-CD31 and
anti-
-SMA antibodies were reacted with avidin-biotin complex (Dako),
and those for CD68 and ECE-1 were reacted with peroxidase-antiperoxidase (Dako). Sections were then treated with 0.035% diaminobenzidine (Fluka) for 30 min, counterstained with hematoxylin (according to Mayer), and mounted. Control reactions without the primary antibody showed no nonspecific staining (not shown).
ISH and immunohistochemistry labeling were evaluated by different
investigators (G. Egidy, L. Juillerat-Jeanneret, and F. Pinet), each
blind to the others' assessment. Results were summarized on a
four-point scale (as shown in Table 2), although the techniques used
provided only semiquantitative evaluation.
RT-PCR analysis.
Total RNA was isolated from primary culture of human dermal fibroblast
cells (a gift from M. Benathan, CHUV, Lausanne, Switzerland) and from
normal human neonatal colon fibroblasts, CCD-18Co (ATCC CRL1459) using
the protocol of Chomczynski and Sacchi (3). cDNA was
prepared with 1 µg of total RNA and 10 pmol of oligo(dT) using
Moloney murine leukemia virus reverse transcriptase (Gibco BRL)
according to the manufacturer's instructions. PCR was performed using
3 µl of cDNA solution and 1.25 U of Taq polymerase
(Boehringer Mannheim) according to the manufacturer's instructions.
Control reactions for RT-PCR analysis were carried out with
non-reverse-transcribed RNA samples. No amplification was observed for
any of the RNA samples tested (not shown). Specific primers (10 pmol, Table 1) for ECE-1
(35), PPET-1 (15), ETA
(13), and ETB (33) receptors and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (40)
were added. The primers chosen were designed to avoid false positive reactions from genomic DNA contamination. Thirty cycles were carried out, consisting of denaturation at 94°C (30 s), annealing at 58°C (PPET-1, ETA, and ETB) or 55°C (ECE-1 and
GAPDH) (30 s), and extension at 72°C (30 s) with a final extension
step of 10 min at 72°C. Amplified products were analyzed on a 1.5%
agarose gel.
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RESULTS |
ET system mRNAs in human colon.
Tissue samples from 18 patients were analyzed, and the results shown
are representative of all the cases. Samples collected from the cecum
and distal colon were also not significantly different in their
distributions of the ET system.
All the components of the ET system were first looked for in the large
vessels of the human colon submucosa (Fig.
1). ECE-1 (Fig. 1A) and PPET-1
(Fig. 1C) mRNAs were detected in CD31-positive endothelial
cells (Fig. 1B). Smooth muscle vascular cells surrounding the vessels, which were immunostained for
-SMA (Fig. 1E),
also contained ECE-1 mRNA but at a lower concentration than in
endothelial cells. Smooth muscle cells were labeled for ETA
(Fig. 1G) and ETB mRNAs (Fig. 1I).
Receptor mRNA was not clearly detected in the endothelium of these
vessels (Fig. 1, G and I). Submucosa connective
tissue was not labeled by any probe. None of the sense probes
hybridized specifically with any structure, as shown for ECE-1 (Fig.
1D), PPET-1 (Fig. 1F), ETA (Fig.
1H), and ETB (not shown) receptors.

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Fig. 1.
Components of the endothelin (ET) system in human colon
submucosa. In situ hybridization was performed in consecutive sections
of normal human colon tissue with the antisense probes for
ET-converting enzyme (ECE-1; A), prepro-ET-1 (PPET-1;
C), ETA (G) and ETB
(I) receptors and the sense probes for ECE-1 (D),
PPET-1 (F), ETA (H), and
ETB (not shown) receptors. The photographs are presented in
dark-field illumination. Immunohistochemistry was performed to identify
endothelial cells using anti-CD-31 (B) and smooth muscle
cells with anti- -smooth muscle actin (SMA) (E)
antibodies. PPET-1 and ECE-1 mRNAs were mainly present in endothelial
cells (arrows), and ETA and ETB mRNAs were in
smooth muscle cells (small arrows). Scale bar: 50 µm.
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Myenteric ganglia also contained the ET system. PPET-1 mRNA was clearly
present in neurons (Fig. 2A),
ECE-1 mRNA (Fig. 2B) and ETB mRNA (Fig.
2F) were found in neurons and glial cells, and
ETA mRNA was present only in glial cells (Fig.
2E); all cells showed no immunostaining for CD31 (Fig.
2C) and
-SMA (Fig. 2D). The circular and
longitudinal muscle layers (Fig. 2D) were labeled more
faintly with all the antisense probes.

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Fig. 2.
ET system components in the myenteric plexus. In situ
hybridization performed in adjacent sections of myenteric plexus of
cecum colon with the antisense probe for PPET-1 (A), ECE-1
(B), and ETA (E) and ETB
(F) receptors are presented in bright-field illumination.
Immunohistochemistry was performed with anti-CD31 (arrow on endothelial
cell; C) and anti- -SMA (D), staining
longitudinal and circular smooth muscle cells. Neurons (arrows) were
labeled by PPET-1, ECE-1, and ETB probes. Glial cells
(arrowheads) were labeled with antisense probe for ECE-1 and
ETA and ETB receptors. Scale bar: 20 µm.
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ISH for PPET-1 mRNA gave a diffuse signal in the crypt epithelium of
normal intestinal mucosa (Fig.
3A). A few
crypts were strongly labeled with the PPET-1 probe (Fig.
3B), as were the epithelial cells within one crypt. ECE-1
mRNA was found in crypt epithelial cells (Fig. 3, C and
D) at a similar intensity from one crypt to another and an
apparent gradient toward the lumen in the opposite sense to the
proliferation marker MIB-1 (Fig. 3I). Unlike PPET-1 and
ECE-1, ETA mRNA was detected only in the lamina propria in
-SMA-positive cells (Fig. 3J), at the top of the crypts
(Fig. 3E) and deeper in the mucosa (Fig. 3F). In
contrast, ETB transcripts were detected only by ISH using
frozen sections with no consistent labeling in paraffin sections (Fig.
3, G and H). Although we did not intend to
quantify the ISH signals, receptor mRNA seemed much less abundant than
mRNA for PPET-1 and ECE-1, with ETB mRNA being the weakest,
considering that ETA- and ETB-hybridized sections were exposed for twice as long as PPET-1 and ECE-1 probes.

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Fig. 3.
ET system components in longitudinal and transverse
sections of human colon mucosa. In situ hybridization performed with
the antisense probes for PPET-1 (A, B), ECE-1
(C, D), ETA (E,
F), and ETB (G, H) in
serial sections are presented in dark-field illumination. Photographs
of longitudinal sections (A, C, E,
G, I) are from consecutive sections, whereas
transverse sections (B, D, F,
H, J) are from different fields of distal colon
in both cases. Immunohistochemistry with anti-MIB-1 (proliferation
marker; I) and anti- -SMA (smooth muscle cell marker;
J) antibodies demonstrates the absence of pathology and the
integrity of the regions studied. Small arrows indicate the
immunohistochemistry signal. Arrow in B indicates Peyer's
patch. PPET-1 and ECE-1 mRNA were found in the crypts; ETA
receptor mRNA was mainly in the core of the crypts, whereas
ETB was only faintly detected on paraffin sections. Scale
bar: 100 µm.
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Examination at higher magnification showed PPET-1 mRNA in tall columnar
cells along the crypts (Fig.
4A) and in CD68-positive lamina propria macrophages (Fig. 4F). Lymphocytes and some
fibroblasts were labeled with the PPET-1 probe (Fig. 4D).
Contrary to the endothelial cells in the submucosa, the microvascular
endothelium of the mucosa was rarely positive for PPET-1 mRNA. ECE-1
mRNA was abundant in the crypt epithelium and in the endothelium of stromal microvessels (Fig. 4B), which were immunostained for
CD31 (Fig. 4C). The enterocytes at the tips of crypts were
also labeled for ECE-1 (Fig. 4E), as were resident
macrophages (Fig. 4F). ECE-1 was detected
immunohistochemically in crypts, with the signal on the luminal surface
of the crypt and cuff epithelium and in vessels of the mucosa and
submucosa (not shown).

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Fig. 4.
Cellular distribution of PPET-1 and ECE-1 mRNAs in human
colon mucosa. In situ hybridization with the antisense probe for PPET-1
(A, D) and ECE-1 (B, E) is
shown in the crypt area (A-C) and luminal stroma area
(D-F) of an ascending colon sample. Crypt epithelial
cells (arrows) were strongly labeled by PPET-1 (A) and ECE-1
(B) probes. ECE-1 probe also labeled endothelial cells
(arrowheads) identified with the anti-CD31 antibody (C). In
the stroma, cells labeled with PPET-1 probe (D) were
identified as fibroblasts and macrophages (small arrows); the latter
cells were identified in consecutive sections using anti-CD68 antibody
(F). There was a moderate number of ECE-1 labeled cells in
the stroma (E). Scale bar: 20 µm.
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ETA mRNA was confined to
-SMA-positive cells (Fig.
5B) adjacent to the basal
membrane of enterocytes (Fig. 5, A and D). Some ETA-labeled cells corresponded to CD68-immunoreactive
resident macrophages (Fig. 5E). There was little
ETB mRNA in paraffin-embedded specimens; it was confined to
scattered endothelial cells, myofibroblasts, and mononuclear cells
(Fig. 5C) but was clearly detected in frozen sections (Fig.
5F). A diffuse signal over the epithelium apparent in some
sections was not considered to be specific because it also covered the
lumen of the crypts, although there was localized labeling in a few
columnar cells per section (Fig. 5, C and F).

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Fig. 5.
Cellular distribution of ETA and
ETB receptor mRNAs in human colon mucosa. In situ
hybridization with the antisense probes for ETA
(A, D) and ETB (C,
F) receptors carried out on embedded paraffin (A,
C) and frozen (D, F) sections from a
sigmoid colon sample. Immunohistochemistry was performed with
anti- -SMA (smooth muscle cell marker) (B) and anti-CD68
(panmacrophage marker) (E) antibodies in consecutive
sections to identify the cells labeled with ETA and
ETB probes. ETA mRNA was strongly present in
myofibroblasts (arrows) (A, D). The few cells
containing ETB mRNA were identified as epithelial cells
(small arrow), myofibroblasts (arrow), and endothelial cells
(arrowhead) (C, F). Scale bar: 20 µm.
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Localization of ET binding sites in the human colon by
autoradiography.
The presence and distribution of ETA and ETB receptors in
human colon were determined by autoradiography of
125I-labeled ET-1 bound to frozen samples from eight
patients. All eight cases gave identical results (Fig.
6). Panels A and E
of Fig. 6 show total 125I-ET-1 binding, and panels
D and H show the nonspecific binding that remained
after displacement with BQ-123 plus S6c (or ET-1, not shown), which was
uniformly low. 125I-ET-1 bound specifically to the colon
mucosa rather than to the submucosa, where only the vessels were
labeled (Fig. 6E). Binding was very high in the lamina
propria of the mucosa and minimal over epithelium (Fig. 6, A
and E), in accordance with the results of ISH (Figs. 3 and
5).

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Fig. 6.
Autoradiographic 125I-ET-1 binding sites in
human colon. 125I-ET-1 binding was performed in frozen
consecutive sections of ascending colon. Dark-field illuminations are
shown of transverse sections of mucosa (A-D) and
longitudinal sections including submucosa (E-H).
A and E: total binding after incubation with 100 pM 125I-ET-1. B and F:
ETA binding. Consecutive sections incubated as in
A and E in the presence of 0.2 µM S6c as
competitor. C and G: ETB binding.
Sections incubated as in A and E in the presence
of 1 µM BQ-123 as competitor. D and H:
nonspecific binding incubated as in A and E in
the presence of both 1 µM BQ-123 and 0.2 µM S6c. Scale bar: 100 µm.
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Receptor subtypes were identified by competition with an
ETA-selective antagonist (BQ-123) and two ETB
agonists, S6c (ETB analog) and ET-3 (natural ligand with
high affinity for ETB and low affinity for
ETA). Total binding was partially displaced by S6c (Fig. 6,
B and F) and by BQ-123 (Fig. 6, C and
G), indicating the presence of both receptor subtypes.
Figure 6B shows ETA binding in which the
selective ETB ligand S6c competed for a small proportion of
125I-ET-1 binding; only residual binding was left when the
competing agent was BQ-123 (Fig. 5C), confirming a higher
proportion of ETA than ETB receptors. However,
longitudinal sections showed 125I-ET-1 binding along the
crypts and at their base to be mainly displaced by S6c (Fig.
6F) as well as 10 nM ET-3, together with weak competition by
BQ-123 (Fig. 6G), indicating ETB receptor subtypes in this location. Most of the
125I-ET-1 binding in the periphery of
large-caliber vessels was displaced by BQ-123 (Fig. 6G), and
the residual signal was displaced by S6c, demonstrating that there are
ETB receptors in the media and adventitia of vessels (Fig.
6, F and G).
ET-1 system in human fibroblasts.
The unexpected presence of both ET receptors in fibroblasts was checked
by analyzing their presence in isolated human fibroblasts by RT-PCR.
Two types of human fibroblasts were used, primary cultures of dermal
fibroblasts and the intestinal subepithelial myofibroblast cell line
CDD-18Co. RT-PCR was performed on total RNA from both cell types. The
amplification primer sequences and expected sizes of the amplified
fragments are shown in Table 1. Both dermal and intestinal human
fibroblasts contained the whole ET system, with a higher concentration
in the CDD-18Co cells (Fig. 7) than in
the dermal cells (not shown).

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Fig. 7.
Components of the ET system in primary cultures of human
colon subepithelial myofibroblasts. RT-PCR analysis for PPET-1, ECE-1,
ETA and ETB receptors, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed using
the specific primers described in Table 1 and total RNA from human
colon fibroblasts, CDD-18Co, and human dermal fibroblasts (not shown).
Amplification after 30 cycles was obtained for each gene at the
expected size, 341 (PPET-1), 675 (ETA), 400 (ETB), 622 (ECE-1), and 649 (GAPDH) bp. Molecular weight
marker (MW) is X174 DNA/Hae III.
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DISCUSSION |
We have determined the distribution of the ET system in the normal
human colon. Evidence is accumulating that ETs act locally rather than
as circulating peptides, so that ECE-1 must be present in the same cell
or close to its substrates for ETs to be effective local mediators
(47). This study has examined the distribution of the ET
system in 18 human colon samples by ISH, 125I-ET-1 binding,
and immunohistochemistry. The data presented are representative of all
cases, whether the samples were collected from the cecum or distal
colon. They indicate that PPET-1, ECE-1, and both ET-1 receptors are
synthesized in situ in the normal human colon, which is consistent with
ET-1 acting locally rather than as a hormone. The relative cellular
distributions of ET components are summarized in Table
2.
Distribution of ET system components.
The morphology of the colon mucosa is heterogeneous (30),
but although the mRNA whose content varied most was PPET-1, this did
not correlate with the source of the material (cecum or sigmoid colon).
The overall distribution of PPET-1 is in good agreement with the
published ET-1 immunohistochemistry in the human colon (14), which has shown that PPET-1-containing cells at the
base of the crypts may be enterochromaffin cells, which contain Big ET-1 in the fetal human gut (9). Although there is not a
great deal of ECE-1 in the colon epithelium compared with that in the lung epithelium (20), it was reliably detected in vessels
of all calibers, including the mesenteric artery, mucosal capillaries, and lymphatic capillaries, both by ISH and immunohistochemistry. This
confirms previous results (20). There was mRNA for ECE-1 but not for ET-1 in the microvascular endothelial cells of the stromal
mucosa; this reflects the presence nearby of other ET precursors,
mainly ET-2 (5) or other substrates such as VIP or
neurotensin (9), recently demonstrated to be cleaved by ECE-1 (16).
Receptors for ET-1 were widely detected in the colon lamina propria as
reported by Inagaki et al. (14). To our knowledge this is
the first study of an ET-1 receptor subtype in human colon at the mRNA
or protein level. ETA receptors were mainly expressed in
pericryptal myofibroblasts. By contrast, ETB receptors were present heterogeneously. The discrepancy in the ETB mRNA
between the signals observed by ISH in frozen and paraffin-embedded
sections may reflect differences in mRNA stability during tissue
processing (J. D. Aubert, personal communication). The higher
signal of ETB-specific binding at the serosal side of the
mucosa compared with ETB mRNA by ISH could be accounted for
by the presence of the receptor protein in ganglion nerve fibers. We
have clearly shown the presence of ETB mRNA in neurons and
associated cells of myenteric plexus.
Possible roles of ET-1 system in normal gastrointestinal tract.
In addition to the well-characterized neural components of ET-1 action
in the rat gut (6), the role of ET-3 and ETB
in the formation of mouse enteric neurons (45) and the
presence of ET receptors in ganglia, pericryptal, vascular, immune, and some epithelial cells suggests several possible functions for this peptide.
The colon is an important site of water absorption and ion transport,
and ET-1 has been shown capable of regulating the natriohydric balance
(19, 22). We detected ET receptors in
subepithelial cells (Table 2), including myofibroblasts, which are
thought to play a role in mucosal contraction and the differentiation and proliferation of colon cells (24). It has been
proposed that myofibroblast contraction affects epithelial restitution and the propulsion of absorbed material in the lamina propria (43). ET receptors have also been detected in smooth
muscle cells of the vascular type, probably regulating vasoconstriction (4). Okabe et al. (31) showed a direct
contractile effect of ETs on circular smooth muscle cells of guinea pig
cecum. ET-1 has been shown to stimulate mitogenesis and survival of
Swiss 3T3 fibroblasts (39) and rat endothelial cells
(34) as well as the migration of endothelial cells
(48). These data suggest that ET-1 might be a colon
survival factor that acts on stromal cells. The presence of both
ETA and mainly ETB mRNAs in human fibroblasts
from dermal and colonic origin corroborates the experiments performed
by Wu et al. (45), who elegantly demonstrated the proliferative effect of ET-3 on smooth muscle cells from mouse gut
embryonic mesenchyme. In fact, the critical developmental role of
ET-3/ETB in the innervation of the distal colon seems to
involve fetal smooth muscle cells; this would prevent the premature differentiation of crest-derived precursors, favoring their migration to colonize the distal bowel (45).
The ET system may also be involved in the immune response in the colon.
Ehrenreich et al. (7) demonstrated the production of ETs
by human macrophages, suggesting a role for ETs in the microenvironment
of tissue macrophages. Because macrophages are found in close proximity
to vascular smooth muscle cells and fibroblasts that possess ET
receptors, these cells are potential targets for the actions of
macrophage-derived ETs. This hypothesis is strengthened by the findings
that bosentan, a mixed antagonist for ET receptors, reduces injury in a
rat model of colitis (12) and that in Crohn's disease
ET-1 immunoreactivity is increased in colon tissue (29).
In conclusion, the finding of the ET system in the human enteric
nervous system makes it possible to consider ET as a neuropeptide in
the human intestine (14). The presence of ETB
in neurons and glial cells enables us to hypothesize that they are also
involved in the formation of enteric neurons, as has been shown for the mouse (45). In the submucosa, the ET-1 system seems to be
a "classical" type, playing a paracrine role between endothelial cells and smooth muscle cells in the control of vascular function. The
main local source of ET-1 seems to be the epithelial cells, colocalized
with ECE-1, suggesting that ET could be a survival factor implicated in
epithelial restitution. The presence of ET receptors in myofibroblasts
suggests a role in the contractility of smooth muscle cells. The
various cellular localizations of ET components suggest that this
system is also implicated in the modulation of intestinal motility,
defense, and secretion.
 |
ACKNOWLEDGEMENTS |
We thank Drs. E. Saraga, L. Guillou, J. Benathar, and P. Chaubert
for help in selecting colon specimens and routine histological and
immunohistological examination and for very helpful discussions and M. Benathan for the generous gift of human fibroblast cultures. G. Egidy
and P. Korth received funds from La Fondation Cino et Simone del Duca
(G. Egidy) and La Fondation pour la Recherche Medicale (G. Egidy, P. Korth). This work was supported in part by the Ministère de la
Recherche et de l'Enseignement (ACC-SV9), the Ministère des
Affaires Etrangères, and the French Embassy in Switzerland and by
grants from the Swiss National Science Foundation (Grant 32.045908.95)
and the Swiss League against Cancer (Grant SKL 353-9-1996).
 |
FOOTNOTES |
Present address of P. Korth: Hoechst Roussel Vet, D-65203 Wiesbaden, Germany.
Address for reprint requests and other correspondence: F. Pinet, INSERM Unit 36, Collège de France, 3 rue d'Ulm,
75005 Paris, France (E-mail: florence.pinet{at}college-de-france.fr).
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.
Received 4 November 1999; accepted in final form 10 February 2000.
 |
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