1 Department of Internal Medicine II and 2 Department of Pharmacology and Toxicology, Technical University of Munich, 81675 Munich, Germany
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ABSTRACT |
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In functional experiments, the nitric oxide (NO)
donor
N-morpholino-N-nitroso-aminoacetonitrile
or the cGMP analog 8-(4-chlorophenylthio)-cGMP caused a
concentration-dependent, tetrodotoxin-resistant relaxation of
precontracted strips from rat small intestine. The inhibitory effect of
both substances was completely blocked at lower concentrations and was
significantly attenuated at higher concentrations by the selective
cGMP-dependent protein kinase (cGK) antagonist KT-5823 (1 µM). cGK-I
was identified by immunohistochemistry in circular and longitudinal
muscle, lamina muscularis mucosae, and smooth muscle cells of the villi
and in fibroblast-like cells of the small intestine. Additionally,
there was staining of a subpopulation of myenteric and submucous plexus
neurons. Double staining for neuronal NO synthase (nNOS) and cGK-I
demonstrated a colocalization of these two enzymes. Western blot
analysis of smooth muscle preparations and isolated nerve terminals
demonstrated that these structures predominantly contain the cGK-I
isoenzyme, whereas the cGK-I
expression is about threefold less. The
isoform cGK-II was entirely confined to mucosal epithelial cells. These
results show that cGK-I is expressed in different muscular structures
of the small intestine and participates in the NO-induced relaxation of
gastrointestinal smooth muscle. The presence of cGK-I in NOS-positive
enteric neurons further suggests a possible neuronal action site.
gastrointestinal tract; nitric oxide; signal transduction; KT-5823; rat; guinea pig; immunohistochemistry; cGMP-dependent protein kinase
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INTRODUCTION |
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RELAXATION OF GASTROINTESTINAL smooth muscle is mainly
mediated by nonadrenergic, noncholinergic inhibitory mechanisms. ATP, vasoactive intestinal polypeptide (VIP), and nitric oxide (NO) have
been suggested as putative mediators (7, 10, 18). In various systems,
NO acts on smooth muscle cells to induce hyperpolarization and
relaxation (13, 38, 41). The mechanisms through which these inhibitory
cellular effects are mediated include either direct effects of NO on
cellular structures, such as stimulation of ion channels (6) or
activation of protein kinase C (8), or effects mediated via stimulation
of soluble guanylyl cyclase (3, 24, 45). NO binds to the heme ring of
soluble guanylyl cyclase, causing activation of this enzyme (39) and
leading to the synthesis and intracellular rise of cGMP.
Analogs of cGMP can mimic the inhibitory effects of NO on isolated
smooth muscle strips (26) and cause transmitter release in isolated
nerve terminals (NT) (2), similar to NO. cGMP may act on ion channels either directly or via modulation of phosphodiesterases or
cGMP-dependent protein kinases (cGK) (39, 44). Three different isoforms
of cGK have been identified so far (17). The isoforms I and I
are
present in vascular smooth muscle, lung, cerebellum, platelets, and
heart (17, 27). The isoform II is expressed in secretory epithelium
(22, 32) as well as in various regions of the brain (31, 42).
The purpose of the present study was, first, to demonstrate in functional studies that cGK is involved in the NO-induced relaxation of small intestinal smooth muscle; second, to investigate which isoforms of cGK are present in various layers and different structures of the small intestine; and third, to localize these forms by immunohistochemistry.
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MATERIALS AND METHODS |
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Functional studies. The experimental model and protocol for investigation of inhibitory response used has been previously described (1). In brief, male Wistar rats (400-500 g) were killed by intraperitoneal injection of phenobarbital sodium (100 mg/kg). The terminal ileum was immediately removed and kept in oxygenated Krebs-Ringer bicarbonate solution (KRS). Six segments of full-thickness strips were prepared from the terminal ileum (length 1.5-2 cm) and fixed to a hook on a holder. Holders with tissue were placed in a jacketed organ bath containing 3 ml of KRS [(in mM) 115.5 NaCl, 1.16 MgSO4, 1.16 NaH2PO4, 11.1 glucose, 24.9 NaHCO3, 2.5 CaCl2, and 4.16 KCl] gassed with 95% O2-5% CO2 and maintained at 37°C by circulating water through the jackets. The free end of one segment was connected with a thread to an isometric force transducer (Swegma force displacement transducer SG 4-500); 1 g of tension was applied to the muscle, and the preparation was allowed to equilibrate for at least 30 min. Changes in the tension were amplified by Hellige couplers and recorded on a Rikadenki chart recorder. The segment was precontracted by addition of the muscarinic receptor agonist carbachol (CCH, 1 µM).
The effect of the NO donor N-morpholino-N-nitroso-aminoacetonitrile (SIN-1; 10 µM-1 mM) or the cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP; 10 µM-1 mM) was tested when the CCH-induced response had reached a plateau, which was usually 30 s after addition of CCH. After each concentration of SIN-1 and 8-pCPT-cGMP, the strips were washed and stimulated again with CCH (1 µM). KT-5823 (1 µM) and KT-5720 (1 µM) were applied 10 min before application of CCH. These concentrations of both blockers have been used in other systems and have been shown to be effective and selective (33, 34). In additional experiments, the inhibitory effect of the guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) against the SIN-1-induced relaxation was tested. After a concentration response curve, ODQ (1 µM) (16) was added 15 min before the concentration response curve of the SIN-1-induced relaxation was repeated. In separate experiments, the inhibitory effect of SIN-1 or 8-pCPT-cGMP on CCH-induced contraction was tested for stability by repeating this protocol several times. The inhibitory action of both agents remained stable for >3 h with this protocol.Isolation of proteins of LM and CM layers.
Protein isolation was performed from muscularis externa of rat and
guinea pig small intestine [longitudinal muscle (LM)/circular muscle (CM), whole mount preparation]. The muscle layers were further subdivided in LM with attached myenteric plexus (LM/MP) and CM
layer (after removal of mucosa). The tissue was minced into small
pieces with scissors and homogenized with a fourfold volume of
buffer
A [(in mM) 12.2 K2HPO4,
7.8 KH2PO4,
2 EDTA, and 2 benzamidine, pH 7, 4°C] in a Polytron PT20
homogenizer at ~1,500 rpm for 25 s (5 × 5 s, cooled on ice in
between homogenizations). The homogenate was centrifuged at 25,000 g for 30 min, and the pellet was
rehomogenized with buffer
B
(buffer
A + 1% Triton X-100). After a second
centrifugation step, the supernatants were combined and stored at
20°C.
Isolation of smooth muscle cells and NT. Smooth muscle cells were isolated from the LM/MP of guinea pig small intestine as described previously (4). Segments of ~5 cm were slipped over a 3-ml glass pipette, the mesenteric attachment was gently scraped off with forceps, and the LM/MP layer was carefully pulled off the CM layer. The tissue was cut into small pieces in a medium containing (in mM) 120 NaCl, 2.6 KH2PO4, 4.0 KCl, 2.0 CaCl2, 0.6 MgCl2, 25 HEPES, and 14 glucose and 2% essential amino acid mixture, pH 7.4. After two periods of incubation (20 min at 31°C) in a medium containing 0.05% collagenase (Worthington CLS type II) and 0.01 soybean trypsin inhibitor, the tissue was filtered through a 224-µm mesh, washed several times with collagenase-free medium, and incubated for spontaneous cell dispersion in collagenase-free medium gently bubbled with 95% O2-5% CO2. The dispersed cells were harvested by filtration through a 500-µm mesh.
Rat enteric NT were isolated by differential centrifugation as described previously (29) from the LM/MP layer isolated from the serosal side as described above. The tissue was minced with scissors and homogenized with a Polytron PT20 homogenizer at ~1,500 rpm for 15 s. The tissue homogenate was centrifuged at 800 g for 10 min, and the supernatant was collected (postnuclear supernatant) and then subjected to various differential centrifugation steps (3,500 g for 10 min, 120,000 g for 60 min, and 10,000 g for 10 min). The resulting pellet of the last centrifugation step (P2) was the enriched NT. Protein concentrations were determined with a commercial kit using bovine plasmaWestern blot analysis.
For Western blot analysis, the cells and the isolated NT were treated
with ultrasound (4 strokes of 15 s, cooled on ice between treatments).
Isolated cells were transferred to sample
buffer 1 (62.5 mM
Tris · HCl, 2% SDS, 10% sucrose, 10%
-mercaptoethanol, and 0.02% bromphenol blue, pH 6.8), and isolated
NT as well as protein extracts were transferred to sample
buffer
2 (62.5 mM Tris · HCl, 2% SDS, 10% glycerol, 10%
-mercaptoethanol, and 0.02% bromphenol blue, pH 6.8). The proteins
were separated by SDS-PAGE on 7.5% slab gels using a 3% stacking gel
in a Bio-Rad minigel apparatus according to the procedure of Laemmli
(30). Protein bands were visualized by Coomassie blue staining or
blotted onto a nitrocellulose membrane (Bio-Rad) using Tris-glycine-SDS
buffer (50 mM Tris, 380 mM glycine, 0.05% SDS, and 20% methanol).
After blocking the membrane with 5% dry milk, we probed blots with
selective antibodies for cGK-I
, cGK-I
, and cGK-II. The cGK-I
and -I
antibodies were raised against the
NH2-terminal domain of the corresponding enzyme (27). The peptide antibody for cGK-II was raised
against the COOH-terminal domain of the kinase (P. Ruth, unpublished
results). The detection was performed either with alkaline
phosphatase-labeled second antibody (Bio-Rad) or horseradish peroxidase-labeled second antibody and enhanced chemiluminescence (ECL
system; Amersham, Braunschweig, Germany).
RNA isolation and PCR amplification.
RNA from rat small intestine was isolated from the LM/MP layer and from
the mucosa, based on the method of Chirgwin et al. (12) and using a
commercially available RNA isolation kit (Stratagene, Heidelberg,
Germany). We placed 0.5 g of tissue in 5 ml of denaturation solution
and subsequently homogenized it with a Polytron PT20 homogenizer at
~1,500 rpm for 20 s (4 × 5 s) on ice. Total yield from 0.5 g of
starting tissue was 60 µg of RNA from the LM/MP layer and 1,500 µg
from the mucosa. One microgram of RNA from each tissue was subjected to
DNase treatment (15 min at room temperature, 1 U DNase I; GIBCO BRL,
Eggenstein, Germany) and transcribed in complementary DNA by using
avian myeloblastosis virus RT and
oligo-p(dT)15 primer (Boehringer
Mannheim, Mannheim, Germany) at 25°C over 10 min and 42°C over
60 min. The synthesized cDNA was stored at 20°C until PCR
experiments were performed.
Immunohistochemistry. Segments of the proximal rat small intestine were removed, opened along the mesenteric border, and fixed in 4% freshly depolymerized paraformaldehyde in 0.1 M Tris-buffered saline (TBS, pH 7.4) at 4°C overnight. After several washes in TBS, the tissue was placed in TBS supplemented with 30% sucrose at 4°C overnight. Frozen sections 10 - 30 µm thick were cut on a cryostat and melted onto glass slides. Activity of endogenous peroxidase was blocked with 0.3% methanol and 3% hydrogen peroxide in TBS. After three rinses in TBS, 10% normal goat serum (NGS) and 1% L-lysine were applied for 90 min. Afterwards, sections were incubated with primary antisera specific for cGK-I or -II (36) diluted 1:1,000 in TBS supplemented with 5% NGS overnight in a humid chamber at room temperature. After the sections were washed, they were incubated with biotinylated goat anti-rabbit serum (Vectastain; Vector, Burlingame, CA) followed by horseradish peroxidase-labeled avidin-biotin complex (Vectastain) diluted in carbonate buffer (11) to prevent nonspecific staining of mast cells. After several additional rinses, peroxidase was revealed by a 3,3'-diaminobenzidine substrate kit (Vectastain). Negative controls were performed without primary antiserum.
For double staining with neuronal NO synthase (nNOS) and cGK-I antibodies, the sections were blocked with 10% NGS and 1% L-lysine and incubated with anti-nNOS (Transduction Laboratories, Lexington, KY) diluted 1:200 in TBS supplemented with 5% NGS overnight at room temperature in a humid chamber. After the sections were washed, positive reactions were visualized by goat anti-mouse IgG antibody coupled with FITC immunofluorescence (Vectastain). This incubation was followed by photography and subsequently by staining for cGK-I as described.Drugs. CCH was obtained from Boehringer Mannheim, SIN-1 was obtained from Cassella-Riedel (Frankfurt, Germany), 8-pCPT-cGMP was obtained from Biolog Life Science Institute (Bremen, Germany), ODQ was obtained from Biotrend (Cologne, Germany), KT-5823 and KT-5720 were from Calbiochem (Bad Soden, Germany), essential amino acid mixture was from GIBCO BRL, collagenase CLS type II was from Worthington Biochemicals (Freehold, NJ), and soybean trypsin inhibitor, EDTA, benzamidine, and Triton X-100 were from Sigma (Munich, Germany). All other chemicals were purchased from Merck (Darmstadt, Germany).
Statistics. For analysis of data, the contraction level of CCH before addition of SIN-1 or 8-pCPT-cGMP was determined. Relaxation induced by SIN-1 or 8-pCPT-cGMP in the presence or absence of the respective blockers (KT-5823, KT-5720, and ODQ) was expressed relative to the contraction level before the addition of SIN-1 or 8-pCPT-cGMP. Data of the functional experiments are given as means ± SE; n indicates the number of independent observations in different muscle strips. Each protocol was repeated with ileal segments of at least three different animal preparations. ANOVA for repeated measures with a subsequent post hoc test (Newman-Keuls) was used for comparison of the functional data, and values of P < 0.05 were considered significant.
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RESULTS |
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Influence of cGK on smooth muscle relaxation. After prestimulation of LM strips from rat small intestine with the muscarinic agonist CCH (1 µM), addition of the membrane-permeable cGMP analog 8-pCPT-cGMP (10 µM-1 mM) caused a concentration-dependent relaxation, which reached 65.0 ± 2.8% at 1 mM (n = 8). In the presence of the specific cGK antagonist KT-5823 (1 µM), the inhibitory effect of the cGMP analog 8-pCPT-cGMP was significantly attenuated (P < 0.05). At the highest concentration tested, the inhibitory effect of 8-pCPT-cGMP was further reduced by a combination of KT-5823 (1 µM) and the selective cAMP-dependent protein kinase (cAK) blocker KT-5720 (1 µM) (Fig. 1A).
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Identification of specific isoforms of cGK by Western blot analysis
and RT-PCR.
For demonstration of the presence of different isoforms of cGK, protein
extracts of rat and guinea pig small intestine muscle and isolated
myenteric NT of rat small intestine were analyzed with specific
antibodies. The isoform I could be detected in all muscle
preparations of both species studied. It was present in the
full-thickness preparations (LM/CM) and in preparations of the LM/MP
and CM layers (Fig. 2,
A and
B). cGK-I
was also detected in isolated longitudinal smooth muscle cells of the guinea pig
(Fig. 2A). Additionally, the isoform
I
was shown to occur in isolated NT of rat small intestine (Fig.
2A). Probing of protein extracts of
LM and CM layers with an antibody directed against cGK-I
also
resulted in a positive signal (Fig.
2C), but this signal was about
threefold less intense compared with the isoform I
[cGK-I
:
~44 ng/mg protein in LM/CM, 62 ng/mg protein in LM/MP, and below the
detection limit (<35 ng/mg protein) in CM; cGK-I
: ~134 ng/mg
protein in LM/CM, 196 ng/mg protein in LM/MP, and 112 ng/mg protein in
CM (Fig. 2, B and
C; see MATERIALS AND
METHODS for calculation)]. The molecular mass of
the detected isoforms I
and I
corresponded, at ~75 kDa, to the
pure recombinant cGK-I
and -I
, respectively (Fig.
2).
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Immunohistochemical localization of cGK-I and -II in rat small intestine. The localization of the two enzymes was further investigated immunohistochemically with antibodies specific for cGK-I or -II in segments of rat small intestine. Additionally, to detect colocalization of the two enzymes, the segments were probed with an antibody for nNOS before incubation with the antibody for cGK-I. The antibody specific for cGK-I showed positive staining with different types of smooth muscle cells within the entire wall of rat small intestine, e.g., smooth muscle cells in the CM and LM layer (Fig. 4A), in the lamina muscularis mucosae (Fig. 4, A and C), and running with the longitudinal axis of the villi (Fig. 4B). There was no staining of mucosal epithelial or submucosal cells (Fig. 4, A and C). However, two additional types of cell which showed a positive reaction with the cGK-I antibody were subepithelial and pericryptal myofibroblast-like cells (Fig. 4, D and E) and subepithelial stellate, fibroblast-like cells (Fig. 4B). In the submucosal plexus and MP, there was positive staining for cGK-I of ~15% of the neurons present (Fig. 4, A and C). When double staining for nNOS and cGK-I was performed, neurons as well as nerve processes showed positive staining for both enzymes (Fig. 5, A and B), demonstrating a colocalization of nNOS and cGK-I. The coexpression of both enzymes, nNOS and cGK-I, might suggest a neuromodulatory action of NO via cGK-I. These data demonstrate for the first time that cGK-I is expressed not only in intestinal smooth muscle but also in fibroblast-like cells and in enteric neurons.
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DISCUSSION |
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We demonstrated that both the cGMP analog 8-pCPT-cGMP and the NO donor SIN-1 produced a concentration-dependent relaxation of rat ileal longitudinal smooth muscle. At lower concentrations of SIN-1 and 8-pCPT-cGMP, this inhibitory effect was abolished by the specific cGK blocker KT-5823 at a concentration that was demonstrated in various smooth muscle cell preparations, including gastrointestinal smooth muscle, to interact specifically with cGK (34, 35, 47). This indicates that the inhibitory effects of the NO donor SIN-1 and the cGMP analog were mediated by an activation of the cGK. This is also consistent with the finding that the SIN-1-induced inhibitory effect at low concentration is dependent on an activation of the guanylyl cyclase. The inhibitory effect of SIN and 8-pCPT-cGMP at high concentrations was significantly attenuated by KT-5823 and further reduced by a combination of KT-5823 and the specific cAK blocker KT-5720 at a concentration that was shown to selectively interact with cAK in smooth muscle cells (33).
It has been demonstrated that NO- and cGMP-dependent mechanisms can release VIP, another inhibitory neurotransmitter (2, 19). This NO-induced release of VIP also occurred in isolated enteric synaptosomes, suggesting a presynaptic site of actions that is resistant to TTX (2). Thus this release of VIP and subsequent activation of cAK could contribute to the inhibition observed. However, because this release was abolished by KT-5823 (19), it is unlikely that this effect contributes to the residual relaxation in the presence of the cGK blocker. Our results suggest that high concentrations of both SIN-1 and 8-pCPT-cGMP could lead to a cross-activation of the cAK, as postulated in other systems (23), which is partially responsible for the inhibitory effect observed at these concentrations. Whether these high concentrations reflect physiological conditions remains speculative and cannot be answered from our study.
In reference to immunohistochemistry, positive staining for cGK-I was
detected in the contractile elements within rat small intestine,
supporting the important role in the relaxation of these cells as shown
by the functional experiments. cGK-I was detected in the CM and LM
layers of the muscularis externa, in the lamina muscularis mucosae,
and, according to Markert et al. (32), in smooth muscle cells in the
lamina propria mucosae. This latter type of cells is thought to act as
a motility mechanism for the villi ("villi pump") that might be
of importance for mixing the chyme and intensifying contact
with the digestible and resorbable substances. Western blot analysis of
proteins isolated from whole thickness muscle layer preparations or
from LM and CM layers of guinea pig and rat small intestine probed with
selective antibodies for cGK-I and -I
further corroborates this
distribution of cGK-I. A semiquantitative Western blot analysis of
cGK-I
and -I
showed that the signal of cGK-I
was about
threefold less compared with cGK-I
, suggesting a predominant role of
cGK-I
in gastrointestinal smooth muscle.
Additionally, cGK-I was localized with immunohistochemistry in fibroblast-like cells, which form an interconnected contractile network in the lamina propria mucosae (21, 25). We could demonstrate different types of these cells, which have been characterized as subepithelial stellate, fibroblast-like cells, pericryptal myofibroblast-like cells, and subepithelial myofibroblast-like cells (5, 28). The exact function of these cells remains speculative, but the positive staining with the cGK-I antibody might indicate that these cells could also play a role in motility of the villi (14).
There was also a positive staining with the cGK-I antibody in a subset
of ~15% of the neurons in the submucous plexus and MP, which was
further confirmed by Western blot analysis of proteins isolated from NT
of rat small intestine with the cGK-I antibody. Immunohistochemically, cGK-I was colocalized with the neuronal isoform
of NO synthase. On the other hand, NO-synthesizing neurons identified
by immunostaining showed a colocalization with VIP in the rat small
intestine (15). There is evidence for an NO-dependent VIP release in
the rat and guinea pig small intestine that can be mimicked by cGMP
analogs (2) and is mediated by activation of cGK, because it was
abolished by KT-5823 (19). Thus our structural and Western blot data
are in good agreement with these functional findings.
However, in species like the dog and guinea pig, cGMP immunoreactivity and NOS or NADPH diaphorase immunoreactivity are not colocalized within the same neuron, whereas cGMP and VIP immunoreactivity are colocalized (40, 46). This suggests that NO might act as a neuromodulator not only within the same neuron but also between different neurons. Additionally, other sources of NO such as interstitial cells of Cajal and smooth muscle cells have to be considered (9, 20, 35).
cGK-II is suggested to be involved in the regulation of intestinal ion transport and fluid secretion (43). In accordance with this function and with other groups (22, 32), the immunohistochemical experiments showed that the enzyme was entirely confined to mucosal cells, with an increase in staining intensity from the crypts toward the top of the villi. There was no evidence for the presence of cGK-II in muscular elements of the gut wall. Because cGK-II is expressed in high concentrations in the CNS (31, 42), we were especially interested as to whether this isoform would also be present in the enteric nervous system. However, there was no evidence for cGK-II in enteric neurons either by immunohistochemistry, Western blot analysis of proteins from enriched enteric NT, or RT-PCR of RNA isolated from the LM/MP layer containing the MP. Thus, in contrast to the CNS, there was no evidence for the presence of cGK-II in the enteric nervous system.
In conclusion, in the present study, we demonstrated an important
functional role of the activation of cGK for intestinal smooth muscle
relaxation in response to the NO donor SIN-1 and the cGMP analog
8-pCPT-cGMP. We characterized the presence and distribution of the cGK
isoforms in the small intestine. cGK-I was present in all contractile
elements, and the isoenzyme cGK-I was the predominant form. Because
of its neuronal localization, this cGK isoform could have an additional
neuromodulatory role. In contrast, the cGK-II isoform was confined to
mucosal epithelium, and, in contrast to the brain, there was no
evidence for its presence in neuronal elements.
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ACKNOWLEDGEMENTS |
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We thank I. Eiglmeier and S. Kerling for expert technical assistance concerning the immunohistochemical experiments.
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FOOTNOTES |
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This study was supported by Deutsche Forschungsgesellschaft Sonderforschungsbereich 391 C5.
Preliminary results of this study were presented at the 1997 annual meeting of the American Gastroenterological Association (Washington, DC).
Address for reprint requests: H.-D. Allescher, Dept. of Internal Medicine II, Technical Univ. of Munich, Ismaningerstr. 22, 81675 Munich, Germany.
Received 19 May 1997; accepted in final form 29 May 1998.
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