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INSERM, U-45, Hôpital Edouard Herriot (F.M., E.N.-G., P.P., J.C.C.), 69437 Lyon; Laboratoire de Physiologie et Pharmacologie, Université de Savoie (S.P.), 73376 Le Bourget du Lac; and Unité d’Ecologie et de Physiologie du Système Digestif, Institut National de la Recherche Agronomique (F.L., P.P., J.C.C.), 78352 Jouy-en-Josas, France
Address all correspondence and requests for reprints to: Dr. Jean-Claude Cuber, INSERM, U-45, Hôpital Edouard-Herriot, Pavillon Hbis, 69437 Lyon Cedex 03, France. E-mail: cuber{at}lyon151.inserm.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the presumed important role of guanylin in the homeostasis of water and electrolytes, little is known about the factors that modulate guanylin secretion. A recent study showed that it is markedly enhanced in response to salt loading (10), suggesting that luminal factors may induce guanylin release. In addition, guanylin-producing cells are located in the vicinity of endocrine cells, immune cells, and enteric nerves, which could secrete guanylin-releasing factors. The cholinergic agonist carbachol was identified as a potent stimulant of guanylin release in a model of isolated rat colonic mucosa mounted in Ussing chambers (11). The colonic wall contains many other candidates that may influence guanylin secretion. Therefore, the first aim of this study was to investigate in detail the potential influence of various neurotransmitters, inflammatory mediators, and hormonal peptides on the secretion of guanylin immunoreactivity (G-IR). Our second aim was to test the possibility that guanylin could be released from the gut into the bloodstream under certain circumstances. The rationale behind this question relies on the observation that proguanylin-derived peptides circulate in blood (12) and are released to both sides of the isolated epithelia of rat mucosa (11).
To specifically address these two questions, we used an isolated vascularly perfused rat colon preparation (13). This model maintains the polarity of peptide secretion and allows separate sampling of the luminal and vascular effluents. Additionally, the guanylin-producing cells may be submitted to well defined luminal, neural, and blood-borne stimuli in a manner that eliminates influences potentially encountered in vivo.
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Materials and Methods |
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Surgical procedure
The procedure used to prepare an isolated vascularly perfused
rat colon was described previously (13). Briefly, male Wistar rats
(250–350 g) were anesthetized with pentobarbital sodium (50 mg/kg,
ip), and the abdomen was opened with a midline incision. Colonic loops
(10 cm in length) were prepared and rinsed twice with 10 ml isotonic
saline, flushed with 20 ml air, and ligated at both ends. A metal
cannula and a SILASTIC brand cannula (Dow Corning, Midland, MI) were
then quickly inserted into the superior mesenteric artery and portal
vein, respectively. The arterial perfusion started immediately at a
rate of 2.5 ml/min with Krebs-Henseleit buffer (2 mM
CaCl2, 6 mM KCl, 3.18 mM
NaH2PO4, 104 mM
NaCl, 1 mM MgSO4, and 41.6
mM NaHCO3, pH 7.4) containing 25%
washed bovine erythrocytes, 3% BSA, 5 mM glucose, and 1%
azonutril 25 (vol/vol). The preparation was removed and transferred to
a bath containing isotonic saline at 37 C.
Experimental protocol to study luminal release of G-IR
The experiments consisted of a 6-min equilibration period,
followed by a 30-min period of stimulation. Immediately after the
equilibration period, the loops were filled by injection with 1.0 ml
prewarmed saline (37 C). All vascularly perfused drugs and peptides
were dissolved in the Krebs-Henseleit buffer supplemented with 3% BSA.
They were delivered at a rate of 0.25 ml/min through a catheter close
to the vascular inflow. The drug concentrations represented the final
concentrations in the arterial inflow cannula. The control group
underwent a 6-min equilibration period, followed by a 30-min period
during which Krebs-Henseleit buffer supplemented with 3% BSA was
administered to the vascular inflow at a rate of 0.25 ml/min. In some
experiments, 1 µM TTX or 10 µM atropine was
infused for a 20-min equilibration period and continued for the 30-min
period of secretagogue perfusion. These experiments had a total
duration of 50 min. At the end of the experimental period, loops were
cut at both ends, and the luminal fluid content was carefully
collected, sonicated, and frozen at -20 C for subsequent determination
of G-IR by RIA.
Experimental design to study the release of G-IR in the portal
vein
The isolated, vascularly perfused rat colon was prepared as
described above and transferred to a bath containing isotonic saline at
37 C. The venous effluent was immediately collected as 2-min fractions
(5-ml blood samples) in tubes containing 250 µl 200 mM
EDTA. Plasma was rapidly separated by centrifugation and frozen at -20
C. The experiments consisted of a 20-min basal period, followed by a
30-min period of stimulation and a subsequent 10-min recovery control
period. Guanylin release was induced by vascular infusion of either
bombesin or bethanechol; the lumen was continuously perfused with
isotonic saline at a rate of 50 µl/min.
Because G-IR levels were low in the portal blood, the individual fractions were pooled to provide two consecutive samples, representing 6-min collection fractions during the basal period and five consecutive 6-min fractions collected immediately after the start of bombesin or bethanechol infusion. Nine-milliliter plasma samples of each individual pool were loaded onto C18 Sep-Pak cartridges (Waters Corp., Milford, MA) at a rate of 1 ml/min. After washing with 0.05% trifluoroacetic acid (TFA), guanylin was eluted with 1.5 ml 80% acetonitrile containing 0.05% TFA. The samples were dried and reconstituted in 1 ml RIA buffer. After Sep-Pak extraction, the recovery of guanylin added to plasma samples to final concentrations of 250 and 1000 pM was 82.3 ± 11.3% (n = 4).
RIA
Antiserum 6C was obtained from a New Zealand White rabbit after
repeated injection of rat guanylin-(101–115) conjugated to bovine
albumin through ethyl-carbodiimide condensation and used in the assay
at a final dilution of 1:7500. Rat guanylin was radioiodinated using
the lactoperoxidase technique. Briefly, 5 µg synthetic peptide were
dissolved in 10 µl 0.5 M ammonium acetate (pH 5.5), added
with 1 mCi Na125I, 5 µg lactoperoxidase, and 5
µl 0.06% hydrogen peroxide for 15 min. The radiolabeled peptide was
purified by reverse phase HPLC (C18 µBondapak)
with a linear gradient of acetonitrile (10–50%, vol/vol) in 0.1%
TFA. The flow rate was 1 ml/min. The specific activity of the
radioactive ligand was about 2000 Ci/mmol. Synthetic rat guanylin was
used as a standard. The reactivity of the antiserum was 100% for rat
guanylin, 5% for human guanylin and
Tyr0-guanylin, and <0.1% for STa and human or
rat uroguanylin. The detection limit and ID50
were 15 and 120 pM, respectively. The recovery of exogenous
guanylin was 83% over the range 20–300 fmol/tube. The guanylin
antiserum detected, in dose-dependent fashion, G-IR contained in
diluted extracts of rat intestinal mucosa (jejunum, ileum, and colon)
and a perfusate collected from isolated vascularly perfused rat colon.
The displacement curves for the G-IR contained in these samples were
parallel to the synthetic guanylin standard line.
Gel chromatography
Acetic extracts of the colonic wall, colonic perfusate, and
portal effluents after Sep-Pak treatment were permeated on a 1.5
x 100-cm Sephadex G-50 superfine column equilibrated in 0.05
M phosphate buffer (pH 7.5) containing 5% horse serum and
0.2 mg/ml sodium azide. The flow rate was 8 ml/h at 4 C. The column was
calibrated with bovine albumin, 22Na, and
synthetic rat guanylin. Fractions of 2.8 ml were collected and stored
at -20 C for subsequent RIA.
Calculations and statistics
Data in all figures are presented as the mean ±
SE. The comparison between mean values was performed using
Student’s t test. P < 0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, intraarterial infusion of
10-4 M bethanechol provoked a
dramatic release of G-IR (389 ± 66 pmol/30 min; n = 8;
P < 0.05). The first significant response was observed
with 10-5 M bethanechol
(126 ± 13 pmol; n = 5; P < 0.05
vs. control group). The bethanechol-induced guanylin release
was blocked upon intraarterial infusion of 10-5
M atropine (data not shown).
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). The
bombesin-induced release of G-IR was strongly reduced by TTX [151
± 25 pmol/30 min (n = 7); control with bombesin alone, 318
± 33 pmol/30 min (n = 7); P < 0.05], whereas
atropine (10-5 M) had no
significant effect on the bombesin-evoked release of G-IR (239 ±
62 pmol/30 min; n = 6).
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Release of G-IR by proinflammatory agents
As fluid electrolyte secretion is often observed during the
inflammatory process of the gut, we examined the effect of
proinflammatory agents on guanylin secretion. Intraarterial
administration of dimethyl-PGE2 (1.4 x
10-5 M) or interleukin-1ß
(2.5 x 10-10 M) induced a 3-
to 4-fold increase in guanylin secretion compared with that in the
control group, whereas bromolasalocid, a degranulator of connective and
mucosal mast cells, induced a modest and statistically insignificant
release of G-IR (Fig. 3).
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). In
contrast, PMA (10-7 ) or ionophore A23187
(10-6 M), alone or in combination,
did not increase G-IR secretion (data not shown).
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).
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, placement of 1 ml isotonic saline with
5 nmol synthetic guanylin in the lumen of the isolated vascularly
perfused rat colon induced a slow and gradual increase in portal G-IR.
This pattern of G-IR appearance in portal effluent is reminiscent of a
passive diffusion process and was markedly different from that
described above upon stimulation with bethanechol or bombesin, which is
evocative of an active secretory process.
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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Guanylin has been isolated as a 15-amino acid peptide from intestinal extracts (2). On the basis of the human and rat complementary DNA sequences, a 94-amino acid proguanylin of 10.4 kDa was predicted to be produced after cleavage of the signal peptide in the 115-amino acid preproguanylin (5, 15, 16, 17). This pro form and an 8.7-kDa molecular variant were detected in intestinal extracts of several species (14, 18, 19). The present study reveals the presence of the 10.4-kDa proguanylin in rat colonic extracts. An immunoreactive form that eluted at gel chromatography in a position similar to that of authentic guanylin was also revealed in colonic extracts. Besides guanylin-15, several native C-terminal forms of rat guanylin, including guanylin-16 and guanylin-14, an N-terminal truncation of guanylin-15, have been previously reported in intestinal extract and perfusate, respectively (14, 20). Our chromatographic experiments do not distinguish among the three. However, the observation that Tyr0-rat guanylin poorly cross-reacts in our RIA suggests that the N-terminal part of guanylin-15 is crucial for antibody recognition. The development of antiserum specifically directed against the N-terminal part of guanylin-14 and -16 is required to fully answer this question. In the gel permeation study, a third immunoreactive peak eluted between the position of proguanylin and guanylin-15, corresponding to a predicted size of 3–4 kDa on the basis of the elution constant. In HPLC performed by Yamaguchi et al. (14), an immunoreactive form intermediate in size between proguanylin and guanylin-15 was revealed in rat intestinal extracts.
A circulating form of guanylin has been purified from human hemofiltrate. Mass spectral analysis of this peptide showed the molecular mass to be 10.3 kDa, which corresponds to the proguanylin from position 22 to the C-terminus of the peptide predicted from the complementary DNA sequence (12). As guanylin is expressed in tissues other than the gut, including the pars tuberalis-specific cells and gonadotrophs of rat adenohypophysis (4), the exact origin of circulating guanylin cannot be stated. In a recent study performed with isolated rat colonic mucosa mounted in a Ussing chamber, proguanylin was shown to be released at the apical side, but not at the basolateral side, of the epithelium in the basal state. Low amounts of guanylin-15 were released from the same tissue on both sides of the Ussing chamber (11). In contrast, the present study shows that large amounts of guanylin were released in the lumen of the isolated, vascularly perfused rat colon, whereas minute amounts of the peptide were secreted in the portal effluent. Additionally, proguanylin was not detectable in either the luminal perfusate or the portal effluent. As the antiserum used in the present study recognized proguanylin immunoreactivity in the colonic extract, it is unlikely that luminal proguanylin does not cross-react in the assay unless the processing and/or sorting of this proform in the luminal and portal effluent are accompanied by profound conformational changes strongly reducing its binding to the antiserum. An alternative explanation is that in the model used by Martin et al. (11), the stress induced by microdissection of the colonic mucosa and mounting in the Ussing chamber produced a discharge of proguanylin from intracellular stores. The preferential accumulation of the peptide immunoreactivity at the apical side of the epithelium in this study is compatible with the extensive data indicating that peptides are preferentially released into the luminal solution in these models. Interestingly, by measuring biologically active guanylin release from rat colon explants, Li et al. (21) revealed two biologically active peaks by HPLC. The major one coeluted with synthetic guanylin-15. Taken together, these data suggest that the short forms of guanylin may be released from intestinal stores of guanylin. The possibility that proguanylin is coreleased with guanylin in the luminal and portal effluents remains an open question that requires the isolation of sufficient proguanylin to determine whether our guanylin RIA is sensitive enough to measure proguanylin in the luminal perfusate and the portal effluent. Interestingly, luminal placement of synthetic guanylin induced a slow and gradual increase in portal G-IR. About 2% of the total amount of peptide administered into the colonic lumen was recovered in the portal effluent at the end of the 30-min experimental period. This indicates that small amounts of guanylin could be transported from the lumen of the colon to the circulation. Although our chromatographic study indicated that this immunoreactive material coeluted with the synthetic peptide, additional work is required to identify the nature of this immunoreactivity.
Little is known about the factors that modulate guanylin secretion. The guanylin-producing cells are located in the vicinity of enteric nerves, and cholinergic innervation is abundant in the gut mucosa. The potential implication of this cholinergic network in the regulation of guanylin release was therefore investigated with the cholinergic muscarinic agonist bethanechol. A 6-fold increase in guanylin secretion was observed in the luminal perfusate in response to bethanechol. This was accompanied by a significant augmentation of guanylin immunoreactivity in the portal effluent. Over the 30-min stimulation period with intraarterial infusion of bethanechol, the release of guanylin immunoreactivity into the luminal effluent was about 40-fold higher than that in the portal effluent. In the isolated rat mucosa, the cholinergic agonist carbachol also produced a marked increase in guanylin release (11). Taken together, these results suggest that the release of guanylin is under vagal control.
The neuropeptide gastrin-releasing peptide (bombesin) is found in the enteric nervous network. It stimulates secretion from the endocrine I, L, and N cells (13, 22, 23). Our recent data indicate that this peptide also markedly increases the discharge of colonic mucins (24). This work presents the novel finding that bombesin is capable of eliciting a pronounced release of guanylin, thus underlying the idea that bombesin-containing nerves of the gut play a key role in essential functions of the intestinal epithelium. Interestingly, the bombesin-induced release of guanylin was significantly reduced upon TTX treatment, but was not modified by atropine. Overall, these data indicate that bombesin evokes guanylin secretion through the activation of enteric noncholinergic nerves.
VIP is a strong stimulant of intestinal water and electrolyte secretion (25), an effect thought to be mediated through activation of the cAMP-dependent pathway. The guanylin release observed upon VIP perfusion suggests that VIP-induced hydroelectrolytic secretion could involve guanylin release in combination with a direct effect of VIP on the enterocytes. The observation that forskolin stimulates guanylin release indicates that the release of this peptide may be dependent on activation of the cAMP pathway.
The inflammatory process is generally accompanied by a loss of fluid and electrolytes in the lumen, which involves the production of a variety of mediators, such as interleukins, PGs, and nitric oxide-generating compounds (26). The involvement of guanylin in inflammatory responses has not yet been described. Here, we identified interleukin-1ß, PGE2, and sodium nitroprusside as potent stimulants of guanylin release, indicating that guanylin may contribute significantly to the loss of fluid and electrolytes generally observed in the inflammatory process. Interestingly, bromolasalocid-induced mast cell degranulation did not significantly increase guanylin release. These cells release a variety of compounds, including histamine, serotonin, and mast cell protease-2. Further investigations are required to delineate the individual contributions of these mediators to guanylin release.
The surgery for preparation of isolated colon requires frequent handling of the bowel. These maneuvers perturb the movements of fluid and electrolytes across the epithelium. The measurement of fluid accumulation in the lumen, therefore, does not represent a reliable parameter to be presented in this study. However, we observed that guanylin secretion grossly correlated with fluid secretion upon stimulation with the various secretagogues.
In conclusion, the present study identified several mediators of the enteric and immune systems as potent modulators of guanylin release. This peptide may therefore play a pivotal role in control of the homeostasis of intestinal water and electrolytes in physiological and pathological situations by a paracrine/luminocrine mechanism. Additionally, the identification of guanylin immunoreactivity in the portal effluent raises the possibility that intestinal guanylin could also operate as a hormonal peptide.
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Acknowledgments |
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Footnotes |
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Received February 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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