Endocrinology Vol. 141, No. 7 2594-2599
Copyright © 2000 by The Endocrine Society
Release of Guanylin Immunoreactivity from the Isolated Vascularly Perfused Rat Colon1
F. Moro,
F. Levenez,
E. Nemoz-Gaillard,
S. Pellissier,
P. Plaisancie and
J. C. Cuber
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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The intestinal peptide guanylin regulates the electrolyte/water
transport in the intestinal epithelium. The aim of the present study
was to investigate the mechanisms that modulate its secretion in the
isolated vascularly perfused rat colon by using a specific guanylin
RIA. Intraarterial infusion of bethanechol (10-4
M) or bombesin (10-7 M) elicited a
significant 6-fold increase in the release of guanylin immunoreactivity
(G-IR) in the lumen. Bombesin-stimulated G-IR secretion was strongly
reduced by tetrodotoxin, whereas atropine had no effect. VIP
(10-7 M) induced a moderate release of G-IR,
whereas substance P, calcitonin gene-related peptide, peptide YY,
somatostatin, and neurotensin were without effect.
Dimethyl-PGE2 (1.4 x 10-5 M)
or interleukin-1ß (2.5 x 10-10 M)
induced a 3-fold increase in G-IR in the lumen, whereas the
degranulator compound bromolasalocid did not stimulate guanylin
secretion. Forskolin (10-5 M) or sodium
nitroprusside (10-4–10-3 M)
induced a significant release of G-IR. In contrast, PMA
(10-7 M) or ionophore A23187
(10-6 M) did not modify basal secretion of
G-IR. Upon stimulation of guanylin release with bombesin or
bethanechol, an increase in G-IR in the portal effluent was also
detected. The release of G-IR in the portal effluent was 40-fold lower
than that of G-IR into the luminal perfusate. Additionally, analysis
with gel chromatography revealed that the immunoreactive material
released in the lumen or in the portal effluent coeluted with the
15-amino acid peptide originally isolated from rat intestine. In
conclusion, the present data suggest that the enteric nervous system
and immune cells may modulate guanylin release from the rat colon. The
release of guanylin in the lumen and portal effluent suggests that this
peptide may exert both luminal/paracrine and hormonal effects.
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Introduction
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INFANT AND TRAVELER’S diarrheas are often
caused by Escherichia coli heat-stable enterotoxins (STa)
that bind to and activate the guanylyl cyclase receptor located on the
apical membrane of the gut epithelium. The consequence of guanylyl
cyclase receptor activation is an increase in intracellular cGMP levels
and a subsequent phosphorylation of the cystic fibrosis transmembrane
conductance regulator, resulting in net water and chloride secretion
(for review, see Ref. 1). Using a bioassay to identify activators of
the STa receptor present in the intestinal mucosa, Currie et
al. isolated the peptide guanylin, which is predominantly
expressed within the colonic mucosa (2). Together with the structurally
related uroguanylin, which is primarily expressed in the mucosa of the
small intestine, guanylin is thought to form a local system that
modulates intestinal water and salt transport (1). Interestingly, these
peptides were also detected in other tissues, including the lung and
the hypothalamo-hypophyseal system (3, 4), but their physiological
role is not clearly established. The precise cellular source of
guanylin (and uroguanylin as well) is still a matter of controversy.
Immunohistochemical and in situ hybridization studies have
localized guanylin in several cell types, including Paneth cells,
enterocytes, goblet cells, and enterochromaffin cells (5, 6, 7, 8, 9).
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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Materials
BSA was purchased from Biovalori (Cassen, France). Azonutril 25,
a mixture of amino acids, was obtained from Laboratoires Roger Bellon
(Neuilly-sur-Seine, France). Bromolasalocid was provided by
Roche Discovery Welwyn (Welwyn Garden City, UK). EDTA was
supplied by Merck & Co., Inc. (Nogent-sur-Marne, France).
125Iodine was supplied as sodium iodine from
Amersham Pharmacia Biotech (Les Ulis, France). Human and
rat guanylins were purchased from Bachem (Bubendorf,
Switzerland) and Saxon Biochemicals GmbH (Bachem,
Hanover, Germany). Human uroguanylin was obtained from Peninsula Laboratories, Inc. (Merseyside, UK). Rat uroguanylin and
Tyr0-guanylin were gifts from Dr. Nakazato
(Miyazaki, Japan). All other reagents were obtained from
Sigma (St. Louis, MO).
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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Release of G-IR in the colonic lumen by neurotransmitters
The guanylin-producing cells are in contact with intramural
nerves. We therefore studied the effects of various neurotransmitters
on guanylin release. The mean release of G-IR in the control group was
66 ± 14 pmol/30 min (n = 10). As shown in Fig. 1
, 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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Figure 1. Effects of intraarterial infusion of bethanechol
on G-IR release from the isolated vascularly perfused rat colon. Values
are the mean ± SE. *, P < 0.05
vs. control group.
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Vascular infusion of bombesin induced a dose-dependent increase in the
release of luminal G-IR over the range
10-8–10-7 M.
Upon stimulation with 10-7 M
bombesin, the secretion of G-IR was about 5-fold higher than that in
the control group (Fig. 2). 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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Figure 2. Effects of increasing doses of bombesin on
guanylin secretion in the isolated vascularly perfused rat colon. Data
are the mean ± SE. *, P < 0.05
vs. control group.
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Vascular infusion of 10-7 M VIP
elicited a significant increase in G-IR in the colonic lumen (167
± 33 pmol/30 min; n = 7; P < 0.05 vs.
control group). In contrast, VIP (10-8
M), calcitonin gene-related peptide
(5 x 10-9 M),
substance P (10-7 M),
somatostatin (10-7 M),
neurotensin (10-11 M), or
PYY (10-10 M) did not
modify the basal secretion rate of G-IR (data not shown).
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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Figure 3. Intraarterial administration of bromolasalocid,
dimethyl-PGE2 (dmPGE2), or interleukin-1ß
stimulates the release of G-IR release in the lumen of the isolated
vascularly perfused rat colon. Values are the mean ±
SE. *, P < 0.05 vs.
control group.
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Postreceptor activation of the release of luminal guanylin
Intraarterial infusion of 10-5
M forskolin or sodium nitroprusside
(10-5–10-4
M) induced a 3-fold increase in luminal G-IR compared with
that in the control group (Fig. 4). 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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Figure 4. Effects of intraarterial infusion of the
postreceptor-activating agents forskolin and sodium nitroprusside on
guanylin secretion in the colonic lumen. Data are the mean ±
SE. *, P < 0.05 vs.
control group.
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Release of G-IR in the portal effluent
To study the possibility that guanylin could be release in the
circulation, we measured G-IR levels in the portal effluent. The mean
basal level of G-IR in 18 preparations was close to the detection limit
of the assay and amounted to 135 ± 12 fmol/6 min. Bombesin
(10-7 M) or bethanechol
(10-4 M) induced a sharp rise in
portal G-IR levels within 6 min (10-fold increase over basal value).
Portal G-IR levels then tended to decline until the end of stimulation
(Fig. 5).
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Figure 5. Effects of intraarterial administration of
10-4 M bethanechol (n = 6) or
10-7 M bombesin (n = 6) on the release of
guanylin in the portal effluent of the isolated vascularly perfused rat
colon (femtomoles per 6 min). Results are the mean ±
SE.
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As the levels of G-IR were considerably lower in the portal effluent
than in the luminal perfusate, the possibility that these small amounts
of guanylin could result from transepithelial, passive diffusion of the
apically released peptide was evaluated. As shown in Fig. 6, 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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Figure 6. G-IR in the portal effluent of the isolated
vascularly perfused rat colon in response to a luminal load of 5 nmol
synthetic guanylin. Results are expressed as picomoles per 2 min
(mean ± SE of six experiments).
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Nature of the immunoreactive material released in the lumen and the
portal effluent
Gel chromatography revealed three immunoreactive peaks in rat
colonic extracts. One immunoreactive form eluted in the position of
authentic guanylin (Kav = 0.76). Another eluted
in a volume similar to that of the previously described 10-kDa
proguanylin (14). A third immunoreactive peak was intermediate in size
(Kav = 0.60). In contrast, a single peak
coeluting with the synthetic peptide was revealed in the eluate of
bethanechol-stimulated guanylin release in both the lumen and the
portal effluent (Fig. 7).
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Figure 7. Gel chromatography pattern of the G-IR material in
extracts of the colon (A), in the luminal perfusate upon intraarterial
infusion of bethanechol, (B) and in portal effluents after Sep-Pak
concentration (see Materials and Methods) of
bethanechol-stimulated rat perfused colon (C).
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Discussion
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Studies providing direct evidence of guanylin release from the
colonic epithelium by neurotransmitters and neuropeptides are scarce.
This work demonstrates that several transmitters of the enteric nervous
system, as well as compounds involved in the inflammatory process are
potent stimulants of guanylin release. In addition, our data support
the idea that guanylin secretion occurs into both the luminal space and
the capillary network of the colon.
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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We are grateful to Kurt Schillinger for correction of the
English.
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Footnotes
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1 This work was supported by La Fondation de la Recherche
Médicale (to F.M.). 
Received February 15, 2000.
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Abstract
Introduction
