Vol. 277, Issue 5, G960-G966, November 1999
Marked increase of guanylin secretion in response to salt
loading in the rat small intestine
Toshihiro
Kita,
Kazuo
Kitamura,
Junichiro
Sakata, and
Tanenao
Eto
First Department of Internal Medicine, Miyazaki Medical College,
Miyazaki 889-1692, Japan
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ABSTRACT |
Guanylin and uroguanylin are intestinal peptides
that stimulate guanylate cyclase C and cause chloride secretion. These
peptides show topological instability due to two disulfide bonds. The
disulfide bonds were reduced and
S-carboxymethylated to cleave the
bonds and obtain stable and sole derivatives. We established a new and reliable RIA system for the stable derivatives from both peptides. With
the use of this system, the response of the peptides to salt loading of
the rat small intestine was evaluated. The lumen of the small
intestines of Sprague-Dawley rats was perfused in vivo with
Krebs-Ringer solution containing different concentrations of salt or
mannitol. Mature guanylin, proguanylin, and mature uroguanylin were
found in the perfusate in the basal state. The highest salt loading
(200 mM NaCl for 20 min) increased the guanylin secretion about
threefold (1.9 ± 0.2 vs. 5.4 ± 0.5 pmol/min), with the effect
lasting for 60 min. The uroguanylin secretion was less affected.
Hyperosmolar mannitol also caused a significant but smaller increase of
guanylin secretion. Increased guanylin could lead to increased salt and
water secretion of the intestine; thus members of the guanylin family
have potential roles in the regulation of water and salt metabolism in
the small intestine.
uroguanylin; radioimmunoassay; chloride secretion; reductive
S-carboxymethylation
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INTRODUCTION |
GUANYLATE CYCLASE C (GCC) is present predominantly
throughout the intestine and is known to be a receptor for heat-stable enterotoxins (4, 9). When they bind to GCC, heat-stable enterotoxins
cause a marked increase in intracellular cGMP and activate chloride
secretion, subsequently resulting in acute secretory diarrhea (5, 10).
The 15- to 16-amino acid peptides guanylin and uroguanylin were
recently identified from rat intestinal extract and human urine,
respectively. These peptides are believed to be endogenous ligands for
GCC (2, 15). Both guanylin and uroguanylin mRNA are expressed
throughout the intestine, and observations from immunohistochemical and
in situ hybridization studies strongly suggest that guanylin and
uroguanylin are secreted into the intestinal lumen (18, 20, 26, 29,
30). If guanylin and uroguanylin are secreted into the intestinal
lumen, they may act to stimulate GCC and participate in the regulation
of chloride secretion. However, no information is available regarding
the secretion of guanylin and uroguanylin into the lumen of the small
intestine. In this study, we clarified the basal secretion rate of
these two peptides and the molecular forms of the secreted peptides.
Evidence has accumulated that the guanylin family of peptides is
involved in salt and water homeostasis. The plasma or urine concentration of guanylin or uroguanylin was found to be elevated in
patients with renal and heart diseases (3, 14, 25). The uroguanylin
concentration in human urine was increased after a high-salt diet (13).
A low-salt intake downregulates the guanylin signaling pathway in the
rat distal colon (19). Guanylin and uroguanylin can cause relatively
weak but significant natriuresis in rat and mouse (6, 11). In addition,
guanylin may contribute to sodium handling in the intestinal tract.
Increased chloride secretion to the lumen of the intestine induced by
the guanylin family simultaneously increases the flow of water and
sodium to the lumen to preserve the ion balance. Increased guanylin
secretion may thus increase sodium secretion to the lumen or,
alternatively, depress sodium absorption through the intestinal tract
(28). In the present study, the reactions of guanylin and uroguanylin to acute sodium loading were examined to clarify the potential participation of these peptides in the sodium handling of the intestine.
To determine the guanylin and uroguanylin concentrations in biological
samples, we established a novel and reliable RIA system, since guanylin
and uroguanylin show unique but unfavorable topological stereoisomerism
resulting from the existence of two disulfide bonds in a 15- to
16-amino acid sequence (1, 27). Each member of this set of isoforms has
not only different physical properties but also different types of
immunogenicity and bioactivity (24). Chemically synthesized guanylin
and uroguanylin show dual peaks on reverse phase (RP)-HPLC. More
importantly, one isoform, with one peak on RP-HPLC, can rapidly
transform to another isoform in solution (1). This feature of the
peptides causes serious confusion in the measurement of the
concentration and bioactivity of the peptides. Therefore, we
established a new RIA system for these peptides in which the disulfide
bonds of the peptides are opened by reduction and stabilized by the use
of S-carboxymethylation. Reduced and
S-carboxymethylated (RCM) guanylin or
uroguanylin generated from both standard peptides and biological
samples clearly showed a single peak on RP-HPLC, and whole amounts of
the peptides in biological samples were correctly measured by this system.
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MATERIALS AND METHODS |
Peptides.
Custom-synthesized rat RCM
[Tyr0]guanylin and RCM
[Tyr0]uroguanylin
(YTDECELCINVACTGC) and other peptides were purchased from Peptide
Institute (Osaka, Japan). The correct synthesis was confirmed by amino
acid analysis and sequencing. Each peptide shows a single peak on
RP-HPLC with over 95% purity.
RIA procedure for guanylin and uroguanylin.
Rat RCM [Tyr0]guanylin
and -uroguanylin (15 mg each) were separately conjugated with bovine
thyroglobulin (25 mg) with the use of 5% glutaraldehyde and used as
antigens (16). The antisera for rat guanylin and rat uroguanylin were
raised in New Zealand White rabbits as previously described (22). Rat
RCM [Tyr0]guanylin and
rat RCM
[Tyr0]uroguanylin were
radioiodinated by the lactoperoxidase method (21). The
125I-labeled peptides were
purified by RP-HPLC, and, in the case of guanylin, only a guanylin
monoiodinated at position Tyr0 was used.
The same assay buffer used in the adrenomedullin RIA system (16) was
used for this RIA procedure. The incubation mixture consisted of 0.1 ml
of diluted sample or standard (rat RCM
[Tyr0]guanylin or rat
RCM
[Tyr0]uroguanylin),
0.1 ml of antiserum diluent (final dilution of 1:150,000 for guanylin
and 1:30,000 for uroguanylin), and 0.1 ml of tracer solution (18,000 cpm). Incubation was carried out for 24 h. The bound and free ligands
were separated by incubation with 0.05 ml of second antibody (goat
anti-rabbit 
-globulin) with 0.75 ml of 20% polyethylene glycol for
2 h and centrifugation. The radioactivity in the precipitate was
counted in a gamma counter (ARC-600, Aloka, Tokyo, Japan). All
procedures were performed at 4°C. Samples were assayed in duplicate.
Rat small intestinal perfusion.
Eight-week-old male specifically pathogen-free Sprague-Dawley rats
(n = 27) were purchased from Charles
River (Atsugi, Japan). The rats were housed in a temperature- and
humidity-controlled environment and maintained on standard rat chow
(Nihon CLEA CE-2; 145 µmol sodium/g) and tap water ad libitum for at
least 1 wk before the experiment.
The rats were anesthetized with pentobarbital sodium (50 mg/kg) and
placed on a heating table (37°C) and then underwent laparotomy. Incisions were made in the middle portion of the duodenum and the end
of the ileum. The lumen of the small intestine was rinsed gently with
physiological saline to remove fecal contents. The duodenum and the end
of the ileum were cannulated for luminal perfusion. Luminal perfusion
was started with modified Krebs-Ringer bicarbonate solution at a rate
of 5 ml/min. After a 10-min equilibration period, every 10 min
perfusate was collected on ice for 100 min. The composition of the
modified Krebs-Ringer bicarbonate solution was (in mM) 50 NaCl, 26 NaHCO3, 5 KCl, 1 MgSO4 · 7H2O,
0.03 Na2EDTA, 11 dextrose, and 2.4 CaCl2 · 2H2O
(pH 7.4). The buffer solution was aerated with 95%
O2-balance
CO2 and maintained at 37°C.
After a 20-min control period, rats were perfused with Krebs-Ringer bicarbonate solution containing various concentrations of salt (122, 170, and 200 mM NaCl) or mannitol (257 mM) for 20 min and then perfused
again with ordinary modified Krebs-Ringer bicarbonate solution for
another 60 min. Immediately after collection, the samples (50 ml each)
were manually applied to C18
Sep-Pak columns (Waters, Milford, MA), which were preequilibrated with
0.1% trifluoroacetic acid
(TFA)-H2O. The columns were washed
with 10 ml of 0.1% TFA-H2O, eluted with 10 ml of 60% acetonitrile-0.1%
TFA-H2O, and lyophilized.
Osmolarities of the solutions for the perfusion experiment were
directly measured by cryoscopic methods. With the use of standard curves, the osmolarity of the solution containing mannitol was exactly
matched with the highest salt solution.
RCM method for biological samples.
The lyophilized sample was reconstructed with 0.5 ml of
H2O and centrifuged at 5,000 rpm
for 10 min at 4°C (MRX-151, Tomy, Tokyo, Japan). Fifty microliters
of the supernatant were placed in an Eppendorf tube, mixed with
reaction buffer, and adjusted to a final concentration of 0.5 M
Tris · HCl (pH 8.0), 2 mM EDTA, and 25 mM
dithiothreitol (DTT) (total volume was 125 µl). The tube was flushed
with nitrogen, capped, and placed in a 37°C water bath for 60 min.
The solution was cooled to room temperature, and 12.5 µl of 1 M
iodoacetic acid were added. After 20 min in the dark, 12.5 µl of
concentrated acetic acid were added to acidify the
solution (final volume was 150 µl). The solution was immediately diluted 10 times with the assay buffer and then subjected to the RIA
for guanylin and uroguanylin.
The tissue extracts were prepared as described previously (16), with
minor modifications. The lyophilized samples were reconstructed with
distilled H2O and adjusted to a
concentration of 1 g wet tissue/ml. One hundred microliters of tissue
extract containing 125I-labeled
rat guanylin, which was radioiodinated by the lactoperoxidase method
and purified by RP-HPLC, were converted using the same ratio of
reagents and then analyzed by RP-HPLC. Every 1-min fraction was
collected; the radioactivity of the fractions was monitored by the
gamma counter.
Characterization of guanylin and uroguanylin in the perfusate.
Two Sprague-Dawley rats were perfused with modified Krebs-Ringer
bicarbonate solution for 60 min. The perfusate was extracted with a
Sep-Pak C18 column and lyophilized
as described in Rat small intestinal
perfusion. The sample was reconstructed
with 1 ml of H2O and briefly
centrifuged. Five hundred microliters of the supernatant were treated
by the same method to gain the RCM peptides. The solution was
centrifuged at 9,000 rpm for 5 min, and the resulting supernatant was
applied to a C18 HPLC column. The
column was developed with the following linear gradient: 10% acetonitrile-0.1% TFA-H2O to 60%
acetonitrile-0.1% TFA-H2O in 60 min at a flow rate of 1 ml/min. Every 1-min sample (1 ml each) was
collected, and 5-µl aliquots of the samples were assessed by the RIA.
Samples (n = 24) from the perfusion
study, which represent initial and peak secretions induced by the
highest salt and mannitol loading, were reconstructed with 0.5 ml of
H2O; 0.1 ml of each sample was
then treated by the same RCM method and separately applied to the HPLC
column. Corresponding 1-min samples were collected and lyophilized. The
samples were reconstructed with RIA buffer and subjected to the RIA.
Statistics.
Results are expressed as means ± SE. A repeated-measures ANOVA was
used to assess the significance of changes. After evaluation with
ANOVA, Student's t-test was performed
for the salt or mannitol loading group and the corresponding control
group. P < 0.05 was taken to
indicate a significant difference.
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RESULTS |
Figure 1 shows the RP-HPLC profiles of
synthetic rat guanylin (Fig. 1A)
and RCM rat guanylin (Fig. 1, B and
C). Authentic rat guanylin exists as
a mixture of two compounds, but the RCM reaction converts these
peptides to a single compound that was eluted at a slightly later
position by RP-HPLC compared with authentic rat guanylin. Various
incubation times and ratios (DTT to iodoacetic acid) were examined to
obtain the appropriate method for RCM reaction. Two suitable methods
are indicated in Fig. 1: a 1-h incubation with DTT at 37°C (Fig.
1B) or an overnight incubation with
DTT at 4°C (Fig. 1C) followed by
a 20-min incubation with iodoacetic acid at room temperature. These
methods cause the complete conversion of guanylin to RCM guanylin. The
subsequent amino acid analysis revealed that peak
1 in Fig. 1 is the completely converted guanylin and
peak 2 is the partially converted
guanylin. The ratio of DTT to iodoacetic acid (1:4) is crucial in this
reaction to avoid the formation of partially converted guanylin. This
method achieved an identical complete conversion in human guanylin and
human and rat uroguanylin (data not shown). To determine whether the
RCM reaction was completed in biological samples, we employed the same
method using 125I-labeled rat
guanylin. The radioactive peak of
125I-labeled rat guanylin was
clearly shifted after the RCM reaction (Fig.
2A). The
same peak shifts were observed in the RCM reactions coexisting with
various biological materials (Fig.
2B).
125I-labeled rat uroguanylin also
showed a peak shift in the same conditions but in an opposite direction
(data not shown, see the elution positions of the standard peptides in
Fig. 4).
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Fig. 1.
Reverse-phase (RP)-HPLC of rat guanylin and reduced and
S-carboxymethylated (RCM) guanylin.
One-microgram samples of authentic rat guanylin
(A) or rat guanylin were reduced by
dithiothreitol for 1 h at 37°C
(B) or overnight at 4°C
(C) and followed by a 20-min
incubation with iodoacetic acid at room temperature. A TSK ODS 120A,
4.6 × 150 mm, column was used. Flow rate was 1 ml/min. The
following solvent systems were used:
H2O-10% trifluoroacetic acid
(TFA) (ratio of 100:1) (solvent 1)
and
H2O-CH3CN-10%
TFA (ratio of 40:60:1) (solvent 2).
A solvent
1-to-solvent 2 linear
gradient was followed for 60 min. Peak
1 is completely converted RCM guanylin, and
peak 2 is partially converted RCM
guanylin. A210, absorbance at 210 nm.
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Fig. 2.
RP-HPLC analysis of 125I-labeled
guanylin (Gn) and final products of the RCM reaction.
A:
125I-labeled guanylin (2 × 106 cpm) and final products of the
same amount of 125I-labeled
guanylin converted by RCM reaction were applied to the HPLC column and
developed as shown in Fig. 1. B:
tissue extracts or Tris buffer containing
125I-labeled guanylin (0.5 × 106 cpm) were converted by RCM
reaction, and the final products were applied to the same HPLC system.
Only radioactive regions are shown. Data are representative of 4 (A) and 3 (B) experiments.
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Figure 3 shows standard curves of RIAs.
Half-maximal inhibitions of radioiodinated ligand binding by guanylin
and uroguanylin were observed at 20-30 fmol/tube. The minimum
detection quantity of guanylin or uroguanylin was 1 fmol/tube, and we
usually used 8-250 fmol/tube as a working range. The antisera did
not show cross-reaction with authentic forms of guanylin and
uroguanylin (Table 1). The antisera recognize and
measure both mature guanylin and proguanylin and uroguanylin and
prouroguanylin, respectively. The antiserum for rat RCM uroguanylin
showed little cross-reaction (up to 5%) with rat RCM guanylin at high
concentrations. However, this interference usually did not cause
problems within the working range. The other antiserum for rat RCM
guanylin did not show any cross-reaction to rat RCM uroguanylin (Table
1), so dual measurements for guanylin and uroguanylin can be corrected
in measurements with the RIA for uroguanylin when needed.
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Fig. 3.
Standard curves of RIAs for rat RCM guanylin and RCM uroguanylin. B,
fraction of tracer bound; Bo, fraction of tracer bound in
absence of unlabeled ligand.
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The characterization of rat intestinal perfusate was performed by
RP-HPLC combined with the two RIAs (Fig.
4). Immunoreactive guanylin
consisted of two peaks, in which peak
1 emerged at a position identical to that of RCM rat
guanylin. Peak 2 eluted at a position
close to that of biologically inactive proguanylin. In contrast to
guanylin, immunoreactive uroguanylin showed only a single peak at the
position of RCM uroguanylin.
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Fig. 4.
Characterization of immunoreactive (ir)-guanylin
(A) and uroguanylin
(B) in the perfusate from rat small
intestine by RP-HPLC. Same HPLC system shown in Fig. 1 was used. Arrows
indicate the elution positions of rat guanylin
(1), rat RCM guanylin
(2), rat uroguanylin
(3), and rat RCM uroguanylin
(4).
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In preliminary experiments, initial secretion rates of guanylin and
uroguanylin with isosmolar Krebs-Ringer solution (122 mM NaCl,
osmolarity of 305 mosM) were 1.84 ± 0.23 and 1.39 ± 0.18 pmol/min (n = 5), respectively.
However, the secretion rate of guanylin gradually increased after a
30-min perfusion (data not shown). We surveyed the optimal
concentration of NaCl to obtain a stable secretion rate for guanylin
and finally selected a relatively low concentration of NaCl (50 mM) as
a control solution (osmolarity of 190 mosM). The basal secretions of
both guanylin and uroguanylin in the perfusate from rat small
intestinal tract maintained relatively stable levels during the
experiment (Fig. 5). The amount of secreted immunoreactive guanylin was almost twice that of uroguanylin, which is
consistent with the characterization of the perfusate (Figs. 4 and 5).
After an equilibration period, the 20-min loading of high-salt solution
(osmolarity of 450 mosM) induced a marked increase of guanylin
secretion. This increase occurred after a 10-min loading and lasted
another 60 min. The peak of increased secretion in the high-salt group
reached almost three times that of the control group (5.4 ± 0.5 vs.
1.9 ± 0.2 pmol/min, respectively, at 50 min). Hyperosmolar solution
containing 257 mM mannitol and 50 mM NaCl (osmolarity of 450 mosM) also
caused a significant but lesser increase of guanylin secretion.
Interestingly, the reaction of uroguanylin to the highest salt loading
was limited to a slight increase compared with the reaction of guanylin
(1.46 ± 0.06 vs. 0.92 ± 0.10 pmol/min at 50 min), and
uroguanylin did not react with mannitol loading. Using the RP-HPLC and
the RIA system, we performed characterizations of basal and peak
fractions of the secreted guanylin stimulated by the highest salt or
mannitol loading. This characterization clearly revealed that both
stimulations similarly increased mature guanylin and proguanylin
secretion (Fig. 6). Figure
7 shows the dose-response relationship of
salt loading and guanylin or uroguanylin response, in which the maximal rate of increase compared with initial secretion was plotted against the different concentrations of NaCl used for the stimulation. Guanylin
secretion was increased dose dependently, but uroguanylin secretion
seemed to reach a plateau at isosmolar solution (122 mM NaCl).
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Fig. 5.
Basal (CT), high-salt (200 mM NaCl, HS)-induced, and mannitol (257 mM
mannitol + 50 mM NaCl; MN)-induced secretions of guanylin (Gn) and
uroguanylin (UGn) in the perfusates drained from rat small intestine.
Concentrations of guanylin and uroguanylin in 10-min perfusates were
monitored by specific RIAs. The y-axis
shows secretion rates of the peptides in 5 ml (1-min fraction) of the
perfusate. * P < 0.05, ** P < 0.01 compared with the
corresponding basal secretions.
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Fig. 6.
Characterization of basal and peak fractions of the guanylin stimulated
by high salt (200 mM NaCl) and mannitol (257 mM mannitol + 50 mM NaCl)
loading. Initial (n = 6) and peak
(n = 6) fractions of each group were
separately fractionated by RP-HPLC and monitored by RIA.
** P < 0.01 compared with the
corresponding basal secretions.
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Fig. 7.
Dose-response relationship of salt loading and guanylin or uroguanylin
response. Maximal rate of increase compared with initial secretion was
plotted against different concentrations of NaCl [50 mM (control,
n = 6), 122 mM
(n = 4), 170 mM
(n = 5), and 200 mM
(n = 6)].
* P < 0.05, ** P < 0.01 compared with the
corresponding initial secretions.
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DISCUSSION |
The present paper describes a new RIA system for rat guanylin and
uroguanylin. To our knowledge, this is the first report to describe an
RIA combined with the RCM reaction being applied to small peptides. The
RCM reaction is commonly used for peptide sequence analyses (12). By
consulting a previous study of -immunoglobulin (8), we optimized the
conditions for the RCM reaction in the guanylin family of peptides. The
disulfide bonds of the peptides are easily opened by a relatively mild
reduction using DTT. This may reflect flexible disulfide bonds in
guanylin, which transform between two isomers in solution (17).
Prolonged incubation (e.g., 4 h) at high temperature (e.g., 50°C)
destroyed the sample, regardless of the amount of the peptides (data
not shown). The ratio of DTT to iodoacetic acid (1:4) is also crucial
for this reaction to avoid the formation of partially converted
guanylin. In a previous report, a DTT-to-iodoacetamide ratio of 1:2 was
used (12), but this ratio of the reagents led to a significant amount
of incompletely converted guanylin formation. The RCM reaction was also
completed in extracts of biological materials (Fig. 2). The final
recovery rates of RCM guanylin were slightly higher in extracts from
small intestine compared with those in a peptide-only solution and
extracts from plasma. The cause of this phenomenon is unclear. Some
protective proteins for guanylin may exist in the extracts.
A simple method is usually better than a complicated procedure in an
RIA, since a more complicated method may entail more problems. However,
the unique features of the guanylin family oblige a novel solution for
correct measurements. The topological stereoisomerism of the guanylin
family, in which each isomer has different physical properties,
immunogenicities, and bioactivities (1, 24, 27), may prevent complete
cross-reactions to antibodies. An antibody that recognizes one isomer
may not react with another isomer. To make matters more complex, one
isomer quickly transforms to another isomer during incubation in an
RIA, so that a complete separation of the isomers is basically
impossible. We decided to open the disulfide bonds, which are the
origin of topological stereoisomerism, and make a single compound. The
RCM reaction worked well with guanylin and uroguanylin, and the
established RIA showed high sensitivity because of the artificial
modification for the peptides. This new RIA system should contribute to
the study of guanylin and related peptides.
Using this RIA system, we characterized guanylin and uroguanylin in
perfusate from rat small intestine. Immunoreactive guanylin contained
two peaks that eluted at the position of RCM guanylin and near the
position of proguanylin in RP-HPLC. Interestingly, immunoreactive
uroguanylin consisted of only a single peak, which emerged at the
position of RCM uroguanylin (Fig. 3). Coupled with the previously
reported bioassay (15), we have already completed the isolation and
sequencing of bioactive guanylin and uroguanylin in the perfusate,
where almost the same amounts of bioactive guanylin-14 (NTCEICAYAACTGC)
and uroguanylin-15 (TDECELCINVACTGC) were yielded (unpublished data).
This observation, together with the same elution positions for RCM
guanylin or RCM uroguanylin on RP-HPLC, strongly suggested that
immunoreactive guanylin (peak 1) and
immunoreactive uroguanylin are bioactive and are mature guanylin and
uroguanylin, respectively. Peak 2 in
Fig. 4 was not detected by the bioassay, which means that it is
biologically inactive. According to a previous report (25),
peak 2 is most likely to be
proguanylin. Uroguanylin may act as a endocrine mediator for the kidney
and is thought to be secreted to both the luminal and basolateral sides
of the intestine (7). The difference in molecular forms observed in the
perfusate may reflect differences in the secretion behavior of
uroguanylin and guanylin. Further study is required to clarify the
basolateral secretion of uroguanylin.
The basal secretion rates of immunoreactive guanylin and immunoreactive
uroguanylin were almost 2 and 1 pmol/min, respectively. Immunoreactive
guanylin seemed to contain almost the same amounts of mature guanylin
and proguanylin, and thus almost the same amounts of mature guanylin
and uroguanylin should be secreted into the perfusate. Guanylin and
uroguanylin showed different distributions of expression in the
longitudinal axis of the gastrointestinal tract (31), but the present
study demonstrated that almost equal amounts of mature peptides were
secreted from the total small intestine. Guanylin and uroguanylin
showed bioactivities at a concentration in the
10
9 M range with T84 cells
(15), and the basal secretion rate of both peptides (1 pmol/min) could
be enough to cause chloride secretion from the small intestine.
The main target of this study was to clarify the secretions of guanylin
and uroguanylin into the lumen of the small intestine and the reaction
of the guanylin family to salt loading. It must be emphasized that
guanylin and uroguanylin and their receptor GCC are predominantly
expressed in the gastrointestinal tract and that the peptides act on
the luminal surface of the intestine (11, 18, 20, 25, 26, 28-31).
The gastrointestinal tract has not received much attention as a
regulator of water and electrolytes. The identification of the guanylin
family may shed light on the role of the gastrointestinal tract in
water and electrolyte regulation. In addition, an endocrine link seems
to exist between the intestine and kidney via uroguanylin (7).
In the present study, stable and large basal secretions of guanylin and
uroguanylin from rat small intestine were observed. Moreover, it was
clearly demonstrated that salt loading to the lumen of the small
intestine causes a dose-dependent increase of guanylin and a lesser but
significant increase of uroguanylin. Interestingly, guanylin reacts,
although not significantly, with isosmolar solution; moreover, the
reaction of uroguanylin seems to reach a plateau at the isosmolar
solution. Because the osmotic pressures of the contents (e.g., foods,
saliva, and gastric juice) of the small intestine are relatively low,
isosmotic pressure could be enough to induce a increase of guanylin and
uroguanylin secretions from the small intestine. Guanylin also reacts
with hyperosmolar solution containing mannitol. This observation
suggested that guanylin secretion is partially driven by the osmotic
pressure of the contents of the small intestine. The increases of
guanylin and uroguanylin in response to salt or hyperosmolar loading
could be expected to induce increased chloride secretion to the lumen of the intestine and simultaneously increase the flow of water and
sodium to the lumen to maintain the ion balance. This would primarily
contribute to maintaining local osmotic homeostasis in the small
intestine because induced water secretion could dilute hyperosmolar
content in the small intestine. On the other hand, guanylin reacted
more to salt loading than to mannitol loading and uroguanylin reacted
with only salt loading (Fig. 5). This observation strongly
suggested that salt itself, besides osmotic pressure, causes a specific
reaction in the secretion of guanylin family members. Increased
guanylin secretion could depress sodium absorption and increase
chloride secretion through the intestinal tract (28). Uroguanylin may
also act as a natriuretic factor in the kidney. This observation raises
the possibility that the guanylin family acts as regulatory
peptides of sodium and water homeostasis in the intestine.
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FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. Kita,
First Dept. of Internal Medicine, Miyazaki Medical College, 5200 Kihara
Kiyotake, Miyazaki 889-1692, Japan (E-mail:
t-kita{at}po.sphere.ne.jp).
Received 3 November 1998; accepted in final form 6 August 1999.
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REFERENCES |
1.
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