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Third Division, Department of Internal Medicine, Miyazaki Medical College (M.N., H.Y., S.M.), Miyazaki 889-1692, Japan; National Cardiovascular Center Research Institute (Y.D., M.M., K.K.), Osaka 565-8565, Japan; Department of Physiology and Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill (M.F.G.); and Peptide Institute Inc. (N.C.), Osaka 562-8686, Japan
Address all correspondence and requests for reprints to: Masamitsu Nakazato, M.D., Ph.D., Third Department of Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. E-mail: nakazato{at}post.miyazaki-med.ac.jp
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Introduction |
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Human uroguanylin has been isolated from the urine and opossum and rat uroguanylin from the urine and small intestine (5, 6, 15, 16). Uroguanylin circulates in the blood (17, 18, 19). Iv administration to the mouse has a natriuretic effect (20). Together with the presence of the GC-C transcript in human and rat kidney (21), these findings suggest that uroguanylin serves as an endocrine factor; an "intestinal natriuretic peptide" that carries information from the intestine to the kidney. To better understand its physiological functions and pathophysiological significance in water and electrolyte homeostasis, we generated an antiserum specific for rat uroguanylin then developed an RIA, after which we isolated endogenous molecular forms of uroguanylin and determined their amino acid sequences. Three uroguanylin molecules, a 15-amino acid uroguanylin (uroguanylin-15), an 18-amino acid uroguanylin (uroguanylin-18), and a 9.4-kDa prouroguanylin were identified. The antiserum recognized the first two small peptides equally on a molar basis, but not prouroguanylin because its tertiary structure probably interfered with the binding of the antibody to this large molecule. To determine the tissue content and ratios of the three molecular forms, we produced uroguanylin-18 from prouroguanylin by trypsin digestion before doing the RIA. We also investigated immunohistochemically the cellular origin of uroguanylin with three independent antisera, one for uroguanylin-15 and two for N-terminal regions of prouroguanylin.
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Materials and Methods |
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Preparation of antisera
To generate an antiuroguanylin antiserum, synthetic rat
uroguanylin-15 (2 mg) was conjugated with bovine thyroglobulin (15 mg)
in 200 µl of a 5% glutaraldehyde solution. The reaction mixture was
dialyzed four times against 3 liters of 0.9% sodium chloride. Amino
acid analysis of the conjugate showed that one thyroglobulin molecule
was coupled with an average 280 uroguanylin molecules. The antigenic
conjugate solution (1.5–3 ml) was emulsified with an equal volume of
Freund’s complete adjuvant and administered to New Zealand white
rabbits. Booster injections were given every 2 weeks, and the animals
bled 7 days after each injection. Two antisera (6910 and 6912) raised
against N-terminal regions of rat prouroguanylin are described
elsewhere (23).
RIA procedure
Synthetic rat Tyr0-uroguanylin-15 was radioiodinated
by the lactoperoxidase method. The 125I-labeled peptide was
purified on a TSK ODS SIL 120A column (Tosoh Co. Ltd., Tokyo, Japan) by
reverse-phase high performance liquid chromatography (RP-HPLC). The RIA
incubation buffer was 50 mM sodium phosphate (pH 7.4) that
contained 0.25% BSA treated with N-ethylmaleimide, 80
mM NaCl, 25 mM EDTA·2 Na, 0.05%
NaN3, and 0.1% Triton X-100. A diluted sample or a
standard peptide solution (100 µl) was incubated for 24 h with
100 µl of the antiserum diluent (final dilution 1/10,000). The tracer
solution (16,000 cpm in 100 µl) was added, and the mixture again
incubated for 24 h. The bound and free ligands were separated in
23% polyethyleneglycol solution (500 µl). All the procedures were
done at 4 C. The cross-reactivity of the antiserum was examined using
rat guanylin and 94-amino acid rat proguanylin that had been isolated
from the intestinal mucosa (24), and human uroguanylin (Fig. 1
).
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). Fractions 58–63 that contained
ir-uroguanylin were pooled and lyophilized. The residue was dissolved
in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.05%
Triton X-100 then subjected to immunoaffinity chromatography on an
antirat uroguanylin IgG-bound Affi-Gel 10 column (gel volume: 4 ml) as
described below. The adsorbed peptides were eluted with a solution of 1
M CH3COOH containing 10% CH3CN and
0.002% Triton X-100. The eluate was subjected to RP-HPLC on a
TSK-ODS-SIL 120A column under the conditions described in the legend to
Fig. 3A. All fractions were analyzed by
RIA for uroguanylin immunoreactivity. The two
uroguanylin-immunoreactive peptides were purified by RP-HPLC on a µ
Bondasphere C-18 300 Å column (Waters, Milford, MA) (Fig. 3, B and C).
They were subjected to a gas-phase protein sequencer (Model 476A,
PE Applied Biosystems, Foster City, CA) linked on-line to
a PTH analyzer (Model 120A, PE Applied Biosystems). Amino
acid sequences thus determined are shown in Fig. 4.
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Quantification and chromatographic characterization of
ir-uroguanylin in tissues and urine
The tissues (
1 g wet wt each) listed in Table 1 were resected immediately after
decapitation of five 8-week-old male Sprague Dawley rats
that had fasted overnight. The jejunum was resected 10–20 cm from the
pyloric ring, the ileum 20–30 cm above the terminal ileum, and the
colon 5–15 cm below the terminal ileum. Twenty-four hour urine samples
were obtained from the same five rats. All the rats were kept in an
air-conditioned room and given standard laboratory chow and water
ad libitum.
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, A and B). Five
milliliters of rat urine was applied to a Sep-Pak C-18 cartridge, then
the eluate was separated by RP-HPLC (Fig. 5C). One-half of each RP-HPLC
fraction was quantified by the RIA without trypsin digestion, and the
other half by the RIA after trypsin digestion. Authentic rat
uroguanylin-15 and -18 were chromatographed with the same HPLC system.
The ir-fractions eluting at about 31 min (Fig. 5A) were subjected to
gel permeation chromatography on a TSKgel G2000 SW column (Fig. 5D) and
all fractions were analyzed by RIA after trypsin digestion. The
recoveries of rat uroguanylin during extraction were determined by
adding rat uroguanylin-15 (1 ng) or
125I-Tyr0-rat uroguanylin-15 (5,000 cpm) to the
tissue homogenates then measuring the immunoreactivity or radioactivity
of the Sep-Pak cartridge eluate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The antiserum had 100%
cross-reactivity with isolated uroguanylin-18, but no cross-reactivity
with rat guanylin, rat proguanylin, or human uroguanylin. The dilution
curves for the extracts of rat jejunum and urine paralleled the
standard curve, and the respective intra and interassay coefficients of
variation were 4.1% and 3.8% at 50% binding. The recoveries of the
"cold" and radiolabeled uroguanylin-15 added to the tissue
homogenates in the extraction done with a Sep-Pak C-18 cartridge
respectively were 91.8 ± 0.4% (SEM) and 88.6 ±
0.7% (SEM).
Isolation and sequence determination of uroguanylin-15 and
uroguanylin-18
Two uroguanylin molecules were purified from small intestinal
mucosa as single peptide peaks using Sephadex G-75 gel filtration (Fig. 2) and immunoaffinity chromatography, followed by two steps of RP-HPLC
(Fig. 3). The elution position of the peptide in fractions 17–19 in
Fig. 3A was identical to that of authentic rat uroguanylin-15. The
yield of this peptide was 23 pmol and that of the other ir-peptide 22
pmol on the basis of immunoreactivity. Amino acid sequence analysis
verified that the former peptide was uroguanylin-15 and the latter
uroguanylin-18 with three N-terminal-extended amino acids (Fig. 4).
Identification of ir-uroguanylin molecules in tissues and
urine
In our study of gel filtration without trypsin digestion, we
detected uroguanylin-15 and -18 in rat intestine, but not their
precursor form. Because rat guanylin has a 10.4-kDa precursor form as a
major molecule in the intestine (24) and on the assumption that rat
uroguanylin-18 must be a monobasic processing product of its precursor,
we analyzed the uroguanylin molecules in the jejunum, kidney, and urine
using a combination of RP-HPLC and trypsin digestion. The kidney sample
was loaded on a medium-sized HPLC column because its uroguanylin
content was much lower than that in the jejunum. Two ir-peptides,
uroguanylin-15 (arrow 1) and uroguanylin-18 (arrow
2), were detected on the chromatograms of the jejunum and kidney
when the HPLC fraction samples were not trypsinized (Fig. 5, A and B,
closed circles). We detected the presence of rat uroguanylin
precursor by digesting the HPLC fraction samples with trypsin then
analyzing the products using the RIA (Fig. 5, A and B, open
circles). This revealed a new immunoreactive peak (arrow
3 in Fig. 5), eluting later than uroguanylin-18. To further
characterize this peptide of arrow 3, it was subjected to
gel permeation chromatography. Samples of the fractions then were
trypsinized and subjected to the RIA (Fig. 5D). Uroguanylin
immunoreactivity was detected at the position corresponding to
Mr 9.4K. This 9.4-kDa precursor produced uroguanylin-18
after trypsin digestion (data not shown). The molar ratios of
uroguanylin-15, uroguanylin-18, and the precursor were 1:2:5 in the
jejunum and 5:1:7 in the kidney. Only uroguanylin-15 was detected in
the urine (Fig. 5C).
Tissue content and urine concentration of ir-uroguanylin
Uroguanylin is present in the gastrointestinal tract from the
stomach to the colon, as well as in the kidney, the highest value being
in the jejunum (Table 1). Uroguanylin was found immunohistochemically
in the pancreas (as described below), but its content in this tissue
was below the detectable level (0.05 pmol/g wet wt). No uroguanylin was
detected in the other tissues examined including the esophagus, heart,
liver, lung, spleen, cerebrum, and cerebellum. The urine concentration
of uroguanylin in 5 normal rats was 345 ± 30 fmol/ml (mean
± SEM).
Immunohistochemistry of uroguanylin-positive cells
We used three independent antisera, one for uroguanylin-15 and two
for N-terminal regions of prouroguanylin, in immunohistochemistry.
Because the two antiprouroguanylin antisera display indistinguishable
staining patterns (23), only results obtained with 6912 are shown in
the figures. Uroguanylin-immunoreactive cells are moderately abundant
in the duodenum (Fig. 6A), most abundant
in the jejunum (Fig. 6B), infrequent in the ileum (Fig. 6C), and vary
rare in the colon. These cells are predominant in the midvilli of the
small intestine along the crypt-villus axis (Fig. 6, A and B).
Uroguanylin-immunoreactive cells are round, basket-shaped, or tall
flask-shaped cells with a dense accumulation of uroguanylin in the
luminal cytoplasm and a long, thin basal process immunoreactive for
uroguanylin (Fig. 6D). Most uroguanylin-positive cells in the intestine
are prouroguanylin-positive and also reacted with serotonin antibody in
serially cut sections (Fig. 6, E–G). In the stomach,
uroguanylin-immunoreactive cells are present in the epithelium of the
basal half of the oxyntic mucosa (Fig. 6H), but not in the cardia or
antrum. These cells also reacted with chromogranin A (Fig. 6I) and
prouroguanylin (data not shown). None of the uroguanylin-immunoreactive
cells showed somatostatin or gastrin immunoreactivity (data not shown).
Uroguanylin and prouroguanylin immunoreactivities are present in the
pancreatic islet (Fig. 6, J and K), where colocalization of uroguanylin
and insulin occurred in a double-immunostaining section (Fig. 6L). In
the kidney, uroguanylin-15 is present in the distal tubules, but not in
the proximal tubules or glomeruli (Fig. 6 M). These cells
showed no immunoreactivity for prouroguanylin (Fig. 6N). No uroguanylin
immunoreactivity was detected in the tissues studied when normal rabbit
serum or antiserum absorbed with excessive synthetic uroguanylin-15 was
used (Fig. 6O).
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Discussion |
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), rat uroguanylin-15 must
be the final processing product that activates GC-C. All mature forms
of uroguanylin and guanylin in humans and the rat are produced by
cleavage of the peptide bond between the -4 and -3 positions from the
first cysteine residue in these molecules (Fig. 4). This processing
produces two intramolecular disulfide bonds indispensable for
bioactivity at the same position in these peptides. However, there is
no common amino acid sequence at the position where this processing
occurs in the uroguanylin and guanylin precursors (Fig. 4).
Crystallization of the precursors and analysis by x-ray diffraction
should provide direct structural information about their conformations
as well as clues to the contribution of the substrate structure to this
processing.
Rat uroguanylin-18 had been isolated from the duodenum by the use of a
cGMP functional assay with T84 cells (16). The peptide is considered a
monobasic processing product of its precursor because the amino acid
sequence in the vicinity where cleavage occurs is consistent with the
following consensus sequences that govern monobasic cleavage (26, 27):
an arginine, a target for monobasic cleavage, is located next to the
N-terminus of uroguanylin-18 within the prouroguanylin sequence (Fig. 4); a basic amino acid is always present at the -3, -5, or -7
position (the 3rd, 5th, or 7th amino acid preceding the monobasic
cleavage site), in this case Lys at the -3 position; short-side chain
amino acids occur at the -2 and +1 positions, in this case Ala at the
-2 and Thr at the +1; cysteine and aromatic amino acids (Trp, Tyr, and
Phe) are never present at the -1 position, in this case Leu at the
-1; and an aliphatic amino acid, Ile in this case, is present at the
+2 position.
We identified a 9.4-kDa uroguanylin precursor in the intestine and kidney. Hamra et al. (28) have purified prouroguanylin from opossum intestine by using pretreatment of prouroguanylin with chymotrypsin to elicit cGMP responses in T84 cells. Although we did not determine the N-terminal sequence of the rat uroguanylin precursor, its molecular size is consistent with that of the 85-amino acid rat prouroguanylin (Mr = 9405.3) that is generated by cleavage of the putative 21-residue signal peptide from preprouroguanylin. Moreover, the precursor produced uroguanylin-18 by trypsin digestion, indicative that it is prouroguanylin. The antiserum recognized uroguanylin-15 and -18 on a molar basis, but not prouroguanylin. This could be due to elements of tertiary structure that interfere with the binding of the antibody to this large molecule. We therefore determined the tissue content and ratio of the endogenous molecular forms of uroguanylin after trypsin digestion. Prouroguanylin accounted for about half the ir-uroguanylin molecules in the intestine and kidney, as is the case for rat proguanylin in the intestine (24).
Uroguanylin is present not only in the intestine but also in the stomach, kidney, and pancreas. Uroguanylin is most abundant in the proximal small intestine, its content and the number of uroguanylin-positive cells decreasing toward the colon. These findings agree with its messenger RNA (mRNA) distribution and abundance (16, 25, 29). A previous report (23) and this study indicate that uroguanylin-containing cells in intestinal epithelia are enterochromaffin (EC) cells. The EC cell, the most abundant type of enteroendocrine cell, is widely distributed in the intestine. When stimulated, the EC cell releases serotonin and substance P both apically (into the lumen) and basolaterally (into circulation) (30). Uroguanylin also is released from the intestine bidirectionally (unpublished data) and is thought to function in a luminocrine (luminal secretion), endocrine, and/or paracrine fashion. Identification of the EC cell as the site of uroguanylin biosynthesis suggests a feasible mechanism for delivering uroguanylin to luminally oriented GC-C as well as to remote tissues such as the kidney via the circulation.
Uroguanylin-containing cells in the gastric fundus appear to be endocrine cells on the basis of their morphological features and colocalization with chromogranin A. These uroguanylin-containing cells had no immunoreactivity for gastrin or somatostatin. The expression of GC-C and a truncated, GC-C-like mRNA that has a 159-nucleotide deletion has been reported in the mucosa of both rat stomach and intestine (31). Further identification of the cellular source of uroguanylin in the stomach should help clarify the cGMP-mediated-regulation of gastric ion transport.
Uroguanylin-15 is present immunohistochemically in the renal distal tubules whose principal function is to regulate the reabsorption of sodium and chloride from the glomerular filtrate. The distal tubules did not react with two antiprouroguanylin antisera. This could result from receptor-mediated uptake of bioactive uroguanylin-15 from the glomerular filtrate. We reported that the 24 h urinary excretion of uroguanylin of persons on a high-salt diet (10 g/day) was significantly higher than that of persons on a low-salt diet (7 g/day) (21). There was a significant positive correlation between the urinary excretions of ir-uroguanylin and Na+, Cl-, K+, or cGMP in these persons, but not between the plasma levels of ir-ANP and ir-BNP and the urinary excretions of Na+, Cl-, and K+. Iv injection of 75 ng of opossum uroguanylin, but not guanylin, to mice induced 3-fold urinary sodium excretion and 2-fold diuresis as compared with the control vehicle (20). Uroguanylin therefore is a prime candidate for a substance that could link the intestine and kidney in an endocrine pathway that regulates renal salt metabolism.
Uroguanylin was found in B-cells of the pancreatic islets by double immunostaining with antiinsulin antiserum. We also detected uroguanylin mRNA in rat pancreatic islets by using the RT-PCR (unpublished data). The peptides produced in the "gastro-entero-pancreatic (GEP) endocrine system" function to control or modulate all the processes linked to the digestion and absorption of nutrients and water (32). Uroguanylin may be a constituent of the GEP endocrine system because of its cellular source and biological activity. Alternatively, uroguanylin in the pancreas may be merely a vestige of the evolutional process.
Our findings have furnished new insights into the potential roles of uroguanylin via intracellular cGMP in various organs and tissues. Further investigations of the uroguanylin contents of the tissues, plasma, urine, and intestinal perfusate under various physiological and pathophysiological conditions should provide information on what mechanisms govern the biosynthesis and secretion of this peptide.
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Footnotes |
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Received March 11, 1998.
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), rat
uroguanylin-18 (•), rat guanylin-15 (
) rat proguanylin (
),
human uroguanylin-16 (
), rat jejunum extract (
) and rat urine
extract (
). The numeral one in the dilution curves of the jejunum
and urine respectively denotes 10 mg wet wt and 0.5 ml.
