Structure and activity of uroguanylin and guanylin from the
intestine and urine of rats
Xiaohui
Fan1,2,
F. Kent
Hamra1,2,
Roslyn M.
London1,2,
Sammy L.
Eber1,2,
William J.
Krause2,
Ronald H.
Freeman2,
Christine E.
Smith3,
Mark G.
Currie3, and
Leonard R.
Forte1,2
1 Truman Veterans Affairs
Medical Center and 2 Departments
of Pharmacology, Pathology and Anatomical Sciences and Physiology,
School of Medicine, Columbia 65212; and
3 Searle Research and Development,
St. Louis, Missouri 63167
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ABSTRACT |
Uroguanylin and guanylin are related peptides
that activate common guanylate cyclase signaling molecules in the
intestine and kidney. Uroguanylin was isolated from urine and duodenum
but was not detected in extracts from the colon of rats. Guanylin was
identified in extracts from small and large intestine but was not
detected in urine. Uroguanylin and guanylin have distinct biochemical
and chromatographic properties that facilitated the separation,
purification, and identification of these peptides. Northern assays
revealed that mRNA transcripts for uroguanylin were more abundant in
small intestine compared with large intestine, whereas guanylin mRNA
levels were greater in large intestine relative to small intestine.
Synthetic rat uroguanylin and guanylin had similar potencies in the
activation of receptors in T84 intestinal cells. Production of
uroguanylin and guanylin in the mucosa of duodenum is consistent with
the postulate that both peptides influence the activity of an
intracellular guanosine 3',5'-cyclic monophosphate signaling pathway that regulates the transepithelial secretion of
chloride and bicarbonate in the intestinal epithelium.
guanylate cyclase; guanosine 3',5'-cyclic
monophosphate; kidney; heat-stable enterotoxin; human T84 intestinal
cells
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INTRODUCTION |
GUANYLIN AND UROGUANYLIN are small peptides that
activate membrane receptor-guanylate cyclase signaling molecules in the
intestine, kidney, and other epithelia (reviewed in Ref. 8). These
receptors are localized to apical membranes of cells lining the
gastrointestinal tract (6, 20, 23, 30) and renal proximal tubules (9, 10, 21). Heat-stable enterotoxin (ST) peptides secreted by enteric
bacteria that cause traveler's diarrhea act as molecular mimics of
uroguanylin and guanylin (4, 6, 8, 14). Intracellular accumulation of
the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) influences the phosphorylation state and putative chloride (Cl
) channel activity of
the cystic fibrosis transmembrane conductance regulator protein, which
may serve as an efflux pathway for
Cl
secretion from the
intestinal mucosa (8). The net effect of receptor activation in the
intestine is to stimulate the transepithelial secretion of
Cl
and
, thus increasing fluid secretion
and modulating the intraluminal pH (4, 6, 11, 14, 17).
Guanylin was first isolated from the intestine of rats as a 15-amino
acid peptide containing four cysteines with two disulfide bonds that
are required for bioactivity (4). Then, cDNAs encoding preproguanylins
of 115-116 amino acids containing the COOH-terminal guanylin
peptides were isolated (31, 32). Guanylin mRNA is highly expressed in
the ileum and colon, with considerably lower amounts found in the
duodenum and jejunum (31, 32). The cellular sites of guanylin
production in the intestinal mucosa are reported to include goblet
cells and absorptive cells (22, 23, 26). Uroguanylin was
initially isolated from opossum urine (14). A search for the tissue
source of urinary uroguanylin resulted in the purification of
prouroguanylin and uroguanylin from large intestine (13, 15). Recently,
cDNAs encoding preprouroguanylin were isolated from opossum, human, and
rat intestinal cDNA libraries (1, 5, 16, 25, 28, 29). The bioactive
uroguanylin peptides found in urine are located at the COOH terminus of
prouroguanylin.
In the present study, we isolated uroguanylin from urine and duodenum
of rats to investigate the structure and biological activity of
uroguanylin in this species. Uroguanylin and guanylin were identified
by their unique chromatographic properties, by NH2-terminal sequence analyses,
and by the effects of medium pH on the relative potencies of the
bioactive peptides. The bioactive peptide in the urine is uroguanylin,
whereas guanylin and uroguanylin were both isolated from the duodenum.
Only guanylin was purified from the large intestine.
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MATERIALS AND METHODS |
Purification of uroguanylin from urine.
Three batches of urine (2-3 liters) pooled from 12 male
Sprague-Dawley rats were used to isolate and identify uroguanylin with
chromatographic methods that have been previously described (13-15). Briefly, urine was collected daily from rats housed in metabolic cages, pooled, and stored at
20°C. After thawing,
the urine was centrifuged at 10,000 g
for 20 min. Trifluoroacetic acid (TFA), 0.1%, was added to the
supernatant, and the sample was then applied to
C18 Sep-Pak cartridges and eluted
with 40% acetonitrile and 0.1% TFA. The eluted polypeptides were
dried and resuspended in 50 mM ammonium acetate and then
chromatographed using a 2.5 × 90-cm column of Sephadex G-25 gel.
Fractions eluted from the G-25 column and in subsequent purification
steps were bioassayed using T84 cells by measurement of cGMP
accumulation as previously described (8, 9). The active fractions were pooled, dried in a Speed-Vac, resuspended in 0.1% TFA, and loaded onto
C18 Sep-Pak cartridges. The
peptides were eluted with a gradient of 10%, followed by 30% and then
60% acetonitrile solutions containing 0.1% TFA. The bioactive
peptides were eluted with the 30% acetonitrile-0.1% TFA solution, and
this fraction was dried, resuspended in 10% acetonitrile and 0.1%
TFA, and applied to a C18
semipreparative high-performance liquid chromatography (HPLC) column
(Waters semipreparative µBondapak, 7.8 mm × 30 cm). The
peptides were eluted with a gradient of 10% acetonitrile-0.1% TFA to
30% acetonitrile-0.1% TFA over a period of 180 min. The peaks of
bioactive peptides were pooled, dried, resuspended in
H2O with 0.8% ampholytes
[pH range 3-10 (Bio-Rad)], and subjected to
preparative isoelectric focusing (Rotorfor, Bio-Rad). The fractions
containing bioactivity were combined, applied to a
C18 HPLC column (Waters analytic
µBondapak, 3.9 mm × 30 cm), and eluted with a gradient of 5%
acetonitrile-10 mM ammonium acetate (pH 6.2) to 25% acetonitrile-10 mM
ammonium acetate (pH 6.2) over 180 min. The peak of bioactive peptides was subjected to a second purification procedure with the same C18 analytic HPLC column, but with
the acetonitrile gradient containing 0.1% TFA instead of ammonium
acetate. The bioactive peptides were then applied to a
C8 microbore column and eluted
with a gradient of 0.33% of acetonitrile and 0.1% TFA per minute as
previously described (4, 14, 15). The purified peptides were subjected to automated Edman NH2-terminal
sequencing, as previously described (4, 14, 15).
Purification of peptides from the mucosa of colon and duodenum.
The mucosa (100 g wet weight) was scraped from colons by use of a glass
microscope slide and then boiled in 10 volumes of 1 M acetic acid for
10 min, homogenized, and centrifuged at 10,000 g for 20 min. The supernatant was
extracted with C18 Sep-Pak
cartridges followed by Sephadex G-25 column fractionation, as described
above. The bioactive peptide fractions from the gel column were
combined and fractionated a second time with
C18 Sep-Pak cartridges. The peptides were eluted using 5, 10, 15, 20, 25, 40, and 60% acetonitrile solutions containing 0.1% TFA. The bioactive peptide fractions (i.e.,
25% acetonitrile) were pooled and subjected to isoelectric focusing as
described above. The final purification of the active peptides was
accomplished by HPLC by use of a series of
C18 columns as we have described.
Fifty-five grams wet weight of mucosa were scraped from the duodenum
(proximal one-third of the small intestine), and the bioactive peptides
were purified using the same methods as described above for the
peptides isolated from colonic mucosa, except that the isoelectric
focusing step was not used.
Northern assays of uroguanylin and guanylin mRNA.
Total RNA was prepared (RNeasy kit, Qiagen) from the mucosa of
individual intestinal segments, and 20 µg of each RNA preparation were subjected to electrophoresis in formaldehyde-agarose gels and then
transferred to nylon membranes (Bio-Rad). The blots were hybridized
with rat uroguanylin and
-actin cDNAs or rat guanylin plus
-actin
cDNAs (27). Prehybridization was for 1 h with QuickHyb (Stratagene, La
Jolla, CA) at 68°C, which was followed by hybridization for 2 h at
68°C with each cDNA probe labeled by random priming (Boehringer
Mannheim). The blots were washed twice with 2× standard sodium
citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) for 15 min at room
temperature and once with 0.2× SSC and 0.1% SDS for 15 min at
60°C. The exposure to film was for 24 h at
80°C with
intensifying screens. Rat uroguanylin cDNA (nucleotides 117-292) was produced by polymerase chain reaction (PCR) amplification from
intestinal mRNA-cDNA (1, 27). This cDNA was isolated and sequenced to
confirm that it matched the uroguanylin expressed sequence tag (EST) of
rat uroguanylin with 100% identity. A rat guanylin cDNA (nucleotides
1-531) was generously provided by Dr. Roger Weigand (Monsanto,
St. Louis, MO).
Cell culture.
T84 cells were obtained from Dr. Jim McRoberts (Harbor-University of
California, Los Angeles, CA) at passage 21. Cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 medium
(1:1), supplemented with 5% fetal bovine serum, 60 mg penicillin/ml,
and 100 mg streptomycin/ml.
cGMP bioassay in T84 cells.
T84 cells were cultured in 24-well plastic dishes, and cellular cGMP
levels were measured in control and agonist-stimulated cells by
radioimmunoassay (12-15). Briefly, column fractions of the
synthetic peptides, uroguanylin and guanylin, were suspended in 200 µl of DMEM containing 20 mM
N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), pH 7.4 or 5.5 buffer, consisting of DMEM, 20 mM 2-(N-morpholino)-ethanesulfonic acid
(MES, pH 5.5), and 1 mM isobutylmethylxanthine (IBMX). The solutions
containing bioactive peptides were then added to cultured cells and
incubated at 37°C for 40 min. After incubation, the reaction medium
was aspirated and 200 µl of 3.3% perchloric acid were added per well
to stop the reaction and extract cGMP. The extract was adjusted to pH
7.0 with KOH and centrifuged, and 50 µl of the supernatant were used
to measure cGMP.
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RESULTS |
Purification of bioactive uroguanylin from urine.
Uroguanylin was purified from rat urine using
C18 Sep-Pak and gel filtration
chromatography, preparative isoelectric focusing, and a series of
reverse phase (RP)-HPLC steps (4, 13-15).
After the isolation of bioactive peptides with
C18 cartridges, a second
chromatographic step with a Sephadex G-25 column yielded a single peak
of peptides that stimulated cGMP accumulation in T84 cells (data not
shown). This peak of bioactive peptides eluted at a position identical
to that previously found for opossum uroguanylin (14, 15). Preparative
isoelectric focusing separates the more highly acidic uroguanylin from
guanylin (14, 15). The bioactive peptides eluting from Sephadex G-25
columns were subjected to preparative isoelectric focusing, and the
active peptides migrated to the most acidic region, eluting at pH
values of 2.4-3.7 in fractions 1-3 (Fig.
1). This peptide fraction from urine
stimulated cGMP accumulation in the T84 cells to a greater magnitude
when the medium pH was 5.5 compared with the stimulation at pH 7.4. The
profile of pH dependence for agonist activity in T84 cells is
consistent with this urine peptide being uroguanylin (12). This peptide
fraction was then combined and subjected to RP-HPLC by use of
C18 columns and a gradient of
5-25% acetonitrile containing 10 mM ammonium acetate, pH 6.2 (13-15). Under these RP-HPLC conditions, guanylin elutes at
16-18% acetonitrile, whereas uroguanylin elutes at 10-11%
acetonitrile. Fractions 1-3 from the isoelectric focusing purification step (Fig. 1) were combined for RP-HPLC under these conditions. A single peak of bioactive peptides eluted at 10% acetonitrile and 10 mM ammonium acetate, an elution pattern consistent with this peptide being uroguanylin (Fig.
2). This peak of bioactive peptides was
purified further using the same
C18 column by RP-HPLC with an
acetonitrile gradient containing 0.1% TFA (20, 21). The bioactive
peptides were eluted at 21% acetonitrile and were combined for
microbore RP-HPLC (data not shown). After further purification with
RP-HPLC with a C8 microbore column
(Fig. 3), the bioactive peptides in the
shaded portion of the ultraviolet absorbance tracing were combined and
subjected to NH2-terminal sequence
analysis (5, 20). A partial sequence of E/DXXELXINVAXTGX (X is unknown)
was obtained because of the low quantity of peptides remaining at this
stage of purification. The partial amino acid sequence obtained for the
rat urine peptide is similar to the corresponding residues reported for
opossum and human forms of bioactive uroguanylin isolated from urine
(14, 18) and identical to the deduced sequence from a uroguanylin EST
cDNA isolated from rat intestine (1). An acidic residue of either
glutamate or aspartate was observed at the position where glutamate is
found in opossum uroguanylin and where aspartate occurs in human
uroguanylin (14, 18). The amino acids identified by sequence analysis consisting of ELXINVAXTGX are identical to the corresponding residues found in opossum uroguanylin. Taken together, these findings suggest that uroguanylin is the major bioactive peptide appearing in the urine
of rats.

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Fig. 1.
Isolation of uroguanylin from rat urine by isoelectric focusing. Rat
urine was first chromatographed with
C18 Sep-Pak cartridges followed by
gel filtration chromatography with Sephadex G-25, as described in
MATERIALS AND METHODS. Active
fractions from the G-25 column were pooled, lyophilized, and subjected
to isoelectric focusing. Fractions were assayed using the T84 cell
guanosine 3',5'-cyclic monophosphate (cGMP) accumulation
bioassay under conditions of MES, DMEM at pH 5.5 (open bars), or HEPES
and DMEM at pH 7.4 (solid bars).
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Fig. 2.
Purification of uroguanylin from rat urine by reverse-phase
high-performance liquid chromatography (RP-HPLC). Active fractions from
the isoelectric focusing step were combined and subjected to RP-HPLC
using a C18 analytic column.
Peptides were eluted with a gradient from 5% acetonitrile containing
10 mM ammonium acetate to 25% acetonitrile containing 10 mM ammonium
acetate over a period of 180 min. Bioassay was conducted with T84 cells
in HEPES and DMEM at pH 7.4. Bioactive peptides eluted from this column
at 10% acetonitrile.
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Fig. 3.
Purification of uroguanylin by RP-HPLC from urine. Ultraviolet (UV)
absorbance of the last RP-HPLC step using a
C8 microbore column. Arbitrary
units for UV absorbance are used. Peak
4 (shaded area) contains the bioactive peptides eluted;
this fraction was subjected to sequence analysis. A residue of either
glutamate or aspartate was observed at the first position, and the
second position was not determined. The other 4 positions marked as X
correspond to the conserved cysteine residues within this family of
peptides. The partial amino acid sequence that was obtained is shown at
top.
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Purification of uroguanylin and guanylin from intestine.
Isolation of uroguanylin and prouroguanylin from colon and small
intestine and uroguanylin mRNA expression in the intestinal mucosa of
other species suggests that the intestine of rats may be a source of
uroguanylin in urine (5, 13, 15, 16). To investigate this possibility,
we isolated bioactive peptides from the mucosa of colon and duodenum
from rats. Extracts of colonic mucosa were prepared and purified by
C18 chromatography, followed by
Sephadex G-25 chromatography as described above. A single peak of
bioactive peptides was observed eluting from the Sephadex G-25 column
(data not shown). The active peptide peak was combined and subjected to
preparative isoelectric focusing. The bioactive peptides eluted in
fractions 1-3 with pH values of 2.6-3.5 (Fig. 4). At this stage of purification, the
peptide components from rat colon exhibited a property similar to that
of guanylin, because the colon peptides stimulated cGMP accumulation in
T84 cells to a greater level at the medium pH of 7.4 compared with the
cGMP responses at pH 5.5 (12). When the active fractions were combined and subjected to RP-HPLC with a
C18 analytic column, the bioactive peptides eluted at 15.5% acetonitrile (Fig.
5). This characteristic elution profile for
rat guanylin (4) indicates that the active peptides isolated from the
colonic mucosa of rats are predominantly guanylin. This peak of
guanylin-like peptides was purified further by use of
C18 RP-HPLC with an acetonitrile
gradient containing 0.1% TFA and finally by microbore RP-HPLC with a
C8 column as described above. This
peptide fraction was then subjected to
NH2-terminal sequence analysis,
and the 15-residue peptide PNTCEICAYAACTGC was obtained. This is the
same amino acid sequence as that obtained when guanylin was originally
isolated from the jejunum of rats (4).

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Fig. 4.
Isolation of guanylin from colonic mucosa by isoelectric focusing.
Extracts of colonic mucosa were chromatographed with
C18 Sep-Pak cartridges and then
fractionated on a Sephadex G-25 column before bioactive peptides were
subjected to isoelectric focusing. Each fraction was assayed with T84
cells in MES, DMEM at pH 5.5 (open bars), or HEPES and DMEM at pH 7.4 (solid bars).
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Fig. 5.
Purification of guanylin from colonic mucosa by RP-HPLC. Bioactive
fractions from the isoelectric focusing step were applied to a
C18 RP-HPLC analytic column and
eluted with acetonitrile/ammonium acetate, as described in Fig. 2.
Fractions were bioassayed using T84 cells in HEPES and DMEM adjusted to
pH 8.0 with 50 mM sodium bicarbonate. Peak of bioactive peptides eluted
from this column at 15.5% acetonitrile is similar to that of authentic
guanylin.
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The duodenum may produce uroguanylin, because the content of guanylin
mRNA in duodenum of rats is considerably lower than the mRNA levels of
colon, and the duodenum has substantial cGMP responses to these
peptides (20, 21, 31). Bioactive peptides were isolated from the mucosa
of rat duodenum, and two separate peaks of peptide bioactivity eluted
at different positions within the internal volume of Sephadex G-25
columns (Fig. 6). When these fractions were
bioassayed using T84 cells, we found that peak 1 stimulated cGMP accumulation greater at pH 5.0 than
at pH 8.0 (uroguanylin-like) and that peak
2 stimulated cGMP accumulation greater at pH 8.0 than
at pH 5.0 (guanylin-like). The very low stimulation of cGMP
accumulation observed for the peak 1 aliquot at pH 8.0 and the correspondingly low stimulation for the
peak 2 aliquot at pH 5.0 may be
explained by the relatively low concentrations of these peptides in the
aliquots from the columns that were bioassayed. Peak
1 (uroguanylin) was pooled and further purified by
C18 RP-HPLC by use of a
5-25% acetonitrile gradient containing ammonium acetate. The
bioactive peptides eluted at 11% acetonitrile, which is consistent with this peptide being uroguanylin (Fig.
7). Moreover, this peptide stimulated cGMP
accumulation greater at the medium pH of 5.0 than at pH 8.0, which is
also a property found in the uroguanylin peptides. To summarize, the
chromatographic elution profile using RP-HPLC and the pH
dependency for activation of receptor guanylate cyclases (GCs) of
this peptide from the duodenum mucosa are characteristic properties of
uroguanylin. An insufficient quantity of the purified uroguanylin-like
peptide was available for
NH2-terminal sequence analysis;
thus confirmation of these findings by elucidation of the peptide's
sequence was not possible.

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Fig. 6.
Separation of uroguanylin-like and guanylin-like peptides from duodenum
by gel filtration chromatography. Mucosa from duodenum was heated at
100°C in 1 M acetic acid; then extracts were fractionated with
C18 Sep-Pak before application to
a Sephadex G-25 column. Fractions were assayed using the T84 cell cGMP
accumulation bioassay in MES and DMEM at pH 5.0 (dashed line) and in
HEPES and DMEM adjusted to pH 8.0 with 50 mM sodium bicarbonate (solid
line).
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Fig. 7.
Isolation of uroguanylin from duodenum by RP-HPLC.
Peak 1 from the Sephadex G-25 gel
filtration column step was combined and subjected to
C18 semipreparative RP-HPLC and
fractionated with a gradient of acetonitrile containing 10 mM ammonium
acetate, as described in Fig. 2. Eluted fractions were assayed by T84
cell cGMP stimulation bioassay in MES and DMEM at pH 5.0 (dashed line)
and in HEPES and DMEM adjusted to pH 8.0 with 50 mM sodium bicarbonate
(solid line). Major peak of bioactive peptides eluted from this column
at 11% acetonitrile, consistent with the chromatographic properties of
uroguanylin.
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A partial cDNA EST encoding the COOH-terminal portion of prouroguanylin
was isolated from the duodenum of zinc-deficient rats (1). This
information facilitated the production of a uroguanylin cDNA by use of
reverse transcription of RNA from rat duodenum and the PCR to amplify
this form of uroguanylin cDNA. The uroguanylin cDNA was cloned and
sequenced to confirm its identity and then used as a cDNA probe in
Northern assays to assess the relative abundance of uroguanylin mRNA
compared with guanylin mRNA in the intestine. Uroguanylin transcripts
of ~0.75 kilobase (kb) were detected throughout the intestinal tract,
but the highest levels were found in the duodenum and jejunum of small
intestine (Fig. 8). Lower levels of
uroguanylin mRNA were observed in ileum and the cecum and colon
compared with duodenum and jejunum. Guanylin mRNA transcripts of ~0.6
kb were detected throughout the intestinal tract, with the highest mRNA
levels observed in cecum and colon compared with the levels in small
intestine. The lowest guanylin mRNA levels were found in the duodenum
relative to other segments of intestine. Progressively greater levels
of guanylin mRNA were found along the longitudinal axis of the small
intestine from duodenum to ileum, with the greatest mRNA levels
observed in the cecum and colon.

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Fig. 8.
Distribution of uroguanylin and guanylin mRNA in the intestine. Total
RNA of 20 µg from mucosa of rat proximal small intestine (Prox. SI),
middle small intestine (Mid. SI), distal small intestine (Dist. SI),
cecum, and colon were loaded on each lane.
A: arrows mark single transcripts for
-actin mRNA of 1.9 kilobase (kb) and uroguanylin mRNA of 0.75 kb;
B: arrows indicate single transcripts
for -actin mRNA of 1.9 kb and guanylin mRNA of 0.6 kb.
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Analysis of the EST for uroguanylin derived from rat intestine (1)
confirmed that the partial amino acid sequence obtained for the urinary
peptide was consistent with the sequence predicted by the uroguanylin
EST. Thus a synthetic peptide was prepared on the basis of the amino
acid sequence TDECELCINVACTGC, and the potency of this peptide was
compared with the potencies of rat guanylin and a truncated form of
uroguanylin, CELCINVACTGC, by use of the T84 cell bioassay (4, 14).
Uroguanylin and guanylin had similar potencies in the activation of
receptor GCs in T84 cells, but the truncated form of uroguanylin was
substantially less potent (Fig. 9). These
data indicate that the
NH2-terminal residues found in the
bioactive uroguanylin peptide consisting of TDE increase the potencies
of this peptide agonist for activation of receptor GCs on T84 cells
compared with the potency of the truncated 12-residue form of
uroguanylin. However, the 12 amino acids in the truncated uroguanylin
analog containing the peptide domain between the first and last
cysteine residues with two intramolecular disulfide bonds represent a
core structure that is required for biological activity in this assay.

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Fig. 9.
Bioactivity of synthetic uroguanylin and guanylin in T84 cells. Values
are representative of 3 experiments conducted with cultured T84 cells
and are means of duplicate assays at each peptide concentration. ,
Rat guanylin (PNTCEICAYAACTGC); , rat uroguanylin (TDECELCINVACTGC);
, 12-residue portion of uroguanylin (CELCINVACTGC). Disulfide bonds
in these synthetic peptides occur between 1st to 3rd and 2nd to 4th
cysteine residues. Medium is DMEM at pH 7.4 for this assay.
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DISCUSSION |
Uroguanylin was isolated from both urine and duodenum mucosa of rats
and identified by its unique biochemical and pharmacological properties
(12-15). Uroguanylin is present in the urine of rats, as it is in
the urine of the opossum and human species (14, 18). Guanylin was
isolated from mucosa of both the duodenum and large intestine, but
active guanylin peptides were not detected in urine. Sequence analysis
of uroguanylin from rat urine revealed that the eight residues obtained
were identical to those found in opossum uroguanylin. One of the two
NH2-terminal acidic amino acids
unique to uroguanylin was not clearly defined (Glu or Asp), and the
other acidic amino acid was not determined. The amino acid sequence of
rat uroguanylin has been recently elucidated by the isolation of cDNA
clones encoding preprouroguanylin and by purification of uroguanylin
from duodenum and NH2-terminal sequence analysis (1, 26, 29). These studies revealed that the sequence
of the 15 COOH-terminal residues for rat uroguanylin is
TDECELCINVACTGC, which agrees with the partial sequence that we
obtained in the present study. A synthetic peptide prepared according
to this sequence activated the T84 cell receptor GC with potency and
efficacy similar to the activation elicited by synthetic rat guanylin.
The reason bioactive guanylin is not found in rat or human urine is
unclear, but it may be the susceptibility of guanylin in the tubular
filtrate to cleavage and inactivation by proteases within renal
tubules. Guanylin is inactivated by chymotrypsin, which cleaves the
peptide bond COOH terminal to the aromatic residues of guanylin
peptides (13, 15). In contrast, uroguanylin and Escherichia coli ST are resistant to
chymotrypsin because these peptides have asparagine residues instead of
tyrosine or phenylalanine (13-15, 18). Plasma uroguanylin and
guanylin may enter the tubular filtrate by glomerular filtration (5,
19). Also, recent evidence suggests that the kidney produces
uroguanylin, because mRNAs encoding this peptide are expressed in the
kidney (29). The presence of bioactive uroguanylin in urine is
significant because renal receptors for uroguanylin are localized on
the apical membranes of proximal tubular cells (9, 10, 21). Circulating
uroguanylin enters the tubules by glomerular filtration to gain access
to these receptors. Activation of the receptor GCs in the perfused rat
kidney elicits a natriuresis, kaliuresis, and diuresis (unpublished observations). Thus uroguanylin may serve in an endocrine pathway to
regulate renal function in vivo (unpublished observations; 8-10).
Recent evidence that low-sodium diets downregulate the guanylin
receptor-GC signaling pathway in the rat colon suggests that the
guanylin signaling pathway may participate in the maintenance of salt
and water homeostasis (24).
In rats, guanylin mRNA levels appear to be most abundant in colon and
ileum, with intermediate mRNA levels in jejunum and the lowest mRNA
levels in duodenum (25, 27, 31). In the present experiments, bioactive
guanylin was isolated from colonic mucosa but bioactive uroguanylin was
not detected. This finding is consistent with the high levels of
guanylin mRNA that were detected using Northern assays with total RNA
from colon and cecum in this study compared with the lower uroguanylin
mRNA levels of large intestine. The lower abundance of uroguanylin
mRNAs in the colon and cecum provides one explanation for our inability to detect bioactive uroguanylin in extracts of large intestine. Isolation of uroguanylin and guanylin from duodenum in this study suggests that both peptides are present and may regulate the activity of receptor-GC signaling molecules in this segment. Whereas Northern assays suggest that uroguanylin mRNA expression is greater than guanylin mRNA expression in the duodenum, both peptides are present in
the mucosa of the duodenum in concentrations sufficient for purification and identification of this bioactive peptide (25, 27, 29).
Other studies have also found a similar pattern of expression of
guanylin and uroguanylin mRNA levels along the longitudinal axis of the
intestinal tract of rats (25, 27, 29).
Uroguanylin and guanylin markedly stimulate the transepithelial
secretion of both Cl
and
anions in the duodenum (11, 17). Exposure of the apical surface of duodenum to these peptides elicits a
stimulation of short-circuit current consisting of both
Cl
and
transport components. Both
peptides may be released from enterocytes into the luminal microdomain at the surface of this epithelium where binding of the peptides to
receptor-GCs occurs, thus activating these signaling molecules and
regulating anion secretion via intracellular cGMP. Because the potency
of uroguanylin is markedly enhanced when the intraluminal pH is acidic
and an acidic pH markedly decreases the potency of guanylin, it may be
postulated that the secretion of uroguanylin is increased when acidic
chyme is delivered from the stomach to the duodenum (12, 13, 15). The
relative potencies of guanylin and uroguanylin for activation of
intestinal receptor GCs and the stimulation of transepithelial
Cl
secretion are markedly
influenced by mucosal acidity (12). In the present study, uroguanylin
isolated from rat duodenum (or urine) stimulates cGMP accumulation in
T84 cells to a greater magnitude at a medium pH of 5.5 than at pH 7.5. Guanylin isolated from the intestine increased cGMP to a greater level
in T84 cells at pH 7.5 than at pH 5.5. Thus uroguanylin and guanylin
isolated from rat intestine exhibit properties similar to those
previously defined for the homologous peptides derived from human
subjects and opossums (12, 13, 15). Evolution of the unique primary structures for uroguanylin and guanylin may have occurred to
allow different peptide hormones that function
cooperatively to regulate intestinal
Cl
and
secretion during digestion. The
lumen of the intestine and the mucosal (microclimate) surface is
acidified when chyme containing HCl enters the duodenum. Under this
condition, uroguanylin may be a more effective agonist for regulating
receptor-GC activity than is guanylin. When the mucosal surface of the
duodenum becomes alkalinized through enhanced
secretion, the affinity of
guanylin for binding to receptor GCs would be increased, thus
facilitating the binding of guanylin to receptors and activation of
these signaling molecules (12, 13, 15). Thus uroguanylin and guanylin
may participate in a cGMP signaling pathway controlling intestinal
Cl
and
secretion (4, 6, 11, 14, 17).
In summary, uroguanylin was isolated from urine and the duodenum of
rats. Bioactive guanylin was not detected in urine, but this peptide
was isolated from colon and duodenum. Uroguanylin mRNA levels were
greater in small than in large intestine, whereas the levels of
guanylin mRNA transcripts were greater in large than in small
intestine. Synthetic rat uroguanylin was approximately equipotent to
rat guanylin in the activation of cGMP production in T84 intestinal
cells when assessed at the medium pH of 7.4. Uroguanylin and guanylin
may both participate in the regulation of
Cl
and
secretion via the intracellular
second messenger cGMP.
 |
FOOTNOTES |
Address for reprint requests: L. R. Forte, Dept. of Pharmacology,
M-515 Medical Sciences Bldg., School of Medicine, Univ. of Missouri,
Columbia, MO 65212.
Received 14 March 1997; accepted in final form 30 July 1997.
 |
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