Structure and activity of uroguanylin and guanylin
`from the intestine and urine of rats
`
`XIAOHUI FAN,1,2 F. KENT HAMRA,1,2 ROSLYN M. LONDON,1,2
`SAMMY L. EBER,1,2 WILLIAM J. KRAUSE,2 RONALD H. FREEMAN,2
`CHRISTINE E. SMITH,3 MARK G. CURRIE,3 AND LEONARD R. FORTE1,2
`1
`2
`
`3
`
`Fan, Xiaohui, F. Kent Hamra, Roslyn M. London,
`Sammy L. Eber, William J. Krause, Ronald H. Freeman,
`Christine E. Smith, Mark G. Currie, and Leonard R.
`Forte. Structure and activity of uroguanylin and guanylin
`from the intestine and urine of rats.
`273
`(
`36): E957²E964, 1997.ÑUroguanylin
`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 guano-
`sine 3!,5!-cyclic monophosphate signaling pathway that regu-
`lates the transepithelial secretion of chloride and bicarbonate
`in the intestinal epithelium.
`guanylate cyclase; guanosine 3!,5!-cyclic monophosphate; kid-
`ney; heat-stable enterotoxin; human T84 intestinal cells
`
`

`

`E958
`
`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`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
`€Bondapak, 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 3²10 (Bio-Rad)], and subjected to
`preparative isoelectric focusing (Rotorfor, Bio-Rad). The frac-
`tions containing bioactivity were combined, applied to a C18
`HPLC column (Waters analytic €Bondapak, 3.9 mm " 30 cm),
`and eluted with a gradient of 5% acetonitrile-10 mM ammo-
`nium 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).
`
`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
`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.
`
`Total
`RNA was prepared (RNeasy kit, Qiagen) from the mucosa of
`individual intestinal segments, and 20 €g of each RNA
`preparation were subjected to electrophoresis in formalde-
`hyde-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 68ÊC, which was followed by hybridization for 2
`h at 68ÊC 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 60ÊC. The exposure to
`film was for 24 h at !80ÊC with intensifying screens. Rat
`uroguanylin cDNA (nucleotides 117²292) was produced by
`polymerase chain reaction (PCR) amplification from intesti-
`nal mRNA-cDNA (1, 27). This cDNA was isolated and se-
`quenced to confirm that it matched the uroguanylin ex-
`pressed sequence tag (EST) of rat uroguanylin with 100%
`identity. A rat guanylin cDNA (nucleotides 1²531) was gen-
`
`erously provided by Dr. Roger Weigand (Monsanto, St.
`Louis, MO).
`
`T84 cells were obtained from Dr. Jim McRob-
`erts (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), supple-
`mented with 5% fetal bovine serum, 60 mg penicillin/ml, and
`100 mg streptomycin/ml.
`
`T84 cells were cultured in
`24-well plastic dishes, and cellular cGMP levels were mea-
`sured in control and agonist-stimulated cells by radioimmuno-
`assay (12²15). Briefly, column fractions of the synthetic
`peptides, uroguanylin and guanylin, were suspended in 200
`€l of DMEM containing 20 mM -(2-hydroxyethyl)piperazine-
`!-(2-ethanesulfonic acid) (HEPES), pH 7.4 or 5.5 buffer,
`consisting of DMEM, 20 mM 2-(
`-morpholino)-ethanesul-
`fonic acid (MES, pH 5.5), and 1 mM isobutylmethylxanthine
`(IBMX). The solutions containing bioactive peptides were
`then added to cultured cells and incubated at 37ÊC for 40 min.
`After incubation, the reaction medium was aspirated and 200
`€l 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 €l of the supernatant
`were used to measure cGMP.
`
`RESULTS
`
`Uroguanylin was purified from rat urine using C18
`Sep-Pak and gel filtration chromatography, prepara-
`tive isoelectric focusing, and a series of reverse phase
`(RP)-HPLC steps (4, 13²15).
`After the isolation of bioactive peptides with C18
`cartridges, a second chromatographic step with a Sepha-
`dex 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 2.4²3.7
`in fractions 1²3 (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 5²25% acetonitrile containing 10 mM ammo-
`nium acetate, pH 6.2 (13²15). Under these RP-HPLC
`conditions, guanylin elutes at 16²18% acetonitrile,
`whereas uroguanylin elutes at 10²11% acetonitrile.
`Fractions 1²3 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 uro-
`guanylin (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%
`
`Downloaded from journals.physiology.org/journal/ajpendo (165.001.202.250) on January 18, 2022.
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`

`

`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`E959
`
`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).
`
`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 ultravio-
`let absorbance tracing were combined and subjected to
`NH2-terminal sequence analysis (5, 20). A partial se-
`quence of E/DXXELXINVAXTGX (X is unknown) was
`obtained because of the low quantity of peptides remain-
`ing 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 intes-
`tine (1). An acidic residue of either glutamate or
`aspartate was observed at the position where gluta-
`mate 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
`
`Fig. 3. Purification of uroguanylin by RP-HPLC from urine. Ultravio-
`let (UV) absorbance of the last RP-HPLC step using a C8 microbore
`column. Arbitrary units for UV absorbance are used.
`(shaded
`area) contains the bioactive peptides eluted; this fraction was sub-
`jected to sequence analysis. A residue of either glutamate or aspar-
`tate 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.
`
`residues found in opossum uroguanylin. Taken to-
`gether, these findings suggest that uroguanylin is the
`major bioactive peptide appearing in the urine of rats.
`
`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 chromatog-
`raphy 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 focus-
`ing. The bioactive peptides eluted in fractions 1²3 with
`pH values of 2.6²3.5 (Fig. 4). At this stage of purifica-
`tion, 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
`
`Fig. 2. Purification of uroguanylin from rat urine by reverse-phase
`high-performance liquid chromatography (RP-HPLC). Active frac-
`tions 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.
`
`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 focus-
`ing. 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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`

`

`E960
`
`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`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 sub-
`jected to NH2-terminal sequence analysis, and the
`15-residue peptide PNTCEICAYAACTGC was ob-
`tained. This is the same amino acid sequence as that
`obtained when guanylin was originally isolated from
`the jejunum of rats (4).
`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
`stimulated cGMP
`accumulation greater at pH 5.0 than at pH 8.0 (urogua-
`nylin-like) and that
`stimulated cGMP accumula-
`tion greater at pH 8.0 than at pH 5.0 (guanylin-like).
`The very low stimulation of cGMP accumulation ob-
`served for the
`aliquot at pH 8.0 and the
`correspondingly low stimulation for the
`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.
`(urogua-
`nylin) was pooled and further purified by C18 RP-HPLC
`by use of a 5²25% 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 chro-
`
`Fig. 6. Separation of uroguanylin-like and guanylin-like peptides
`from duodenum by gel filtration chromatography. Mucosa from
`duodenum was heated at 100ÊC 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 accumula-
`tion 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 bicarbon-
`ate (solid line).
`
`matographic 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 elucida-
`tion of the peptide's sequence was not possible.
`A partial cDNA EST encoding the COOH-terminal
`portion of prouroguanylin was isolated from the duode-
`num of zinc-deficient rats (1). This information facili-
`tated 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 con-
`firm 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 kilo-
`
`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/ammo-
`nium 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.
`
`Fig. 7. Isolation of uroguanylin from duodenum by RP-HPLC.
`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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`

`

`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`E961
`
`base (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 jeju-
`num. Guanylin mRNA transcripts of !0.6 kb were
`detected throughout the intestinal tract, with the high-
`est 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 lev-
`els of guanylin mRNA were found along the longitudi-
`nal axis of the small intestine from duodenum to ileum,
`with the greatest mRNA levels observed in the cecum
`and colon.
`Analysis of the EST for uroguanylin derived from rat
`intestine (1) confirmed that the partial amino acid
`sequence obtained for the urinary peptide was consis-
`tent with the sequence predicted by the uroguanylin
`EST. Thus a synthetic peptide was prepared on the
`basis of the amino acid sequence TDECELCINVAC-
`TGC, 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 recep-
`tor 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.
`
`DISCUSSION
`
`Uroguanylin was isolated from both urine and duode-
`num mucosa of rats and identified by its unique bio-
`chemical and pharmacological properties (12²15). Uro-
`guanylin 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 duode-
`num and large intestine, but active guanylin peptides
`were not detected in urine. Sequence analysis of urogua-
`nylin 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
`
`Fig. 8. Distribution of uroguanylin and gua-
`nylin mRNA in the intestine. Total RNA of 20 €g
`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.
`: arrows mark
`single transcripts for #-actin mRNA of 1.9 kilo-
`base (kb) and uroguanylin mRNA of 0.75 kb;
`:
`arrows indicate single transcripts for #-actin
`mRNA of 1.9 kb and guanylin mRNA of 0.6 kb.
`
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`

`

`E962
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`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`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 find-
`ing 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 pep-
`tides 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 HCO3! anions
`
`in the duodenum (11, 17). Exposure of the apical
`surface of duodenum to these peptides elicits a stimula-
`tion of short-circuit current consisting of both Cl! and
`HCO3! transport components. Both peptides may be
`
`released from enterocytes into the luminal microdo-
`main at the surface of this epithelium where binding of
`the peptides to receptor-GCs occurs, thus activating
`these signaling molecules and regulating anion secre-
`tion via intracellular cGMP. Because the potency of
`uroguanylin is markedly enhanced when the intralumi-
`nal 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 stimu-
`lation 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 intes-
`tine exhibit properties similar to those previously de-
`fined for the homologous peptides derived from human
`subjects and opossums (12, 13, 15). Evolution of the
`unique primary structures for uroguanylin and gua-
`nylin may have occurred to allow different peptide
`
`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 uro-
`guanylin (TDECELCINVACTGC); #, 12-residue portion of urogua-
`nylin (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.
`
`determined. The amino acid sequence of rat urogua-
`nylin has been recently elucidated by the isolation of
`cDNA clones encoding preprouroguanylin and by purifi-
`cation 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 ob-
`tained in the present study. A synthetic peptide pre-
`pared 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. Gua-
`nylin is inactivated by chymotrypsin, which cleaves the
`peptide bond COOH terminal to the aromatic residues
`of guanylin peptides (13, 15). In contrast, uroguanylin
`and
`ST are resistant to chymotrypsin
`because these peptides have asparagine residues in-
`stead of tyrosine or phenylalanine (13²15, 18). Plasma
`uroguanylin and guanylin may enter the tubular fil-
`trate by glomerular filtration (5, 19). Also, recent
`evidence suggests that the kidney produces urogua-
`nylin, because mRNAs encoding this peptide are ex-
`pressed in the kidney (29). The presence of bioactive
`uroguanylin in urine is significant because renal recep-
`tors for uroguanylin are localized on the apical mem-
`branes 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 obser-
`vations). Thus uroguanylin may serve in an endocrine
`pathway to regulate renal function in vivo (unpub-
`lished observations; 8²10). Recent evidence that low-
`
`Downloaded from journals.physiology.org/journal/ajpendo (165.001.202.250) on January 18, 2022.
`
`

`

`STRUCTURE AND ACTIVITY OF UROGUANYLIN AND GUANYLIN
`
`E963
`
`hormones that function cooperatively to regulate intes-
`tinal Cl! and HCO3! 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
`HCO3! 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 HCO3! 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 urogua-
`nylin 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. Urogua-
`nylin and guanylin may both participate in the regula-
`tion of Cl! and HCO3! secretion via the intracellular
`
`second messenger cGMP.
`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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