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The Truman Veterans Administration Medical Center (X.F., Y.W., R.M.L., S.L.E., L.R.F.) and the Departments of Pharmacology (X.F., Y.W., R.M.L., S.L.E.), Pathology and Anatomical Sciences (W.J.K.), and Physiology (R.H.F.), Missouri University School of Medicine, Columbia, Missouri 65212
Address all correspondence and requests for reprints to: Dr. Leonard R. Forte, Department of Pharmacology, University of Missouri School of Medicine, M-515 Medical Sciences Building, Columbia, Missouri 65212. E-mail: Leonard-R.-Forte{at}muccmail.missouri.edu
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Guanylin was purified from rat jejunum as a small, heat-stable peptide that stimulates cGMP accumulation in T84 human intestinal cells (1). Preproguanylin cDNAs were then isolated, demonstrating that the precursor molecule is a protein of 115–116 amino acids in the rat, human, and mouse intestine (14, 15, 16, 17). Guanylin messenger RNAs (mRNAs) are more abundant in the large intestine than in the small intestine in rats and humans (14, 15). Immunoreactive guanylin has been localized to several cell types, including goblet cells, absorptive enterocytes, and enterochromaffin cells (18, 19, 20, 21). Lower levels of guanylin mRNA were also detected by Northern analyses in RNA preparations obtained from adrenal gland, kidney, and uterus/oviduct of rats (21). Guanylin and proguanylin were isolated from the hemodialysates of patients with chronic renal failure, indicating that this peptide is secreted into the circulation in patients with this disease (22, 23). Proguanylin and/or guanylin have not been isolated and identified at the molecular level from the plasma of normal animals or human subjects (24). Therefore, it is unclear whether guanylin is a circulating peptide hormone under normal physiological conditions.
Uroguanylin was initially isolated from the urine of opossums, and the primary structure was elucidated by N-terminal sequence and mass spectrometry analyses applied to the 13-, 14-, and 15-residue forms of the bioactive peptides (2). Subsequently, uroguanylin was isolated from human (25) and rat urine (26), and both uroguanylin and prouroguanylin were purified from the opossum intestine (27, 28). Complementary DNAs encoding the opossum (24) and human (29, 30) forms of preprouroguanylin were recently isolated that encode 109- and 112-amino acid preprouroguanylin polypeptides, respectively. Northern analyses demonstrated that both uroguanylin and guanylin mRNAs occur throughout the small and large intestines of opossums (24). Uroguanylin mRNAs are most abundant in the cecum and colon, whereas guanylin mRNAs are highest in the jejunum and ileum of this species. In addition, uroguanylin (but not guanylin) mRNA transcripts were detected in RNA from atria and ventricles of the heart. In rats, uroguanylin mRNAs are greatest in the small intestine, and guanylin mRNAs are most abundant in the large intestine (9, 14, 31). An active form of uroguanylin and the inactive prouroguanylin precursor were both isolated from plasma, revealing that uroguanylin is a circulating peptide in normal animals (24). In patients with chronic renal failure, the plasma levels of uroguanylin increase substantially, indicating that the kidney may clear uroguanylin from the circulation (32, 33). An endocrine link between intestine and kidney was proposed for uroguanylin acting in vivo to regulate kidney function (34). Recent demonstrations of the natriuretic, kaliuretic, and diuretic activities of uroguanylin in both the opossum in vivo and in perfused kidneys of the rat in vitro are important elements contributing to the hypothesis that uroguanylin influences salt and water homeostasis as an intestinal natriuretic hormone (35, 36).
Consistent with the expression pattern for guanylin and uroguanylin mRNAs, the receptor-GC signaling molecules for these peptide agonists are also found on enterocytes lining the intestine. Receptors for uroguanylin, guanylin, and bacterial ST peptides were identified previously through application of several biochemical techniques, including [125I]ST receptor autoradiography in situ, cGMP accumulation responses in vitro and in vivo to the agonist peptides, as well as Northern and in situ hybridization histochemistry analyses detecting GC-C mRNA in the stomach and intestine (6, 7, 8, 9, 19). In vitro receptor autoradiography and cGMP bioassays demonstrated that receptor-GCs also occur in proximal tubules of the kidney, hepatocytes of liver, seminiferous tubules of testis, and epithelial cells lining the gall bladder, bile duct, and trachea of opossums (7, 8, 37). Subsequently, Northern analyses and reverse transcription (RT)-PCR amplification methods detected GC-C mRNA in the stomach, adrenal gland, brain, perinatal liver, placenta, and testis of rats and in the human and bovine airway epithelia (9, 21, 38). The intestinal receptor-GCs are thought to be activated by intraluminally secreted guanylin and/or uroguanylin as first messengers in paracrine and/or autocrine signaling pathways (34). Such a mechanism is supported by the demonstration that both guanylin and uroguanylin accumulate in solutions perfused through the lumen of the small intestine of rats in vivo (39). Regulation of receptor-GCs by guanylin and uroguanylin in the kidney may occur via circulating uroguanylin and prouroguanylin entering the tubules by glomerular filtration. Also, uroguanylin and/or guanylin may be produced in tubular cells and secreted into the filtrate within close proximity to target cells in the nephron in a fashion similar to the intrinsic mechanisms found in the intestine.
In the present study, we report the isolation of cDNAs encoding opossum preproguanylin and the catalytic domain of a receptor-GC expressed in OK cells (GC-OK), transiently express the preproguanylin cDNA in COS-1 cells to assess the bioactivity of proguanylin, compare the biological activities of synthetic preparations of guanylin and uroguanylin in the kidney cortex and OK cells, and compare the expression of guanylin, uroguanylin, and GC-OK mRNAs in tissues within the digestive, renal, central nervous, reproductive, and immune/lymphoid organ systems. Uroguanylin, guanylin, and GC-OK mRNAs are widely expressed in tissues outside the intestinal tract of the opossum. Additional studies were conducted using the duodenum, kidney, testis, spleen, and cerebellum to confirm that the molecular machinery required for this signal transduction pathway occurs in these tissues. Thus, uroguanylin and guanylin may regulate cellular cGMP production in target cells within many tissues and organs of the opossum.
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Materials and Methods |
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Cloning of opossum preproguanylin cDNAs
Complementary DNAs encoding preproguanylin were produced using
RT of intestinal RNAs to prepare cDNAs followed by rapid amplification
of cDNA ends (RACE)-PCR. Total RNA was extracted from opossum colon
using the RNeasy kit (Qiagen, Chatworth, CA). The amounts of RNA
isolated were measured, and the quality of the RNAs were verified by
agarose gel electrophoresis and ethidium bromide staining.
Oligo(deoxythymidine)15-primed cDNAs were synthesized from
3 µg total RNA using reverse transcriptase (Superscript II, Life
Technologies, Gaithersburg, MD). Two of the primers used, SHTN-22
[5'-GGAGA(TC)TT(TC)TCCTA(TC)CCTCTGG-3'] and SHTC-22
[5'-GC(AG)AAGGCACAGAT (CT)TCACATG-3'] were derived from the
N-terminal amino acid sequence (GDFSYPLE) and C-terminal residues
(TCEICAFA) of opossum proguanylin, respectively (2, 27). These
oligonucleotide primers generated a 210-bp cDNA product (nucleotides
75–284; Fig. 1A
) when colon cDNA was
used as template and 30 cycles of the PCR were carried out at 93 C for
1 min, 58 C for 1 min, and 72 C for 1.5 min using Taq DNA
polymerase (U.S. Biochemical Corp., Cleveland, OH). The PCR-generated
cDNA products were isolated by electrophoresis using 1% agarose gels,
purified (Gel Extraction kit, Qiagen), and ligated into the plasmid
vector pCRII (TA Cloning kit, Invitrogen, San Diego, CA), and molecular
cDNA clones were sequenced. Automated sequencing was performed by the
DNA Core Laboratory of the Missouri University Molecular Biology
Program. Two independent clones were sequenced to confirm that the PCR
product encoded the expected portion of proguanylin.
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A 5'-RACE System (Life Technologies) was employed to extend this proguanylin cDNA to synthesize the 5'-end of the preproguanylin mRNA. The cDNAs prepared by RT from ileum RNAs were primed with a guanylin-specific oligonucleotide primer, 5'-TGGAAAGCCTGGGCTGCA-3'. An anchor homopolymetric dC tail was then added to the 3'-end of the cDNA using terminal deoxynucleotidyl transferase and deoxy-CTP. The first round of PCR amplification was carried out using Taq DNA polymerase, an anchor primer provided with the system, 5'-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGII-GGGIIG-3', and a second guanylin-specific oligonucleotide primer, 5'-GGGTGCAGATGGGTTTCAAG-3'. Five percent of the PCR mixture was transfered into the second round of PCR. The primers for this PCR were a third guanylin-specific oligonucleotide, 5'-GAAGATTCGGGTGGGCGCAC-3', and a general primer containing the 5'-portion of the anchor primer 5'-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3'. The resulting 203-bp cDNA product was cloned by ligation into pCRII and then sequenced. The conditions for both 3'-RACE-PCR and 5'-RACE-PCR were 30 cycles at 93 C for 1 min, 60 C for 1 min, and 72 C for 1.5 min. At least two independent clones from 5'-RACE-PCR and 3'-RACE-PCR were sequenced to determine whether PCR-induced errors had occurred in the cDNA sequences.
Expression of preproguanylin in COS-1 cells
The preproguanylin cDNA containing 24 nucleotides of the
5'-untranslated region, the 300 nucleotides of open reading frame
(ORF), and 41 nucleotides of the 3'-untranslated region was generated
using RT-PCR from opossum ileum cDNA. This cDNA was ligated into the
pCDM8 expression vector at HindIII and XhoI sites
(pCDM8-Guan), and the construct was confirmed by sequencing. COS-1
cells cultured in 100-mm dishes were transfected with 3 µg pCDM8
vector or pCDM8-Guan using the diethylaminoethyl-dextran procedure
(41). Conditioned medium samples were collected 48 and 72 h after
transfection.
For the chymotrypsin pretreatment experiments, the medium samples were heated at 100 C for 30 min in 1 M acetic acid, which has been shown to release the C-terminal guanylin peptide, thus activating the prohormone (27, 28). The resulting guanylin peptides were isolated from the medium using C18 Sep-Pak cartridges (octadecylsilane, Waters Associates, Milford, MA) as previously described (2, 27, 28). The peptides eluting from the C18 cartridges with 25% acetonitrile-0.1% trifluoroacetic acid (TFA) were dried and reconstituted in 10 mM HEPES, pH 8.0, and then pretreated with 3 µg chymotrypsin (Sigma Chemical Co., St. Louis, MO)/100 µl or with vehicle as previously described (24, 27, 28). The subsequent peptide fractions were bioassayed using the cGMP accumulation response to guanylin in cultured human T84 intestinal cells.
The peptides in conditioned medium samples were also extracted with C18 cartridges without heating the samples at 100 C. Under these conditions, the proguanylin molecule is preserved, whereas heating proguanylin under the conditions described above cleaves this polypeptide to produce the C-terminal guanylin peptide (27). The polypeptides in conditioned medium from COS-1 cells were eluted from C18 cartridges first with 25% acteonitrile, 0.1% TFA and then with 50% acetonitrile-0.1% TFA. A portion of these peptide fractions was then bioassayed in T84 cells in the native state, and another aliquot was treated for 30 min at 100 C in 1 M acetic acid. The samples were then bioassayed in T84 cells (1, 2).
Cloning of the GC-OK cDNA
Total RNA was made from cultured OK cells as described above.
Two PCR primers, OK-GC primer (5'-GAAACCCTTCCGCCCAGAT-3') and
OK-DEG-REV primer (5'-CCTCTTCCCTTTAAGTA(CT)GT-3'), were designed from
regions flanking a highly conserved segment of the rat and human GC-C
catalytic domain (4, 5). A PCR product of the expected size of about
0.9 kilobase (kb) was amplified from the OK cell template cDNA after 30
cycles at 93 C for 1 min, 50 C for 1 min, and 72 C for 2 min using
Taq DNA polymerase. The PCR-generated cDNA product was
isolated by molecular cloning and sequenced as described above.
RT-PCR analysis of GC-OK mRNAs
Total RNA preparations from opossum tissues were used in cDNA
synthesis for RT-PCR amplification of the OK-GC cDNA. The PCR
amplification reaction conditions were 30 cycles at 93 C for 1 min, 55
C for 1 min, and 72 C for 1.5 min. The primers were OK51 primer
(5'-ACAACGAGAGTTACATGGACACC-3') and OK31 primer
(5'-GGATTCCATCCT-TGAGGCTGTG-3'), which were expected to amplify a cDNA
product of 599 bp. The PCR products were fractionated on 1% agarose
gels and visualized with ethidium bromide.
Northern assays of GC-OK mRNAs
Total RNA was prepared as described above, and 20 µg of each
RNA preparation were subjected to electrophoresis in
formaldehyde-agarose gels and then transferred to nylon membranes (MSI,
Westboro, MA). The blots were hybridized with the 0.9-kb OK-GC cDNA and
ß-actin cDNA. Prehybridization was performed for 2 h with
ExpressHyb (Clontech, Palo Alto, CA) at 68 C, followed by hybridization
for 4 h at 68 C, with each cDNA probe labeled by random priming
(Boehringer Mannheim, Indianapolis, IN). The blots were washed twice
with 2 x SSC (standard saline citrate)-0.1% SDS for 15 min at
room temperature. Exposure to x-ray film was performed at -80 C with
intensifying screens.
RT-PCR and Southern analyses
Total RNA was prepared, and first strand cDNAs were synthesized
as described above. The PCR primers for uroguanylin corresponding to
nucleotides -21 to -2 (5'-GGAACAAGACTGGCAGACAC-3') and nucleotides
266–247 (5'-GCTCTGAAGATGTTGGCAGC-3') of the opossum preprouroguanylin
cDNA sequence were synthesized to amplify a cDNA product of 287 bp (24, 40). PCR cycles at 93 C for 1 min, 61 C for 1 min, and 72 C for 1.5 min
were repeated 35 times. The PCR primers for preproguanylin corresponded
to nucleotides 126–144 (5'-CCGTGAAGAAACTCAAGGA-3') and nucleotides
255–238 (5'-TGGAAA-GCCTGGGCTGCA-3') of the opossum preproguanylin
cDNA sequence. These oligonucleotides were used to amplify a 130-bp
cDNA product. Guanylin PCR conditions were the same as those for
uroguanylin PCR, except the annealing temperature was 58 C instead of
61 C. The PCR-generated cDNA products were visualized by agarose gel
electrophoresis and ethidium bromide staining. After agarose gel
electrophoresis, the cDNAs were transferred to nylon membranes
(Bio-Rad, Hercules, CA). The blots were prehybridized for 15 min
(QuikHyb, Stratagene, La Jolla, CA) and then hybridized for 1 h at
68 C with 1 x 106 cpm/ml 32P-labeled
uroguanylin or guanylin cDNA probes. The blots were washed twice at
room temperature, followed by a final wash for 30 min in 0.2 x
SSC-0.1% SDS at 60 C.
In vitro receptor autoradiography
Kidney and testis from opossums were frozen with liquid
N2 and stored at -80 C for sectioning in a cryostat
maintained at -20 C. Adjacent sections were mounted on opposite ends
of gelatin-coated slides, which were then stored at -80 C. Tissue
sections were thawed and allowed to dry for 10 min at room temperature
before incubation with 50 µl DMEM, pH 5.5, and 0.5% BSA at 37 C for
15 min. One section was incubated with this solution plus 1000 cpm
[125I]ST/µl to assess total binding of this radioligand
to receptors, whereas the adjacent section was incubated with 1000 cpm
[125I]ST/µl plus 1 µM Escherichia
coli ST (Sigma Chemical Co.) to assess nonspecific binding, as
previously described (8, 9, 37). After incubation, the sections were
initially washed with a stream of cold PBS and then washed three times
for 5 min each time in ice-cold PBS. The sections were dried in air and
placed in contact with Kodak X-Omat AR x-ray film (Eastman Kodak,
Rochester, NY) for initial detection of [125I]ST. The
slides containing tissue sections were then coated with Kodak NTB-2
emulsion (Eastman Kodak), dried overnight, and sealed in light-tight
boxes. The emulsion-coated sections were stored at 4 C for 2–3 weeks
until they were developed using standard photographic development and
fixation.
Preparation of [125I]ST
E. coli ST was iodinated with 125I using
the Iodogen method (Pierce Chemical Co., Rockford, IL).
[125I]ST was purified with a C18 column under
reverse phase conditions using HPLC as previously described (7, 8).
Synthesis of uroguanylin, guanylin, and ST peptides
The opossum uroguanylin (QEDCELCINVACTGC), opossum guanylin
(SHTCEICAFAACAGC), and E. coli ST (CCELCCNPACAGC) peptides
were synthesized by the solid phase method on an Applied Biosystems
model 431A peptide synthesizer (Foster City, CA) and purified by
reverse phase C18 chromatography as previously described
(2).
Cell culture
T84 cells (human colon carcinoma, passage 21, obtained from Dr.
James McRoberts, Harbor-University of California-Los Angeles Medical
Center, Torrance, CA) were cultured in DMEM and Ham’s F-12 medium
(1:1) containing 5% FBS, and 60 µg penicillin and 100 µg
streptomycin/ml, as previously described (1, 2). Opossum kidney (OK-E)
cells were cultured in DMEM and Ham’s F-12 medium (1:1) as previously
described (7).
cGMP bioassay in opossum kidney slices, OK cells, and T84 cells
OK or T84 cells were cultured in 24-well plastic dishes, and
cellular cGMP levels were measured in control and agonist-stimulated
cells by RIA (7, 8, 9). In brief, the synthetic uroguanylin, guanylin, and
ST peptides or the recombinant guanylin/proguanylin peptides expressed
in COS-1 cells were suspended in 200 µl DMEM containing 20
mM HEPES, pH 7.4, and 1 mM
isobutylmethylxanthine. T84 or OK cells were washed twice with 200 µl
DMEM before addition of the peptides. The solutions containing the
bioactive peptides were then added to the cultured cells and incubated
at 37 C for 40 min. After incubation, the reaction medium was
aspirated, and 200 µl 3.3% perchloric acid were added/well to stop
the reaction and extract cGMP. The extract was adjusted to pH 7.0 with
KOH and centrifuged, and 50 µl supernatant were used to measure cGMP.
Slices of kidney cortex were prepared using a Stadie-Riggs microtome
(Thomas Scientific, Swedesboro, NJ) and 50 mg tissue (wet weight) were
incubated in 200 µl DMEM, HEPES, and isobutylmethylxanthine as
described above for the cultured OK and T84 cells and reported
previously for kidney slices (7, 8). The renal slices were incubated
for 40 min at 37 C, and the tissue plus medium cGMP were extracted with
perchloric acid and measured by RIA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) coupled with 5'- and 3'-RACE-PCR to
extend this cDNA to include parts of the 5'- and 3'-untranslated
domains of the preproguanylin transcript. The composite cDNA sequence
is 688 bp, with an open reading frame encoding a protein of 100 amino
acids (Fig. 1A). The N-terminus of preproguanylin begins with a
20-amino acid hydrophobic signal peptide, which precedes the previously
reported N-terminal residues of
V21TVQDGDFSYPLE33 for proguanylin that was
isolated from opossum intestine (24). The bioactive guanylin peptides
that we isolated from urine and intestinal mucosa
(S86HTCEICAFAACAGC100) are found at the
C-terminus of this protein, followed by a stop codon (2). Analysis of
the preproguanylin structure reveals 40% identity between opossum
preproguanylin and preprouroguanylin (Fig. 1B), indicating that these
proteins are related in a family of guanylin-like gene products. Two
highly conserved regions between proguanylin and prouroguanylin are at
the proguanylin N-terminus represented by
L32ESVKKLKDL41 and the C-terminal bioactive
peptide, C89EICAFAACAGC100. Opossum
preproguanylin is similar to rat and human preproguanylins, sharing
57% and 60% identity, respectively (14, 15, 16, 17).
Opossum preproguanylin cDNA molecules were ligated into the eukaryotic
expression vector, pCDM8, and transfected into COS-1 cells. Previous
experiments demonstrated that the 15-residue guanylin peptide is
separated from proguanylin by eluting the adsorbed peptides from
C18 cartridges with 25% acetonitrile to elute guanylin and
then with 50% acetonitrile to elute proguanylin (27, 28). In addition,
guanylin is cleaved from proguanylin and activated by heating the
prohormone at 100 C in 1 M acetic acid (27). Guanylin was
separated from proguanylin in conditioned medium in these experiments
using C18 chromatography. The peptides eluted with 25%
acetonitrile did not stimulate cGMP accumulation in T84 cells when
tested before and after heating (data not shown), suggesting that the
transfected COS-1 cells did not secrete an active form of guanylin.
When the peptides eluted by 50% acetonitrile were bioassayed, we
observed that heating this fraction at 100 C in 1 M acetic
acid greatly increased its bioactivity in T84 cells (Fig. 2A). This shows that COS-1 cells
transfected with pCDM8-Guan secrete inactive proguanylin into the
medium. Another experiment confirmed that the bioactivity was
attributed to guanylin by first heating the conditioned medium at 100 C
in 1 M acetic acid before isolating guanylin by
C18 chromatography. The bioactivity of this peptide was
markedly reduced by pretreatment with chymotrypsin, which inactivates
guanylin, thus indicating that proguanylin is secreted from transfected
COS-1 cells (Fig. 2B).
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). This
close similarity in the primary structures suggests that the GC-OK
protein is an opossum homolog of the GC-C enzyme that is activated by
guanylin, uroguanylin, and ST in the intestine and stomach (3, 4, 5, 9).
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shows the cDNA products that were
produced when RNA samples were prepared and then reverse transcribed to
synthesize cDNAs to use as templates for the PCR. In the central
nervous system, a uroguanylin cDNA of the expected 287 bp was detected
in the cerebellum of brain when the cDNAs on agarose gels were stained
with ethidium bromide. In contrast, a guanylin cDNA product of the
expected size of 130 bp that was amplified by the RT-PCR was observed
throughout these brain regions, including cerebral cortex, cerebellum,
hypothalamus, midbrain, medulla, and olfactory bulb, as well as in the
spinal cord. For the other organs shown, cDNAs corresponding to mRNAs
encoding both guanylin and uroguanylin were observed in stomach,
duodenum, kidney, liver, and spleen. We confirmed that only uroguanylin
mRNA is expressed in the heart using this RT-PCR method (24). Moreover,
we detected no mRNA expression for either guanylin or uroguanylin in
the lung.
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shows the hybridization of specific
32P-labeled cDNAs to the cDNAs that were amplified by
RT-PCR using Southern blots of the gels illustrated in Fig. 4A. The
guanylin cDNA probe hybridized to guanylin cDNAs, and the uroguanylin
cDNA probe hybridized to uroguanylin cDNAs amplified by the PCR. In
addition, Southern analyses provided a more sensitive assay to detect
the guanylin and/or uroguanylin cDNAs that were amplified by the
PCR. Guanylin and uroguanylin cDNA products that were not detected by
staining the cDNAs in agarose gels with ethidium bromide (Fig. 4A)
could readily be detected by Southern hybridization analyses, revealing
that low levels of uroguanylin mRNA transcripts are present in the
midbrain, olfactory bulb, and hypothalamus (Fig. 4B). Moreover,
Southern analyses confirmed that uroguanylin and guanylin mRNA
transcripts in the lung are below the detection limit of this assay and
that guanylin mRNA is undetectable in the heart.
Figure 5 provides a summary of the
tissues that were examined for uroguanylin and guanylin mRNA expression
by RT-PCR and Southern assays. Each tissue was used to prepare RNA from
the listed organs/tissues from at least two different animals for the
complete RT-PCR steps to amplify the specific cDNAs. To avoid PCR
amplification from genomic DNA, a parallel cDNA synthesis reaction
without reverse transcriptase was used as a negative control. No cDNAs
were detected under this control condition. Uroguanylin and guanylin
mRNAs were found in the small and large intestines, as previously
reported using Northern analyses (24). Other parts of the digestive
system, such as salivary glands, pancreas, liver, and gastric mucosa,
also express guanylin and uroguanylin mRNAs. In the renal system, we
observed that the kidney as well as cultured OK cells and urinary
bladder express both guanylin and uroguanylin mRNA transcripts.
Uroguanylin and guanylin mRNAs were readily detected by RT-PCR assays
using RNA-cDNA from tissues of the immune-lymphoid system, including
spleen, thymus, and lymph nodes. In the reproductive organs, guanylin
and uroguanylin mRNAs were detected in uterus, ovary, testis, and
prostate, whereas guanylin (but not uroguanylin) mRNA was detected in
the lactating mammary gland, and neither of these peptide transcripts
was observed in the oviduct. The adrenal gland contained mRNAs for both
guanylin and uroguanylin, whereas the retina exhibited only uroguanylin
mRNA. Thus, guanylin and uroguanylin mRNAs are widely expressed in
the opossum.
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). This is the expected size of
GC-C-like mRNA transcripts based on previous reports (4, 5). In
addition to the receptor-GC-OK transcripts, mRNAs for uroguanylin were
detected in RNA preparations from duodenum, spleen, and testis (data
not shown). Uroguanylin or guanylin mRNA transcripts were not detected
by Northern assays using the total RNA preparations made from kidney,
OK cells, and cerebellum. RT-PCR assays with primers specific for
amplification of the GC-OK catalytic domain amplified a cDNA of the
expected size of 0.6 kb when RNAs from duodenum, kidney, OK cells,
testis, spleen, and cerebellum were used to prepare the template cDNAs
(Fig. 6). Thus, the receptor-GC-OK mRNA is expressed in all of these
tissues in addition to guanylin and/or uroguanylin mRNAs.
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). Glomeruli did
not bind [125I]ST. Because of the relatively low
resolution of this method, it is possible that some of the
receptor-bound [125I]ST may also be associated with cells
in the distal tubules or cortical collecting tubules. The addition of
either unlabeled ST, guanylin, or uroguanylin inhibited the binding of
[125I]ST to the renal receptors, indicating a common set
of binding sites for these peptides in the opossum kidney (data not
shown).
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). These peptides were approximately
equipotent in the activation of GC-OK receptors, causing increases in
cGMP levels of cultured OK cells elicited by the maximally effective
concentrations of peptide agonists. E. coli ST was more
potent than the endogenous peptide agonists, as observed previously
with T84 intestinal cells (1, 2). When renal cortical slices were used
in this bioassay, we observed that uroguanylin was more potent than
guanylin and ST was more potent than either endogenous peptide. In both
cultured OK cell and kidney cortex preparations, uroguanylin, guanylin,
and ST peptides are full agonists, with parallel concentration-response
curves for the activation of apical membrane GC-OK signaling
molecules.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The preproguanylin cDNA isolated in this study contains an open reading frame encoding a protein of 100 amino acids, which is shorter than the 115- or 116-amino acid precursors found in the human, rat, and mouse (14, 15, 16, 17). Opossum preproguanylin is similar in primary structure to the other preproguanylins, sharing 55–60% identity at the amino acid level. The 80-amino acid length of intestinal proguanylin, the N-terminal V21TVQDGDFSYPLE33 peptide domain, and the C-terminal S86HTCEICAFAACAGC100 peptide structure for the bioactive guanylin peptides were identified by N-terminal sequence and mass spectrometry analyses of proguanylin and guanylin molecules previously isolated from opossum intestine (2, 27, 28). The present experiments confirmed and extended those findings by analysis of the predicted structure of preproguanylin deduced from this cDNA. The 688-bp preproguanylin cDNA isolated in these experiments is close to the approximately 0.8-kb length of preproguanylin mRNAs identified in this study and in previous Northern hybridization assays (24). The polyadenylase consensus site, AATAAA, occurs at nucleotides 641–646 of this cDNA. Additional nucleotides may be present in the preproguanylin mRNA 5' to the 25 nucleotides obtained for the 5'-untranslated region.
Prior experiments also revealed that proguanylin made within the intestinal mucosa is inactive unless the protein is cleaved by pretreatment with V8 protease to yield a 16-residue form of guanylin that activates receptor-GC signaling molecules located on the apical surface of T84 cells (27). Heating the intestinal form of opossum proguanylin at 100 C in 1 M acetic acid also resulted in cleavage of proguanylin to activate the prohormone. The form of proguanylin secreted by transfected COS-1 cells in the present study was also inactive in the T84 cell bioassay until this polypeptide was heated at 100 C in 1 M acetic acid. Moreover, prouroguanylin expressed in COS-1 cells was inactive until a C-terminal form of uroguanylin was released from the precursor by proteolytic cleavage with chymotrypsin (24). Human and rat forms of proguanylin were also inactive until the C-terminal residues constituting the active guanylin peptides were released by cleavage through the use of proteases or by heating proguanylin in acid (16, 21). Examination of the highly conserved domains in the opossum proguanylin and prouroguanylin molecules reveal 10 residues near the N-terminus of proguanylin (L32ESVKKLKDL41) and prouroguanylin (L35DSVKKLDEL44) with similarity of primary structure. Similar homologous domains occur in the human and rat proguanylin and prouroguanylin molecules (14, 15, 16, 29, 30, 31). This region in the prohormones may function as an inhibitory domain, thus accounting for the inactive nature of proguanylin and prouroguanylin molecules. These highly conserved domains may lie in close proximity to the C-terminal active peptide domain when the precursor proteins are folded in native conformations. An alternate possibility is that these highly conserved domains represent potential bioactive peptides that exert regulatory actions in the intestine or other tissues where proguanylin and/or prouroguanylin are produced, secreted, and cleaved by endogenous proteases.
An experimental model for the paracrine/autocrine regulation of target cell function via intracellular cGMP is found in the intestinal epithelium (reviewed in Ref.42). Proguanylin and prouroguanylin are made in the gastrointestinal mucosa as revealed by this study and previous reports (9, 14, 15, 16, 17, 27, 28, 29, 30, 31). These prohormones are secreted into the intestinal lumen, where they are cleaved and activated by converting enzymes, or posttranslational processing occurs before secretion of the bioactive peptides. Recent experiments have shown that both guanylin and uroguanylin accumulate in luminal perfusates of the small intestine perfused in vivo (39). These bioactive peptides then interact with and regulate the enzymatic activity of receptor-GC proteins, such as GC-C, that have ligand-binding domains exposed on the mucosal surface of target enterocytes (1, 2, 4, 5, 6, 7, 8, 9). Increased levels of intracellular cGMP regulates the transepithelial, electrogenic secretion of chloride through cGMP-dependent protein kinase-mediated phosphorylation and activation of apical CFTR molecules in target cells within the intestinal mucosa (13, 43, 44). The potential magnitude of this salt and fluid secretion pathway in the intestine is emphasized by the disorder of traveler’s diarrhea, which is caused by bacterial ST peptides that mimic the actions of guanylin and uroguanylin and elicit secretory diarrhea of a remarkable magnitude (6, 10, 42). Therefore, a major physiological role for guanylin and uroguanylin in the gastrointestinal tract is likely to involve the regulation of salt and water secretion during digestion by an intrinsic cGMP signaling pathway.
Another intestinal transport function that is influenced by these enteric peptides is bicarbonate secretion, which is stimulated by the activation of receptor-GCs in the duodenum (11, 12). Uroguanylin is probably a major regulator of receptor-GC activity in the duodenum because its mRNA expression in this segment appears greater than that of guanylin mRNA (8, 14, 15, 24, 26, 31). Also, uroguanylin is a considerably more potent and effective secretogogue than guanylin in stimulating anion secretion when the mucosal surface is exposed to acidic conditions (12, 27, 28, 45). The contents of the duodenum are acidified during digestion due to entry of the highly acidic chyme from the stomach. In this physiological condition, uroguanylin may stimulate the secretion of bicarbonate from target enterocytes, thus buffering and protecting the mucosal surface from the deleterious effects of mucosal HCl (11, 12). Such an action of uroguanylin may be particularly important in the portion of the duodenum proximal to the pancreatic duct where duodenal ulcers are commonly found. It should be stressed that uroguanylin-stimulated anion secretion in the proximal duodenum is reduced in transgenic animals with impaired CFTR genes, but anion secretion responses to uroguanylin are not completely lost in the duodenum of the CF mouse (12). This indicates that uroguanylin regulates anion secretion in this segment of the intestine via CFTR-independent as well as CFTR-dependent mechanisms. The transport mechanism(s) responsible for the CFTR-independent secretion of bicarbonate is presently unknown.
A second epithelial tissue that is regulated by uroguanylin and/or guanylin is the kidney, as documented in this and previous studies (7, 8, 37). Activation of tubular receptor-GCs in the kidney elicits natriuresis, kaliuresis, and diuresis (34, 35, 36). The local release of uroguanylin and/or guanylin into the filtrate from renal cells that produce these peptides may regulate the transport of sodium and potassium in the nephron via cGMP in a manner similar to that described for the intestinal epithelium. The transporter molecules affected by these peptides via cGMP in the kidney are not known, but cGMP-dependent protein kinase II and CFTR mRNAs are both expressed in the kidney (43, 46, 47). The cGMP-dependent protein kinase II may be the receptor for intracellular cGMP, with the CFTR protein serving as a target for kinase-mediated phosphorylation, leading to the stimulation of chloride secretion and/or an inhibition of sodium reabsorption. These tubular processes could be regulated by the paracrine and/or autocrine actions of locally produced uroguanylin and/or guanylin. Our finding that mRNAs encoding uroguanylin and guanylin are expressed in the opossum kidney are consistent with this postulate. Other observations that mRNAs for these cGMP agonists are found in the rat kidney provide additional evidence of a potential intrarenal signaling pathway (14, 31). Thus, renal production of uroguanylin and/or guanylin provides agonist peptides for an intrinsic cGMP signal transduction pathway that may regulate renal tubular function in vivo (35, 36).
An endocrine pathway may also exist for uroguanylin that links the intestine with the kidney via circulating uroguanylin and prouroguanylin (24, 34). Prouroguanylin and/or uroguanylin appear to be secreted into the bloodstream, because both prouroguanylin and uroguanylin have been isolated from opossum plasma (24). The gastrointestinal tract as well as other organs may be sources of plasma uroguanylin and prouroguanylin. These circulating peptides can be filtered at glomeruli to gain access to the receptor-GC signaling molecules that are localized on brush border membranes of renal tubular cells (7, 8, 37). The diuresis, natriuresis, and kaliuresis elicited by the iv injection of ST into opossums in vivo or perfusion of the isolated rat kidney with uroguanylin or guanylin in vitro are consistent with this hypothesis (35, 36). Glomerular filtration and renal clearance of circulating uroguanylin and guanylin and their precursors appear to be a major elimination pathway for the circulating peptides as well, because their plasma levels increase dramatically in patients with chronic renal failure (22, 23, 32, 33). Thus, uroguanylin and/or guanylin may serve in an endocrine axis as natriuretic, kaliuretic, and diuretic peptides. A natriuretic hormone was postulated to exist by Carey and his colleagues, who demonstrated that oral administration of a sodium chloride load caused a much larger natriuresis than did iv administration of the same quantity of salt (48, 49). An endocrine model was formulated, consisting of a factor that may be released from the digestive system into the circulation upon the consumption of a high salt meal. Delivery of this natriuretic substance to the kidney via the bloodstream would stimulate urinary salt excretion in times of dietary sodium surfeit. Uroguanylin is a candidate to serve in such an endocrine axis linking the intestine and the kidney to regulate urinary salt excretion during postprandial periods of salt absorption by the digestive tract (34). In this fashion, uroguanylin could regulate the activity of transmembrane receptor-GC signaling molecules that are expressed in the opossum kidney and localized on the apical surface of renal proximal tubules (7, 8, 37).
A common theme in the intestine and kidney appears to be the control of salt and water transport by uroguanylin and guanylin. The expression of these peptides and their cognate receptor-GC, GC-OK, in other tissues within the central nervous, reproductive, and immune/lymphoid organ systems suggests that ion transport could also be regulated by this cGMP signaling pathway as a putative physiological mechanism in these tissues. Our findings in the present study confirm previous reports that several extraintestinal epithelia in the opossum express receptors for this class of agonist peptides (7, 8, 37). The new information presented in this communication extends this cGMP signal transduction pathway to a number of additional tissues in which the molecular machinery potentially exists for an intrinsic mechanism for regulation of target cell function by guanylin- and/or uroguanylin-mediated activation of cell surface receptor-GC enzymes. Signaling via this cGMP pathway in lymphoid tissues is consistent with an earlier description of cGMP accumulation responses to E. coli ST in a cultured basophilic leukemia cell line (50). Lymphocytes within the spleen, thymus, and lymph nodes of the opossum may express uroguanylin, guanylin, and/or GC-OK mRNA transcripts. These findings suggest that guanylin and/or uroguanylin may influence intracellular cGMP levels in several different tissues within the digestive, renal, central nervous, reproductive, and immune/lymphoid organ systems. The specific cellular targets for the cGMP agonist peptides and the physiological functions influenced by these newly discovered peptides in various organs of the body provide fertile ground for future investigations.
It is possible that unique physiological actions of guanylin and/or uroguanylin may exist in the opossum, Didelphis virginiana, or other marsupials that may not be present in placental mammals. A major difference between eutherian (placental) and metatherian (marsupial) mammals lies in their biology of reproduction. Marsupials have a very short period of intrauterine gestation, and the newborns complete their fetal development attached to mammae, usually in a specialized pouch of the mother. Thus, it is conceivable that the guanylin/uroguanylin signaling pathway could be uniquely functional within the female reproductive organs and/or mammary glands of marsupial mammals. This possibility seems less likely for the cellular functions regulated by the cGMP signaling pathway in the seminiferous tubules of testis. The male opossum is unlikely to have major differences in its reproductive mechanisms from any other mammal, eutherian or metatherian. In other respects, especially in its diet, the opossum is similar to the hominids, placental mammals that evolved as opportunistic hunter/scavengers with an omnivorous diet. A common ancestor to both placental and marsupial mammals is estimated to have lived during the early Cretaceous period, about 130 million yr before the present time (51). Recent evidence suggests that the signaling pathway for the guanylin family of peptides appeared much earlier during vertebrate evolution. This conclusion is supported by the recent identification of guanylin-like peptides in the intestine of fish that activate the human receptor-GC found in T84 cells and the correlated demonstration that receptors-GCs exist in the intestinal mucosa of fish that are activated by mammalian guanylin (52). It is likely that the basic features of this signal transduction pathway emerged quite early in vertebrate evolution and are also highly conserved between fish and man. From this perspective, a reasonable hypothesis may be formulated that predicts that information derived from the opossum will be generally applicable to other mammals, including Homo sapiens.
In conclusion, this study has provided new insights into the potential roles of guanylin and uroguanylin as regulatory peptides for cGMP signaling in several different organs and tissues of the opossum. Both paracrine/autocrine and endocrine signal transduction pathways appear to be involved in the mechanisms of action of uroguanylin and guanylin. Defining both the cellular and organ functions that are regulated by these peptides via intracellular cGMP in the central nervous, reproductive, and immune/lymphoid systems of the body will require additional studies designed to identify the cells within these tissues that produce the agonist peptides and/or express the cell surface receptor-GC signaling molecules. Cellular functions other than the previously documented actions of uroguanylin and guanylin to regulate ion transport in the intestine and kidney should also be considered for these newly discovered peptide hormones.
Received May 9, 1997.
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  A. Sindic and E. Schlatter Mechanisms of action of uroguanylin and guanylin and their role in salt handling Nephrol. Dial. Transplant., November 1, 2006; 21(11): 3007 - 3012. [Full Text] [PDF]
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