J Gastroenterol 2001; 36:219-225
`
`Review
`
`Journal o
`Gastroenterology
`© Springer-Verlag 2001
`
`Guanylin family: new intestinal peptides regulating electrolyte and
`water homeostasis
`
`Masamitsu NAKAZATO
`
`Third Departmentof Internal Medicine, Miyazaki Medical College, Miyazaki 889-1692, Japan
`
`The regulation of intestinal salt and water transportis
`critical to the maintenanceof fluid volume. Control of
`this life-sustaining activity is mediated by the concerted
`actions of hormones, neurotransmitters, and locally
`acting factors. Guanylin and uroguanylin are novel
`peptides that were first isolated from rat jejunum and
`opossum urine, respectively. They bind to and activate
`guanylyl cyclase-C (GC-C) receptors to regulate intesti-
`nal and renalfluid and electrolyte transport through the
`second messenger, cyclic guanosine 3’,5'-monophos-
`phate (GMP). Heat-stable enterotoxins produced by
`pathogenic bacteria have close structural similarities to
`guanylin and uroguanylin, and they use this mimicry to
`act on GC-C, causing life-threatening secretory diar-
`rhea. Guanylin primarily is restricted to the intestine,
`whereas uroguanylin is present in the stomach,kidney,
`lung, and pancreas, in addition to intestine. Guanylin
`and uroguanylin are secreted into the intestinal lumen
`and blood in response to sodium chloride administra-
`tion. These peptides function in salt and water transport
`in the intestine and kidney by luminocrine and endo-
`crine actions. The guanylin family is involved in the
`pathophysiology of some gastrointestinal, renal, and
`heart diseases.
`
`guanylin,
`enterotoxin,
`heat-stable
`Key words:
`uroguanylin, guanylyl cyclase, salt homeostasis, intesti-
`nal natriuretic factor
`
`
`Introduction
`
`Diarrheal diseases are a leading cause of morbidity
`and mortality in humans, causing up to 50% of infant
`
`Received: August 21, 2000 / Accepted: November 17, 2000
`Reprint requests to: M. Nakazato
`
`deaths in developing countries.! Heat-stable enterotox-
`ins (STs) elaborated by various pathogenic bacteria,
`including enterotoxigenic Escherichia coli and Yersinia
`enterocolitica, bring about acute diarrhea, and also
`cause‘travelers diarrhea’ or‘turista’. STs are 15- to 30-
`amino acid peptides. In 1990, STs were shownto bind to
`intestinal receptor-guanylyl cyclase (GC-C) in the brush
`border membrane,” which subsequently leads to the
`activation of guanylyl cyclase.* Human GC-C,a 1050-
`amino acid protein and rat GC-C, a 1053-amino acid
`protein, have an extracellular domain, a transmem-
`brane domain, and intracellular protein kinase-like and
`guanylyl cyclase catalytic domains. Includedin this type
`of plasma membraneform of guanylyl cyclase are GC-
`A and GC-B, which are receptors of the natriuretic
`peptide family that functions in the regulation of body
`fluid balance. The paradox of a bacterial toxin acting
`on a mammalian receptor remained unclear until the
`following recent discoveries were made.
`
`Discoveries of guanylin and uroguanylin
`
`Just as opiates derived from the poppy predicted
`the existence of endogenous opium-like regulatory
`peptides, the isolation of bacterial ST peptides and
`elucidation of their cyclic guanosine 3’,5'-monophos-
`phate (CGMP)-regulating activity foreshadowedthe dis-
`covery of endogenous ligands for GC-C in mammals.
`STs act on GC-C, produce a second messenger, cGMP,
`and elicit the stimulation of Cl- secretion and the inhi-
`bition of Na* and H,O absorption, thereby causing
`secretory diarrhea.‘ Cells transfected with GC-C cDNA
`were proposedas a meansto search for the endogenous
`ligand by monitoring cGMPproduction, butcells that
`had a high sensitivity and a maximum response to STs
`were unavailable. Currie et al. found that T,, cells, a
`humancolon cancercell line, had the desired ability for
`the monitoring of cGMP production by STs, and an
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`220
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`M.Nakazato: Guanylins regulate electrolyte and water homeostasis
`
`human guanyl in
`
`rat guanylin
`
`1
`
`15
`
`Pro-Gly-Thr-Cys-Glu-Ile-Cys-Ala-Tyr-Ala-Ala-Cys-Thr-Gly-Cys
`
`--- “Al arn rn rrr rrr rrr rrr rrr rrr rcs rrr errs cscs
`
`human uroguanyl in
`
`Asn-Asp-Asp--------- Leu----- Val-Asn-Val------------rrrrr rrr Leu
`
`rat uroguanylin
`
`Thr-Asp-Glu--------- Leu----- lfe-Asn-Val--~------- 55555 r rr rne
`
`115
`
`115
`
`112
`
`106
`
`E.coli
`
`enterotoxin Asn-Ser-Ser-Asn-Tyr-Cys--------- Leu----- Cys-Asn-Pro--------- Thr--------- Tyr
`Lo
`
`Fig. 1. Primary structures of guanylin and uroguanylin. Dotted lines indicate the same amino acids as those in humanguanlyin,
`and brackets indicate disulfide bond linkage. Guanylin and rat uroguanylin are 15-aminoacid peptides, and human uroguanylin
`is a 16-aminoacid peptide. The numbers of amino acid residues of precursor proteins are shown on the right
`
`unidentified endogenous ligand. Indeed, T,, cells pos-
`sessed the sensitivity to detect 10pM ST, a maximum
`30 000-fold increase in cGMPto ST,and specificity for
`ST-like agonists. Using this assay combined with multi-
`step chromatography, Currie’s group isolated a cognate
`endogenousligand for GC-C from rat jejunum.’ This
`peptide wasdesignated guanylinafter its binding to GC-
`C. One year after the discovery of guanylin, another
`guanylin-like peptide, called uroguanylin, was isolated
`from the urine and intestinal mucosa of an opossum
`(Didelphis virniana, an American marsupial), using the
`same methodology.® Uroguanylin then wasidentified in
`the intestines and urine of humansandrats.** Guanylin
`and uroguanylin are 15- to 16-amino acid peptides(Fig.
`1). These two are novel peptides that have no sequence
`identities to other known peptides. Human and rat
`uroguanylin has two additional acidic residues that are
`not found in guanylin.
`As expected, the primary structures of STs are quite
`similar to those of the guanylin family (Fig. 1). Human
`guanylin shares 7 of 15 amino acids with Escherichia
`coli ST, and human uroguanylin shares 9 of 16 residues
`with it (47% and 56% similarity, respectively). STs are
`thought to use this mimicry to act on GC-C. A major
`structural difference between the guanlyin family pep-
`tides and STs is the numberof cysteines and disulfide
`bonds: guanylin and uroguanylin have four cysteines
`and two disulfide bridges compared with STs, with six
`cysteines and three disulfides. These disulfides are indis-
`pensable for optimal peptide potencies in the stimula-
`tion of cGMPproduction in vitro. The potency of the
`stimulation of guanylyl cyclase activity, in descending
`order,
`is STs, uroguanylin, and guanylin.*’? An addi-
`tional pair of cysteine residues in STs may contribute
`to their apparently higher potencies.+? Guanylin and
`uroguanylin are small molecules and have two intra-
`molecular disulfide bonds. This structural characteristic
`produces two topological isomers, a right-handed spiral
`
`form and a left-handed spiral form.'*!! One form is
`bioactive to stimulate cGMP production, but the other
`is not. These isomersare interconvertible; however, the
`biological significance and mechanism of the inter-
`conversion are still unclear.
`
`Gene sequenceandtissue distribution
`
`Based on the primary structure of guanylin and
`uroguanylin, cDNAs encoding their precursors were
`isolated.’**° Guanylin and uroguanylin mRNAsin hu-
`mans and rats have approximately 600 bases. Mature
`guanylin and uroguanylin peptides are located at the
`carboxy terminal ends of their precursor proteins.
`The sequence identity between human guanylin and
`uroguanylin is 20%,
`that between human and rat
`guanylin is 64%, and that between human andrat
`uroguanylin is 63%. Ten-kilodalton proguanylin is a
`major guanylin molecule in the intestinal mucosa and
`plasma of humans andrats, but it has no biological
`activity until cleaved by proteolytic enzymesto release
`the biologically active 15-amino acid peptide.?!* Ten-
`kilodalton prouroguanylin is also a major molecular
`form in the intestine of humans and rats, whereas
`mature 16-amino acid uroguanyin is a major form in the
`urine of both species.”
`Uroguanylin and guanylin genes are arranged in a
`tail-to-tail array on the short arm of human chromo-
`some 1p33-p34 and on mouse chromosome 4.207425
`The respective intergenic distances between the two
`genes in the human and mouse genomesare approxi-
`mately 6.5 and 8kb. The intergenic region between the
`human uroguanylin and guanylin genes has an Alu se-
`quence and a (CA/TG),, microsatellite sequence,
`suggesting that these two genes are produced by gene
`duplication. The genes of both human guanylin and
`uroguanylin consist of three exons and two introns
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`M. Nakazato: Guanylins regulate electrolyte and water homeostasis
`
`221
`
`within an overall length of 2.5kb. The 5’ flanking region
`has TATA and CAAT boxes. The genes also have
`multiple binding sites for promoter-specific transcrip-
`tion factor, activator protein-1, activator protein-2,
`and a cyclic adenosine 3’,5'-monophosphate (cAMP)-
`regulated enhancer element.
`Northern blot and reverse transcription-polymerase
`chain reaction (RT-PCR) analyses have revealed that
`guanylin mRNAis most abundant in the intestine, and
`there are small amounts in the kidney, adrenal gland,
`uterus, and oviduct.’The distribution of guanylin
`mRNAintheintestine is characterized by relatively low
`levels in the duodenum and jejunum and much higher
`levels in the ileum and colon. Two radioimmunoassays
`(RIAs) for guanylin and uroguanylin, respectively, were
`developed.’Guanylin peptide is distributed widely
`from the duodenum to colon in both rats and humans,
`the highest contents being in the ileum and proximal
`colon. The plasma concentration of immunoreactive
`guanylin was 31.2 + 3.0fmol/ml (mean + SE)in normal
`individuals and was markedly higher in patients with
`renal
`insufficiency, which may be due to impaired
`catabolism of guanylin. Patients with carcinoid tumors
`that cause secretory diarrhea had elevated levels of cir-
`culating guanylin.2”? The tumors contained guanylin, as
`detected by immunohistochemical study. Although se-
`rotonin is considered to be a major cause of diarrhea in
`patients with carcinoid tumors, guanylin released from
`the tumors may also be responsible for the stimulation
`of intestinal fluid secretion. Northern blot analysis and
`RIA showedthat uroguanylin mRNAandpeptide were
`expressed in the stomach, small and large intestines,
`kidney, heart, and lung of humans andrats.*!*° The
`highest values were found in the upper small intestine.
`The plasma concentration of bioactive uroguanylin was
`5.0 + 0.3fmol/ml in normal individuals, and it was
`higher in patients with renal
`insufficiency.’ Plasma
`uroguanylin concentration in patients with heart failure
`was also significantly higher than that in normal con-
`trols, and increased with the severity of heart failure.
`Plasma uroguanylin levels in the coronary sinus and
`anterior interventricular vein were higher than that in
`the aorta, indicating that uroguanylin is secreted from
`the heart in heart failure. Uroguanylin has natriuretic
`activity, as described below. The possible function of
`uroguanylin in the regulation of body fluid balance in
`heart failure needs further investigation.
`
`Cellular localization
`
`The specific cellular sites of guanylin mRNA produc-
`tion in the intestine are a source of considerable debate,
`with conflicting reports.5?*° A recent immunohisto-
`chemical finding indicates that the enterochromaffin
`
`(EC) cells of guinea pig stomach and small intestine are
`a potential source of guanylin.° On the other hand,
`uroguanylin-producing cells have been well defined.
`Uroguanylin-immunoreactive cells in the rat intestine
`are round, basket-shapedortall flask-shaped cells with
`a dense accumulation of uroguanylin in the luminalcy-
`toplasm and a long, thin basal process immunoreactive
`for uroguanylin.”?! Uroguanylin-positive cells in the
`rat intestine were identified as EC cells because they
`reacted with serotonin antibody. The ECcell, the most
`abundant type of enteroendocrinecell, is widely distri-
`buted in the intestine. When stimulated, the EC cell
`releases serotonin and substance P both apically (into
`the lumen) and basolaterally (into the circulation).*
`Uroguanylin also is
`released from the intestine
`bidirectionally and is
`thought
`to function in a
`luminocrine (luminal secretion), endocrine, and/or
`paracrine fashion. The ECcell appears to have a fea-
`sible mechanism for delivering uroguanylin to luminally
`oriented GC-C,as well as to remote tissues such as the
`kidneyvia the circulation.
`Uroguanylin-producingcells in the rat stomach were
`identified as enterochromaffin-like (ECL) by im-
`munocytochemical methods and in situ hybridization
`cytochemistry using gastric mucosal cells isolated by
`counterflow elutriation.** ECL cells release histamine,
`leading to the stimulation of gastric acid secretion from
`parietal cells. Seven distinct endocrine cells: EC, ECL,
`D, D1, P, G, and X cells, have been identified ultrastruc-
`turally and immunohistochemically in rat and human
`gastric mucosa.***° Very recently, X cells have been
`clarified to produce ghrelin, a novel peptide that stimu-
`lates the release of growth hormone from the pitu-
`itary.*”*8 X cells thus can be abbreviated as Grcells.
`ECLcells are small cells (8- to 10-nm in diameter) that
`contain cytoplasmic vesicles with eccentric electron-
`dense cores. ECL cells are scattered in the oxyntic
`glands, often in direct contact with parietal cells.
`Patients with Zollinger-Ellison syndrome, presenting
`with gastrinoma, peptic ulcer, and ECL hyperplasia,
`had markedly high plasma concentrations of uro-
`guanylin. Further investigation of the cGMP-mediated
`gastric ion transport mechanism is a fascinating topic
`that could lead to better understanding of the regulation
`of gastric acid secretion.
`Uroguanylin and GC-C mRNAare present in B
`cells of the pancreatic islets, but the involvement of
`uroguanylin in the regulation of glucose metabolism has
`not been determined. The peptides produced in the
`“gastro-entero-pancreatic (GEP) endocrine system”
`function to control or modulate all the processes linked
`to the digestion and absorption of nutrients and water.”
`Uroguanylin may be a constituent of the GEP endo-
`crine system becauseofits cellular source and biological
`activity.
`
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`222
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`M.Nakazato: Guanylins regulate electrolyte and water homeostasis
`
`Escherichia coli
`Yersinia enterocolitica
`
`!
`
`enterotoxin
`
`guanylin or
`uroguanylin
`
`Guanylin or
`
`Nat’, H2O
`CFTR
`
`inhibition
`
`
`
`
`
`
`Uroguanylin-
` GTP
`
`
`cGMP
`Producing Cell
`
`
`enterocyte
`
`vessel
`Cl”
`guanylin or uroguanylin
`
`Cl”
`
`the guanylyl
`for
`Fig. 2. A model
`cyclase-C (GC-C) signaling pathway
`in intestinal Cl--secreting cells. Lumi-
`nally secreted guanylin and urogu-
`anylin activate GC-C in nearbycells.
`These peptides are also secreted into
`the bloodstream to function in an
`endocrine fashion. Cl” enters across
`the basolateral (serosal) membrane.
`Cyclic
`guanosine
`3’,5’-monophos-
`phate
`(cGMP)
`activates
`cGMP-
`dependent protein kinase II (PKG
`II). A key substrate for phosphoryla-
`tion by PKG II is the apical mem-
`brane-localized CFTR protein. CFTR
`serves as a channel for Cl- and HCO,”
`secretion across the apical (mucosal)
`membrane of epithelial cells. G7P,
`Guanosine 5’-triphosphate
`
`stimulation
`
`Kinase
`(PKGII)
`
`Molecular mechanism of guanylin and
`uroguanylin action
`
`A model for the GC-Csignaling pathway in intestinal
`Cl--secreting cells is shown in Fig. 2. GC-C is more
`abundantin the small intestine than in the large intes-
`tine. Moreover, a gradient of receptor levels was ob-
`served in small intestine, with the highest amounts in
`the upper portions of crypts and in the lowerparts of
`villi adjacent to the crypts.“ A truncated, GC-C-like
`mRNAthat has a 159-nucleotide deletion was found in
`the mucosa of rat stomach and intestine.*! The physi-
`ological implications of this truncated GC-C remain to
`be elucidated. Intestinal cells produce preproguanylin
`or preprouroguanylin. Luminal secretion of active
`peptides can activate GC-C in nearbycells of this epi-
`thelium in a luminocrine fashion. Proguanylin and
`prouroguanylin are secreted into the bloodstream to
`function in an endocrine fashion. cGMP activates
`cGMP-dependent protein kinase II (PKG II), which
`phosphorylates the apical membrane-localized CFTR
`protein. CFTRis present immunohistochemically in the
`epithelia of the intestine, stomach, airway, small pancre-
`atic ducts, renal tubules, and the sweat duct. CFTR is
`one member of a large family of ABC proteins that
`transport small molecules across the cell membrane in
`an ATP-dependentfashion. Guanylin and uroguanylin
`were verified to stimulate Cl- secretion in T,, cells, as
`measured byincreasesin the shortcircuit current, using
`an Ussing chamber.°* Cl-
`is secreted through the
`CFTR channel pathway across the apical (mucosal)
`membrane.
`
`transduction system and physiological
`The signal
`implications of guanylin and uroguanylin were also
`investigated by using transgenic and knockout mice.
`The mutation of CFTR genes, resulting in either loss of
`the protein or modification of its activity, underlies the
`genetic disease of cystic fibrosis. CFTR knockout mice
`had marked reductions of intestinal Cl- and HCO,-
`secretion responses to guanylin and uroguanylin.*4
`Mice deficient in PKG II wereresistant to E. coli.* PKG
`II knockout mice also developed dwarfism that was
`caused by a severe defect in endochondral ossification
`at
`the growth plates. GC-C-null mice had no STa-
`stimulable guanylyl cyclase activity.“ Gavage with STa
`resulted in marked fluid accumulation within the intes-
`tine of wild-type and heterozygous suckling mice,
`but GC-C-null animals were resistant. Infection with
`enterotoxigenic bacteria that produce STaled to diar-
`rhea and death in wild-type and heterozygous mice,
`whereas the null mice were protected. Diets including
`high carbohydrate, fat, or protein, or drinking water
`including high K* or Na* did not severely affect the
`GC-C-null animals.
`A wide range of mucosal acidity occurs within the
`intestinal lumen during digestion. At an acidic mucosal
`pH of 5.0, uroguanylin was 100-fold more potent than
`guanylin, but at an alkaline pH of 8.0, guanylin was
`more potent
`than uroguanylin in stimulating intra-
`cellular cGMP production and transepithelial chloride
`secretion.*”7 Uroguanylin and guanylin appear
`to
`cooperatively regulate the guanylyl cyclase activity of a
`common set of mucosal receptors in a pH-dependent
`fashion.
`
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`M. Nakazato: Guanylins regulate electrolyte and water homeostasis
`
`223
`
`Uroguanylin as a candidate for intestinal
`natriuretic factor
`
`When dietary sodium chloride is low, the regulation
`of salt homeostasis is maintained by mineralcorticoids.
`However, contemporary diets commonly have excess
`sodium;
`thus, mechanisms are required to achieve
`salt homeostasis during sodium surfeit. An intestinal
`natriuretic factor has been sought, because the oral
`ingestion of sodium chloride causes a dramatic increase
`in urine salt excretion, whereas the same amount ad-
`ministered intravenously haslittle effect on renal salt
`excretion.
`The intravenous administration of uroguanylin to
`mice induced diuresis, natriuresis, and kaliuresis.*
`Guanylin was less potent than uroguanylin and STa.
`When uroguanylin was administered into the renal
`artery, it was filtered through the glomerulus and then
`activated the GC-C/CFTR system localized in the
`tubules, thereby causing natriuresis and an increase
`in cGMPexcretion.*° Urinary uroguanylin excretion in
`persons who took a high-salt diet was significantly
`higher than that in persons with a low-salt diet.*! The
`magnitude of uroguanylin excretion was proportional to
`increases in urinary Na* and cGMPexcretion in sub-
`jects receiving high-salt diets. The oral administration of
`salt to rats augmented uroguanylin mRNAlevels in the
`intestine and kidney. Uroguanylin secretion from the
`intestine in response to salt was studied in vitro. In
`isolated vascularly and luminally perfused rat intestine,
`uroguanylin produced in the intestine was secreted
`mainly in the lumen, but in part in the blood, in re-
`sponse to high-concentration sodium chloride adminis-
`tration. These findings taken together indicate that
`uroguanylin is a prime candidate for a substance that
`could link the intestine and kidney in an endocrine
`pathway that regulates renal salt metabolism. Uro-
`guanylin secretion from the gastrointestinal tract or
`other organs such as the heart could be increased
`secondary to renal Na* retention as a compensatory
`mechanism involving enhanced circulating levels of
`uroguanylin functioning as a natriuretic and diuretic
`hormone. Thus, plasma uroguanylin may be elevated
`to help maintain body salt and water balance in both
`normal physiological circumstances and in diseases such
`as congestive heart failure and nephrosis,*° in which
`Na* and water retention cause edema. In contrast to
`uroguanylin, guanylin gene expression wasnotaffected
`by a high-salt diet? A low-salt diet reduced the ex-
`pression of guanylin to 30%-40% of the level found
`in control animals. The guanylin pathway is thought
`to be down-regulated as an adaptive response to salt
`restriction.
`In conclusion, membrane guanylyl cyclase-C, GC-C,
`is an intestinal receptor for guanylin and uroguanylin
`
`that is responsible for the stimulation of Cl- and HCO,~
`secretion into the intestinal
`lumen. Guanylin and
`uroguanylin are produced mainly in the gastrointestinal
`epithelial cells to serve in a luminocrine mechanism for
`the regulation of gastrointestinal fluid and electrolyte
`secretion. Uroguanylin also serves in an endocrine axis
`linking the intestine and kidney, where its natriuretic
`and diuretic actions contribute to the maintenance of
`Na? balance after the oral ingestion of NaCl. Enteric
`bacteria secrete peptide toxin that mimics guanylin pep-
`tides, activating GC-C, to produce secretory diarrhea.
`Guanylin peptides are involved in the pathophysiology
`of such gastrointestinal diseases as diarrhea and peptic
`ulcer, and in chronic renal diseases or congestive heart
`failure,
`in which guanylin and/or uroguanylin levels
`in the circulation and/or urine are elevated. Guanylin
`peptides function in the regulation of salt and water
`homeostasis in the gastrointestinal tract and kidney.
`
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