Brazilian Journal of Medical and Biological Research (1999) 32: 1329-1336
`ISSN 0100-879X
`
`Guanylin peptides: cyclic GMP
`signaling mechanisms
`
`L.R. Forte! ,
`R.H. Freeman,
`W.J. Krause* and
`R.M. London! 2
`
`'Harry S. Truman Veterans’ Hospital, Departments of
`Pharmacology, *Physiology and ‘Pathology and Anatomical Sciences,
`School of Medicine, Missouri University, Columbia, MO, USA
`
`Key words
`« Kidney
`« Intestine
`
`« Guanylate cyclase
`« Chloride secretion
`¢ Sodium excretion
`
`Correspondence
`L.R. Forte
`
`Department of Pharmacology
`School of Medicine
`Missouri University
`M-515 Medical Sciences Building
`Columbia, MO 65212
`USA
`Fax: + 1-573-884-4558
`E-mail: Irf@ missouri.edu
`
`Presentedat the Meeting
`“NOBrazil, Basic and Clinical
`Aspects of Nitric Oxide”,
`Foz do Iguacu,PR,Brazil,
`March 10-13, 1999.
`
`Received May 28, 1999
`Accepted June 22, 1999
`
`Abstract
`
`Guanylate cyclases (GC) serve in two different signaling pathways
`involving cytosolic and membrane enzymes. Membrane GCsare
`receptors for guanylin and atriopeptin peptides, two families ofcGMP-
`regulating peptides. Three subclasses ofguanylin peptides contain one
`intramolecular disulfide (lymphoguanylin), two disulfides (guanylin
`and uroguanylin) and three disulfides (E. coli stable toxin, ST). The
`peptides activate membrane receptor-GCsandregulate intestinal Cl-
`and HCO;secretion via cGMP in target enterocytes. Uroguanylin and
`STalsoelicit diuretic and natriuretic responses in the kidney. GC-C is
`an intestinal receptor-GC for guanylin and uroguanylin, but GC-C
`may not be involved in renal cGMP pathways. A novel receptor-GC
`expressed in the opossum kidney (OK-GC) has been identified by
`molecular cloning. OK-GC cDNAsencodereceptor-GCsin renal
`tubules that are activated by guanylins. Lymphoguanylin is highly
`expressed in the kidney and heart where it may influence cGMP
`pathways. Guanylin and uroguanylin are highly expressedin intestinal
`mucosato regulate intestinal salt and water transport via paracrine
`actions on GC-C. Uroguanylin and guanylin are also secreted from
`intestinal mucosa into plasma where uroguanylin servesas an intesti-
`nal natriuretic hormoneto influence body Na* homeostasis by endo-
`crine mechanisms. Thus, guanylin peptides control salt and water
`transport in the kidney and intestine mediated by cGMP via membrane
`receptors with intrinsic guanylate cyclase activity.
`
`Introduction
`
`Guanylin, uroguanylin and lymphogua-
`nylin are heat-stable peptides that regulate
`the enzymatic activity of cell-surface guany-
`late cyclase signaling molecules. Guanylin
`wasthe first endogenouspeptide identified
`and wasisolated as a 15 aminoacid peptide
`from rat intestine that stimulates cGMPpro-
`duction in T84 intestinal cells (1). Urogua-
`nylin was next isolated from opossum urine
`
`as biologically active, 13, 14 and 15 amino
`acid peptides using the T84 cell cGMPbio-
`assay (2). Lymphoguanylin wasrecently iden-
`tified using a PCR-based homology cloning
`strategy to isolate cDNAs from opossum
`spleen and other lymphoid tissues, which
`encode a 109 amino acid precursorthat is
`most similar in its primary structure to pre-
`prouroguanylin (3). Guanylin and uroguany-
`lin have two intramoleculardisulfide bonds,
`but lymphoguanylin has only one disulfide
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`1330
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`L.R. Forte et al.
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`bond. A synthetic form of lymphoguanylin
`also activates guanylate cyclase (GC)-recep-
`tors in human T84 intestinal and opossum
`kidney (OK)cells. The family of guanylin
`regulatory peptides share similarities both in
`primary structures and biological activities
`with the heat-stable enterotoxin (stable toxin,
`ST) peptides secreted by strains of enteric
`microorganismsthat cause a watery form of
`diarrhea similar to that seen in cholera (4).
`Thus, bacteria-derived ST peptides are mo-
`lecular mimics ofguanylin peptidesacting to
`stimulate fluid and electrolyte secretion into
`the lumen ofthe intestine by activation of
`native guanylin receptors located on the api-
`cal surfaces of enterocytes lining the intes-
`tine. Biologically active peptides in the
`guanylin family of proteins are found at the
`COOH-terminiof longer precursor polypep-
`tides that are secreted as biologically inac-
`tive prohormonesor protoxins (5-8). Pro-
`teolytic enzymes serve as converting en-
`zymesto activate the proguanylinsor proSTs,
`but these important enzymes have not been
`identified thus far.
`
`Physiological actions of guanylin
`peptides
`
`Physiological actions of the endogenous
`guanylin peptides include the regulation of
`intestinal fluid secretion during digestion,
`and neutralization of HCI in the duodenum
`and of organic acids derived from enteric
`bacteria in the large intestine (9,10). Guany-
`lins and STs stimulate the electrogenic se-
`cretion of both chloride and bicarbonate an-
`ions, which provides the physiological driv-
`ing force to accomplish fluid secretion into
`the intestinal lumen. Controlofthese intesti-
`nal functions by guanylin peptides is medi-
`ated via intracellular cGMP through activa-
`tion of cGMP-dependentprotein kinase II
`(cG-kinase IT) and/or cAMP-dependentpro-
`tein kinase II (cA-kinaseII) with subsequent
`phosphorylation of the cystic fibrosis trans-
`membrane conductance regulator (CFTR)
`
`protein (11,12). CFTR and GC-receptors are
`localized together in apical plasma mem-
`branes of target enterocytes. The first GC-
`receptor for guanylin family peptides was
`identified at the molecularlevel (i.e., GC-C)
`by molecular cloning ofcDNAsencoding an
`intestinal membraneprotein that binds !*°I-
`ST with high affinity and is activated by ST
`(13). Transgenic suckling mice with dis-
`abled GC-C genes have a markedreduction
`in the intestinal fluid secretion responseto E.
`coli ST,indicating that GC-C is responsible
`for a major componentofthe intestinal ac-
`tions of guanylin peptides to enhance fluid
`secretion (14,15). However, about 10% of
`specific '*5I-ST binding to receptor sites on
`intestinal membranesstill remain functional
`in GC-C-knock out (GC-C-KO)animals (15).
`This suggests that an additional gene or mul-
`tiple genes encoding GC-receptors for
`guanylin agonists exist
`in the mouse ge-
`nome.
`
`Bacterial STs were the first peptides
`shown to activate GC-receptor signaling
`molecules in the intestine, thus causing se-
`cretory diarrhea (4). Moreover,the intestinal
`GC targets for ST peptides in the intestine
`were considered unique and not existing
`outside the intestinal epithelium until we
`discovered that E. coli ST also stimulates
`GC-receptors found on the surface of OK
`and potoroo kidney (PtK-2) cell lines (Fig-
`ure 1; Ref. 16). It can be seen from oneofour
`original experiments that these two kidney
`cell lines have remarkable cGMPresponses
`to E. coli ST, but only small cGMP re-
`sponses to atriopeptin-A and no detectable
`responsesto the nitric oxide donor, sodium
`nitroprusside. The additional demonstration
`that kidney cortex has specific GC-receptors
`that are activated by ST implies that this
`class of cell surface GC-receptors plays a
`muchbroaderphysiological role in the body
`than was previously recognized (17-19). It
`should be emphasized that these findings
`were made well before the first guanylin
`peptides were isolated (1,2). Putative “ST
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`Guanylin peptides and cGMP
`
`1331
`
`receptors” were also foundin other epithelia
`of the opossum in addition to the receptors
`localized to brush border membranes (BBM)
`of epithelial cells lining the intestinal tract
`and within renal tubules (16-19). The exist-
`ence of renal, hepatic, airway andtesticular
`“ST receptors” predicted that endogenous
`ST-like peptides exist to regulate the activity
`of the extra-intestinal as well as intestinal
`GC-receptors. These seminal experiments
`led directly to the subsequent isolation of
`guanylin and uroguanylin peptides from in-
`testine and urine, respectively (1,2). Both
`guanylin and uroguanylin are produced in
`the intestine, but uroguanylin is the major
`bioactive peptide found in urine, which con-
`tains either no guanylin or very small a-
`mounts of the peptide (2,20-22). Uroguany-
`lin and ST stimulate the enzymatic activity
`of renal tubular GC-receptors and increase
`the urinary excretion of sodium, potassium
`and waterin both the perfused rat kidney ex
`vivo and the mouse in vivo (16-19,23-25).
`Guanylin is less potent in the stimulation of
`urinary Na* and water excretion compared
`to either uroguanylin or ST, but guanylin
`does have marked kaliuretic activity in the
`perfused rat kidney (24). Thus, uroguanylin
`hasbiological activity consistent with a pep-
`tide hormonethat influences renal function
`by regulating the urinary excretion of so-
`dium chloride as a physiological mechanism
`that contributes to the maintenance of Na*
`balance in the body. A natriuretic peptide
`such as uroguanylin waspredictedto exist in
`the digestive system forrelease into the blood-
`stream for the purpose of stimulating the
`urinary excretion of NaCl followinga salty
`meal (26,27). Uroguanylin is a prime candi-
`date for this “intestinal natriuretic hormone”
`becauseit is produced at extraordinarily high
`concentrations in the upper small intestine
`and is released following a high salt meal
`(28-32). Uroguanylin mRNAsare most abun-
`dant
`in the small
`intestine compared to
`guanylin mRNA levels, which peak in the
`large intestine. Uroguanylin and prourogua-
`
`nylin are also foundin the circulation andit
`is likely that the gastrointestinal (GI) tract is
`a main source of the plasma peptides (7,33,
`34). Secretion of uroguanylin from GI mu-
`cosa into the plasmain responseto oral NaCl
`may explain the prolonged increase in uri-
`nary sodium excretion that occurs following
`a high salt meal (26,27).
`
`Identification of a kidney
`GC-receptor for uroguanylin
`
`Wesoughtto elucidate the primary struc-
`ture of a membrane GC expressed in cul-
`tured OK cells and in the opossum kidney
`becausethe prior discovery ofthis renal GC-
`receptor wasa stimulusthatultimately led to
`the isolation of guanylin and uroguanylin
`(1,2,16-19). A PCR-based cloning strategy
`was used to isolate 3762-bp cDNAs from
`RNA/cDNAsexpressed in OK cells and
`opossum kidney cortex (35,36). Transfec-
`tion and expression of the OK-guanylate
`cyclase (OK-GC) cDNA into COS and
`HEK293 cells producesa cell surface GC-
`receptor of~160 kDasize that is activated by
`uroguanylin, guanylin and E. coli ST pep-
`tides. OK-GC cDNAcontains an open read-
`ing frame encoding a 1049 residue mature
`
`70000
`
`60000
`
`>
`=
`hee
`© 40000
`S
`E 30000 +
`<
`=
`S 20000
`3 1000
`750
`500
`250
`
`OK
`
`Basal
`
`ST ANP-A Na-NP
`
`13000
`
`12000
`11000
`wn
`3 10000
`oO
`2 9000
`=
`eg
`8000
`& 7000
`a
`6000
`Q
`500
`250
`
`0
`
`PtK-2
`
`Basal
`
`ST ANP-A Na-NP
`
`Figure 1 - Activation of surface receptors on OK and Ptk-2 cells by Escherichia coli stable
`toxin (ST) and atriopeptin-A. Confluent cells were treated for 15 min at 37°C in DMEM-
`HEPES-MIX with 0.5 uM ST, 0.1 uM atriopeptin-A (ANP-A) or 1 mM sodium nitroprusside
`(Na-NP). The data are reported as the mean of 4 experiments with opossum kidney (OK)
`cells and 2 experiments with potoroo kidney (PtK-2) cells.
`
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`1332
`
`L.R. Forte et al.
`
`protein belonging to the family ofmembrane
`GC-receptor signaling molecules. OK-GCis
`similar to other membrane GCproteins con-
`taining NH)-terminal agonist-binding do-
`mains, a single membranespanandintracel-
`lular kinase-like and GC catalytic domains.
`OK-GCis 72, 76 and 75% identical in its
`overall structure comparedto the intestinal
`BBM-localized GC-C receptorsfor guanylin
`peptides found in rats, humans andpigs,
`respectively (13,37,38). The catalytic do-
`mains of OK-GC and GC-C receptorsofrat,
`human and porcine intestine share 92, 94
`and 95% identity, respectively. The most
`highly variable region of membrane GC-
`receptors occurs within the NH,-terminal
`ligand-binding domains of these proteins.
`OK-GCshares only 55-59% identity in this
`domain when compared to GC-C intestinal
`receptors for guanylin peptides. The GC-C
`receptors of the rat are more closely related
`to human and pig GC-Cin the ligand-bind-
`ing domainswiththese proteins sharing ~70%
`identity in this region. Thus, OK-GCis a
`distinctive renal GC-receptorthat, along with
`the intestinal GC-C receptor, provides two
`distinctive molecular subtypes of GC-recep-
`tors for a growing family of membranere-
`ceptors for the guanylin peptides.
`OK-GC mRNAlevels were measured in
`total RNA prepared from tissues of opos-
`sums by Northern and RT-PCR. A 3.8-kb
`mRNA was detected in kidney, OK cells,
`urinary bladder, adrenal gland, heart and
`intestine. Lower levels of OK-GC mRNA
`were detected in renal medulla comparedto
`cortex. Tissues with lower levels of OK-GC
`mRNAare urinary bladder, adrenal gland
`and both the ventricles andatria of the heart.
`In the GI tract, high levels ofOK-GC mRNA
`were measured in both the small and large
`intestine. Thus, OK-GC mRNAis highly
`expressedin the kidney cortex and intestinal
`mucosa with mRNAtranscripts occurring at
`lowerlevels in a numberof other organs.
`OK-GCis a candidate forthe renal tubu-
`lar receptor that is activated by uroguanylin
`
`and/or guanylin peptides that signals via
`cGMPto regulate the urinary excretion of
`sodium, potassium and water (23-25). OK-
`GCisthe first kidney receptor for guanylin
`family peptides to be fully defined at the
`molecular level. Prior to the identification of
`GC-receptor signaling molecules for E. coli
`ST in the OK and PtK-2 cells and in the
`opossum kidney,
`it was thought that ST-
`stimulated GCs were restricted to intestinal
`mucosa (16-19). Thus, identification of OK-
`GC in OKcells and opossum kidney opened
`up a new field of inquiry that culminated
`recently with the discoveries of guanylin,
`uroguanylin and lymphoguanylin peptides
`(1-3). These peptides activate OK-GC and
`may serve as endogenous agonists for this
`membrane GC-receptor. The OK cell line
`has the differentiated properties of renal
`proximaltubules and receptor autoradiogra-
`phy experiments with !*I-ST have clearly
`shownthat specific binding sites for this
`uroguanylin-like radioligand are found in
`cells of both convoluted and straight por-
`tions of proximal tubules (16-19). High lev-
`els of!?°I-ST-labeled receptorsare also found
`in BBMsisolated from the kidney andintes-
`tine, indicating that this GC-receptoris pref-
`erentially localized to apical plasma mem-
`branes of both kidney andintestinal target
`cells. It is likely that OK-GC serves as a
`physiological receptor for uroguanylin, which
`is the major bioactive guanylin family pep-
`tide in urine (2,20-22). However, guanylin
`wasalso isolated from opossum urine indi-
`cating that this peptide may influence renal
`function in vivo via cGMP(2). Active uro-
`guanylin and inactive prouroguanylin pep-
`tides circulate in plasma, thus providing a
`source for the urinary forms of bioactive
`uroguanylin in opossum, humanandrat urine
`(2,20-22). The kidney also expresses uro-
`guanylin and guanylin mRNAs, which of-
`fers the possibility that an intra-renal signal-
`ing pathwayexists for the guanylin peptides
`as well as providing another potential source
`of uroguanylin in the urine (35,39). Physi-
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`Guanylin peptides and cGMP
`
`1333
`
`ologically, uroguanylin may regulate kidney
`function via an endocrine axis linking the
`intestine to the kidney and/or through an
`intra-renal paracrine mechanism. Both pos-
`sibilities involve the activation of OK-GC as
`a key signaling molecule in renal tubular
`target cells that possess the guanylate cy-
`clase-cGMPsignaling machinery.
`
`Identification of lymphoguanylin
`
`ing frame within the lymphoguanylin cDNAs
`encode a 109 aminoacid polypeptidethat is
`84% identical to preprouroguanylin and 40%
`identical to preproguanylin (7,35). At the
`COOH-terminusof the 109 aminoacid pre-
`prolymphoguanylin is a 15 aminoacid pep-
`tide that is 80% identical to uroguanylin, but
`shares only 40% identity with guanylin (Fig-
`ure 3). Illustrated for comparison are the
`structures of opossum lymphoguanylin, uro-
`guanylin, guanylin and an E. coli ST peptide,
`which form three different subclasses of
`guanylin peptides based on the number of
`intramolecular disulfides found within the
`active peptides. A major difference within
`lymphoguanylin is the tyrosine!residue
`because guanylins and uroguanylins have
`
`A third memberofthe guanylin family of
`peptides was sought since biologically ac-
`tive uroguanylin peptides wereisolated from
`opossum urine (2). This inquiry culminated
`in the isolation ofcDNAsencoding opossum
`preprolymphoguanylin, a third and unique
`memberof the guanylin family of endoge-
`nousregulatory peptides (3). The identifica-
`tion of lymphoguanylin stems from recent
`SHTCEICAFAACAGG|CO2
`experiments showing that mRNAtranscripts
`for guanylin and/or uroguanylin and their
`GC-receptors are expressed broadly intis-
`sues of the digestive, renal-urinary, cardio-
`109
`;
`95)
`vascular, reproductive,
`lymphoid-immune
`sHN W/EPROSueTNTocece.cwmactsy| co;
`and central nervous organ systems (7,35).
`Moreover, hybridization assays revealed the
`existence of a novel mRNAtranscript that
`hybridizes with both uroguanylin and
`guanylin cDNA probes, but is substantially
`longer than either the 1.2-kb uroguanylin or
`0.8-kb guanylin mRNAs(3). Weidentified a
`third guanylin-related mRNAtranscript by
`molecular cloning using a homology cloning
`strategy based on the PCR with cDNAsiso-
`lated from opossum tissues. The deduced
`polypeptide was named preprolymphogua-
`nylin because the first cDNAsisolated were
`derived from several different lymphoid or-
`gans and the encoded polypeptide is similar
`in primary structure to preprouroguanylin
`and preproguanylin (Figure 2). Sequence
`analyses of multiple independent cDNA
`clones reveals that the lymphoguanylin
`cDNAis 92.7% identical to the correspond-
`ing nucleotide sequences for preprourogua-
`nylin reported previously (7). An open read-
`
`t/a
`
`86
`
`1 °10
`
`95
`109
`/Ecece co;
`
`.N
`
`Figure 2 - Prepropeptide structures for guanylin, uroguanylin and lymphoguanylin. The
`amino acid sequencesofthe active peptides are shownusingthesingle letter abbreviation
`of the amino acids within the COOH-terminal regions of each opossum polypeptide.
`
`ClassI
`E. coli ST
`
`ClassII
`Guanylin
`
`Uroguanylin
`
`NS S NY C CE LCCNP ACT GC Y
`
`IC A F AACA GC
`S H T C E
`es
`NV AC TGC
`
`QEDCELCI
`
`Class III
`Lymphoguanylin
`
`Ee ew bey
`
`NMAC TG Y
`
`Figure 3 - Comparisonof the primary structures of subclasses within the guanylin family of
`biologically active peptides. The peptide subclasses are provided from top to bottom in the
`orderof their discovery as: Class | peptides containing 3 disulfides, class Il peptides with 2
`disulfides andclassIII peptides that have only 1 intramolecular disulfide bond. The single
`letter abbreviation is used to designate amino acid residues in each peptide. E. coli ST, E.
`coli stable toxin.
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`1334
`
`L.R. Forte et al.
`
`cysteine residues at this position (1,2). The
`disulfide bonds formed betweenthefirst and
`third and second to fourth cysteines in the
`peptide chain were thought to be required
`for biological activity of guanylin and uro-
`guanylin. The replacement ofcysteine!with
`the tyrosine!residue is a novel molecular
`change within the guanylin family of pep-
`tides. Lymphoguanylin is uroguanylin-like
`because it has two glutamate residuesin its
`NH,-terminal domain. Opossum uroguany-
`lin has glutamate and aspartate residues and
`all other uroguanylin molecules in mamma-
`lian species have acidic residues at these
`positions (2,20,29,30,39). Shared between
`lymphoguanylin and uroguanylinis an inter-
`nal asparagine residue, which is also found
`in the bacterial ST peptides. Guanylin pep-
`tides have an aromatic amino acid at this
`position (1,2,6,7,35). The third difference is
`the methionine!™ substitution in lymphogua-
`nylin for the valine!“ of uroguanylin.
`A lymphoguanylin peptide was synthe-
`sized and oxidized to form an intramolecular
`disulfide bond between cysteine”® and cys-
`teine!, with cysteine!” protected. Synthetic
`lymphoguanylin containing a single disul-
`fide stimulates cGMP production in human
`T84 intestinal cells, but its potency is less
`than uroguanylin or guanylin (3). All three
`guanylin peptides are full agonists in the
`stimulation of intestinal GC-C expressed in
`humanintestinal T84 cells (1-3). When the
`potencies of these peptides were examined
`in OK cells, we observed that the OK-GC
`receptors werealso activated by lymphogua-
`nylin.
`Lymphoguanylin mRNAtranscripts of
`~1.6 kb were detected in total RNA prepara-
`tions using Northern hybridization assays
`with a lymphoguanylin cDNA probe (3).
`Although lymphoguanylin cDNAswereiso-
`lated first from lymphoid tissues, we were
`surprised to discover that tissues with the
`most abundant lymphoguanylin mRNAsare
`the atria and ventricles of heart as well as
`kidney cortex. We also detected ~1.6 kb
`
`mRNAsfor lymphoguanylin using Northern
`assays in RNA from spleen, thymus and
`testis. Spleen appears to have the most abun-
`dantlevels oflymphoguanylin mRNAwithin
`tissues ofthe lymphoid/immunesystem. RT-
`PCR wasusedto amplify the mRNA-cDNAs
`of spleen, thymus, lymph nodes,circulating
`white blood cells, bone marrow,cerebellum
`and testis. Lymphoguanylin cDNAs were
`cloned and sequenced to confirm that lym-
`phoguanylin mRNAsare expressed in thy-
`mus, lymph nodes, circulating white blood
`cells, bone marrow,spleen, cerebellum, kid-
`ney, OKcells, testis, ovary and heart of the
`opossum.
`
`Conclusion
`
`Three different guanylin regulatory pep-
`tides, guanylin, uroguanylin and lymphogua-
`nylin, together with their cognate receptors,
`GC-C and OK-GC,provide signal transduc-
`tion machinery for cGMP-mediated regula-
`tion of cellular function in the kidney,intes-
`tine and otherepithelia. Combinatorial com-
`plexity is achieved by mixing and matching
`certain subtypes of GC-receptors with cell-
`specific expression of one or more guanylin
`peptides for local control of cGMPlevels in
`nearby target cells. A modelforthis type of
`local regulation by a paracrine mechanismis
`best exemplified by the guanylin-cGMP-ion
`transport mechanismsthat have beenrigor-
`ously demonstrated in the intestinal mucosa.
`An endocrine axis for uroguanylin serving
`as an intestinal natriuretic hormone involved
`in the maintenance of body sodium balance
`has emergedas well. The recent discovery of
`lymphoguanylin coupled with evidencethat
`additional subtypes ofmembrane GC-recep-
`tors exist for guanylin peptides suggest that
`signal transduction pathwaysutilized by the
`guanylin family of regulatory peptides are
`considerably more complicated than previ-
`ously suspected.It is likely that novel physi-
`ological actions of the guanylin regulatory
`peptides will be discovered as more details
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`Guanylin peptides and cGMP
`
`1335
`
`are learned concerning the cellular and mo-
`lecular mechanisms of action of these im-
`portant peptide hormones.Physiological roles
`for guanylin peptides in the regulation of
`target cell function via intracellular cGMP
`are likely to be documentedin the future for
`the immune, reproductive, cardiovascular and
`
`central nervous organ systems. This will
`complement the physiological roles of
`guanylin and uroguanylin that have been
`documented thus far in the regulation of
`fluid and electrolyte transport within the in-
`testine and kidney.
`
`References
`
`1. Currie MG, Fok KF, Kato J, Moore RJ,
`Hamra FK, Duffin KL & Smith CE (1992).
`Guanylin: an endogenous activatorof in-
`testinal guanylate cyclase. Proceedings of
`the National Academyof Sciences, USA,
`89: 947-951.
`SL,
`LR, Eber
`2. Hamra
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