Proc. Natl. Acad. Sci. USA
`Vol. 94, pp. 2705-2710, March 1997
`Pharmacology
`
`Regulation of intestinal uroguanylin/guanylin receptor-mediated
`responses by mucosal acidity
`
`F. Kent Hamra**t#, SAMMYL. EBER*t, DAvID T. CHINT, MARK G. CURRIETS, AND LEONARD R. ForTE*14
`
`*Truman Veterans Affairs Medical Center and tDepartments of Pharmacology and Biochemistry and Molecular Biology Program, Missouri University, Columbia,
`MO65212; and $Searle Research and Development, St. Louis, MO 63167
`
`Communicated by Philip Needleman, Monsanto Company, St. Louis, MO, January 2, 1997 (received for review March 27, 1996)
`
`Guanylin and uroguanylin are intestinal
`ABSTRACT
`peptides that stimulate chloride secretion by activating a
`commonset of receptor— guanylate cyclase signaling molecules
`located on the mucosalsurface of enterocytes. High mucosal
`acidity, similar to the pH occurring within the fluid micro-
`climate domain at
`the mucosal surface of the intestine,
`markedly enhances the cGMP accumulation responsesof T84
`humanintestinal cells to uroguanylin. In contrast, a mucosal
`acidity of pH 5.0 renders guanylin essentially inactive. T84
`cells were used as a model epithelium to further explore the
`concept that mucosal acidity imposes agonist selectivity for
`activation of the intestinal receptors for uroguanylin and
`guanylin, thus providing a rationale for the evolution of these
`related peptides. At an acidic mucosal pH of5.0, uroguanylin
`is 100-fold more potent than guanylin, but at an alkaline pH
`of 8.0 guanylin is more potent than uroguanylin in stimulating
`intracellular cGMP accumulation and transepithelial chlo-
`ride secretion. The relative affinities of uroguanylin and
`guanylin for binding to receptors on the mucosal surface of
`T84 cells is influenced dramatically by mucosalacidity, which
`explains the strong pH dependency of the cGMP andchloride
`secretion responses to these peptides. The guanylin-binding
`affinities for peptide—-receptor interaction were reduced by
`100-fold at pH 5 versus pH 8, whereas the affinities of
`uroguanylin for these receptors were increased 10-fold by
`acidic pH conditions. Deletion of the N-terminal acidic amino
`acids in uroguanylin demonstrated that these residues are
`responsible for the increase in binding affinities that are
`observed for uroguanylin at acidic pH. We conclude that
`guanylin and uroguanylin evolved distinctly different struc-
`tures, which enables both peptides to regulate,
`in a pH-
`dependent fashion,
`the activity of receptors that control
`intestinal salt and water transport via cGMP.
`
`Guanylin and uroguanylin are structurally related peptides
`that were isolated from intestinal mucosa and urine (1-5). A
`receptor for guanylin and uroguanylin that has beenidentified
`at the molecular level is a transmembrane form of guanylate
`cyclase, termed GC-C (6). This membrane protein wasorigi-
`nally discovered as an intestinal receptor for the heat-stable
`toxin (ST) peptides, which are secreted intraluminally by
`enteric bacteria that cause traveler’s diarrhea (7). Bacterial ST
`peptides are related in primary structure to uroguanylin and
`guanylin,
`thus acting as molecular mimics of the enteric
`peptide hormones (reviewed in refs. 8 and 9). Membrane
`receptor—guanylate cyclases are found on the luminal surface
`of enterocytes throughout the small and large intestine and in
`other epithelia (10-13). Binding of peptide agonists to an
`extracellular domain of the receptoractivates the intracellular
`
`The publication costs of this article were defrayed in part by page charge
`payment. This article must therefore be hereby marked “advertisement”in
`accordance with 18 U.S.C. §1734 solely to indicate this fact.
`Copyright © 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA
`0027-8424/97/942705-6$2.00/0
`PNASis available online at http://www.pnas.org.
`
`catalytic domain producing the second messenger cGMP
`within target enterocytes (1-6). Intracellular cGMPstimulates
`transepithelial chloride secretion by regulating the phosphor-
`ylation state and chloride channelactivity of the cystic fibrosis
`transmembrane conductance regulator, an apical protein that
`is located with the receptors for uroguanylin, guanylin, and ST
`peptides (14-16).
`Isolation of uroguanylin from opossum urine (2) followed by
`the cloning of a colon cDNA that encodes opossum pre-
`prouroguanylin (17) revealed that the uroguanylin and gua-
`nylin genes are evolutionarily related (18-20). Furthermore,
`the mRNAsandprecursorproteins for both uroguanylin and
`guanylin are expressed together throughout the mucosa of
`small and large intestine along with their receptors (5, 11,
`17-20). This raised a question of whether the differences in
`primarystructure between guanylin and uroguanylin evolved
`to regulate intestinal salt and water transport
`through a
`cooperative mechanism using common receptor—guanylate
`cyclase signaling molecules located on the mucosal surface of
`the intestine.
`During the isolation of uroguanylin, guanylin, and their
`prohormoneprecursors, we observed that acidic column re-
`agents markedly attenuated the cGMPresponses of T84 cells
`to guanylin, but enhanced the responses to uroguanylin (4, 5).
`This pH dependencyfor activation of guanylate cyclase was
`successfully used to detect guanylin and uroguanylin during
`their separation andpurification from intestinal mucosa. The
`possibility was then considered that the primary structures of
`guanylin and uroguanylin could have evolved to regulate the
`enzymatic activity of a commonset of receptors over the wide
`range of mucosalacidity that occurs within the intestinal lumen
`during digestion (21-24). We report here that high mucosal
`acidity rendered guanylin ineffective as a cGMP agonist and
`chloride secretogogue, whereasan acid pH markedly enhanced
`the potency of uroguanylin. A mucosal pH of8.0 substantially
`increased the potency of guanylin but decreased the potency of
`uroguanylin. These changes in agonist potencies were ex-
`plained by corresponding directionalshifts in the affinities of
`guanylin and uroguanylin for binding to receptors at pH 5.0
`versus 8.0. Uroguanylin and guanylin cooperatively regulate
`the guanylate cyclase activity of a common set of mucosal
`receptors in a pH-dependentfashion,thus providing an enteric
`signaling pathway for the intrinsic, paracrine regulation of
`intestinal salt and water transport.
`
`MATERIALS AND METHODS
`
`cGMP Accumulation Assay in T84 Cells. T84 cells were
`cultured in 24-well plastic dishes, and the cGMPlevels were
`
`Abbreviation: ST, heat-stable toxin.
`+Present address: Howard Hughes MedicalInstitute and Department
`of Pharmacology, University of Texas Southwest Medical Center,
`5323 Harry Hines Boulevard, Dallas, TX 75235-9050.
`To whom reprint requests should be addressed at: Department of
`Pharmacology, School of Medicine, University of Missouri, Colum-
`bia, MO 65212. e-mail: Leonard R. Forte@muccmail.missouri.edu.
`MSN Exhibit 1021 - Page 1 of 6
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`Pharmacology: Hamraetal.
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`Proc. Natl. Acad. Sci. USA 94 (1997)
`
`measuredin control and agonist-stimulated cells by radioim-
`munoassay(2). Synthetic peptides were suspendedin 200 plof
`each of two assay buffers; pH 8.0 buffer [Dulbecco’s modified
`Eagle’s medium (DMEM)/20 mM N-(2-hydroxyethyl)pipera-
`zine-N’-(2-ethanesulfonic acid) (Hepes)/50 mM sodium bi-
`carbonate, pH 8.0/1 mMisobutylmethylxanthine (IBMX)] and
`pH 5.0 buffer [DMEM/20 mM 2-(N-morpholino)ethanesul-
`fonic acid (Mes), pH 5.0/1 mM IBMX]. T84 cells were washed
`twice with 200 pl of the respective pH 8.0 and pH 5.0 buffers
`before addition of the peptides. These solutions containing the
`bioactive peptides were then added to T84 cells and incubated
`at 37°C for 40 min. Following incubation, the reaction medium
`wasaspirated, and 200 pl of 3.3% perchloric acid was added
`per well to stop the reaction and extract cGMP.Theextract was
`adjusted to pH 7.0 with KOH,centrifuged, and 50 pl of the
`supernatant was used to measure cGMP.
`Measurement of Short Circuit Current in T84 Cell Mono-
`layers. T84 cells were raised on collagen-coated permeable
`filters and mounted in custom-made Ussing chambers for
`measurementof transepithelial chloride secretion as described
`(15, 25). The buffer in the basolateral reservoir was a Krebs—
`Ringer solution (pH 7.4) (25 mM sodium bicarbonate), con-
`taining 10 mM glucose. Buffers in the apical reservoir were
`Krebs-Ringer solutions containing 10 mM glucose and ad-
`justed to either pH 5.5 (25 mM Mes,minus sodium bicarbon-
`ate) or pH 7.8 (60 mM sodium bicarbonate). The pH of
`basolateral and apical reservoir buffer solutions was main-
`tained by bubbling 95% O2/5% CO, through the medium
`(except for the apical reservoir at pH 5.5, which did not use a
`bicarbonate/CO, buffering system). The signals for short
`circuit current (Isc) and potential difference across the epi-
`thelium were measured every 20 sec, digitized with an ADC-1
`data recording system (Remote Measurement Systems, Seat-
`tle) and stored for later analysis. The Isc observed with T84
`cells cultured on permeablefilters has been shownto be caused
`by the net secretion of chloride across T84 cell monolayers
`whencells were treated with various chloride secretogogues,
`including guanylin, uroguanylin, and Escherichia coli ST (1, 2,
`
`15, 26).Competitive Radioligand-BindingAssay in T84 Cells. Com-
`
`petitive radioligand-binding experiments were performed with
`intact, T84 cells cultured in 24-well plastic dishes using meth-
`ods that were previously described (15), but at medium pH
`values of 5.0 and 8.0. Identical buffer conditions at pH 5.0 and
`pH 8.0 were used in the competitive radioligand-binding assays
`as those used in the cGMP accumulation bioassays. !I-ST-
`(1-19) was used as the radioligand (2, 15). Concentration-
`response curves for cGMP accumulation and competitive
`radioligand-binding curves performed with each agonist were
`analyzed with the computer program PRISM (Graphpad, San
`Diego). A better fit of the binding data was consistently
`obtained with a two-site model as comparedwith a single-site
`model for all agonists at either pH 5 or pH 8 (15). The
`concentrations at which specific binding of the radioligand at
`each bindingsite was inhibited by 50% ICs», were obtained by
`nonlinear regression of the untransformed competition bind-
`ing data. The apparent equilibrium dissociation constants, Ki,
`for the competing ligands were calculated from the computed
`ICs9 values using the previously reported estimates of the
`affinity of the radioligand in these cells, Kg ~15 nM (27): Kj =
`ICs0/1 + (L/Ka), where L equals the radioligand concentra-
`tion. It should be noted that the calculated ICs9 and K; values
`are essentially identical because the concentration of the
`radioligand used in these studies (~120 pM) was a small
`fraction of the reported binding affinity of the radioligand.
`Synthesis of Uroguanylin, Guanylin, and ST Peptides. Hu-
`man uroguanylin (NDDCELCVNVACTGCL) and human
`guanylin (PGTCEICAYAACTG(O), and the opossum formsof
`uroguanylin®-!°? (QEDCELCINVACTGC), uroguany-
`lin°8-1°° (CELCINVACTGC), and E. coli ST-(5-17) (CCEL-
`
`CCNPACAGC)were synthesized by the solid-phase method
`on an Applied Biosystems model 431A peptide synthesizer and
`purified by reverse-phase Cig chromatography as previously
`(1-3). The structure and mass of synthetic peptides were
`verified by electrospray mass spectrometry, gas-phase se-
`quence analysis, and amino acid composition analysis.
`Cell Culture. T84 cells (passage 21 obtained from Jim
`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% fetal bovine serum
`and 60 yg of penicillin plus 100 wg of streptomycin per ml as
`described (2, 15).
`
`RESULTS
`
`Therelative potencies of the synthetic forms of human urogua-
`nylin and guanylin for stimulation of cGMP accumulation in
`intact T84 intestinal cells were assessed at medium pH values
`of 5.0 and 8.0, which represent the extremes of microclimate
`pH found at the mucosalsurface of the intestine. Experiments
`using opossum uroguanylin and guanylin provided additional
`insights into the optimal medium pHvaluesusedin this study
`(4). The potency of guanylin foreliciting cGMP accumulation
`responses in T84 cells was 10-fold greater when tested at pH
`8.0 compared with its potency at pH 5.0 (Fig. 14). In contrast,
`the potency of uroguanylin was reduced by 10-fold at pH 8.0
`compared with its potency at pH 5.0 (Fig. 1B). We previously
`reported that E. coli ST-(5-17) was 2- to 3-fold more potentat
`acidic pH compared with alkaline pH (4). Under acidic
`conditions, uroguanylin was 100-fold more potent than gua-
`nylin. At pH 5.0, 3000 nM guanylin was required to stimulate
`~200-fold increases in cellular cGMP, whereas 30 nM urogua-
`nylin elicited this magnitude of cGMPresponse. However, this
`rank order of potency was reversed at pH 8.0 with guanylin
`becoming 3-fold more potent than uroguanylin. For example,
`30 nM guanylin caused ~50-fold increases in cGMPlevels,
`whereas 100 nM uroguanylin was required at pH 8.0. Thresh-
`old stimulation of cGMP levels was observed with ~0.1 nM
`uroguanylin and ~10 nM guanylin at pH 5.0, whereasat pH 8,
`~0.3 nM guanylin and ~3 nM uroguanylin were required to
`stimulate cGMPincreases by at least 2-fold over the basal
`cGMPlevels. Thus, variations in mucosal pH similar to those
`that may occur within the intestinal lumen during digestion
`markedly and differentially influenced the cGMPresponsesof
`T84 cells to guanylin and uroguanylin.
`We further tested the effects of mucosal acidity on the
`relative potencies of guanylin, uroguanylin, and ST-(5-17) for
`stimulation of chloride secretion across monolayers of T84
`cells cultured on permeable filters and mounted in Ussing
`chambers. T84 cells secrete chloride in the serosal to mucosal
`direction and the magnitude of chloride transport can be
`measured as the input current required to maintain a trans-
`epithelial potential difference equal
`to zero (short circuit
`current, Isc). Guanylin, uroguanylin, and E. coli ST stimulate
`the Isc of T84 cells when added to the mucosal bath of Ussing
`chambers (1-3, 15, 26). In these experiments, the basolateral
`(serosal) surfaces of T84 cells were maintained at pH 7.4,
`whereasthe apical (mucosal) surface was maintained at either
`pH 5.5 or pH 7.8. The potency of guanylin for stimulation of
`chloride secretion was markedly increased at an apical pH of
`7.8 compared with pH 5.5 (Fig. 2 Upper). In contrast, urogua-
`nylin was considerably more potent in the stimulation of
`chloride secretion when the apical pH was 5.5 compared with
`its potency at pH 7.8 (Fig. 2 Lower). Fig. 3 compares the
`relative potencies of guanylin, uroguanylin, and ST-(5-17) for
`stimulating chloride secretion at a mucosal pH of 5.5 compared
`with pH 7.8 over a wide range of agonist concentrations.
`ST-(5-17) and uroguanylin act similarly by stimulating greater
`increases in chloride secretion at a mucosal pH of 5.5 com-
`pared with the stimulation observed at pH_7.8 (Fig. 3). The
`2 of 6
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`c2°= o T
`
`Proc. Natl. Acad. Sci. USA 94 (1997)
`
`2707
`
`© pH 5.5
`
`Guanylin
`
`* pH 7.8
`
`ST (5-17)
`
`Isc,UA/cm2
`
`ST (5-17)
`
`Uroguanylin
`
`ime, (min)
`
`Fic. 2. Effects of mucosal pH onthe stimulation of Isc in T84 cells
`by guanylin and uroguanylin. T84 cells were cultured on collagen-
`coated membranes and mounted in Ussing chambersas described. At
`the arrows, the indicated concentrations of guanylin (Upper), urogua-
`nylin (Lower), and E. coli ST-(5—17) were addedto the apical reservoirs
`containing medium at either pH 5.5 (©) or pH 7.8 (@). Forall
`experiments, the basolateral reservoir contained medium at pH 7.4.
`Data are representative experiments ofat least five separate experi-
`ments performed with each peptide at the mucosal pH valuesof 5.5
`and pH 7.8. Concentrations of guanylin and uroguanylin greater than
`100 nM were not used in these experiments because they consumed
`excessive amounts of the peptides.
`
`affinity for binding to the low affinity site when tested at pH
`8.0 compared with pH 5.0 (Fig. 4 Top). In contrast, parallel 9-
`to 10-fold increases in affinities for the high and low affinity
`sites were observed when uroguanylin was tested at pH 5.0
`versus pH 8.0, indicating that mucosal acidity increased the
`affinity of uroguanylin for binding to receptors (Fig. 4 Middle).
`In agreementwith a previous report (27), mucosal acidity had
`little influence on the binding affinities for ST-(5-17) inter-
`action with the receptors on T84 cells (Fig. 4 Bottom). The
`remarkable effects of mucosal pH on cGMPaccumulation and
`chloride secretion responses to these peptides may be ex-
`plained by pH-dependentshifts in the affinities of uroguanylin
`and guanylin for binding to guanylate cyclase effector mole-
`cules on the apical surface of this modelintestinal epithelium.
`The uniqueacidic residues at the N terminusof uroguanylin
`were postulated to be involved in the increased potencies and
`binding affinities for uroguanylin in the interaction of this
`peptide with T84 cell receptors at acidic versus alkaline pH. A
`truncated form of opossum uroguanylin®-!was synthesized
`without the N-terminal Gln®—Glu®°-Asp”aminoacidstotest
`the hypothesis that conformational changes in these residues
`95-109
`
`wane ©° NiSN'Exhibit1031--Page3 of 6
`MSNv. Bausch - IPR2023-00016
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`Pharmacology: Hamraetal.
`
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`[Uroguanylin], Log (M)
`
`Fic. 1. Comparison of cGMP accumulation responses to the
`synthetic forms of humanguanylin and uroguanylin in T84 cells under
`acidic and alkaline conditions. T84 cells were treated with the indi-
`cated concentrations of guanylin (A) or uroguanylin (B) for 40 min at
`a mucosal pHof5.0 (0) or pH 8.0 (@). Data are the meanof duplicate
`experiments performed with each agonist at each pH value. Concen-
`trations of guanylin and uroguanylin greater than 10 uM and 1 uM,
`respectively, were not used because it consumedexcessive amounts of
`the peptides.
`
`rank order of potencies for agonist-mediated stimulation of
`chloride secretion was ST > uroguanylin > guanylin at acidic
`pH andST > guanylin > uroguanylin at an alkaline pH (Fig.
`3). The relative potencies of uroguanylin, guanylin, and ST-
`(5-17) in the stimulation of transepithelial chloride secretion
`across monolayers of T84 cells at acidic versus alkaline pH
`matched their relative potencies for stimulation of cGMP
`levels under these conditions.
`Modulation by mucosalacidity of the relative affinities of
`uroguanylin and guanylin for binding to a commonset of
`receptors(1, 2, 15) could accountforthe effects of medium pH
`on the cGMPand chloride secretion responseselicited by these
`peptides in T84 cells. This hypothesis was tested using com-
`petitive radioligand-binding assays in cultured T84 cells with
`125]-ST-(1-19) as the radioligand (2, 15). Uroguanylin, guany-
`lin, and ST-(5-17) fully inhibited the binding of !*I-ST-(1-19)
`to apical receptors on T84 cells when tested at medium pH
`values of 5.0 and 8.0. Examination of the radioligand-binding
`data using computer-assisted fitting of curves to a two-site
`model (15) confirms that T84 cells have both high- and
`low-affinity bindingsites for each ligand. The Kj values for each
`peptide at medium pHvaluesof 5.0 versus 8.0 are found in the
`legend to Fig. 4. Guanylin had a 100-fold increase in affinity
`for binding to the high affinity site and a 30-fold increase in
`
`

`

` Proc. Natl. Acad. Sci. USA 94 (1997)
`
`OpH 5.0
`1006
`
`75
`
`50
`
`25.
`
`-12 -11 -10 -9
`
`-8
`
`-7
`
`-6
`
`-5
`
`@ pH 8.0
`
`
`
`2708
`
`AIsc,HA/cm?
`
`Pharmacology: Hamraetal.
`
`pH 5.5
`O ST (5-47)
`O Uroguanylin
`4 Guanylin
`
`pH 7.8
`@ ST (5-17)
`= Uroguanylin
`4 Guanylin
`
`125|-STBound,%ofBo
`
`= OooO J)
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`40~60
`[Peptide], Log (M)
`
`[Peptide], nM
`
`Fic. 3. Comparison of the relative potencies of uroguanylin,
`guanylin, and E. coli ST-(5-17) in the stimulation ofIsc in T84cells at
`the mucosal pH values of 5.5 (Upper) and pH 7.8 (Lower). Peptides
`were addedto the apical reservior at 10-15 minintervals starting with
`the lowest concentrations shown, followed by successive additions of
`the peptide. The basolateral reservior was maintained at pH 7.4. Data
`are the mean of five experiments performed with each agonist.
`Horizontal bars indicate the SEM for each point.
`
`creased potency of uroguanylin at acidic versus alkaline pH (4,
`17). The truncated peptide, uroguanylin’*!°, stimulated
`cGMPaccumulation in T84cells similarly at pH 5.0 compared
`with pH 8.0 (Fig. 5). The potency of this peptide wasactually
`increased at alkaline pH, thus demonstrating that the trun-
`cated uroguanylin®®-!peptide possessed this characteristic
`pharmacological property found with the guanylin peptides
`(Fig. 1, ref. 4). The 15 amino acid form of opossum urogua-
`nylin®S-!, containing the N-terminal gIn’> and the twoacidic
`residues, glu°® and asp®’, was substantially more potent at pH
`5.0 compared with pH 8.0. The opossum uroguanylin?™!
`peptide and humanuroguanylin share this pH dependencyfor
`agonist potency (Fig. 1). We previously reportedsimilar effects
`of acid pH onthe potency of opossum uroguanylin’®! that
`did not have the N-terminal gln® residue, but retained the two
`acidic amino acids (4). The presence or absence of the
`N-terminal glutaminein the opossum form of uroguanylin did
`not influence the characteristic enhancement of agonist po-
`tency elicited by the mucosal pH of 5.0.
`In competitive radioligand-binding experiments, uroguany-
`lin°8-1bound to cell-surface receptors on T84 cells with
`similar affinities at pH 5.0 and pH 8.0 (Fig. 5). In this
`radioligand-binding assay, the K; values for uroguanylin’>-'
`binding to the high affinity site were 0.14 nM at pH 8.0
`compared with 0.19 nM at pH 5.0 and the Kj values for
`uroguanylin?’interaction with the low affinity site were 345
`nM atpH 8.0 versus 404 nM at pH 5.0. Thus, uroguanylin?*"'
`did not exhibit an increase in the affinity of this peptide for
`
`Fic. 4. Effects of medium pHontherelative affinities of guanylin,
`uroguanylin, and E. coli ST-(5-17) for binding to receptors on T84
`cells. Binding of !251-ST-(1-19) to intact T84 cells was determined in
`the presence of the indicated concentrations of guanylin (Top),
`uroguanylin (Middle), and ST-(5—17) (Bottom)as described. The values
`shownare the composite data (mean +SEM)from three experiments
`performed in duplicate with each peptide at pH 5 and pH 8 and are
`expressed as the total binding of !?I-ST-(1-19) in the absence of a
`competing ligand. Nonspecific binding was measured using 1 uM
`ST-(5-17). Competitive radioligand binding curves are computer-
`derivedbestfits of the binding data to a two-site model (15). Kj values
`obtainedfor the high and low affinity sites were: guanylin, pH 5 ~102
`nM and 2.3 uM, pH 8 ~1 nM and 77 nM; uroguanylin, pH 5 ~1 nM
`and 70 nM, pH 8 ~10 nM and 615 nM; ST-(5-17), pH 5 ~94 pM and
`7 nM,pH 8 ~440 pM and 17 nM.
`
`binding to receptors on T84cells at acidic versus alkaline pH.
`This observation is consistent with the similar potencies mea-
`sured at pH 5.0 compared with pH 8.0 for the cGMPaccu-
`mulation response to uroguanylin?®!. We conclude that the
`uniqueacidic amino acidsat the N terminusof uroguanylin are
`required for the increased bindingaffinities, and accordingly,
`the enhanced potencies of uroguanylin in the stimulation of
`target cell responses underthe acidic conditions of pH 5.0-5.5
`maintained at the mucosal surface of T84 cells in this model
`epithelium.
`
`DISCUSSION
`
`At the surface of the intestinal mucosa, between the apical
`plasma membranesof enterocytes and a protective layer of
`hydrated mucin, the ligand-binding domains of a commonset
`of receptors for uroguanylin and guanylin extend into an
`aqueous(microclimate) zonethat hasa variable pH (6, 10-13).
`It is in this microdomain of changing mucosalacidity where the
`luminally secreted agonists uroguanylin and guanylin (28) bind
`to and activate the intestinal receptor—guanylate cyclase sig-
`MSNExhibit 1021 - Page 4 of 6
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`Pharmacology: Hamraetal.
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`Proc. Natl. Acad. Sci. USA 94 (1997)
`
`2709
`
`
`
`(Uroguanylin95-109], Log (M)
`
`200.
`400
`
`@ pH 8.0
`© pH 5.0
`
`0
`1-100 9-8-7 KK
`
`ee
`
`receptors undergo pH-dependentshifts by as much as 100-fold.
`Thus, variation in mucosal acidity within the physiological
`limits observed at the surface of the intestinal mucosa (21-24)
`influences the activation of receptors by uroguanylin and
`guanylin.
`Modulation of receptor—guanylate cyclase activity and chlo-
`ride secretion by uroguanylin would be most effective in
`regions of the intestine where the luminal microclimate do-
`main is acidic, whereas the actions of guanylin would be
`augmented whenalkaline pH occurs at the mucosal surface.
`Acidic conditions occur intraluminally in the proximal small
`intestine and proximal colon during digestion (21-24). Gastric
`5 300
`3.
`emptying introducesinto the lumen of the duodenumahighly
`acidic chyme (22), thus increasing mucosalacidity and poten-
`tially enhancing the cGMP accumulation and chloride secre-
`tion responsesof the intestine to uroguanylin, while rendering
`guanylin ineffective. The observation that uroguanylin mRNA
`is relatively abundant in the opossum duodenum compared
`with guanylin mRNAis consistent with a physiological role for
`uroguanylin in the proximal small intestine (17). Guanylin
`mRNA levels are also lower in the duodenum of other
`mammals compared with the mRNAlevels in the ileum and
`colon (18). Luminal pH within the colon can becomeacidic
`due to the production of short chain fatty acids by enteric
`microorganisms (23, 24). High levels of uroguanylin and
`guanylin and their mRNAsare expressed in the mucosalining
`the cecum and colon(4, 5, 11, 17, 18). Increased acidity in the
`lumenofthe large intestine due to microbial metabolism could
`lower the microclimate pH, thus increasing the affinity of
`uroguanylin and reducing the affinity of guanylin for binding
`to and activation of receptors. In addition, alkalinization of the
`microclimate domain at
`the mucosal surface is achieved
`through bicarbonate secretion from the pancreas into the
`duodenum, and/or from epithelial cells lining the small and
`large intestine (22, 24). Alkalinization would enhance the
`potency of guanylin, while attenuating responses to urogua-
`nylin. The intraluminal secretion of guanylin and uroguanylin
`provides an intrinsic mechanism for control of salt and water
`transport underthe variable acidity conditions occurring in the
`microclimate domain that bathes the mucosal surface of
`enterocytes lining the intestinal tract (5, 11, 17-20).
`A striking difference in the primarystructure of uroguanylin
`compared with guanylin is the appearance of two acidic amino
`acidsat the N terminusof uroguanylin (Fig. 6). All uroguanylin
`peptides have aspartate or glutamate residues at these posi-
`tions (8, 9). Deletion of the N-terminal residues (GIn®—Glu°*—
`Asp?’) of opossum uroguanylin®-!converted the truncated
`uroguanylin®*-!™ into a uroguanylin analogue that possessed
`the pharmacological property that is characteristically ob-
`served in the guanylin subfamily of peptide agonists. The
`truncated uroguanylin®’-!© was actually somewhat more po-
`tent at pH 8.0 than at pH 5.0. We conclude that the N-terminal
`acidic residues of uroguanylin are required for the increased
`binding affinities, and therefore, the enhanced potency of
`uroguanylin for activation of receptors underacidic conditions.
`It
`is likely that acidic conditions influence the ionization
`and/or conformationalstate of the uroguanylin molecule as a
`molecular mechanism for the increased biological activity of
`uroguanylin in this circumstance. Presently, we have no infor-
`
`33L
`ao2
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`a=9o
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`9ar
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`a5
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`3a—9
`
`[Uroguanylin98-109] , Log (M)
`
`Fic. 5. Effects of uroguanylin?-!° and uroguanylin?®-!° on
`cGMPaccumulation andthe affinities of uroguanylin?’—!? for binding
`to receptors on T84 cells at pH 5.0 versus pH 8.0. The data are
`representative experiments with duplicate assays. Each experiment
`was performedat least three times with similar results. The conditions
`are the same as those given in the Materials and Methods and the
`legends to Figs.
`1 and 4.
`(Top) Stimulation of T84 cell cGMP
`accumulation by opossum uroguanylin?>-!©. (Middle) Stimulation of
`cGMPaccumulation by opossum uroguanylin?®-!9. (Bottom) Inhibi-
`tion of !°]-ST binding to receptors on T84cells by opossum urogua-
`nylin98-109,
`
`naling molecules. Using a modelintestinal epithelium, we
`demonstrated that potential changes in mucosal acidity can
`differentially influence the relative potencies of uroguanylin
`and guanylin for activation of these receptors located on the
`apical surface of intestinal cells (1, 2, 15, 26). Mucosalacidity
`markedly increases the potency of uroguanylin, while render-
`ing guanylin ineffective in the stimulation of cGMP accumu-
`lation and transepithelial chloride secretion. In sharp contrast,
`a mucosal pH of 8.0 substantially increases the potency of
`guanylin, while diminishing the potency of uroguanylin. This
`striking effect of mucosal pH on agonist potencies was ex-
`plained by the correspondingshifts in affinities of guanylin and
`uroguanylin for binding to receptors on T84 cells at
`the
`mucosal pH values of 5.0 versus pH 8.0. As a result, the
`affinities of guanylin and uroguanylin for binding to these
`
` Uroguanylin
`
`Guanylin
`
`E. coli ST(5-17)
`
`c
`
`Cc
`
`Cc
`
`c
`
`E
`
`cE
`
`CE
`
`
`
` c
`
`c
`
`A
`
`A
`
`¢C¢
`
`G¢
`
`T GCL
`
`T
`
`G ¢
`
`C¢
`
`C
`
`N
`
`P
`
`A GA GEC
`
`Fic. 6. Comparison of the primary structures of uroguanylin, guanylin, and E. coli ST. Underlined aminoacids indicate the identical residues
`shared between uroguanylin, guanylin, and ST-(5-17). Shaded boxes highlight the differences in primary structure between uroguanylin and
`MSNExhibit 1021 - Page 5 of 6
`guanylin.
`MSNv. Bausch - IPR2023-00016
`
`

`

`2710
`
`Pharmacology: Hamraetal.
`
`Proc. Natl. Acad. Sci. USA 94 (1997)
`
`mation concerning the residues in guanylin that contribute to
`the remarkable increase in affinities for interaction with
`receptorsat alkaline compared with acidic pH. Although these
`properties of uroguanylin and guanylin were discovered using
`the T84 cell line as a model epithelium, current studies have
`demonstrated that the shortcircuit current responses of mouse
`duodenum and cecum to uroguanylin in vitro are markedly
`increased when the luminal pH is acidic compared with the
`responses observed at a mucosal pH of7.4 (29). This prelim-
`inary observation with uroguanylin using a native epithelium
`mounted into Ussing chambersis consistent with the increased
`affinity of uroguanylin for binding to and subsequentactiva-
`tion of the apical membrane receptor—guanylate cyclases of
`T84 cells revealed by experiments presented in this commu-
`nication.
`E. coli ST-(5-17) binds with extraordinarily high affinities to
`the uroguanylin/guanylin receptors on the apical surface of
`T84 cells and potently stimulates cGMPproduction and chlo-
`ride secretion at both alkaline and acidic pH. Theinteractions
`of ST peptides with these receptorsislittle affected by mucosal
`pHin this model epithelium. Enteric bacteria have evolved a
`single peptide toxin that serves as a molecular mimic for both
`of the intestinal hormones, uroguanylin and guanylin. The
`remarkable potencies of ST peptides compared with the
`potencies of the enteric hormonesis causedby higheraffinities
`for ST binding to the intestinal receptors for uroguanylin and
`guanylin. Bacteria have created superagonist peptide toxins
`and this pharmacological property contributes to the remark-
`able toxicities of ST peptides in the molecular and cellular
`mechanism underlying Travelers diarrhea (8, 9, 30, 31).
`Weconclude that uroguanylin and guanylin cooperatively
`regulate a signaling pathway that modulatesintestinal salt and
`water transport via an intrinsic, paracrine mechanism involving
`cGMPas a second messenger. Uroguanylin is a highly potent
`agonist under high mucosalacidity, a condition that renders
`guanylin ineffective. Conversely, guanylin is highly potent
`under low mucosalacidity, conditions that reduce the potency
`of uroguanylin. An influence of intraluminal pH on urogua-
`nylin and guanylin actions may also occur in other epithelia
`such as the renal tubule. Thefiltrate bathing tubularcells lining
`the nephron also becomes acidic under normal conditions,
`thus potentially modulating the interaction of uroguanylin with
`renal receptors (13, 32, 33), which may influence the urinary
`excretion of sodium chloride (34). Finally, this study empha-
`sizes that potentially novel regulation mechanisms may exist
`whereby normalconstituents (such as the H* concentration)
`of