Original Paper
`
`
`
`Acta Anat 1995:153:210-219
`
`Distribution of Escherichia coli
`Heat-Stable Enterotoxin/Guanylin/
`Uroguanylin Receptors in the
`Avian Intestinal Tract
`
`COMO O COLE OSE OEOOEOEEEE OOOO RO OESOASOSEES ODED OOEED ECHOES OOOOOOOCESEO TOO ORDEEOEHAEORESEOESES
`
`Abstract
`Pathogenic strains of enteric bacteria secrete small heat-stable toxins (STs) that
`activate membrane guanylyl cyclase receptors found in the intestine. The intes-
`tinal peptide agonists, guanylin and uroguanylin, are structurally related to STs.
`Receptors for '*]-ST were found throughout the entire length of the intestinal
`tract ofall the birds examined. These receptors were restricted to intestinal epi-
`thelial cells covering villi and forming intestinal glands and were not observed in
`other strata of the gut wall. The most intense labeling of receptors by '*I-ST
`occurred in the region of the microvillus border of individual enterocytes. There
`appeared to be a decrease in receptor density distally along the length of the
`small intestine, although labeling of receptors by “I-ST was observed through-
`out
`the small
`intestine and colon. Cellular CGMP accumulation responses to
`Escherichia coli ST and rat guanylin in the domestic turkey and duck were
`greater in the proximal small intestine compared to the distal small intestine or
`colon. Brush border membranes (BBM) isolated from the mucosa of proximal
`small intestine of turkeys exhibited agonist-stimulated guanylyl cyclase activity.
`The rank order potency for enzyme activation was E. coli ST > uroguanylin >
`guanylin, Competitive radioligand binding assays using '“I-ST andturkey intes-
`tine BBM revealed a similar rank order affinity for the receptors that was exem-
`plified by the K, values of ST 2.5 nM, uroguanylin 80 nM and guanylin 2.6 pM.
`It may be concluded that functional receptors for the endogenous peptides. gua-
`nylin and uroguanylin, occur in the apical membranes ofenterocytes throughout
`the avian intestine. The receptor-guanylyl cyclase(s) of proximal small intestine
`were preferentially activated by uroguanylin relative to guanylin, but both
`endogenous peptides were less potent than their molecular mimic, E. coli ST.
`
`WJ. Krause®
`R.H. Freeman*
`S.L. Eber* 4
`FE-K. Hamra‘
`KF. Fok*
`M.G. Currie®
`L.R. Forte**
`
`Departments of
`Patholagy and Anatomical Sciences,
`Physiology, and
`Pharmacology. School of Medicine.
`Missouri University. Columbia, Mo.,
`Harry S. Truman Memorial VA
`Medical Center. Columbia. Mo.. and
`Searle Research and Development,
`St. Louis. Mo.. USA
`
`Key Words
`Enterocytes
`Guanylyl cyclase
`Cyclic GMP
`Avian species
`
`introduction
`
`diarrhea in children as well as in laboratory and domestic
`animals [Sack et al., 1975: Donta et al., 1977; Burgess et
`Heat-stable enterotoxins (STs) are peptides produced by—al... 1978; Giannella, 1981: Black et al., 1982]. The apical
`pathogenic strains of bacteria such as Yersinia enterocoli-
`plasmalemma(microvillus border) of enterocytes lining the
`tica or Escherichia coli that are a major cause of secretory—intestinal tract and forming the intestinal glands (crypts of
`
`a©
`
`Reveived:
`November 28, 1994
`Accepted:
`May §9, 1948
`
`William 3. Krause
`Department of Patholagy und Anatomical Sciences
`School of Medicine
`University of Missouri MSN Exhibit 1033 _ Page 1 of 10
`Columbia, MO 65212 (OSAI
`
`©1995 S. Karger AG. Basel
`
`MSNv. Bausch - IPR2023-00016
`
`

`

`Leberkiihn) of man and several other mammalian species
`have been shown to contain specific, high-affinity binding
`sites for these peptides [Forte et al.. 1989: Krause et al..
`1990, 1994] . These STs share receptors with two recently
`described endogenous peptides: guanylin and uroguanylin
`(Currie ct al., 1992: Hamra et al.. 1993; Kita et al.. 1994].
`The ST/guanylin/uroguanylin receptor has been shown to
`be an intestinal
`isoform of membrane guanylyl cyclase
`(GC-C) that belongs to a family of cell surface proteins
`which catalyze the production of cyclic 3'.S'guanosine
`monophosphate (CGMP) [Schulz et al., 1990; Forte and
`Currie, 1995]. [t is by the increase in production of cGMP
`that STs, guanylin or uroguanylin influence cellular func-
`tion. Increasedintracellular levels of this second-messenger
`molecule can activate a cAMP-dependent protein kinase
`which stimulates intestinal epithelial cell Cl
`secretion
`[Forte ct al., 1992; Tien et al., 1994]. The net effect of bac-
`terial STs, guanylin, and uroguanylin in the mammalian
`intestinal tract is to promote CI secretion andto inhibit Na*
`absorption [Field et al.. 1978; Rao ct al.. 1981; Guandalini
`et al., [982: Currie et al., 1992: Hamra et al., 1993]. We
`previously reported that '“I-ST labeled receptors on entero-
`cytes throughout the intestinal tract of man and other mam-
`mals [Krause et al.. 1994]. Proximal small
`intestine had
`the greatest apparent density of these receptor-guanylyl
`cyclases that serve as common receptors for bacterial STs
`and the endogenous peptide hormones, guanylin and
`uroguanylin.
`Receptors for E. coli ST and ST-stimulated guanylyl
`cyclase activity have been reported to occur in brush border
`membranes isolated from chicken intestine [Katwa and
`White, 1992]. The present study examined the intestinal
`tract of several species ofbirds, to better define the distribu-
`tion of the ST/guanylin receptors in enterocytes along both
`the length and vertical axis (villus/erypt unit) of the avian
`intestinal tract utilizing “I-ST as a radioligand for these
`receptors and cGMPresponses to these peptide agonists in
`vitro |Forte et al.. 1988: Krause et al., 1990, 1994]. We
`report here that all birds that were examined had ST/gua-
`nylin receptors localized to the enterocytes throughout the
`intestinal tract and that agonist-stimulated CGMP accumu-
`lation responses of intestinal mucosa were greatest in the
`proximal small
`intestine of turkeys and ducks. Brush-
`border membranes (BBM) isolated from the mucosaoftur-
`key proximal small
`intestine exhibited guanylyl cyclase
`activity that was stimulated by ST > uroguanylin > guanylin
`in rank order potency. A similar order ofrelative affinities
`was observed in competitive radioligand binding assays
`using these BBMs suggesting that the receptors were prel-
`crentially activated by ST > uroguanylin > guanylin.
`
`Materials and Methods
`
`Receptor Autoradiography
`Specimens of small
`intestine and colon were gathered from a
`variety ofavian species (lable 1) and frozen in liquid nitrogen as soon
`as possible after death. After freezing. the tissues were stored at
`- 80°C until used. The frozen specimens were sectioned at 14 pmina
`cryostat maintained at —20°C. Twocut sections were mounted onto
`opposite ends ofgelatin-coated slides, air-dried and stored at -80°C
`until used. Each slide was then incubated with 50 pl of Dulbecco's
`modified Eagle's medium (DMEM), pH 5.5, containing 0.5% bovine
`serumalbumin (BSA) at 37°C for 15 min as previously described
`[Forte et al.. 1989: Krause et al.. 1990. 1994}. To measure the total
`binding for this radioligand. SO ul DMEM containing 1.000 cpm'I-
`ST/ul was added to onetissue section, The adjacent section was incu-
`bated with the same concentration ofradioligand plus | pM unlabeled
`ST to assess nonspecific binding of ““I-ST. Additional sections also
`were incubated with 10 pM ofrat guanylin to determine if guanylin
`inhibited binding of “I-ST to intestinal receptors. “I-ST,
`,, was cho-
`senas the radioligand because iodination of Tyr 9 in guanylin appears
`to interfere with the biological activity ofthis radioligand resulting in
`poor binding of ““I-guanytin to receptors on cultured T,, humanintes-
`tinal epithelial cells. For this reason. a radiolabeled form of guanylin
`was not used. Thus. ST peptides currently are the best radioligands for
`identifying the tissue location of guanylin/uroguanylin receptors. Fol-
`lowing an incubation of 1S min at 37°C. the slides were washed with
`a gentle stream of cold phosphate-buffered saline (PBS) and then
`washed 3 additional times by placing theminto 50 ml ofice-cold PBS
`for 3 min. The sections were air-dried prior to being coated with
`Kodak NTB-2 or NTB-3 emulsion, dried again, then sealed in light-
`tight boxes and stored at 4°C for 3-4 weeks until developed. Follow-
`ing routine photographic development andfixation, the sections were
`coverslipped and examinedby bright and dark field microscopy.
`
`Cyclic GMP Acciulation Bioassay
`Proximal, middle and distal segments of the small intestine as well
`ats a segment of midcolon from domestic turkeys (Nicholas broad-
`breasted strain) and domestic ducks (Peking white strain) were dis-
`sected and mucosa prepared by scraping the intestinal mucosafree,
`and washing the tissue gently once in. 0.9% NaCl and twice in DMEM.
`20 mM HEPES, pH 7.4. To measure ST and guanylin stimulation of
`cGMP production, mucosal suspensions (60 mg wet weight) were
`placed in 0.2 ml DMEM (pH 7.4) containing 20 mM HEPESbuffer
`(pH 7.4) at $°C. The tissue was incubated for 40 min at 37°C with
`either 1 uM E. coli ST. 10 uM rat guanylin or vehicle that was added to
`DMEM-HEPEScontaining | mM isobutylmethylxanthine (IBMX).
`Perchloric acid was then added to a final concentration of 3.3%. cells
`were centrifuged and the resultiag supernatants neutralized with [NV
`KOH. The supernatant solution was used to measure CGMP concen-
`tration by radioimmunoassay as reported previously |Forte et al.
`1988].
`
`Competitive Radioligand and Binding Assays
`BBM were thawed and centrifuged at 16.000 ¢ for 15 min, then
`resuspended in a solution containing 20 mM Tris-HCI. pH 7.8. 150
`mM NaCl. and |! mM EDTA. The binding assay consisted of 35 yl of
`the same buffer. 20 ul "1-ST (S0,000- 100,000 cpm), 20 ul of peptide
`ligand in H.O, and 25 ui of BBM (1$—20 pg protein). Incubation was
`at 37°C for | h. Then 3 ml of cold phosphate-buftered saline (PBS)
`was added and this solution filtered using Whatman GE/F filters
`
`MSNExhibit 1033 - Page 2 of 10
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`Table 1. Avian species examined tor E. coli ST-specitic binding
`sites in intestinal epithelial cells
`
`Numberof
`animals used!
`
`Species
`
`3
`2
`2
`
`2
`2
`2
`3
`
`6
`
`&
`
`2
`2
`2
`2
`3
`2
`2
`2
`2
`6
`2
`
`2
`
`ST,1
`
`+++
`++
`++
`
`++
`++
`++
`++
`
`++
`
`Emu (Dromaius novachollandie)
`Blue jay (Cyanocitta cristata)
`Red-headed woodpecker
`(Melanerpes ervthrocephalus)
`Purple grackle (Quiscaltus guiscula)
`Starling (Sturnus vulgaris)
`Bob-white quail (Colinus virginianits)
`Ring-necked pheasant
`(Phasianus colchicus torquatus)
`Domestic chicken
`
`++
`
`(Barred rock/leghorn cross)
`Domestic turkey
`(Nicholas broad breasted)
`Mourning dove (Zenaidura macroura)
`Domestic pigeon (Feral)
`Coot (Fulica americana)
`Wood duck (Aix sponsa)
`Ring-necked duck (Aythya collaris)
`Northern shoveller (Anas clypeata}
`Mallard duck (Anes platvrivachos)
`American green-winged teal (Anus crecca)
`Blue-winged teal (Anas discors)
`Domestic duck (Peking white)
`Giant Canada goose
`(Branta canadensis maxima)
`Wilson's snipe (Capella gallinago delicata) ++
`Woodcock ( Philohela minor)
`++
`
`++
`++
`++
`++
`++
`++
`++
`++
`++
`++
`++
`
`cation using a one-precipitation step with {2 mM MgSO,. The BBMs
`were stored frozen at —80°C prior to use. Protein content was mea-
`sured by the method of Bradford [1979].
`
`Measurement of Guanylyl Cyclase Activity
`Enzyme activity was measuredin an assay volume of 100 pl con-
`taining LO ue BBM protein, 50 mM HEPES, pH 7.6, 0.5 mM isobutyl-
`methylxanthine (IBMX),
`1 mM ATP, 10 mM creatine phosphate.
`5 units creatine phosphokinase.
`| m4f GTP and 5 mM MgCl.Incu-
`bation was for 15 min at 37°C. The reaction was stopped by adding
`100 pl of 6%. perchloric acid. Then cach reaction mixture was neutral-
`ized with 10 N KOH andcentrifuged to remove the potassiumper-
`chlorate precipitate. A 25-p] portion was removed to use for the
`estimation of cGMP byradioimmunoassay as previously described
`|Forte et al., 1988]. The data are expressed as pmol cGMP formedper
`ug protein per 15 min. Agonists or vehicle were added at the indicated
`concentration and assayed in duplicate.
`
`Preparationof latestinal Extracts and Bioassay
`About 200g of mucosa isolated tromthe small intestine and colon
`of turkeys was suspended in 2 liters of | M acetic acid. heated at
`100°C for 10 min and then homogenized with a Polytron as previ-
`ously described for opossumintestinal mucosa [Hamraet al.. 1993].
`The homogenate was centrifugedat 10.000 g for 20 min and the super-
`natant was made in 0.1% trifluoroacetic acid (TFA). Extracts were
`isolated using C18 cartridges as previously described |Hamraet al..
`1993]. Bioactive peptides that eluted with 40% acetonitrile and 0.1%
`TFA in H.O were chromatographed on a 2.5 x 90 cm Sephadex G-25
`column as previously described. The bioassay for guanylin/uro-
`guanylin-tike peptides was carried out by removing 0.5 ml from each
`10-ml columnfraction, drying in a Speed-Vac and resuspending each
`fraction in 200 pl of DMEM. This sample was added to one well of a
`24-well culture plate containing confluent T,, cells. Incubation was
`for 40 min at 37°C and cellular CGMP was measured by RIA as previ-
`ously described [Forte et al., 1988: Hamraet al., 1993]. The conditions
`for culture of T,, cells were as previously described [Hama et al..
`1993],
`
`Synthesis of E. coli ST S-17 and Guanylin
`Rat guanylin (PNTCEICAYAACTGC) and £. coli STS-17
`(CCELCCNPACAGC) were synthesized by the solid-phase method
`as previously described [Currie et al.. 1992].
`
`Preparation of °'t-ST
`The iodination of E. coli ST (NSSNYCCELCCNPACTGCY.
`Multiple Peptide Systems, San Diego, Calif.. USA) was carried out
`using the lactoperoxidase procedure described previously | Forte et al.
`1988. 1989]. Purification of '"I-ST was achieved using high-per-
`formance liquid chromatography with a C18 column under reverse-
`phase conditions also as described carlier [Forte ct al.. 1988, 1989].
`Na’ was purchased from DuPont NEN. Wilmington. Del.. USA, as
`the carrier-free radionuclide (14-17 pCi/ug). Lactoperoxidase was
`purchasedin a solid-state form trom Biorad Laboratories. Richmond,
`Calil., USA. E. coli ST, GTP. ATP, creatine phosphate, and creatine
`phosphokinase were purchased from the Sigma Chemical Company.
`St. Louis. Mo., USA. Other reagents and materials were obtainedfrom
`various suppliers.
`
`Intensity ofsilver grains: +++ = strong: ++ = moderate.
`Total number of animals used tor both in vitro autoradiography
`and cGMP accumulation bioassays of intestinal mucosa.
`
`(25 mm diameters) and a vacuum manifold. The filters were washed
`twice with 3 ml cold PBS. Eachfilter had been soakedprior to use with
`0.1% polyethylenimine and washed with 3 ml PBS before filtering the
`BBM reaction mixture. Each filter was then placed into glass tubes
`andradioactivity measured by gammascintillation spectrometry. The
`radioligand binding data were analyzed using the Inplot computer
`programto estimate K, and B,,,, values (Graph Pad Software for Sci-
`ence. San Diego. Calif, USA). The data were fit to a single-site model
`in these experiments,
`
`fsolation of Intestinal BBM
`The proximal one-half of the small intestine from an adult turkey
`was washed in 0.9%NaCl, cut open longitudinally and mucosa
`scraped [ree with a microscope slide. The mucosa was homogenized
`in 7.5 vol of a buffer containing 300 mM D-mannitol. S mM EGTA, [2
`mM Tris-HCI. pH 7.5, per gram of mucosa with a Polytron homoge-
`nizer according to the method ofBiber et al. [1981]. The isolation ofa
`fraction enriched in BBM wascarried throughthe P4 stage ofpurifi-
`
`tw
`
`Ww
`
`Krause/Freeman/Eber/Hamra/Fok/Currie/Forte
`
`ST/Guanylin Receptors in Avian Intestine
`
`MSN Exhibit 1033 - Page 3 of 10
`MSNv. Bausch - IPR2023-00016
`
`a©
`
`
`
`
`
`

`

`
`
`Fig. 1. A portionofintestinal mucosa fromthe proximal small intestine of an emu. The enterocytes covering villi
`(V) and fonningthe intestinal glands (D express ST/guanylin receptors. Elements of the lumina propria and muscularis
`mucosae (M) show little if any receptor labeling. Note the intense labeling in the region of the microvillus border
`(arrows) by “I-ST. Dark field. x297,
`Fig.2. A rcgion of small intestinal wall from the red-headed woodpeckerillustrating the evendistribution of recep-
`tors labeled by '“I-ST in enterocytes ofthe intestinal mucosa. The lumen of the small intestine (L) is shown at the
`extreme right: the muscularis externa (M) at the far left. Dark field. x 119.
`Fig. 3. A portion of small intestine from the purple grackle also shows a relatively even distribution of | ‘I-ST-
`labeled receptors among enterocytes (arrows) of the intestinal mucosa. The lumen (L) is shown near the top; the mus-
`cularis externa (M) near the bottomof the photomicrograph. Dark field. x 119.
`Fig. 4.
`'“I-ST-receptor density in the intestinal mucosaofthe blue-jay, unlike the red-headed woodpecker and pur-
`ple grackle. appears greater in enterocytes covering villi (V) than in entcrocytes forming intestinal glands (1). The intes-
`tinal lumen (L) is oriented to the right: the muscularis externa (M) tothe left of the photomicrograph. Dark field. x 119,
`Fig. 5.
`'-“I-ST-receptor density in enterocytes lining the small intestine of a domestic chicken. The intestinal lumen
`(L) is to the eight; the muscularis externa (M) is to the left. Dark field. x 119.
`Fig.6. A segment of colon taken from a mallard duck illustrates greater '“I-ST-receptor density in enterocytes lin-
`ing the luminal surtace. Note that the most intense labeling occurs in the microvillus border (arrows), Dark field. x [19.
`
`MSNExhibit 1033 - Page 4 of 10
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`
`Nw mn
`
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`128.111.121.42
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`

`

`
`
`50
`
`40 4
`
`305
`
`|| Control
`
`[] st 1pm
`
`=] Guanylin 10 pM
`
`D 2
`
`aa=
`
`90-4
`a 20
`oO
`
`10
`
`I
`
`T
`
`
`
`Colon
`Distal
`Micdle
`Proximal
`
`CL J
`
`
`
`
`
`iH Control
`
`[a] ST 1M
`
`i] Guanylin 10 1M
`
`@ 2ea o 6o
`
`O
`
`Proximal
`
`Middle
`
`Distal
`
`Colon
`
`Fig.8. cGMPproductionbythe intestinal mucosa takenfromdit-
`Fig.7. cGMPproduction bythe intestinal mucosa taken tromdif-
`ferent segments of Peking duck small intestine and colon following
`ferent segments of turkey small intestine and colon following stimula-
`stimulation by buM—coli ST and 10 wrat guanylin. The data are
`tion by | uM £. coli ST and 10 pMrat guanylin. The data are the mean
`ofthree experiments. Small intestine — proximal, oviddle and distal
`the mean oftwo experiments. Small intestine — proximal. middle and
`segments.
`distal segments.
`
`Results
`
`Distribution ofST/Guanylin Receptors
`Examination ofthe distribution of receptors labeled with
`"SI-ST from various segments of the small
`intestine and
`colon of a number of avian species showed that high-atfin-
`ity '*I-ST-binding sites were present and confined to the in-
`testinal epithelium throughout
`the length of the intestinal
`tract (table 1: fig. 1-6). “I-ST binding sites were not ob-
`served in other layers of the gut wall (lamina propria, muscu-
`laris mucosae, submucosa, muscularis externa or serosa).
`The greatest intensity of "I-ST binding occurred along the
`microvillus (striated) border of intestinal epithelial cells.
`Adjacent sections that were incubated with the same concen-
`tration of radioligand plus | pM ofeither unlabeled ST or 10
`uM ofrat guanylin eftectively inhibited '“I-ST binding sug-
`gesting that commonreceptors were labeled by ™I-ST.
`Along the vertical axis (the villus/crypt unit) of the small
`intestine in the birds that were examined. *I-ST binding to
`receptors appeared to be evenly distributed among intesti-
`nal epithelial cells (enterocytes) covering villi and forming
`the intestinal glands (fig. 2, 3, 5). In some birds, such as the
`blue jay. receptor density appeared greatest in enterocytes
`covering villi andlining the intestinal lumen (fig. 4). Recep-
`tor density of enterocytes lining the surface of the colon
`in the mallard duck (fig.6) and Canada goose (data not
`shown) also appeared to be greater than in those entero-
`
`cytes forming the intestinal glands. Receptor density along
`the longitudinal axis of the small
`intestine following in
`vitro receptor autoradiography appearedto be greaterin the
`proximal region in most species,
`To evaluate further the possibility that ST/guanylin re-
`ceptors are more abundant in proximal small intestine, dil-
`ferent segments of the intestinal tract of domestic turkeys
`and ducks were used to measure guanylin and E. coli
`ST-stimulated guanylyl cyclase activity. Agonist-mediated
`activation of the membrane guanylyl cyclase was measured
`by the increased cGMP content of mucosa exposed to these
`peptides. Treatment ofthe intestinal mucosa with 1 uM ST
`elicited a large increase in CGMP levels in the proximal
`small intestine of turkeys with a much reduced cGMP accu-
`mulation response to ST occurring in the distal small intes-
`tine and colon (fig. 7). ST stimulated cGMP production by
`the intestinal mucosa to much higher levels than did 10 pM
`guanylin in all segments. Likewise. CGMP accumulation
`responses to ST in the intestinal mucosa of the domestic
`(Peking) duck were much greater in the proximal small
`intestine as compared to the middle or distal small intestine
`and colon (fig.8).
`In comparison to ST, 10 uM guanylin
`was considerably less effective in stimulating CGMP accu-
`mulation in the mucosa of small
`intestine or that of the
`colon in ducks. The magnitude of cGMP responses in prox-
`imal intestine of turkeys was substantially greater than that
`observed in ducks.
`
`Krause/Freemian/Eber/Hamra/Fok/Currie/Forte
`
`ST/Guanylin Receptors in Avian Intestine
`
`MSNExhibit 1033 - Page 5 of 10
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`

`

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`Fig. 9. Guanyly! cyclase activity of brush border membranesiso-
`lated from turkey small intestine. The data shown in this figure are
`representative of two such experiments with each point assayed in
`duplicate. Synthetic peptides, ST,
`opossum uroguanylin and rat
`guanylin were used in these experiments.
`
`Fig. 10. Comparisonofthe relative affinities of ST. uroguanylin
`and guanylin for receptors in turkey intestine. These dataare the mean
`of two experiments with each point assayed in triplicate for each
`experiment. The radioligand was '“I-ST (E. coli STD). Bo = Total
`bound '“1-ST in the absence of competing ligand.
`
`To characterize further the properties of the guanylyl
`cyclase receptors in turkey intestine. isolated BBM from
`the mucosaof proximal small intestine were used. Agonist-
`stimulated guanylyl cyclase activities were measured to
`compare the relative potencies and efficacies of E. coli ST,
`rat guanylin and opossum uroguanylin. These peptides
`exhibited a rank order of potencies with ST > uroguany-
`lin>guanylin (fig.9). A similar
`rank order of agonist
`potencies has been reported for the human GC-C isoform
`that is expressed in T,, colon carcinoma cells [Hamraet al.,
`1993: Kita et al., 1994]. Thus. turkey small intestine, like
`the human receptor-guanylyl cyclase, has a BBM receptor-
`guanylyl cyclase that appears to prefer uroguanylin relative
`to guanylin. It should be noted that ST is considerably more
`potent than either uroguanylin or guanylin. Our supplies of
`these peptide agonists were insufficient to use concentra-
`tions higher than 10-30 uM in this assay so that maximal
`stimulation of the guanylyl cyclase was not achieved in
`these experiments.
`Evaluation of the pharmacological properties of the
`BBM receptors was extended using competitive radioli-
`gand binding assays to measure the affinities of the recep-
`tors for E. coli ST, opossum uroguanylin and rat guanylin
`(fig. 10). All three peptides inhibited the binding of *I-ST
`to an apparently commonset of binding sites (B,,,, = 6.13
`pmol/mg protein) on turkey intestine BBM. The K, values
`for these peptides were: ST 2.5 nM, uroguanylin 80 nM and
`
`guanylin 2.6 uM. Thus. ST had a 40-fold higher apparent
`affinity for these receptors than did uroguanylin, which had
`an approximate 32-fold higher affinity than guanylin.
`These data are consistent with the relative potencies of
`ST > uroguanylin > guanylin as activators of the BBM
`guanylyl cyclase.
`Guanylin and uroguanylin peptides are found in the
`mammalian intestine [Currie et al., 1992: Hamraet al.
`1993, 1995]. To examine whether these bioactive peptides
`also occur in avian intestine, we prepared an extract of tur-
`key intestinal mucosa and subjected this extract to gel fil-
`tration chromatography (fig. 11). A broad peak ofbioactiv-
`ity eluted in the internal volume of this Sephadex G-25
`column. These fractions activated the humanT,,, cell intes-
`tinal guanylyl cyclase (GC-C) that was used for the bio-
`assay ofturkey intestinal agonists. The dominant peak of
`bioactive fractions eluted before cither uroguanylin or gua-
`nylin, suggesting that these putative peptides from turkey
`intestine may be longer forms of guanylin and/or uroguany-
`lin or have different structures from the peptides isolated
`from mammalian intestine or urine {Currie et al.. 1992;
`Hamraet al., 1993, 1995]. Therefore, intestinal mucosa of
`turkeys contains bioactive agonists that stimulate cGMP
`production in human intestinal cells. It
`is likely that these
`substances are peptides that are structurally similar to uro-
`guanylin. guanylin and ST peptides, the known agonists for
`this class of receptors.
`
`MSN Exhibit 1033 - Page 6 of 10
`MSNv. Bausch - IPR2023-00016
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`Sephadex G-25
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`tract of some mammalian species | Krause et al,,
`testinal
`1994]. Although substantial interspecies variation may oc-
`cur in birds and mammals, a general conclusion can be
`drawn from these experiments indicating that- proximil
`small intestine has the highest
`levels of ST/guanylin/uro-
`guanylin receptor-guanylyl cyclases relative to other re-
`gions of small or large intestine.
`With regard to the vertical axis (the villus/crypt unit)
`of the avian small intestine. ST/guanylin receptor density
`appeared relatively evenly distributed among enterocytes
`covering villi and in those enterocytes forming intestinal
`glands of the majority of birds that were examined. This
`observation is in contrast to that found in the human smal
`intestine as well as several other mammalian species in
`which the receptor density is greatest in enterocytes cover-
`ing the basal one-halfofvilli and in those lining the proxi-
`mal one-half of the intestinal glands (crypts of Liberkiihn)
`[Cohenet al., 1992; Li and Goy, 1993: Krause et al., 1994].
`The ST/guanylin/uroguanylin receptor belongs to the
`guanylyl cyclase family of proteins that differ markedly in
`their selectivity and activation by ligands. Included in this
`group are atrial natriuretic peptides and nitric oxide which
`are endogenousactivators for two particulate forms (GC-A
`and GC-B) and a soluble (GC-S) form of guanylyl cyclase.
`respectively |Drewett and Garbers, 1994]. All
`these en-
`zymes catalyze the production of cGMP andby this mech-
`anism influence cellular function. The ST/guanylin/uro-
`guanylin receptor,
`an_
`intestinal cytoskeletal-associated
`form of membrane guanylyl cyclase (GC-C) is selectively
`activated by the STs [Field et al.. 1978: Guerrant et al..
`1980; Schulz et al.. 1990; Forte and Currie. 1995] as well as
`by guanylin [Currie et al., 1992] and the newly discovered
`peptide, uroguanylin [Hama et al.. 1993; Kita et al., 1994].
`Guanylin and uroguanylin have 15 or 16 amino acids, are
`producedin the intestine and appear to serve as endogenous
`regulators for the intestinal form of guanylyl cyclase. GC-
`C. Enterotoxigenic strains of bacteria cause secretory (i.e.
`travelers) diarrhea by producing molecular mimics of guany-
`lin and/or uroguanylin: thereby activating the GC-C recep-
`tors in enterocytes. Guanylin, uroguanylin and E. coli STs
`bind to the N-terminal, extracellular domain ofthis cell sur-
`face receptor andactivate a C-terminal, intracellular catalytic
`domain causing increased cellular levels of CGMP. Although
`the physiological role of guanylin and uroguanylin in the
`avian intestinal
`tract is unknown at present, both peptides
`stimulate chloride secretion in enterocytes of mammalian
`species [Currie et al.. 1992; Forte et al., 1993: Hamraet al.
`1993: Cuthbert et al.. 1994: Kita et al.. 1994]. Thus. guany-
`tin and uroguanylin may regulate ion transport and fluid
`secretion in both the mammalian and avian intestinal tract by
`
`Krause/Freeman/Fber/Hamra/Fok/Cutrie/Forie
`
`ST/Guanylin Receptors in Avian Intestine
`
`MSNExhibit 1033 - Page 7 of 10
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`Fig. 11. Guanylin/uroguanylin-like activity isolated from the
`intestinal mucosa of turkeys. The bioassay is the CGMP accumula-
`tion response of culture T,. cells to 200 pl of each fraction from a
`2.5x90 cm Sephadex G-25 column. vv = Void volume.
`
`Discussion
`
`Receptors for E. coli ST had previously been identified
`in purified BBM isolated from small
`intestine of the
`chicken |[Katwa and White, 1992]. The current study
`extends those observations and demonstrates that
`the
`ST/guanylin/uroguanylin receptor is localized to entero-
`cytes of the chicken intestine as well as those cells lining
`the intestinal tract of a number of other avian species. Like
`man and other mammalian species examined to date. these
`receptors are localized to the enterocytes lining the intesti-
`nal lumen and forming the intestinal glands. As in the avian
`species investigated in the present study, the greatest recep-
`tor density in enterocytes of mammals occurs in the region
`of the microvillus border [Krause ct al.. 1990, 1994]. There
`is an apparent decrease in receptor density distally along
`the longitudinal axis of the small intestine of some avian
`species as demonstrated by in vitro receptor autoradio-
`graphy. A similar decrease has been observed in man and
`other mammalian species using this methodology [Krause
`et al. 1994]. E. coli ST/guanylin-stimulated guanylyl cy-
`clase activity, as indicated by an increased accumulation of
`cellular cGMP, also suggests that
`the receptor-guanyly!
`cyclase density is greatest in the proximal small intestine as
`compared to distal regions of small intestine or colon in the
`turkey and duck. Similar observations have been made
`using these techniques which showed decreasing gradients
`of receptor density along the longitudinal axis of the in-
`
`

`

`this signaling mechanism. Other epithelial tissues may also
`be targets for these peptides. Receptor-guanylyl cyclase re-
`sponsiveness to ST occurs in kidney, liver andtestes | Forte
`et al.. 1988, 1989: Krause et al., 1990: Laney et al.. 1992].
`Thus, in addition to their intestinal paracrine function, these
`peptides may also function as hormones.
`Receptors for these peptides have now been demon-
`strated in both mammals and birds suggesting that the reg-
`ulation of enterocyte function by guanylin and uroguanylin
`through the cGMP second messenger mechanismis a fun-
`damental signaling pathway that appeared early in verte-
`brate evolution. [t is of interest that intense receptor label-
`ing by '“I-ST occurs in the distal small
`intestine and the
`colon of some birds, yet these regions of the gut had very
`small cGMPresponses to either ST or guanylin. The expla-
`nation for the receptors in the colon anddistal small intes-
`line of these species being less responsive to these agonists
`is unclear, particularly in light of the fact
`that guanylin
`mRNA levels are most abundant in the mammalian colon
`| Wiegandet al.. 1992a, b: Li and Goy. 1993]. Our observa-
`tion that '-1-ST-labeled receptors appear to be abundant in
`the avian colon, which in the turkey and duck hadlittle or
`no cGMPresponses to either ST or guanylin, is consistent
`with the possibility that ST receptors (i.e. binding proteins)
`exist. in the avian distal small intestine and colon. which are
`not guanylyl cyclases. A similar conclusion was made from
`our recent studies of these receptors in the mammalian
`colon [Krause et al.. [994]. Similarly, cultured [EC-6 intes-
`tinal cells have been shown to have specific. high affinity
`binding sites for °“I-ST, but these cells express no GC-C
`mRNA and are devoid of ST-stimulated guanylyl cyclase
`activity [Mannet al., 1993]. A 56-kD protein, isolated from
`the small
`intestine of rats, bound '1-ST but exhibited no
`guanylyl cyclase activity [Hakki ct al.. 1993]. This protein
`may be a proteolytic fragment of GC-C with the C-terminal
`catalytic portion of the receptor missing. However, these
`preliminary findings suggest
`that other receptor proteins
`may occur in the distal small intestine and/or colon of both
`birds and mammals, which bind “I-ST with high affinity.
`but have no guanylyl cyclase activity. A similar cell-surface
`protein occurs which binds atrial peptides with high affinity
`but
`lacks the intracellular catalytic doma