`
`RAPID COMMUNICATIONS
`
`Uroguanylin and Guanylin: Distinct But Overlapping Patterns
`of Messenger RNA Expression in Mouse Intestine
`
`TERESA L. WHITAKER,* DAVID P. WITTE, M. CATHERINE SCOTT,* and MITCHELL B. COHEN*
`Divisions of *Pediatric Gastroenterology and Nutrition and Pediatric Pathology, Children's Hospital Medical Center, University of Cincinnati,
`Cincinnati, Ohio
`
`See editorial on page 1036.
`
`Background & Aims: Uroguanylin and guanylin, endoge-
`nous ligands of the guanylate cyclase C receptor, are
`presumed to mediate uid and electrolyte secretion in
`the intestine. The aim of this study was to characterize
`the expression patterns of uroguanylin and guanylin
`messenger RNA (mRNA) in the mouse intestine. Meth-
`ods: A mouse uroguanylin complementary DNA was am-
`plied from a partial genomic clone, and Northern analy-
`ses and in situ hybridization were performed to localize
`guanylin and uroguanylin mRNA along the duodenal-
`colonic and crypt-villus axes. Results: Uroguanylin
`mRNA was expressed throughout the mouse intestine
`and also in the kidney. Signal intensity was greatest
`in the small intestine for uroguanylin and in the distal
`small intestine and colon for guanylin. In situ hybridiza-
`tion showed uroguanylin mRNA localized predominantly
`in intestinal villi and the corticomedullary junction of
`the kidney, whereas guanylin mRNA was localized in
`both crypts and villi in the small intestine and to super-
`cial epithelial cells in the colon. Conclusions: Mouse
`uroguanylin mRNA expression is discrete from guanylin
`expression in the intestine. The patterns of distribution
`in the intestine and the known pH optima of these
`ligands suggest a complementary role for these secre-
`tagogues.
`
`eat-stable enterotoxin (STa) is a peptide ligand
`!
`elaborated by Escherichia coli and other bacteria.1
`Binding of this ligand to the intestinal receptor guanylate
`cyclase C (GC-C) results in increased intracellular levels
`of guanosine 3!,5!-cyclic monophosphate (cGMP).2 This,
`in turn, activates a cGMP-dependent protein kinase
`(cGKII),3 which phosphorylates the cystic brosis trans-
`membrane conductance regulator (CFTR), resulting in
`net chloride (Cl") and water secretion.4,5 Infection with
`STa-producing E. coli results in secretory diarrhea.1 Two
`endogenous ligands, guanylin and uroguanylin, which
`are structurally related to STa, activate the same signal
`
`transduction pathway, causing net Cl" secretion.4 7
`However, guanylin and uroguanylin are less potent acti-
`vators of GC-C than STa.6,8,9 Guanylin and STa have also
`")
`been shown to stimulate duodenal bicarbonate (HCO3
`secretion.10
`Guanylin was originally isolated from the rat jeju-
`num.7 Although uroguanylin was originally identied in
`opossum and human urine,6,9 similar to guanylin, uro-
`guanylin messenger RNA (mRNA) is predominantly ex-
`pressed in the intestine.11,12 However, the precise location
`of uroguanylin mRNA in the intestine, along the longi-
`tudinal (duodenal-colonic) and crypt-villus axes, has been
`less well studied.11 Uroguanylin mRNA expression has
`also been reported in opossum kidney and heart13 and in
`rat lung, pancreas, and kidney.11
`To determine the functions of these endogenous li-
`gands within the intestine, it is important to localize
`their sites of expression. In this study, we describe clon-
`ing of the mouse uroguanylin complementary DNA
`(cDNA) and characterization of the tissue and cellular
`location of uroguanylin and guanylin mRNA in the
`mouse. We show a distinct, yet overlapping pattern of
`expression for guanylin and uroguanylin in the mouse
`intestine as well as extraintestinal expression of urogua-
`nylin in the kidney.
`
`"$0'.*$+/ $,& "'0)-&/
`
`cDNA Cloning
`
`A single 4-kilobase (kb) Xho1-Xho1 fragment from
`a genomic clone (accession no. U95182) was subcloned into
`pBluescript II SK! (Stratagene, La Jolla, CA). Overlapping
`oligonucleotide primers were used to sequence the ends until
`sequences homologous to the human uroguanylin cDNA14
`
`Abbreviations used in this paper: CFTR, cystic brosis transmem-
`brane conductance regulator; cGKII, guanosine 3!,5!-cyclic mono-
`phosphate dependent protein kinase II; GC-C, guanylate cyclase C;
`RT-PCR, reverse-transcription polymerase chain reaction; STa, heat-
`stable enterotoxin.
`# 1997 by the American Gastroenterological Association
`0016-5085/97/$3.00
`
`MYLAN EXHIBIT - 1051
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`
`
`September 1997
`
`UROGUANYLIN AND GUANYLIN mRNA LOCALIZATION 1001
`
`were observed. Two polymerase chain reaction (PCR) primers
`were then designed for the cDNA ends (5! primer, AGGTGG-
`ACAGCAGAAGGAAG; 3! primer, TGGTGCCTAAGT-
`ATGGAC) and used to amplify a partial 450-nucleotide cDNA
`from strain FVB/NJ mouse intestinal total RNA using reverse-
`transcription (RT)-PCR as described previously.15 Sequencing
`conrmed the delity of the fragment subcloned into PCR
`2.1 (Invitrogen, Carlsbad, CA). Sequences were analyzed using
`MacVector sequence analysis software (Eastman Kodak, Roch-
`ester, NY).
`
`Northern Blot Analysis
`
`Total RNA was isolated from a panel of strain FVB/NJ
`mouse tissues using acid guanidine isothiocyanate phenol
`chloroform extraction.16 Mouse tissues were harvested under a
`protocol approved by the Institutional Animal Care and Use
`Committee. For intestinal tissues, segments were divided into
`the following: duodenum (rst 3 cm distal to stomach), proxi-
`mal jejunum (proximal one third of small intestine), distal
`jejunum (central one third of small intestine), ileum (distal
`one third of small intestine), cecum, proximal colon (proximal
`two fths of large intestine), and distal colon (distal three fths
`of large intestine). Total RNA (20 !g) was electrophoresed
`through a 1.0% agarose/1.9% formaldehyde denaturing gel,
`transferred to a nylon membrane (MagnaGraph; MSI, Westbor-
`ough, MA), and cross-linked to the membrane using UV light
`(Stratalinker; Stratagene) as described previously.17 Blots were
`hybridized with cDNA fragments generated by PCR encom-
`passing nucleotides 288 386 of mouse uroguanylin and nucle-
`otides 118 307 of mouse guanylin.18 Each PCR product was
`subcloned into PCR 2.1, and the resultant plasmids were called
`PCR 2.1 m.uro 98 and PCR 2.1 mgg exon 2. An end-labeled
`oligonucleotide complementary to 18S ribosomal RNA was
`used for quantitation of relative amounts of total RNA loaded
`and normalization of signal intensities.19 Northern blots were
`hybridized overnight at 60"C and were washed under high-
`stringency conditions as described previously.19 Image analysis
`and mRNA quantitation were performed on a surface emission
`scanner (Molecular Dynamics PhosphorImager; Molecular Dy-
`namics, Sunnyvale, CA) using ImageQuant software (Molecu-
`lar Dynamics).
`
`In Situ Hybridization
`
`Tissues were processed for in situ hybridization as de-
`scribed previously.20,21 Briey, fresh tissue was xed in 4%
`paraformaldehyde and then saturated in 30% sucrose before
`being embedded in M1 embedding matrix (Lipshaw, Pitts-
`burgh, PA) and snap-frozen. Cryostat sections were cut at
`10 12 !m, air-dried on slides coated by Vectabond (Vector
`Laboratories, Burlingame, CA), and xed with paraformalde-
`hyde. Prehybridization and hybridization were performed as
`described previously.20,21 [35S]uridine 5! triphosphate labeled
`sense and antisense riboprobes were prepared from linearized
`PCR 2.1 m.uro 98 and PCR 2.1 mgg exon 2 plasmids (de-
`scribed above). Tissue sections were photographed under dark-
`eld and bright-eld illumination.
`
`) Sequence of mouse uroguanylin cDNA. The start site
`Figure 1. (
`and the stop codon are underlined. The poly(A)! signal is
`(
`) Deduced amino acid sequence of mouse uroguanylin
`cDNA. The sequence of the 15 amino acid mature peptide is high-
`lighted (- - -).
`
`#'/1+0/
`
`Cloning of Mouse Uroguanylin cDNA
`
`A single 4-kb genomic fragment was identied,
`which strongly hybridized with a 32P-labeled human uro-
`guanylin cDNA probe (gift of O. Hill14). This fragment
`was subcloned into pBluescript II SK! and further ana-
`lyzed by restriction mapping and direct dideoxynucleo-
`tide sequencing. We were able to identify the 5! and 3!
`ends of the coding sequence from the sequence informa-
`tion. Using primers designed for each, we used RT-PCR
`to amplify 450 nucleotides of mouse uroguanylin cDNA
`from total RNA extracted from mouse jejunum. This
`amplied product was then subcloned into the PCR 2.1
`vector. From direct sequencing of the genomic fragment,
`we predicted the cDNA start site near a TATAA se-
`quence upstream of the coding sequence. The location
`of the poly(A)! signal sequence predicted a full-length
`cDNA of 590 nucleotides.
`The sequence of the full-length mouse uroguanylin
`cDNA (accession no. U90727) and the predicted amino
`acid sequence are shown in Figure 1. The mouse urogua-
`nylin cDNA shares 82% homology with human urogua-
`nylin,14 90% homology with rat uroguanylin,11 and 51%
`homology with mouse guanylin.18 In addition, the uro-
`guanylin cDNA is 99% identical to an unpublished ex-
`pressed sequence tag cloned from mouse kidney.22 Trans-
`lation of the mouse uroguanylin cDNA predicts a 106
`amino acid peptide. The deduced amino acid sequence
`
`/ 5E20$$0026
`
`08-08-97 18:40:09
`
`gasa
`
`WBS-Gastro
`
`
`
`1002 WHITAKER ET AL.
`
`GASTROENTEROLOGY Vol. 113, No. 3
`
`of the mature carboxy-terminal 15 amino acid peptide
`is 80% identical to the predicted human sequence14 and
`93% identical to the rat peptide.11
`
`Northern Analysis
`
`A 98 base pair (bp) fragment extending from
`nucleotides 288 386 was used as a probe for Northern
`blot analysis to localize mouse uroguanylin mRNA. A
`single predominant mRNA species of approximately 600
`bp was recognized with this probe. Signal was present
`in all intestinal tissues examined, with highest concentra-
`tions observed in the duodenum, jejunum, and ileum
`(Figure 2). Extraintestinal expression was also found in
`the kidney (Figure 2). No signal was detected in the
`liver, spleen, heart, lung, brain (Figure 2), stomach, or
`thymus (not shown). As shown in Figure 2, this expres-
`sion pattern is distinct from that observed when the
`same blot was probed with a guanylin cDNA; guanylin
`message was also present in all intestinal tissues but was
`most prevalent in the distal jejunum, ileum, cecum, and
`colon; no extraintestinal expression of guanylin was found
`by Northern analysis. Quantitation of relative signal in-
`tensities for both uroguanylin and guanylin is shown in
`Figure 3.
`
`In Situ Hybridization
`
`To further delineate the crypt-villous and extrain-
`testinal expression pattern of mouse uroguanylin and
`
`guanylin, we performed in situ hybridization of a panel
`of mouse tissues using 35S-labeled sense and antisense
`riboprobes. Both antisense probes showed highly re-
`stricted patterns of expression. In the duodenum, urogua-
`nylin signal was primarily in midvillous epithelial cells
`(Figure 4A, arrow). Signal in isolated crypt cells (arrow-
`heads) was detected by dark-eld illumination with both
`sense (Figure 4B ) and antisense probes (Figure 4A ) and
`is caused by autouorescence of Paneth cell granules.23
`Absence of bona de signal was conrmed by bright-
`eld microscopy (data not shown). Intense uroguanylin
`signal in the proximal jejunum was primarily seen in
`villous epithelium, although a uniform narrow band of
`autoradiographic grains was also present at the base of
`the crypts (Figure 4C ). In distal jejunum and ileum, the
`uroguanylin signal appeared to be restricted to villous
`epithelium (Figure 4D F ). As with the duodenal crypt
`cell expression, the signal in isolated crypt cells in distal
`jejunum and ileum results from Paneth cell autouores-
`cence rather than autoradiographic grains (Figure 4D and
`E ). At the ileocecal boundary, uroguanylin signal was
`only detected in epithelial cells overlying a lymphoid
`aggregate (Figure 4F, arrow). No uroguanylin signal was
`detected in the colon (Figure 4G ), raising the possibility
`that the level of expression of uroguanylin mRNA in
`the colon is below the level of detection of our in situ
`
`Figure 2. Representative Northern analysis of guanylin and urogua-
`nylin message. Total RNA was separated and the blot was probed as
`described in Materials and Methods. The positions of 28S and 18S
`rRNA are indicated along the side of each blot. Both guanylin and
`uroguanylin mRNA were present throughout the intestine. Unlike gua-
`nylin mRNA, which was highest in distal small intestine and colon,
`uroguanylin signal was highest in small intestine with very little signal
`in the cecum and colon. Uroguanylin signal was also found in the
`kidney but not in liver, spleen, heart, lung, or brain. No extraintestinal
`expression of guanylin was detected. For quantitation purposes, the
`blot was reprobed with an oligonucleotide that recognizes 18S rRNA19;
`the results are shown in the lower panel.
`
`)
`Figure 3. Quantitation of relative signal intensities for guanylin (
`and uroguanylin (
`) mRNA in mouse intestine. All quantitation was
`performed with ImageQuant software. Relative signal intensities were
`normalized to 18S ribosomal RNA signal intensity.19 Guanylin values
`are shown as a percentage of cecum signal intensity with cecum set
`at 100%. Uroguanylin values are shown as a percentage of proximal
`jejunal expression with this value set at 100%. Data are mean # SE
`of three separate determinations.
`
`/ 5E20$$0026
`
`08-08-97 18:40:09
`
`gasa
`
`WBS-Gastro
`
`
`
`September 1997
`
`UROGUANYLIN AND GUANYLIN mRNA LOCALIZATION 1003
`
`hybridization experiments or that the signal found by
`Northern analysis represents weak cross-hybridization
`with guanylin mRNA. We also found a discrete band of
`uroguanylin message in the kidney, in cells localized to
`the corticomedullary junction (Figure 4H ), using the
`antisense probe; no signal was seen with the sense probe
`(Figure 4I ).
`In contrast to uroguanylin, authentic and robust gua-
`nylin message was detected in crypt cells in the proximal
`and distal jejunum and ileum (Figure 4J, L, and M).
`Guanylin mRNA was also present in many but not all
`villous cells in the proximal jejunum (Figure 4J ) and in
`all villous cells in the distal jejunum (Figure 4L ) and
`ileum (Figure 4M ). The most intense signal was observed
`in the supercial epithelium of the cecum and colon
`(Figures 4N, O, and P ). Interestingly, guanylin mRNA
`was limited to the supercial epithelium in distal colon
`(Figure 4P ) but was present in the neck and deeper
`colonic glands in proximal colon (Figure 4O ). The
`broader distribution of signal in proximal than in distal
`colon correlated with the increased expression detected
`by Northern analysis (Figures 2 and 3). No signal was
`seen using the sense probe in proximal jejunum (Figure
`4K ) and colon (Figure 4Q ). No guanylin signal was de-
`tected in mouse kidney (data not shown).
`
`!*/%1//*-,
`
`We have cloned a mouse uroguanylin cDNA and
`showed a discrete yet overlapping pattern of expression
`for mouse uroguanylin and guanylin. Although guanylin
`and uroguanylin mRNA are found throughout the mouse
`intestine, their levels of expression vary in different seg-
`ments, and they seem to be expressed by different cell
`types. The highest level of uroguanylin mRNA expres-
`sion occurs in the proximal small intestine; the highest
`level of guanylin mRNA expression occurs in the distal
`small intestine, cecum, and proximal colon. Mouse uro-
`guanylin mRNA is primarily restricted to the villi,
`whereas guanylin mRNA is localized to both crypts and
`villi throughout the small intestine.
`Our observations are consistent with the reported dis-
`tribution of uroguanylin mRNA in rat intestine and
`kidney,11 although the tissue-specic pattern of urogua-
`nylin mRNA expression in the mouse is more restricted
`than previously reported for rats11 and opossums.13 Uro-
`guanylin mRNA was found in human colon; no signal
`was found in the kidney, and the small intestine was not
`investigated.14 The cellular localization of uroguanylin
`has not been previously reported for any species.11,13,14
`We have also shown that guanylin mRNA is expressed
`throughout the mouse intestine, as has been shown in
`rats21 and humans.24 Localization of guanylin mRNA to
`
`both crypt and villous cells in the mouse intestine corre-
`lates with the distribution of guanylin mRNA we have
`observed in human intestine25 but not with the villous
`pattern we observed in the rat.21 This species-specic
`pattern of guanylin mRNA expression parallels the pat-
`tern of GC-C mRNA expression, which is primarily re-
`stricted to villous cells in the rat small intestine19 but
`includes both crypt and villous epithelium in the
`mouse.26
`On the basis of the localization data in this study for
`guanylin and uroguanylin mRNA and previously docu-
`mented distribution patterns for GC-C,26 CFTR,27 and
`cGKII,28 the entire guanylin/uroguanylin-mediated sig-
`nal transduction pathway is probably localized to some
`of the same epithelial cells. Mouse GC-C mRNA is ex-
`pressed in both crypts and villi in the small intestine
`and in the deep crypts and supercial epithelium of the
`large intestine.26 cGKII mRNA expression has not been
`examined in mice, but in rats, cGKII mRNA expression
`extends from the crypts to the upper villous, with the
`highest level of expression in midvillous cells in the small
`intestine. Expression of cGKII mRNA is also found in
`the crypts of the cecum and proximal colon28; no cGKII
`expression is found in the distal colon.28 Mouse CFTR
`mRNA is present in the small intestine and colon. In
`the small intestine, it is predominantly expressed in the
`crypts with a decreasing gradient along the villi, with
`no expression at the villous tips.27 Thus, in the small
`intestine, there is probably colocalization of guanylin
`and/or uroguanylin, GC-C, cGKII, and CFTR mRNA
`within the same cells in the crypts and bottom one third
`of the villi. The entire secretory pathway is also likely
`to be present in guanylin mRNA-expressing cells in the
`cecum and proximal colon. The observation of urogua-
`nylin mRNA expression in epithelial cells overlying
`lymphoid aggregates parallels the distinctive pattern of
`CFTR mRNA expression in epithelial cells in close con-
`tact with lymphoid tissue in human intestine.29 How-
`ever, guanylin, uroguanylin, and GC-C mRNA are also
`present in high concentrations in villous cells where
`CFTR is poorly or not at all expressed,26,27 and guanylin
`and GC-C are present in the distal colon where cGKII
`may not be expressed.28 Therefore, it is possible that
`there are other downstream targets or intermediaries of
`this endogenous receptor-ligand interaction.
`Guanylin and uroguanylin have different pH optima
`of activity, with uroguanylin functional at pH $5.5 and
`guanylin at pH $8.0.30 Also, a pH microclimate exists
`along the villi of the jejunum; in rats, the pH gradient
`changes from pH 6.6 at the villous tip to pH 8.15 in
`the crypts31; it is likely that a similar microclimate exists
`in villi in the mouse. Given this microclimate, urogua-
`
`/ 5E20$$0026
`
`08-08-97 18:40:09
`
`gasa
`
`WBS-Gastro
`
`
`
`1004 WHITAKER ET AL.
`
`GASTROENTEROLOGY Vol. 113, No. 3
`
`/ 5E20$$0026
`
`08-08-97 18:40:09
`
`gasa
`
`WBS-Gastro
`
`
`
`September 1997
`
`UROGUANYLIN AND GUANYLIN mRNA LOCALIZATION 1005
`
`and ) proximal jejunum,
`Figure 4. In situ hybridization of guanylin and uroguanylin riboprobes. Sections of ( and ) mouse duodenum, (
`( and ) distal jejunum, (
`and
`) ileum, (
`) cecum, (
`) proximal colon, (
`and
`) distal colon, and ( and ) kidney were prepared
`as described in Materials and Methods.
`were hybridized with a uroguanylin riboprobe;
`were hybridized with a guanylin riboprobe.
`Uroguanylin:
`shows uroguanylin signal primarily in duodenal midvillous epithelial cells (
`). Signal in isolated crypt cells (
`) was
`detected by dark-eld illumination with both (
`) sense and ( and
`) antisense probes and is a result of autouorescence of Paneth cell
`granules. Absence of bona de signal was conrmed by bright-eld microscopy (not shown). Uroguanylin signal in the (
`) proximal jejunum was
`primarily found in villous epithelium with a thin band of true signal in the crypts; in the (D) distal jejunum and (E) ileum, uroguanylin signal was
`restricted to villous epithelium. ( ) At the ileocecal boundary, true uroguanylin signal was only detected in epithelial cells overlying a lymphoid
`aggregate (
`). As with the duodenal crypt cell expression, the signal in isolated crypt cells in distal jejunum and ileum results from Paneth
`cell autouorescence. No uroguanylin signal was detected in (
`) colon or cecum (not shown). Using the antisense probe, a discrete band of
`uroguanylin message was also observed in the (
`) kidney in cells localized to the corticomedullary junction. ( ) No signal was seen with the
`sense probe. Guanylin: In contrast to uroguanylin, authentic guanylin message was detected in crypt cells in ( ) proximal and (
`) distal jejunum
`and (
`) ileum. Guanylin mRNA was also present in most, but not all, villous cells in ( ) the proximal jejunum and in all villous cells in the distal
`( ) jejunum and (
`) ileum. The most intense signal was observed in the supercial epithelium of the (
`) cecum and ( and ) colon. Guanylin
`mRNA was limited to the supercial epithelium in (
`) distal colon but was present in the neck and deeper colonic glands in (
`) proximal colon.
`No signal is seen in the (
`) proximal jejunum or (
`) colon with a sense, control riboprobe.
`
`nylin activity would be greatest nearer the villous tip,
`whereas guanylin activity would be greatest nearer to
`the crypts. Interestingly, uroguanylin mRNA expression
`occurs primarily in the villi in small intestine where the
`pH microclimate is more likely to be acidic and closer to
`its pH optimum.30 Unlike uroguanylin, guanylin mRNA
`expression is also prominent in the crypts of the small
`intestine, where guanylin is more likely to bind to GC-
`C in a less acidic milieu.
`Guanylin and STa can elicit bicarbonate secretion in
`vitro from rat duodenum.10 Because of the inux of acid
`from the stomach, luminal pH as well as the villous pH
`microclimate in the duodenum are likely to be acidic.
`Although both guanylin and uroguanylin could partici-
`", on the basis of their
`pate in the secretion of HCO3
`relative patterns of expression and pH optima, urogua-
`nylin is probably the predominant ligand for this role.
`The expression of uroguanylin mRNA but not GC-C
`mRNA in mouse kidney26 raises the possibility that this
`ligand also functions as a natriuretic peptide in an endo-
`crine pathway to maintain uid and electrolyte balance
`in an intestinal-renal axis.12 This possibility is supported
`by a demonstration of circulating uroguanylin13 and by
`the pattern of uroguanylin mRNA expression in the
`mouse kidney. This pattern resembles that of erythropoi-
`etin, another circulating hormone, which is expressed at
`the corticomedullary junction.32 However, there are other
`explanations for the presence of uroguanylin mRNA in
`the kidney in the absence of GC-C expression. There
`may be a paracrine signaling mechanism with a receptor
`other than GC-C located in the kidney. Alternatively,
`uroguanylin mRNA expression in the kidney may be a
`vestige of a signaling pathway that is present in other
`mammals33,34 but is no longer functional in the mouse.
`In summary, we examined localization of both urogua-
`nylin and guanylin in the mouse intestine and localiza-
`tion of uroguanylin in the mouse kidney. The overlap-
`
`ping pattern of uroguanylin and guanylin mRNA
`distribution with that of GC-C mRNA26 is consistent
`with these ligands cooperatively regulating salt and water
`metabolism in the intestine via an autocrine or paracrine
`GC-C mediated pathway. It is possible that differences
`in the expression patterns of uroguanylin and guanylin
`correlate with their predominant physiological functions.
`In addition, based on the discrete patterns of mRNA
`expression and different pH optima of activity, guanylin
`and uroguanylin may have different spatially restricted
`functions along the crypt-villus and duodenal-colonic
`axes.
`
`#'('.',%'/
`
`In:
`1. Cohen MB, Giannella RA. Enterotoxigenic
`Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, eds. Infections
`of the gastrointestinal tract. New York, Raven, 1995:691 708.
`2. Schulz S, Green CK, Yuen PST, Garbers DL. Guanylyl cyclase is
`a heat-stable enterotoxin receptor. Cell 1990; 63:941 948.
`3. Pfeifer A, Aszodi A, Seidler U, Ruth P, Hofmann F, Fassler R.
`Intestinal secretory defects and dwarsm in mice lacking cGMP-
`dependent protein kinase II. Science 1996; 274:2082 2086.
`4. Cuthbert AW, Hickman ME, MacVinish LJ, Evans MJ, Colledge
`WH, Ratcliff R, Seale PW, Humphrey PPA. Chloride secretion in
`response to guanylin in colonic epithelia from normal and
`transgenic cystic brosis mice. Br J Pharmacol 1994; 112:31
`36.
`5. Chao AC, de Sauvage FJ, Dong YJ, Wagner JA, Goeddel DV, Gard-
`ner P. Activation of intestinal CFTR Cl" channel by heat-stable
`enterotoxin and guanylin via cAMP-dependent protein kinase.
`EMBO J 1994; 13:1065 1072.
`6. Hamra FK, Forte LR, Eber SL, Pidhorodeckyj NV, Krause WJ, Free-
`man RH, Chin DT, Tompkins JA, Fok KF, Smith CE, Dufn KL,
`Siegel NR, Currie MG. Uroguanylin: structure and activity of a
`second endogenous peptide that stimulates intestinal guanylate
`cyclase. Proc Natl Acad Sci USA 1993; 90:10464 10468.
`7. Currie MG, Fok KF, Kato J, Moore RJ, Hamra FK, Dufn KL, Smith
`CE. Guanylin: an endogenous activator of intestinal guanylate
`cyclase. Proc Natl Acad Sci USA 1992; 89:947 951.
`8. Freeman RH, Forte LR, Hamra FK, Currie MG, Krause WJ. Urogua-
`nylin: a potential natriuretic peptide hormone produced by the
`intestine (abstr). FASEB J 1996; 8:A552.
`9. Kita T, Smith CE, Fok KF, Dufn KL, Moore WM, Karabatsos PJ,
`
`/ 5E20$$0026
`
`08-08-97 18:40:09
`
`gasa
`
`WBS-Gastro
`
`
`
`1006 WHITAKER ET AL.
`
`GASTROENTEROLOGY Vol. 113, No. 3
`
`Kachur JF, Hamra FK, Pidhorodeckyj NV, Forte LR, Currie MG.
`Characterization of human uroguanylin: a member of the guanylin
`peptide family. Am J Physiol 1994; 266:F342 F348.
`10. Guba M, Kuhn M, Forssmann WG, Classen M, Gregor M, Seidler
`U. Guanylin strongly stimulates rat duodenal HCO3" secretion:
`
`proposed mechanism and comparison with other secretagogues.
`Gastroenterology 1996; 111:1558 1568.
`11. Miyazato M, Nakazato M, Matsukura S, Kangawa K, Matsuo H.
`Uroguanylin gene expression in the alimentary tract and extra-
`gastrointestinal tissues. FEBS Lett 1996; 398:170 174.
`12. Forte LR, Fan X, Hamra FK. Salt and water homeostasis: urogua-
`nylin is a circulating peptide hormone with natriuretic activity. Am
`J Kidney Dis 1996; 28:296 304.
`13. Fan X, Hamra FK, Freeman RH, Eber SL, Krause WJ, Lim RW,
`Pace VM, Currie MG, Forte LR. Uroguanylin: cloning of preprouro-
`guanylin cDNA, mRNA expression in the intestine and heart and
`isolation of uroguanylin from plasma. Biochem Biophys Res Com-
`mun 1996; 219:457 462.
`14. Hill O, Cetin Y, Cieslak A, Magert HJ, Forssmann WG. A new
`human guanylate cyclase activating peptide (GCAP-II, urogua-
`nylin): precursor cDNA and colonic expression. Biochem Biophys
`Acta 1995; 12853:146 149.
`15. Mann EA, Cohen MB, Giannella RA. Comparison of receptors for
`heat-stable enterotoxin: novel receptor present
`in IEC-6 cells. Am J Physiol 1993; 264:G172 G178.
`16. Chomczynski P, Sacchi N. Single-step method of RNA isolation by
`acid guanidium thiocyanate phenol chloroform extraction. Anal
`Biochem 1987; 162:156 159.
`17. Laney DW Jr, Mann EA, Dellon SC, Perkins DR, Giannella RA,
`Cohen MB. Novel sites for expression of an
`heat-
`stable enterotoxin receptor in the developing rat. Am J Physiol
`1992; 263:G816 G821.
`18. Sciaky D, Kosiba JL, Cohen MB. Genomic sequence of the murine
`guanylin gene. Genomics 1994; 24:583 587.
`19. Cohen MB, Mann E, Lau C, Henning SJ, Giannella R. A gradient
`in expression of the
`heat-stable enterotoxin re-
`ceptor exists along the villus-to-crypt axis of rat small intestine.
`Biochem Biophys Res Commun 1992; 186:483 490.
`20. Witte DP, Wiginton DA, Hutton JJ, Aronow BJ. Coordinate develop-
`mental regulation of purine catabolic enzyme expression in gas-
`trointestinal and postimplantation reproductive tracts. J Cell Biol
`1991; 115:179 190.
`21. Lewis LG, Witte DP, Laney DW, Currie MG, Cohen MB. Guanylin
`mRNA is expressed in villous enterocytes of the rat small intes-
`tine and supercial epithelia of the rat colon. Biochem Biophys
`Res Commun 1993; 196:553 560.
`22. Marra M, Hillier L, Allen M, Bowles M, Dietrich N, Dubuque T,
`Geisel S, Kucaba T, Lacy M, Le M, Martin J, Morris M, Schellen-
`burg K, Steptoe M, Tan F, Underwood K, Moore B, Theising B,
`Wylie T, Lennon G, Soares B, Wilson R, Waterston R. The WashU-
`
`accession
`
`project. 1996, Genbank
`
`HHMI mouse EST
`#AA139503.
`23. Garrett KL, Grounds MD, Beilharz MW. Nonspecic binding of
`nucleic acid probes to Paneth cells in the gastrointestinal tract
`with in situ hybridization. J Histochem Cytochem 1992; 40:
`1613 1618.
`24. Wiegand RC, Kato J, Huang MD, Fok KF, Kachur JF, Currie MG.
`Human guanylin: cDNA isolation, structure, and activity. FEBS
`Lett 1992; 311:150 154.
`25. Hochman JA, Sciaky D, Witte D, Whitaker T, Cohen MB. The
`guanylin promoter confers intestinal epithelial cell-specic ex-
`pression (abstr). Gastroenterology 1996; 110:A805.
`26. Swenson ES, Mann EA, Jump ML, Witte DP, Giannella RA. The
`guanylin/STa receptor is expressed in crypts and apical epithe-
`lium throughout the mouse intestine. Biochem Biophys Res Com-
`mun 1996; 225:1009 1014.
`27. Zhou L, Dey CR, Wert SE, DuVall MD, Frizzell RA, Whitsett JA.
`Correction of lethal intestinal defect in a mouse model of cystic
`brosis by human CFTR. Science 1994; 266:1705 1708.
`28. Markert T, Vaandrager AB, Gambaryan S, Pohler D, Hausler C,
`Walter U, De Jonge HR, Jarchau T, Lohmann SM. Endogenous
`expression of type II cGMP-dependent protein kinase mRNA and
`protein in rat intestine. J Clin Invest 1995; 96:822 830.
`29. Strong TV, Boehm K, Collins FS. Localization of cystic brosis trans-
`membrane conductance regulator mRNA in the human gastrointesti-
`nal tract by in situ hybridization. J Clin Invest 1994;93:347354.
`30. Hamra K, Fan X, Krause WJ, Freeman RH, Chin DT, Smith CE,
`Currie MG, Forte LR. Prouroguanylin and proguanylin: purication
`from colon, structure, and modulation of bioactivity by proteases.
`Endocrinology 1996; 137:257 265.
`31. Daniel H, Neugebauer B, Kratz A, Rehner G. Localization of acid
`microclimate along intestinal villi of rat jejunum. Am J Physiol
`1985; 248:G293 G298.
`32. Lacombe C, Da Silva JL, Bruneval P, Fournier JG, Wendling F,
`Casadevall N, Camilleri JP, Bariety J, Varet B, Tambourin P. Peritu-
`bular cells are the site of erythropoietin synthesis in the murine
`hypoxic kidney. J Clin Invest 1988; 81:620 623.
`33. Forte LR, Krause WJ, Freeman RH. Receptors and cGMP signal-
`ling mechanism for
`enterotoxin in opossum kidney. Am J
`Physiol 1988; 255:F1040 F1046.
`enterotoxin
`34. Forte LR, Krause WJ, Freeman RH.
`receptors: localization in opossum kidney, intestine, and testes.
`Am J Physiol 1989; 257:F874 F881.
`
`Received March 31, 1997. Accepted May 20, 1997.
`Address requests for reprints to: Mitchell B. Cohen, M.D., Division
`of Pediatric Gastroenterology and Nutrition, 3333 Burnet Avenue,
`Cincinnati, Ohio 45229. e-mail: mitchell.cohen@chmcc.org;
`fax:
`(513) 636-7805.
`Supported in part by grant DK 47318 from the National Institutes
`of Health.
`
`/ 5E20$$0026
`
`08-08-97 18:40:09
`
`gasa
`
`WBS-Gastro
`
`