Humanguanylin Yukari Date, Masamitsu Nakazato, Hideki Yamaguchi, Mikiya Miyazato and Shigeru Matsukura

ORIGINAL ARTICLE

Tissue Distribution and Plasma Concentration of HumanGuanylin Yukari Date, Masamitsu Nakazato, Hideki Yamaguchi, Mikiya Miyazato and Shigeru Matsukura

Guanylin, a peptide homologue of the bacterial heat-stable enterotoxins, is an endogenous activator ofguanylate cyclase C (GC-C). Wedetermined the tissue content and plasma concentration of human guanylin, and its cellular source in the intestine. Humanguanylin is distributed widely from the duodenum to the rectum, the highest content being in the ileum and proximal colon. The plasma concentration of immunoreactive guanylin in the normal individuals tested was 30.3±3.7 fmol/ml (mean±SE) and that in patients with chronic renal failure was elevated with increasing serum creatinine concentration. Guanylin immunoreactivity was detected in the villus epithelial cells in the small intestine and these guanylin-containing cells were increased in number along the cephalocaudal axis of the gut. Guanylin was also present in Paneth cells in the small intestine and superficial epithelial cells in the large intestine. Guanylin mRNAwas detected in the intestine by the reverse transcription-polymerase chain reaction. Guanylin may have paracrine action on neighboring enterocytes, activating intestinal guanylate cyclase and thereby regulating intestinal fluid as well as electrolyte transport through the second messenger, cyclic GMP. (Internal Medicine 35: 171-175, 1996) Key words: guanylate cyclase C, enterotoxin, natriuretic polypeptide, electrolyte and water bal- ance, surface epithelial cell, Paneth cell

Introduction coli,The heat-stableYercinia enterocoliticaenterotoxins and(STs)VibrioproducedcholeraebyareEscherichiaall 15-30 amino acid peptides. These enterotoxins cause acute diarrhea, whichdevelopingreportedlycountriesaccounts(1). STsforbindup toto intestine-specific50%of infant deathsrecep-in tor-guanylateand mediate the cyclasestimulation(GC-C)of Cl~in secretionthe brush andborderthe inhibitionmembrane (2).of Na+Guanylinand H2Oisabsorption,a 15-amino acidtherebypeptidecausingthat secretoryhas recentlydiarrheabeen colonpurifiedcarcinomafrom ratcelljejunumline whoseusingGC-Ca bioassayresponds ofto aSTsT84withhumanan aminoincreaseacidin intracellularsequence homologycyclic withGMP(CGMP)Escherichia(3). It colihas a ST.47% clase,Guanylinstimulatehas beenCl"shownsecretion,to activateand displaceintestinalST bindingguanylateto cy-T84 andcells electrolyte(4). It may transportbe a physiologicaland mayregulatorbe involvedof intestinalin the patho-fluid ingphysiologyof the physiologicalof diarrheal diseases.functions Aandmorepathophysiologicalcomplete understand-sig- requiresnificancedetailedof guanylinanalysisinofwaterits celland andelectrolytetissue distributionhomeostasisin Weprepared humans.an antiserum raised against synthetic rat guanylin bothand establishedhuman and rata radioimmunoassayguanylin, then identified(RIA) thathumancan quantifyguanylin andresultsits precursorsof the regionalin thedistributionintestine andplasmaand plasma(5).concentrationHere we reportof humanreverseguanylin.transcription-polymeraseWeinvestigated its genechainexpressionreaction (RT-PCR).using the Weofalsoguanylindeterminedin theimmunohistochemicallyintestine. the cellular source Materials and Methods TheQuantificationgastrointestinalof thetractguanyiinfrom thecontentstomachin totissuethe colon and kidneydiedwereof resectedischemic atheartautopsydisease.fromTissuesthree patientswere excisedwho andhad volumeimmediatelyof water heatedto inactivatefor 10 intrinsicminutes atproteinases.95-100°C inAftera 10-foldcool- From the Third Department of Internal Medicine, Miyazaki Medical College, Miyazaki Received for publication June 29, 1995; Accepted for publication December 5, 1995 Reprint requests should be addressed to Dr. Masamitsu Nakazato, the Third Department of Internal Medicine, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-16

Internal Medicine Vol. 35, No. 3 (March 1996) 171 Date et al ing the tissue samples to 4°C, CH3COOHand HC1were added MD).Reverse transcription was done for 30 minutes at 42°C to the respective final concentrations of 1 Mand 20 mM.The then incubation for 3 minutes at 94°C to inactivate the reverse tissues then were homogenized in a polytron for 10 minutes, transcriptase. The resulting CDNAwas subjected to PCRampli- after which the homogenate was centrifuged at 20,000 x g for fication with the 2 jiM sense and antisense primers for guanylin 30 minutes at 4°C. The supernatants were applied to Sep-Pak C- and (3-actin, and the 2.5 U Taq DNApolymerase (Perkin-Elmer 1 8 cartridges (Waters, Milford, CA), which were washed with Cetus, Foster City, CA). The sequences of the oligonucleotides 0.5 MCH3COOHand 10% acetonitrile (CH3CN) solution (first sense then antisense) for the guanylin and (3-actin PCRs containing 0. 1% trifluoroacetic acid (TFA), and the peptides were guanylin, 5'-ATGAATGCCTTCCTGCTCTTCGCA-3' were eluted with 60% CH3CNsolution containing 0. 1% TFA. and 5'-TACAGGCAGCGTAGGCACAGATTT-3 ; p-actin, 5'- Cartridge eluates corresponding to 100 mgwet weight were ATCCAGGCTGTGCTATCCCTGTAC-3' and 5'-TCATTG- lyophilized and subjected to the RIA for guanylin. CCAATGGTGATGACCTGG-3'.The PCR conditions were 34 cycles of denaturation at 94°C for 1 minute, annealing at Determination of plasma guanylin concentration 58°C for 2 minutes, and extension at 72°C for 2 minutes. PCR Blood was obtained from 30 normal individuals ( 15 men and products were electrophoresed on a 2%agarose gel (FMC 15 women, aged 18^0 years) and 23 patients (ll men and 12 BioProducts, Rockland, ME)then made visible with ethidium women, aged 37-58 years) with chronic renal failure (CRF) bromideunderultraviolet. whose serum creatinine levels were above 1.5 mg/dl. These patients did not undergo hemodialysis. After the plasma was Immunohistochemistry diluted to one-half with 0.9% saline, it was applied to a Sep-Pak Humangastrointestinal tracts obtained from the same 3 C-18 cartridge which had been pre-equilibrated with 0.9% patients described above were fixed with 3.7% formaldehyde in saline. The cartridge was washedwith saline and 10%CH3CN 10 mMphosphate-buffered saline (PBS, pH 7.2) and, after solution containing 0.1% TFA. The adsorbed peptides were dehydration in a graded ethanol series, they were embedded in eluted with 60% CH3CNsolution containing 0. 1% TFA, after paraffin. Sections cut 3 jam thick were deparaffinized in xylene which the eluate was evaporated and used for the RIA. then rehydrated in a graded ethanol series, after which they were treated with 0.3% hydrogen peroxide for 30 minutes to inacti- RIA for guanylin vate endogenous peroxidases. Nonspecific binding was blocked The antiserum for rat guanylin was raised in NewZealand with normal goat serum. Anti-guanylin antiserum was used at white rabbits by the carbodiimide methodagainst synthetic rat the final dilution of 1/500 in 10 mMPBS containing 1% bovine guanylin conjugated with bovine thyroglobulin (5). It was used serum albumin (BSA) and 5% normal goat serum. The anti- at a final dilution of 1/1 ,000. Tyr°-rat guanylin was radioiodinated serum was allowed to react with the preparations overnight at by the lactoperoxidase method, after which the labeled peptide 4°C in a moist chamber. Sections first were incubated for 90 was purified by reverse-phase high performance liquid chroma- minutes with goat-biotinylated anti-rabbit IgG (Vectastain, tography (RP-HPLC). The half-maximum inhibition by human Vector Lab., Burlingame, CA) then for 45 minutes with peroxi- guanylin on the standard RIAcurve was 1 30 fmol/tube, and the dase-conjugated streptavidin (GIBCO BRL) diluted 1/200 in peptide was detectable at the low level of 1 1 fmol/tube (10% 10 mMPBS containing 1% BSA. They then were stained for 3 replacement). The antiserum did not crossreact with human minutes at room temperature with 20 mgof 3,3'-diamino- uroguanylin, ANP, BNP or CNP. The specificity of the antise- benzidine tetrahydrochloride and 0.006% hydrogen peroxide in rumfor guanylin molecules was confirmed by the analysis of 50 mMTris buffer solution (pH 7.2), after which they were immunoreactive substances in humanplasma and intestinal counterstained with methyl green. In the sequential double tissue extract by the use ofRP-HPLCcoupled with the RIA (5). staining for guanylin vs lysozyme, chromogranin A, serotonin, Therespective intra- and interassay coefficients of variation glucagon, pancreatic polypeptide, vasoactive intestinal polypep- were 3.2%and 4.2%.tide and somatostatin, guanylin first was stained using the streptavidin-peroxidase method, then the specimens were RT-PCRamplification of the guanylin transcript washed with 0.1 Mglycine-HCl buffer (pH 2.2) and stained The total RNAsof the humancolon, gastric antrum and with these proteins and peptides by the streptavidin-alkaline kidney were extracted by the acid-guanidium thiocyanate- phosphatase method using a Labeled Streptavidin Biotin Kit phenol-chloroform method (6). Three units of RNase-free and an AP Substrate Kit III (Vector Lab.). Control studies were DNase I (Pharmacia, Piscataway, NJ), 1 10 U RNase inhibitor, done with normal rabbit serum or anti-guanylin antiserum that 40 mMTris HC1 (pH 7.6) and 6 mMMgCl2 were added to the had been preabsorbed with 1 jig of human guanylin. 2.5 jig RNAsamples to digest the genomic DNAintroduced into the RNAfraction. The first strand CDNAwas synthesized Statistical analysis with 0.4 jig of the RNAsamples that had been treated with Comparison between groups of patients was done by analy- DNase I, 1 10 U RNase inhibitor, 6 mMMgCl2, dNTPmixture sis of variance followed by modified t test ofDunnett. A p value (8 mMeach), 1 x PCR buffer (GeneAmpá"PCRReagent Kit, of less than 0.05 was considered statistically significant. Takara), 7.5 juM oligo (dT)18 primer, and 200 U Superscript RT RNase H" reverse transcriptase (GIBCO BRL, Gaithersburg,

172 Internal Medicine Vol. 35, No. 3 (March 1996) Cell and Tissue Distribution of Guanylin

Results concentrations (r=0.776, p<0.0005). Tissue content and plasma concentration of immunoreac- RT-PCRamplification of the guanylin transcript tive (ir-) guanylin Guanylin gene expression in the humancolon, gastric an- The ir-guanylin contents in the gastrointestinal tract are trum and kidney was examined by RT-PCR. A guanylin tran- shown in Table 1. The highest values are seen in the ileum and script product that corresponded to the predicted size was proximal colon. Noguanylin immunoreactivity was detected in detected at 337 bp in the colon sample (Fig. 2). This guanylin the stomach or kidney, which agrees with the mRNAdata transcript product was not detected in the gastric antrum or described below. The plasma concentration of ir-guanyjin in kidney even though the (3-actin transcript product was detected normal individuals was 30.3±3.7 fmol/ml. Figure 1 lists the in these tissue samples. plasma ir-guanylin concentration in 23 patients with CRFas classified according to their serum creatinine levels. The renal immunohistochemical localization ofguanylin in the intes- failure patients with 1.5-3, 3-6 and >6 mg/dl of serum creati- tine nine concentration showed higher plasma guanylin levels than Guanylin immunoreactivity was detected in the villus epi- normal subjects; 401.8±67.0, 1 ,490.3+248.3 and4,066.0±592.9 thelial cells and Paneth cells in the small intestine and in the fmol/ml, respectively. Plasma guanylin concentrations in the superficial epithelial cells in the large intestine (Fig. 3). Guanylin- patients were statistically correlated with serum creatinine immunoreactivecells were scattered in the duodenumand jejunum, were moderately found in the ileum and rectum, and were abundant in the colon (Fig. 3A-F). Granular immunoreactive products of guanylin were present in the supranuclear Table 1. Tissue Content of Humanir-Guanylin cytoplasm and basal cytoplasm of the cells. In the colonic and rectal superficial epithelial cells, guanylin immunoreactivity Tissue pmol/g wet weight Tissue pmol/g wet weight was diffuse throughout the supranuclear cytoplasm (Fig. 3E, F). Stom ach N D C olon ascen din g 28.1+4.7 Noguanylin immunoreactivity was detected in the appendix D u od en um 9 .4ｱ 1.3 transv erse 27.4+6.5 (Fig. 3D). Granular immunoreactive products of guanylin were Jej unum 8 .9+ 4 .9 descen d in g 13.6+3.8 present in the cells at the deepest part of the base of the small Tleum 32.8ｱ7.4 sigm o id 4.4+1.4 intestinal crypts (Fig. 3C), in which colocalization of guanylin A pp en dix N D K idn ey N D and lysozyme was seen in double-immunostained sections (Fig. 3G); this is evidence that these guanylin-containing cells Results are expressed as mean±SE(n=3). ND: not detected. are Paneth cells. No colocalization ofguanylin and chromogranin A (Fig. 3H) or serotonin, glucagon, pancreatic polypeptide,

p<0.01 Colon Antrum Kidney 5,000 - p<0.05 I 1 -I~ SE _4,000 - H|^^H I N.S. ^^H 3 3,000 - ^^^H ! ^H 1 2,000 - J^^H 1 Y7%A ^1

Figure 2. Representative electrophoretic analysis pattern of the RT-PCRproducts of human guanylin Normal l._ _ I >6 and (3-actin transcripts in the colon, gastric antrum subject Serum creatinine (mg/dl) and kidney. Template RNAs(375 ng) were reverse- transcribed with oligo (dT)18 primer, subjected to PCR Figure 1. Plasma level of ir-guanylin in 23 patients with amplification with both guanylin- and (3-actin-specific chronic renal failure. The respective numbers of the patients who primers then electrophoresed on a 2%agarose gel. The have serum creatinine levels of1.5-3, 3-6 and >6 mg/dl are 8, 6 and RT-PCR product for guanylin is 337 bp and that for p- 9. Analysis of variance resulted in an F value of 16.84, p<0.001. actin is 253 bp. bp: base pair.

Internal Medicine Vol. 35, No. 3 (March 1996) 173 Date et al

Figure 3. Immunohistochemical staining of human guanylin in the duodenum (A), ileum (B, C, G-I), appendix (D), ascending colon (E) and rectum (F). Guanylin immunoreactivity was observed in duodenal (A), ileal (B), colonic (E) and rectal (F) epithelial cells and in Paneth cells (C), but not in the appendix (D). In the double staining for guanylin vs lysozyme (G) and chromogranin A (H), guanylin was stained by the streptavidin-peroxidase method (brown stain), and lysozyme and chromogranin A by the streptavidin- alkaline phosphatase method (blue stain). No guanylin immunoreactivity was detected in the ileal epithelial cells whenantiserum preabsorbed with 1 jig synthetic guanylin was used (I). Original magnification (A-I xl,000).

vasoactive intestinal polypeptide or somatostatin in the same lar mediators and extracellular effectors. Guanylin is an endog- cells was shown in the double-immunostained sections (data enous ligand for the heat-stable enterotoxin receptor and is not shown). No guanylin immunoreactivity was detected in the believed to function in the control of intestinal salt and water ileum whennormalrabbit serumor antiserum preabsorbedwith transport. The present study was undertaken to analyze the excessive synthetic guanylin was used (Fig. 31). guanylin distribution in human intestine and to identify its cellular localization by the use of immunohistochemistry. The Discussion ir-guanylin content was found to be high in the ileum and proximal colon; low in thejejunum, duodenum and distal colon, Three plasma membraneforms ofguanylate cyclase (GC-A, with no guanylin being detected in the stomach or appendix. GC-B, GC-C) are activated by extracellular peptides: GC-Aby This guanylin distribution was consistent with the tissue ex- atrial natriuretic peptide (ANP) and B-type natriuretic peptide pression of GC-Cwhich also was abundant in the lower small (BNP); GC-B by C-type natriuretic peptide (CNP); and GC-C intestine and colon (7). Wefound guanylin to be present in the by guanylin. The general functions of these receptors appear to villus epithelial cells and Paneth cells of the small intestine, and be the regulation of water and electrolyte homeostasis. Intesti- in the superficial epithelial cells in the large intestine. Rat nal ion-transport processes are regulated by various intracellu- guanylin also is present in the villus epithelial cells of the small

174 Internal Medicine Vol. 35, No. 3 (March 1996) Cell and Tissue Distribution of Guanylin intestine, and in the superficial epithelial cells in the large Uehara Memorial Foundation (M. N.). intestine (8). On the other hand, an immunohistochemical study of guinea pig intestine has showed that guanylin is exclusively References confined to enterochromaffin cells (9), but guanylin is not 1) FieldM, Rao MC,Chang EB. Intestinal electrolyte transport and diarrheal present in human and rat enterochromafrin cells. Species- disease. N Engl J Med 321: 879, 1989. specific differences in the cellular source of guanylin expres- 2) Giannella RA, Luttrell M, Thompson M. Binding of Escherichia coli heat-stable enterotoxin to receptors on rat intestinal cells. AmJ Physiol sion may explain these disparate data. 245: G492, 1983. Paneth cells are specialized, small intestine epithelial cells 3) Currie MG,Fok KF, Kato J, et al. Guanylin: an endogenous activator of that contain antimicrobial proteins (lysozyme, cryptdin and intestinal guanylate cyclase. Proc Natl Acad Sci USA89: 947, 1992. defensin), have phagocytic properties and digest certain micro- 4) Forte LR, Eber SL, Turner JT, Freeman RH, Fok KF, Currie MG. organisms and protozoans (10, 1 1). The molecular mechanism Guanylin stimulation of Cl~ secretion in humanintestinal T84 cells via ofguanylin-induced Cl" secretion via CGMPis explained by the cyclic guanosine monophosphate. J Clin Invest 91: 2423, 1993. activation of CGMP-dependent protein kinase which in turn 5) Nakazato M, Yamaguchi H, Shiomi K, et al. Identification of 10-kDa proguanylin as a major guanylin molecule in humanintestine and plasma phosphorylates and opens the cystic fibrosis transmembrane and its increase in renal insufficiency. Biochem Biophys Res Commun conductance regulator (CFTR) Cl~ channel ( 1 2). Cl" predomi- 205: 1966, 1994. nantly is secreted from cells in the crypts (13) and an immuno- 6) Chomczynski P, Sacchi N. Single-step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem histochemical study shows that the CFTRis localized in crypt 162: 156, 1987. epithelial cells (14). It remains to be determined whether or not 7) Krause G, Bayerl A, Heim J-M, Singh S, Gerzer R. Distribution of the guanylin synthesized in Paneth cells contributes to Cl" membrane bound guanylyl cyclases in human intestine. Gut 35: 1250, secretion fromthe crypt cells through the activation of the 1994. CFTR. 8) Lewis LG, Witte DP, Laney DW, Currie MG, Cohen MB. Guanylin The plasma ir-guanylin concentration was found to be el- mRNAis expressed in villous enterocytes of the rat small intestine and superficial epithelia of the rat colon. Biochem Biophys Res Commun196: evated and to parallel the degree of renal insufficiency. This 553, 1993. augmented plasma concentration may be due to reduced guanylin 9) Cetin Y, Kuhn M, Kulaksiz H, et al. Enterochromaffin cells of the clearance in the kidney because 125I- labeled guanylin accumu- digestive system: cellular source ofguanylin, a guanylate cyclase-activat- lated mainly in the kidney after it was administered to the rat ing peptide. Proc Natl Acad Sci USA 91: 2935, 1993. (unpublished data). Wehave reported that the major molecular 10) Erlandsen SL, Chase DG. Paneth cell function: phagocytosis and intra- cellular digestion of spiral microorganisms. J Ultrastruct Res 41: 296, form of human guanylin in the intestine and plasma is 10-kDa 1972. proguanylin and that 15-amino acid guanylin accounts for 2- 1 1) Erlandsen SL, Chase DG. Paneth cell function: phagocytosis and intra- 3%of the guanylin molecules (5). Also in the rat intestine, 94- cellular digestion of spiral microorganisms. J Ultrastruct Res 41: 319, amino acid proguanylin and guanylin- 16 are major molecular 1972. forms ofguanylin and guanylin-15 is minor form (15). The 10- 12) Chao AC, de Sauvage FJ, Dong Y-J, Wagner JA, Goeddel DV, Gardner kDa humanproguanylin is reported to increase the CGMP P. Activation of intestinal CFTRCl~ channel by heat-stable enterotoxin and guanylin via CAMP-dependentprotein kinase. EMBOJ 13: 1065, content in T84 cells ( 1 6) but two studies have reported that there 1994. is no such activity (17, 18). Further investigations of the 13) Welsh MJ, Smith PL, FrommM, Frizzell RA. Crypts are the site of structure-activity relationship of the guanylin molecules are intestinal fluid and electrolyte secretion. Science 218: 1219, 1982. necessary to determine whether the high molecular weight 14) Crawford I, Maloney PC, Zeitlin PL, et al. Immunocytochemical localization of the cystic fibrosis gene product CFTR. Proc Natl Acad Sci USA guanylin circulating in the plasma modulates fluid and electro- 88: 9262, 1991. lyte metabolism in renal failure. 15) Yamaguchi H, Nakazato M, Miyazato M, Kangawa K, Matsuo H, Currie's group isolated a peptide homologous to guanylin Matsukura S. Two novel rat guanylin molecules, guanylin-94 and guanylin- from opossum (Didelphis virginiana) intestinal mucosa and 16, do not increase cyclic GMPproduction in T84 cells: Biochem from humanand opossumurine, which they nameduroguanylin Biophys Res Commun214: 1204, 1995. 16) Kuhn M, Raida M, Adermann K, et al. The circulating bioactive form of (19, 20). This peptide increases CGMPlevels in T84 cells, human guanylin is ahigh molecular weightpeptide ( 10.3 kDa). FEBSLett displaces ST binding to intestinal receptors and stimulates Cl" 318: 205, 1993. secretion (20). Wehave cloned humanCDNAencoding a 17) Schultz S, Chrisman TD, Garbers DL. Cloning andexpression ofguanylin: uroguanylin precursor from an intestinal library and deter- its existence in various mammaliantissues. J Biol Chem267: 16019, mined its nucleotide sequence (unpublished data). Whether 1992. 18) de SauvageFJ, Keshav S, Kuang W-J, GillettN, Henzel W, Goeddel DV. intestinal guanylate cyclase is regulated by these two homolo- Precursor structure, expression, and tissue distribution of humanguanylin. gous peptides is of particular interest. Studies of tissue-specific Proc Natl Acad Sci USA 89: 9089, 1992. expression and the cellular localization of uroguanylin provide 19) Hamra FK, Forte LR, Eber SL, et al. Uroguanylin: structure and activity better understanding of the physiology of intestinal and/or renal of a second endogenous peptide that stimulates intestinal guanylate fluid and electrolyte transport. cyclase. Proc Natl Acad Sci USA90: 10464, 1993. 20) Kita T, Smith CE, Fok KF, et al. Characterization of human uroguanylin: Acknowledgements: This work was supported in part by grant-in-aid from a member of the guanylin peptide family. AmJ Physiol 266: F342, 1 994.

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