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Endocrinology Vol. 140, No. 5 2398-2404
Copyright © 1999 by The Endocrine Society

ARTICLES

Enterochromaffin-Like Cells, a Cellular Source of Uroguanylin in Rat Stomach1

Yukari Date, Masamitsu Nakazato, Hideki Yamaguchi, Kenji Kangawa, Yoshikazu Kinoshita, Tsutomu Chiba, Yoichi Ueta, Hiroshi Yamashita and Shigeru Matsukura

Third Division (Y.D., M.N., Hid.Y., S.M.), Department of Internal Medicine, Miyazaki Medical College, Miyazaki 889-1692; National Cardiovascular Center Research Institute (Y.D., K.K.), Osaka 565-8565; Second Division (Y.K.), Department of Internal Medicine, Shimane Medical University, Shimane 693-8501; Department of Internal Medicine (T.C.), Postgraduate School of Medicine, Kyoto University, Kyoto 606-8507; and Department of Physiology (Y.U., Hir.Y.), University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807-8555, Japan

Address all correspondence and requests for reprints to: Masamitsu Nakazato, M.D., Ph.D., Third Department of Internal Medicine, Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan. E-mail: nakazato{at}post.miyazaki-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Uroguanylin is an endogenous peptide ligand for guanylyl cyclase-C, an apical membrane receptor predominantly located in the gastrointestinal epithelium. It regulates intestinal and renal fluid and electrolyte transport through the second messenger, cyclic GMP. Uroguanylin messenger RNA and the peptide are present in rat stomach, but the cellular source has not been identified. We separated gastric mucosal cells by size into seven fractions (F1–F7) and enriched endocrine cells into F1–F3 using counterflow elutriation. Uroguanylin messenger RNA and peptide were found in F1–F3 by Northern blot analysis and an RIA specific for rat uroguanylin. Uroguanylin-producing cells were identified as endocrine cells by immunocytochemical methods using antisera for uroguanylin, prouroguanylin, and chromogranin A, as well as by in situ hybridization cytochemistry. Double-staining showed that uroguanylin and histamine are colocalized in enterochromaffin-like (ECL) cells that release histamine, leading to the stimulation of gastric acid secretion from parietal cells. Uroguanylin is synthesized in ECL cells. These findings should contribute to elucidating the physiological functions of ECL cells and the cyclic GMP-mediated gastric ion transport mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GUANYLIN AND uroguanylin are 15- or 16-amino acid peptides that regulate intestinal salt and water transport (1, 2, 3, 4, 5, 6) and have a 50% amino acid sequence identity. Two intramolecular disulfide bonds essential for their bioactivities are conserved. These peptides bind to and activate guanylyl cyclase-C (GC-C), an apical membrane receptor localized in the epithelia of the stomach (7), intestine (8), kidney (9), liver (10), reproductive tract (10), airway (3), and pancreas (4). The increase in intracellular cyclic GMP (cGMP) induced by GC-C activates type II cGMP-dependent protein kinase II, thereby phosphorylating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel (11, 12, 13). Recent studies showed that both guanylin and uroguanylin stimulate HCO3- secretion in the duodenum (14, 15). Moreover, uroguanylin (but not guanylin) is more potent and effective in stimulating anion secretion across the proximal duodenum when the mucosal surface is exposed to acidic condition (15, 16).

Rat uroguanylin messenger RNA (mRNA) is very abundant in the upper small intestine; moderately abundant in the stomach, lower small intestine, and kidney; and present in considerably lesser amounts in the pancreas, lung, and testis (7, 17, 18, 19). Intestinal rat uroguanylin was found in enterochromaffin (EC) cells, the most abundant type of enteroendocrine cells, by in situ hybridization and immunohistochemical methods (20, 21). Uroguanylin-immunoreactive cells also were found in the gastric mucosa by an immunohistochemical method (21), but they were infrequent on the formaldehyde-fixed paraffin sections. Counterflow elutriation can be used to separate gastric mucosal cells by size and to enrich endocrine cells (22, 23, 24, 25). We chose this method to identify the uroguanylin-producing cells in the stomach. Using the RIA, Northern blot analysis, and immunocytochemical and in situ hybridization techniques, we showed that rat uroguanylin is synthesized in EC-like (ECL) cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell isolation and separation
Seven-week-old male Sprague Dawley rats (Charles River Japan, Inc., Shiga, Japan) were used in all the experiments. Glandular stomachs were excised from 14 rats that had been anesthetized with pentobarbital before being killed. Gastric epithelial cells were dispersed with pronase (Actinase E; Kaken Pharmaceutical Co. Ltd., Tokyo, Japan) then separated by counterflow centrifugation as described previously (24, 25). Briefly, the dispersed cells were loaded in an elutriation chamber using a masterflex pump (7521; Cole Parmer, Chicago, IL) at the flow rate of 8 ml/min, after which the cells were separated at 1,800 rpm. Seven cell fractions (F1–F7) were obtained at the flow rates of F1, 13.5 ml/min; F2, 16.5 ml/min; F3, 20 ml/min; F4, 24 ml/min; F5, 29 ml/min; F6, 37 ml/min; and F7, 80 ml/min.

Northern blot analysis
Total RNA was obtained from each gastric mucosal cell fraction using an Isogen kit (Nippon Gene, Tokyo, Japan). Twenty micrograms of the total RNA was denatured with 16 µl of 1 M glyoxal and 50% dimethylsulfoxide, then electrophoresed on a 1.2% agarose gel (FMC BioProducts, Rockland, ME) in 10 mM sodium phosphate buffer (pH 7.0), after which the sample was transferred to a Zeta Probe membrane (Bio-Rad Laboratories, Inc., Richmond, CA) and fixed by UV irradiation. The probes used for Northern blot analyses were full-length rat uroguanylin complementary DNA (cDNA) (17), a 0.42-kb cDNA fragment of rat histidine decarboxylase (HDC), and a 0.25-kb cDNA fragment of rat ß-actin. The membrane first was treated for 2 h at 37 C in 6 x SSPE (900 mM NaCl, 60 mM NaH2PO4·H2O, 7 mM EDTA, pH 7.4) containing 40% formamide, 5 x Denhardt’s solution, 0.5% SDS, and 0.1 mg/ml denatured salmon sperm DNA, then hybridized for 18 h at 37 C in an identical solution that contained a 32P-labeled uroguanylin cDNA probe. The RNA blot was washed with 2 x saline-sodium citrate (SSC) (150 mM NaCl, 15 mM sodium citrate, pH 7.0)/0.1% SDS solution at 50 C and exposed to film to detect uroguanylin probe binding. The membrane then was boiled for 20 min at 70 C in 0.1 x SSC solution to strip it of the uroguanylin probe and used for sequential hybridizations with the probes for HDC and ß-actin. Hybridization signals were measured with a Fujix Bio-image analyzer, BAS 2000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). Uroguanylin mRNA levels were calculated, relative to the radioactivity of the ß-actin.

Immunoreactive uroguanylin contents in gastric mucosal cell fractions
Cells (1 x 107) collected by centrifugation from each gastric mucosal cell fraction were heated for 10 min at 95–100 C in a 10-fold vol of water to inactivate intrinsic proteases, then cooled to 4 C. Next, CH3COOH and HCl were added to the respective final concentrations of 1 M and 20 mM, and the cells were homogenized in a Polytron for 5 min. The homogenates were centrifuged at 11,500 x g for 30 min. The supernatants were applied to Sep-Pak C-18 cartridges (360 mg resin/cartridge, Waters Corp., Milford, CA), then washed with 0.5 M CH3COOH, then 10% acetonitrile (CH3CN) solution containing 0.1% trifluoroacetic acid. Peptides were eluted with 60% CH3CN solution containing 0.1% trifluoroacetic acid, then digested with 5 µg trypsin (Sigma Chemical Co., St. Louis, MO) for 3 h at 37 C in 100 µl of 0.1 M Tris-HCl buffer (pH 8.0) to liberate the immunoreactive carboxyterminal 18-amino acid peptide (uroguanylin-18) from prouroguanylin (21). The reaction was terminated by the addition of 10 µg soy trypsin inhibitor (Sigma Chemical Co.) in 300 µl RIA buffer. The resulting samples were analyzed by RIA for uroguanylin, as described previously (21). Antiserum against rat uroguanylin was raised in New Zealand white rabbits by repeated immunization with synthetic rat uroguanylin that had been conjugated with thyroglobulin by the carbodiimide method. A diluted sample (100 µl) was incubated for 24 h with 100 µl of the diluted antiserum (final dilution 1/10,000), then the tracer solution (16,000 cpm in 100 µl) was added, and the mixture was incubated for 24 h. The bound and free ligands were separated using polyethyleneglycol solution. All procedures were done at 4 C, and duplicate samples were assayed. The minimum level of detection of rat uroguanylin was 8 fmol/tube (10% replacement). The respective intra- and interassay coefficients of variation were 4.1% and 3.8%, respectively, at 50% binding. The antiserum does not cross-react with rat guanylin, atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide. It recognizes only uroguanylin-15 and uroguanylin-18 in reverse phase-HPLC analyses of rat urine and intestinal extract (21). The antiserum does not recognize prouroguanylin.

Cytological identification and immunocytochemistry
Centrifugation in a Cytospin-3 apparatus (Shandon, Runcorn, UK) was used to attach the cells (1 x 105) of each fraction to glass slides. The slides were air-dried, then fixed for 30 sec with 80% acetone and 0.74% formaldehyde in 10 mM PBS. For the cytological analysis, some slides were stained with hematoxylin/eosin. For the immunocytochemical study, three separate slides were incubated with 0.1% Triton X/PBS for 10 min, then treated with 0.3% hydrogen peroxide for 30 min to inactive endogenous peroxidases, and incubated with normal goat serum to block nonspecific binding. Next, they were incubated overnight at 4 C in a moist chamber with antirat uroguanylin antiserum (final dilution 1/1,000), antirat prouroguanylin antiserum (6912 in Ref. 20 ; final dilution 1/1,000), antichromogranin A antiserum (DAKO Corp. A/S, Glostrup, Denmark; final dilution 1/100), or antihistamine antiserum (CHEMICON International Inc., Temecula, CA; final dilution 1/100). After being rinsed 3 times with PBS, then incubated for 2 h with goat-biotinylated antirabbit IgG (Vectastain, Vector Laboratories, Inc., Burlingame, CA), they were allowed to react for 60 min with peroxidase-conjugated streptavidin (Gibco BRL, Gaithersburg, MD) diluted 1/200 in 10 mM PBS. They then were stained for 3 min at room temperature with 0.02% 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) and 0.006% hydrogen peroxide in 50 mM Tris HCl buffer solution (pH 7.2) and counterstained with hematoxylin. In the sequential double staining for uroguanylin vs. histamine and SRIF, uroguanylin first was stained by the streptavidin-peroxidase method, after which the slides were washed with 100 mM glycine-HCl buffer (pH 2.2), then stained with antihistamine antiserum or anti-SRIF antiserum (DAKO Corp. A/S; final dilution 1/200) by the streptavidin-alkaline phosphatase method using a Labelled Streptavidin Biotin kit (DAKO Corp. A/S). Control studies were done with normal rabbit serum or antiuroguanylin antiserum that had been absorbed by 10 µg synthetic rat uroguanylin. At least 200–250 cells were counted in different visual fields, and the findings were expressed as the number of positive cells per visual field at the magnification of x400.

In situ hybridization
Preparation of the complementary RNA (cRNA) probe. A 260-bp fragment of rat uroguanylin cDNA (nucleotide number 74–333 in Ref. 17) was produced by RT-PCR amplification of the total RNA extracted from rat jejunum. The PCR product was subcloned into the pCRII vector using a TA cloning kit (Invitrogen, San Diego, CA). The recombinant plasmid was linearized with the restriction enzyme EcoRV to generate the antisense probe and with BamHI to generate the sense probe. Antisense and sense riboprobes were obtained by incubating the linearized-vectors with [{alpha}-35S] uridine triphosphate and RNA polymerase (SP6 polymerase for the antisense probe and T7 polymerase for the sense probe), using an SP6/T7 transcription kit (Boehringer Mannheim, Mannheim, Germany).

In situ hybridization cytochemistry. Cells (1 x 105) of each fraction (F1–F7) were attached to silane-coated slides by centrifugation. The slides were air dried and stored at -80 C until used for the in situ hybridization analysis. The stored slides were allowed to dry for 10 min at room temperature, then fixed in 4% paraformaldehyde in PBS (pH 7.5) for 5 min and washed twice in PBS (pH 7.5). They next were incubated for 10 min in 0.9% saline containing 0.1 M triethanolamine and 0.25% acetic anhydride, dehydrated in a graded ethanol series, and delipidated in 100% chloroform for 5 min, after which they were immersed in 100% ethanol, then 95% ethanol, and were allowed to dry briefly in air. Hybridization was done at 50 C overnight in 45 µl of hybridization buffer containing 55% formamide, as described elsewhere (26). The slides were rinsed in 2 x SSC, then washed for 30 min in two changes of 2 x SSC/50% formamide at 50 C, after which they were incubated at 37 C in 2 x SSC containing 20 µg/ml ribonuclease (RNase) A (Sigma Chemical Co.) to digest any unhybridized probe. After another 45-min wash in three changes of 2 x SSC/50% formamide at 50 C, the slides were dehydrated in a graded ethanol series and air-dried. For autoradiography, slides that had been hybridized with the riboprobes were dipped in K-5 nuclear emulsion (Ilford, Cheshire, UK) diluted 2:3 with distilled water, then exposed in the dark at 4 C for 4 days. After being developed in D-19 (Eastman Kodak Co., Rochester, NY) for 4 min, then fixed with Unifix (Eastman Kodak Co.), the slides were rinsed in water, counterstained with hematoxylin/eosin, and viewed by bright- and dark-field microscopy.

RT-PCR for GC-C and guanylin
Total RNAs extracted from each gastric mucosal cell fraction were used in the RT-PCR analyses for GC-C and guanylin. To digest the genomic DNA, 3 U RNase-free deoxyribonuclease (DNase) I (Pharmacia Biotech, Piscataway, NJ), 110 U RNase inhibitor, 40 mM Tris HCl (pH 7.6), and 6 mM MgCl2 were added to the 2.5 µg RNA samples. The samples then were incubated for 30 min at 37 C, after which they were heated to 90 C for 5 min to inactivate the DNase. The primers specific for the extracellular domain of rat GC-C (8), the guanylin (27), and ß-actin (28) were designed; and their sequences and names are given in Table 1Go. To examine GC-C gene expression, the first-strand cDNA was synthesized with 0.4 µg of an RNA sample that had been treated with DNase, 2.5 µM GCC-AS1 primer, the deoxynucleotide triphosphate mixture (1 mM each), 110 U RNase inhibitor, and 200 U reverse transcriptase (Gibco BRL). RT was done for 30 min at 42 C, followed by incubation for 3 min at 94 C to inactivate the reverse transcriptase. The resulting cDNA was subjected to PCR amplification with 2 µM of the GCC-S and -AS1 primers, and 1.25 U Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). The reaction vol was 25 µl, and the PCR conditions were 35 cycles of denaturation for 30 sec at 94 C, annealing for 30 sec at 53 C, and extension for 60 sec at 72 C. Nested PCR was done with 1 µl of the first-step RT-PCR product and 2 µM of the GCC-S and -AS2 primers under the PCR conditions described above. To examine guanylin gene expression, the first-strand cDNA was synthesized with 0.4 µg of an RNA sample that had been treated with DNase, 5.0 µM oligo (dT)18 primer, the deoxynucleotide triphosphate mixture (1 mM each), 110 U RNase inhibitor, and 200 U reverse transcriptase (Gibco BRL). RT was done under the same condition described above. The resulting cDNA was subjected to PCR amplification with the 2 µM sense and antisense primers for guanylin and ß-actin, and 1.25 U Taq DNA polymerase (Perkin Elmer Cetus). The reaction vol was 25 µl, and the PCR conditions were 35 cycles of denaturation for 30 sec at 94 C, annealing for 30 sec at 58 C, and extension for 60 sec at 72 C. The PCR products were electrophoresed on a 2% agarose gel (FMC BioProducts).


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  		Table 1. Primers for RT-PCR of GC-C, guanylin, and ß-actin

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of uroguanylin and HDC mRNAs and immunoreactive uroguanylin (ir-uroguanylin) of the gastric cell fractions
Chromogranin A-positive cells predominated in small-cell fractions F1–F3 (Fig. 1Go). In contrast, approximately 70% of the cells in F6 and F7 were parietal cells, identified as large cells with an eosinophilic large cytoplasm. Uroguanylin mRNA of 0.6 kb was detected mainly in F1–F3 (Fig. 2AGo). The uroguanylin mRNA levels were calculated after marking the correction for ß-actin, as described in Materials and Methods (Fig. 2BGo). HDC mRNA of 2.3 kb also was present in F1–F3, and ß-actin was detected in all the RNA samples (Fig. 2AGo). The immunoreactive uroguanylin content was highest in F1, decreasing in direction to F7 (Fig. 3Go). The dilution curve for the F1 sample paralleled the standard RIA curve for uroguanylin (Fig. 3Go, inset).



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  		Figure 1. Representative distribution pattern of chromogranin A-positive cells (open bar) and parietal cells (closed bar) in gastric mucosal cell fractions obtained by counterflow elutriation. Chromogranin A-positive cells, detected by the immunohistochemical method, predominate in the small-cell fractions (F1–F3); and parietal cells, identified by cytological analysis, predominate in the large-cell fractions (F6 and F7).

 


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  		Figure 2. Representative Northern blot pattern of uroguanylin, HDC, and ß-actin mRNAs in the gastric mucosal cell fractions (A). The hybridization intensity of uroguanylin was quantified with a Bio-image analyzer, after which the membrane was used for sequential hybridizations with HDC and ß-actin. The uroguanylin mRNA amount was calculated relative to an arbitrary unit of ß-actin (B). Values are the means ± SD (n = 3). ND, Not detected.

 


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  		Figure 3. ir-uroguanylin contents of gastric mucosal cell fractions determined by an RIA. Inset, The standard RIA curve for rat uroguanylin ({circ}) and dilution curve for the F1 sample (•). The numeral one, in the dilution curve, denotes 0.2 x 107 cells.

 
Immunocytochemistry and in situ hybridization cytochemistry
Uroguanylin and prouroguanylin were found by immunocytochemical techniques in small cells whose sizes and numbers were consistent with those of chromogranin A- and histamine-positive cells (Fig. 4Go, A–D). Parietal cells showed no uroguanylin or prouroguanylin immunoreactivity (Fig. 4Go, A and B). Uroguanylin and histamine were extensively colocalized in the double-immunostained specimen (Fig. 4EGo), whereas double staining showed no colocalization of uroguanylin and SRIF (Fig. 4FGo). No uroguanylin immunoreactivity was found when normal rabbit serum or anti-uroguanylin antiserum that had been absorbed by excess synthetic rat uroguanylin was used (Fig. 4GGo). Strong signals for the uroguanylin antisense cRNA probe were present on small cells whose sizes and numbers were the same as those of the uroguanylin-immunoreactive cells (Fig. 4HGo). No hybridization signals were present on the parietal cells. A control experiment with the uroguanylin sense probe produced no signals on the small cells (Fig. 4IGo).



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  		Figure 4. Immunocytochemical (A–G) and in situ hybridization (H and I) studies of ECL cells isolated by counterflow elutriation. Uroguanylin (A), prouroguanylin (B), chromogranin A (C), and histamine (D) immunoreactivities are present in endocrine (arrows), but not in parietal (arrowheads), cells. In the double staining for uroguanylin vs. histamine (E) and SRIF (F), uroguanylin was stained by the streptavidin-peroxidase method (brown stain), and histamine and SRIF by the streptavidin-alkaline phosphatase method (red stain). Uroguanylin is colocalized with histamine but not with SRIF. There is no uroguanylin immunoreactivity when antiuroguanylin antiserum, that had been absorbed by synthetic rat uroguanylin, was used (G). Hybridization signals for the rat uroguanylin antisense cRNA probe are present on ECL cells (H), but no signal is present for the uroguanylin sense cRNA probe (I). Original magnification: A–D and F–I, x800; E, x2000.

 
RT-PCR for GC-C and guanylin
The GC-C and guanylin gene expressions in each gastric mucosal cell fraction were examined by RT-PCR because these mRNAs were not detected by Northern blot analysis (data not shown). A GC-C transcript product corresponding to the predicted size of 263 bp was found in all the fractions (Fig. 5Go). A guanylin transcript product corresponding to the predicted size of 337 bp was detected in F1–F5, and ß-actin in all the fractions (Fig. 5Go).



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  		Figure 5. Electrophoretic analysis of the RT-PCR products of GC-C and guanylin mRNAs in F1–F7. The 263-bp GC-C transcript is present in all the fractions, and the 337-bp guanylin transcript is in F1–F5. The PCR product of ß-actin is present in all the fractions. Control, Primer only, no cDNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Uroguanylin and guanylin are members of a peptide family that regulates fluid and electrolyte transport in the intestine and kidney by activating GC-C. Human and rat guanylin predominate in the intestine, their mRNA and peptide contents being highest in the colon and ileum (2, 3, 29, 30, 31). Rat uroguanylin mRNA and peptide also are abundant in the intestine, being most plentiful in the upper small intestine; however, uroguanylin also is expressed at lower levels in the stomach, kidney, lung, pancreas, and testis (7, 17, 18, 19). These differential distributions suggest that the two peptides have different roles in the regulation of epithelial functions associated with water and electrolyte homeostasis. In our previous immunohistochemical study, uroguanylin was localized to an unidentified population of chromogranin A-positive endocrine cells in rat oxyntic mucosa (21). None of the uroguanylin-immunoreactive cells showed SRIF or gastrin immunoreactivity. We now have made a more complete evaluation of the cellular source of uroguanylin in rat stomach to clarify its contribution to gastric ion transport. The gastric mucosal epithelium consists of chief cells, endocrine cells, parietal cells, mucous cells, and progenitor cells. We enriched the endocrine cells in the small-cell fractions by counterflow elutriation. Results of Northern blot analysis, RIA, in situ hybridization, and immunocytochemistry showed that uroguanylin is confined exclusively to ECL cells. We confirmed the specificity of antiuroguanylin antiserum by identifying uroguanylin-immunoreactive molecules in rat urine and tissue extract using reverse phase-HPLC and gel filtration, as well as the specificity of antiprouroguanylin antiserum by Western blot analysis (20, 21).

At least seven distinct endocrine cells (EC, ECL, D, D1, P, G, and X cells) have been identified ultrastructurally and immunohistochemically in rat and human gastric mucosa (32, 33, 34). ECL cells are small cells (8–10 µm in diameter) containing cytoplasmic vesicles with eccentric electron-dense cores (33). They produce histamine, chromogranin A, pancreastatin, peptide YY, enteroglucagon, and calbindin (35). ECL cells are scattered in the oxyntic glands, often in direct contact with parietal cells. The number of ECL cells in mammal stomachs varies greatly with species. ECL cells constitute 65% of the endocrine cell mass in rat oxyntic mucosa, whereas D cells represent a minor population of only 2–5% (34). Very recently, uroguanylin was found in human gastrointestinal D cells (36). In human oxyntic mucosa, two of the major endocrine cell types are ECL and D cells, which respectively constitute 30% and 22% of the endocrine cell mass (34). Species-specific differences in the cellular source of uroguanylin expression may explain the discrepancy between rat and human data.

In an immunohistochemical study on rat stomach, uroguanylin-immunoreactive cells were infrequent on formaldehyde-fixed paraffin sections (21). Histamine immunoreactivity also was lost in these samples, as found in a previous report in which formaldehyde-fixed paraffin sections had unsatisfactory immunostaining for histamine (37). In the present study, we isolated gastric endocrine cells by counterflow elutriation, fixed them with acetone/formaldehyde solution, then subjected them directly to immunocytochemical analysis. These procedures may preserve the antigenic determinant sites of both uroguanylin and histamine, improve antigen-antibody reactivity, providing excellent uroguanylin and histamine immunostaining.

The major known function of ECL cells is to release histamine in response to gastrin and acetylcholine (38), thereby stimulating gastric acid secretion from parietal cells. London et al. (7) detected GC-C and a truncated, GC-C-like mRNA that has a 159-nucleotide deletion in the mucosa of rat stomach and intestine. They reported that exposure of the gastric epithelium to heat-stable enterotoxin (STa), a bacterially produced analog of uroguanylin, significantly increased tissue cGMP levels and fluid accumulation in the rat stomach. In this study, GC-C mRNA was found in all the elutriation fractions when primers specific for the extracellular domain of rat GC-C were used in RT-PCR, indicating that GC-C may be present in both the endocrine and parietal cells. Furthermore, guanylin mRNA was found in F1–F5, but not in F6 or F7, which implies that guanylin, as well as uroguanylin, may be expressed primarily in the gastric endocrine cells. These findings, taken together, suggest that uroguanylin and guanylin function in the regulation of gastric ion and fluid transport through the autocrine and/or paracrine system. In isolated vascularly and luminally perfused rat intestine, uroguanylin (produced mainly in the intestine) is secreted in the lumen, but (in part) in the blood, in response to sodium chloride administration (unpublished observations). Whether uroguanylin is secreted from ECL cells in the gastric lumen, systemic circulation, or both is unknown. Future investigation of the uroguanylin concentrations of the vascular and luminal effluents from isolated vascularly perfused rat stomach should provide information on the direction in which this peptide is secreted and on the mechanisms that govern its secretion.

Guanylin, uroguanylin, and STa stimulate anion secretion via CFTR that is a molecular target for cGMP-stimulated protein kinases (39, 40, 41). CFTR is present immunohistochemically in the epithelia of the sweat ducts, airway, small pancreatic ducts, kidney tubules, and intestine, where this protein is thought to function as a Cl- channel or as a regulator of channel activity (42). CFTR mRNA is abundant in the small intestine and is present, in a little amount, in the stomach (43). The short-circuit current response to uroguanylin in the proximal duodenum is markedly reduced in the CFTR knockout mice, but it is not completely lost (15). This indicates that uroguanylin may regulate anion secretion via CFTR-independent, as well as CFTR-dependent, mechanisms. Uroguanylin may participate in the gastric ion transport in a manner similar to that of the mechanisms found in the proximal duodenum.

In summary, our findings confirm that rat ECL cells express uroguanylin. Although the ECL cell was identified 26 yr ago, except for the release of histamine, its physiological role and biological significance are poorly defined (44). Patients with Zollinger-Ellison syndrome, presenting with gastrinoma, peptic ulcer, and ECL hyperplasia, have markedly high plasma concentrations of uroguanylin (unpublished observations). Our results indicate the possibility that a better understanding of the physiological functions and pathophysiological implications of uroguanylin in ECL cells will provide information with which to clarify the gastric ion transport mechanism and the pathogenesis of acid-related diseases.


   Acknowledgments
 
We thank Dr. Michael F. Goy (University of North Carolina, Chapel Hill, NC) for providing the prouroguanylin antiserum (6912); Prof. Tatsuo Suganuma (Department of Anatomy, Miyazaki Medical Col- lege, Miyazaki, Japan) for his valuable comments on the immunocyto-chemistry methods used; and Ms. Akiko Kuzuhara for her technical assistance.


   Footnotes
 
1 This study was supported, in part, by grants-in-aid from the Ministry of Education, Science, Sports and Culture and the Ministry of Health and Welfare, Japan, as well as by grants from the Uehara Memorial Foundation, the Yamanouchi Foundation for Research on Metabolic Disorders, The Inamori Foundation, the Society of Molecular Mechanism of the Digestive Tract, and the Salt Science Research Foundation. Back

Received September 10, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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