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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 7 2568-2573
Copyright © 1999 by The Endocrine Society

Original Studies

Coexpression of Mineralocorticoid Receptors and 11ß-Hydroxysteroid Dehydrogenase 2 in Human Gastric Mucosa

Katsuaki Kato, Hironobu Sasano, Shuichi Ohara, Hitoshi Sekine, Shizue Mochizuki, Tomoatsu Mune, Keigo Yasuda, Hiroshi Nagura, Tooru Shimosegawa, Takayoshi Toyota and Zygmunt Krozowski

The Departments of Medicine III (K.K., S.O., H.S., T.S., T.T.) and Pathology (H.S., S.M., H.N.), Tohoku University School of Medicine, Sendai, Japan; The Department of Medicine III (T.M., K.Y.), Gifu University School of Medicine, Gifu, Japan; Baker Medical Research Institute (Z.K.), Melbourne, Australia

Address all correspondence and requests for reprints to: Katsuaki Kato, M.D., the Department of Internal Medicine (III), Tohoku University School of Medicine, 1-1 Seiryomachi, Aoba-ku, Sendai 980, Japan. E-mail: kato-ka{at}mx2.nisiq.net


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The role of mineralocorticoids in human gastrointestinal tract is well established. In the stomach, aldosterone is thought to regulate electrolyte transport associated with gastric acid secretion. In mineralocorticoid target organs, the action of the glucocorticoid inactivating enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) facilitates aldosterone binding to a nonselective mineralocorticoid receptor (MR) in the face of high levels of circulating glucocorticoids. In the present study, we examined 25 specimens of human stomach for the presence of MR and 11ß-HSD2 using a [3H]aldosterone binding assay, Northern blot analysis, RT-PCR, and immunohistochemistry. Specific [3H]aldosterone binding sites were detected in gastric fundic mucosa, but not in the antrum. In fundic mucosa the Kd was 0.72 ± 0.05 nmol/L (mean ± SE), and Bmax was 6.0 ± 1.4 fmol per milligram of protein. Northern blot analysis demonstrated a faint band for MR mRNA at 6.0 kb, although message for 11ß-HSD2 was undetectable. However, RT-PCR demonstrated specific PCR products for both MR and 11ß-HSD2. Immunohistochemistry demonstrated the colocalization of MR and 11ß-HSD2 only in parietal cells. MR-positive cells were further characterized by electron microscopy, confirming the identity of parietal cells. This study shows that parietal cells contain both MR and 11ß-HSD2, suggesting that the human stomach is a novel target organ for mineralocorticoids. Aldosterone may, therefore, regulate biological functions of parietal cells including gastric acid secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUMEROUS studies have elucidated the important role of mineralocorticoids in human gastrointestinal tract function. Aldosterone has been demonstrated to increase luminal membrane Na+ conductance, active Na+ absorption, active K+ secretion, and transmucosal potential difference; all these processes are associated with electrolyte transport systems in the lower gastrointestinal tract (1, 2, 3). The actions of aldosterone are initiated by binding to intracellular receptors (4). The steroid-receptor complexes then translocate to the nucleus, bind to specific DNA recognition elements, and thereby stimulate the transcription of genes mediating ion transport (5). Two receptors for adrenocortical steroid hormones have been identified; mineralocorticoid, or type I receptors (MR), bind aldosterone and glucocorticoids equivalently, whereas glucocorticoid, or type II receptors, bind glucocorticoids specifically (4, 5, 6). A radioisotopic aldosterone binding assay has demonstrated the presence of MR in duodenum, terminal ileum, and colon of rat and man (7, 8, 9, 10, 11).

Selectivity of aldosterone action within target organs is generally thought to be mediated by the action of the microsomal enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2). This enzyme converts the functional form of glucocorticoids (cortisol and corticosterone) to inactive 11-keto-steroids (cortisone and 11-dehydrocorticosterone) (12, 13). Because glucocorticoids circulate at levels 100–1000 times greater than aldosterone, colocalization of 11ß-HSD2 with MR facilitates the selective action of aldosterone on its target organs (12, 13). Recent studies have demonstrated the existence of two isoforms of 11ß-HSD. Type 1 11ß-HSD is widely distributed with highest concentrations in the liver, possesses both 11ß-dehydrogenase and 11-oxoreductase activities, and requires nicotinamide adenine dinucleotide phosphate (NADPH) as cofactors (14). However, the Km of 11ß-dehydrogenase activity is approximately 1 µmol/L, too high to effectively lower intracellular glucocorticoid levels (14). On the other hand 11ß-HSD2 catalyzes only 11ß-dehydrogenation, utilizes NAD+, and exhibits a Km in the low nanomolar range (15). Colocalization of 11ß-HSD2 and MR has been reported in human aldosterone target organs associated with water and electrolyte transport (16).

Gastric acid secretion is accompanied by transmembrane flux of various electrolytes including Na+, K+, Cl-, and HCO3- as in other mineralocorticoid target tissues (17, 18, 19). The electrolyte transport systems associated with gastric acid secretion have been postulated to be regulated by mechanisms similar to those found in aldosterone target organs. However, the human stomach has not been examined for the presence of MR and 11ß-HSD2. Therefore, in the present study we have investigated the presence of MR and 11ß-HSD2 in human stomach utilizing the radioisotopic aldosterone binding assay, Northern blot analysis, RT-PCR, immunohistochemistry, and electron microscopy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue preparation

For the radioisotopic aldosterone binding assay and Northern blot analysis, 10 gastric fundic mucosal specimens and 5 gastric antral specimens were obtained at surgery from 10 gastric cancer patients. For RT-PCR, 5 gastric fundic mucosal specimens and 5 gastric antral specimens were obtained by endoscopic biopsy from 5 subjects. All specimens were immediately frozen and stored at -80 C. Histological examination and immunohistochemistry were performed in 25 tissue specimens obtained from the regions adjacent to the tissues employed for the radioisotopic aldosterone binding assay, Northern blot analysis and RT-PCR. These specimens were fixed in 10% formalin for 48 h at room temperature and embedded in paraffin. Light microscopic examination of these tissue specimens revealed nonatrophic gastric mucosa.

[3H]aldosterone binding assay

Cytosol for the radioisotopic aldosterone binding assay was prepared by the method of Krozowski and Funder (20). Only the mucosal layers were stripped from the tissue samples, rinsed briefly, and homogenized in ice-cold TMSD buffer [100 mmol/L Tris (pH 7.4), 250 mmol/L sucrose, 100 mmol/L Na2MoO4, 2 mmol/L dithiothreitol]. Homogenates were centrifuged at 105,000g for 60 min at 4 C, and the supernatants were stored at -80 C. The concentration of cytosol protein was determined by the method of Bradford (21), with bovine serum albumin as standard. The [3H]aldosterone binding assay was performed as described by Armanini et al. (22) with some modifications (23). Portions (300 µL) of cytosol (protein concentration of 1 mg protein/mL) were incubated for 18 h at 4 C with various concentrations (0.675 nmol/L to 40 nmol/L) of [1,2-3H]aldosterone (56 Ci/mmol) (New England Nuclear, Boston, MA) and a 200-fold molar excess of RU 28362 (24), a specific glucocorticoid receptor antagonist (donated by Roussel-Uclaf, Romainville, France) to prevent binding to glucocorticoid receptors. Nonspecific binding was measured in the presence of a 400-fold molar excess of unlabeled aldosterone (Sigma, St. Louis, MO). Receptor-bound steroid was separated from the cytosol by the addition of 300 µL of an ice-cold suspension of 15% (wt/vol) hydroxyapatite in 50 mmol/L Tris and 10 mmol/L KH2PO4, pH 7.2. After incubation for 30 min at 4 C, the samples were centrifuged at 1000g for 3 min at 4 C, the supernatants were decanted, and the pellets were washed 3 times with 1 mL of a solution containing 10 mmol/L Tris, 5 mmol/L Na2HPO4, 1.5 mmol/L ethylene-diaminetetraacetic acid (EDTA), and 1% Tween 80 (polyoxyethylene sorbitan monooleate, Sigma, St. Louis, MO), pH 7.4. The washed pellets were resuspended in 2 mL of ethanol at room temperature for 60 min and centrifuged at 1000g for 5 min. The supernatants were transferred to scintillation vials and 10 ml of ASC II (Amersham Pharmacia Biotech, Tokyo, Japan) added. Radioactivity was measured with a liquid scintillation counter (PR-2650; Packard, Downers Grove, IL). Specific binding in cytosols from human gastric body and antral mucosa was determined by subtracting nonspecific binding from total binding. Dissociation constants were determined by Scatchard analysis.

Northern blot analysis

Samples for Northern blot analysis were prepared from mucosal specimens stripped from the muscular layer. Total RNA was extracted with ISOGEN (Nippon Gene, Osaka, Japan), followed by poly (A) RNA preparation with Oligotex-dT30 (Takara Shuzo, Tokyo, Japan). The poly (A) RNA concentration of samples was measured by spectrophotometry at 260 nm. Samples of 2 µg of poly (A) RNA were denatured in 17.5% (v/v) formaldehyde + 50% (v/v) formamide in 0.02 mol/L 3-(Morpholiino) propanesulfonic acid (MOPS) + 50 mmol/L NaOAs + 10 mmol/L EDTA (pH 7.0) at 65 C for 5 min, electrophoresed on 1.2% agarose gel in 0.02 mol/L MOPS + 50 mmol/L NaOAs + 10 mmol/L EDTA (pH 7.0), and then blotted to nylon membrane. The radiolabeled probes of human MR cDNA (25) and 11ß-HSD2 cDNA (26) were hybridized in the buffer containing 50% (v/v) formamide, 5 x SSC, 50 mmol/L sodium phosphate, 4 x Denhardt’s solution, 40 µg/mL salmon sperm DNA at 42 C for 18 h. Each probe was labeled by 32P-dATP (Amersham Pharmacia Biotech) with Megasprime DNA labeling system (Amersham Pharmacia Biotech). After hybridization, the membranes were washed in 2 x SSC at room temperature for 10 min, 2 x SSC + 2% SDS at 65 C for 45 min, and 0.1 x SSC at room temperature for 10 min, and exposed to film. Normal human terminal ileum was obtained surgery from the ascending colon cancer patient and was employed as a positive control.

RT-PCR analysis and PCR direct sequence of RT-PCR products

Samples of total RNA for RT-PCR were prepared from freshly frozen specimens obtained from gastric body and antrum by endoscopic biopsy. RNA samples were isolated with RNeasy Midi kit (QIAGEN, Hilden, Germany). First strand cDNA was synthesized from 1 µg of total RNA with the SUPERSCRIPT Preamplification System for First Strand cDNA Synthesis (Gibco BRL Products, Rockville, MD). The PCR primer set for human MR was designed according to Arriza et al. (25), i.e. sense primer was 5'-TTCAGCTACACTGCTTCTGGCA-3'(1237–1258) and antisense primer was 5'-GTACCTTGAGCACCAATCCTGT-3'(1790–1811). The PCR primer set for human 11ß-HSD2 was designed according to Slight et al. (27), i.e. sense primer was 5'-GACCAAACCAGGAGACATTAGC-3'(525–546) and antisense primer was 5'-ATGTAGTCCTTGCCGTAGGC-3'(987–1007). ß-actin was employed as a control for RT-PCR. PCR primer set for ß-actin (XAHR17 and XAHR20 Primer) was purchased from Funakoshi (Tokyo, Japan). Expected lengths of PCR products of MR, 11ß-HSD2, and ß-actin were 574 bp, 483 bp, and 289 bp, respectively. The PCR procedure was performed with GeneAmp PCR System 2400 (Perkin Elmer, Norwalk, CT) under the following conditions: denaturation at 95 C for 30 sec, annealing at 53 C for 1 min, and extension at 72 C for 1 min, 40 cycles in PCR mixture containing dNTP 50 mmol/L, primer 0.5 mmol/L, Taq DNA polymerase (Takara Shuzo, Tokyo, Japan) 2.5 units/100 mL, KCl 50 mmol/L, Tris-HCl 10 mm (pH 8.3), MgCl2 1.5 mmol/L. PCR products were electrophoresed on 2% agarose gel.

To examine the specificity of each PCR product, PCR direct sequence was performed with an ABI Prism Genetic Analyzer (Perkin Elmer, Norwalk, CT). Utilizing the dRhodamine Terminator Cycle Sequencing FS Ready Reaction Kit (Perkin Elmer, Norwalk, CT), unidirectional PCR was performed with either sense or antisense primer, using RT-PCR products of human MR and 11ß-HSD2 as PCR templates. The products of unidirectional PCR were digested with Shrimp Alkaline Phosphatase (Amersham Pharmacia Biotech), purified on Centri-Sep spin columns (Perkin Elmer, Norwalk, CT), then sequenced by ABI Prism 310. The results were compared with reported sequences of MR (25) and 11ß-HSD2 (26).

Antibodies

We used polyclonal antibodies that were generated against human MR and 11ß-HSD2 designated as MINREC4 and HUH23, respectively. The production and characteristics of MINREC4 (28, 29, 30) and HUH23 (31, 16) have been previously reported.

Immunohistochemistry

Specimens fixed in 10% formaldehyde for 18 h at 4 C and embedded in paraffin were used for immunohistochemistry. Paraffin-embedded specimens were cut into 2.5-µm-thick sections and placed on poly-L-lysine coated glass slides. Paraffin was removed from the sections by treatment with xylene and ethanol, after which they were treated with methanol containing 0.3% H2O2 for 30 C min to block endogenous peroxidase, washed with three changes of phosphate-buffered saline for 5 min each, and finally incubated with 10% (vol/vol) normal goat serum for 30 min at room temperature. Immunohistochemistry was performed by the biotin-streptavidin amplification method (Histofine immunostaining system, Nichirei, Tokyo, Japan). The sections were treated consecutively with optimal dilutions of each antiserum (MINREC4; 1:750, HUM23; 2 µg/mL) overnight at 4 C, followed by biotinylated goat antibodies to rabbit immunoglobulin for 30 min at room temperature, and peroxidase-conjugated streptavidin for 30 min at room temperature, with washing in cold phosphate-buffered saline between incubations. After the final wash, sections were immersed for 2 min in a solution containing 0.66 mmol/L 3,3'-diaminobenzidine, 0.03% (vol/vol) H2O2 plus 50 mmol/L Tris (pH 7.6), then counterstained with Mayor’s hematoxylin. For control immunostaining, phosphate-buffered saline, normal rabbit IgG, or pre-immuned serum was used instead of the first antibody.

Electron microscopy

The fixation and embedding procedure for electron microscopy was modified from that of Hogan and Smith (32). Tissue specimens were fixed in 4% paraformaldehyde with 0.5% glutaraldehyde in 0.1 mol/L sodium phosphate (pH 7.4) for 18 h at 4 C (without subsequent treatment with OsO4), washed with phosphate-buffered saline containing 10% (wt/vol) sucrose, dehydrated in gradient ethanol (60% to absolute) and propylene oxide, and embedded in 60.2 mL of epon-araldite plastic containing 17 mL Epon 812, 33 mL dodecenyl succinic anhydrite, 9 mL Araldite CY212, and 1.2 mL dimethylaminomethyl phenol, all of which were purchased from TAAB (Berks, England). They were polymerized at 37 C for 18 h and 60 C for 2 days and were cut into serial sections of 90 nm and 1 µm for electron microscopy and immunohistochemistry, respectively.

For light microscopic immunostaining, the sections were placed on poly-L-lysine coated glass slides and deplasticized according to a modified version of the method of Hogan and Smith (32). The plastic was completely removed from tissue sections by soaking slides overnight at 4 C in an alkaline organic solvent containing equal volumes of 0.5% KOH in methanol, acetone, and benzene prepared immediately before use. The deplasticized sections were then neutralized in 1% acetic acid in methanol for a few minutes, washed several times in methanol, then stained immunohistochemically as described above. The serial ultrathin sections for electron microscopy were stained with uranium and lead, then examined with a Hitachi H300 electron microscope (Hitachi Scientific Instruments, Inc., Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
[3H]aldosterone binding

Specific [3H]aldosterone binding was detected in gastric fundic mucosa. The dissociation constant (Kd) for the ten samples of gastric fundic mucosa examined was 0.72 ± 0.05 nmol/L (mean ± SE), and the maximum number of binding sites (Bmax) was 6.0 ± 1.4 fmol per milligram of protein. The results of individual samples are depicted in Table 1Go. On the other hand, gastric antral mucosa did not demonstrate detectable specific binding of [3H]aldosterone in any of five specimens examined.


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  		Table 1. Patients profile and [3H] aldosterone binding to human gastric fundic mucosa

 
Northern blot analysis of MR mRNA and 11ß-HSD2 mRNA in the normal gastric fundic mucosa

Northern blot analysis of MR mRNA in the normal gastric fundic mucosa demonstrated a faint single band at 6.0 kb, identical with the size observed in a sample of terminal ileum employed as a positive control (Fig. 1Go). However, no significant hybridization signal was observed for 11ß-HSD2 mRNA in the gastric mucosa by Northern blot analysis (data not shown).



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  		Figure 1. Northern blot analysis of MR mRNA. Lane 1, normal gastric fundic mucosa; lane 2, terminal ileum. Faint single bands were detectable at approximately 6.0 kb. Lower panel shows corresponding ß-actin bands as controls.

 
RT-PCR analysis of MR and 11ß-HSD2 mRNA’s

RT-PCR products derived from mRNA of MR and 11ß-HSD2 were detected as single bands located at the expected sizes on 2% agarose gel only in the normal gastric fundic mucosa (Fig. 2Go). Direct sequencing of PCR products gave results consistent with the amplification of MR mRNA (25) and 11ß-HSD2 mRNA (26) (data not shown).



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  		Figure 2. RT-PCR of MR and 11ß-HSD2 mRNAs. Each PCR product derived from MR gene (lane 1), 11ß-HSD2 (lane 3), and ß-actin (lane 4) was electrophoresed on 2% agarose gel. Lane 2 was 100 bp molecular size marker. The expected sizes of PCR products were 574 bp (MR), 483 bp (11ß-HSD2), and 289 bp (ß-actin), respectively. Direct PCR sequencing confirmed the presence of MR and 11ß-HSD2 products.

 
Immunohistochemistry of MR and 11ß-HSD2

The 25 specimens of gastric mucosa examined by immunohistochemistry consisted of 15 fundic mucosa and 10 antral mucosa specimens. Immunohistochemistry of serial tissue sections demonstrated immunoreactivity for both MR and 11ß-HSD2 in parietal cells in gastric fundic glands (Fig. 3Go, A and B). MR immunoreactivity was detected as intense staining in the nuclei and cytoplasm of parietal cells, whereas 11ß-HSD2 immunoreactivity was weaker and localized only to the cytoplasm. Gastric mucosal cells, including surface epithelial cells, chief cells, pyloric gland cells, and other unidentified cell types were negative for MR and 11ß-HSD2 immunostaining. Immunore-activity was not present in sections incubated with pre-immune serum, normal rabbit IgG, or phosphate-buffered saline.



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  		Figure 3. Immunohistochemical staining of human gastric fundic mucosa with MINREC4 polyclonal antibody to the human MR (A) (x200) and HUH23 polyclonal antibody to the human 11ß-HSD2 (B) (x200). Immunostaining was performed on serial sections. Immunoreactivity of MR and 11ß-HSD2 was detected in cells shown histologically to be parietal cells in the fundic glands.

 
Ultrastructural characterization of MR positive cells

Serial electron microscopic analysis of immunohistochemical sections was performed to identify MR positive cells. Electron microscopy showed that MR positive cells were characterized by the following ultrastructural features: 1) the nucleus was spherical and centrally or basally located, 2) microvilli of the intracellular canaliculi were long and of uniform density and width, and 3) elements of the tubulovesicular system were distributed throughout the cytoplasm and were either round or flattened (Fig. 4Go). These ultrastructural features confirmed that the MR positive cells corresponded to parietal cells of human gastric glands (33).



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  		Figure 4. Immunohistochemistry with MINREC4 polyclonal antibody to the human MR (A) and electron microscopy (B, C) of serial ultrathin sections of human gastric fundic mucosa. The area boxed in A (x400) is magnified in B (x1000) and C (x3000). The MR-positive cells were identified as parietal cells by their numerous mitochondria and tubulovesicles. The intracellular canaliculi are filled with microvilli.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study is the first to demonstrate the presence of both MR and 11ß-HSD2 in human stomach. Specific [3H]aldosterone binding sites were detected in the gastric fundic mucosa, but not in the antrum. The mean Kd value for [3H]aldosterone binding MR in gastric fundic mucosal cytosol (0.72 ± 0.05 nmol/L) is consistent with previous studies of [3H]aldosterone binding in human mineralocorticoid-responsive tissues (7, 8, 9, 10, 11, 22, 23, 24). In addition, MR mRNA was detected by both Northern blot analysis and RT-PCR in the gastric fundic mucosa. Furthermore, immunohistochemical and electron microscopic analysis of gastric mucosa revealed that MR immunoreactivity was identified only in the parietal cells located in the gastric fundic mucosa.

The number of aldosterone binding sites in human gastric fundic mucosa in the present study (6.0 ± 1.4 fmol per milligram of protein) is an order of magnitude lower than that reported in colonic mucosa, a well characterized gastrointestinal mineralocorticoid responsive tissue (9, 10, 11), but comparable to the number of sites detected in kidney (24). It should be noted that these results represent minimal values as tissue specimens were obtained from patients with intact adrenals and mineralocorticoid receptor occupied by endogenous aldosterone. In the colon, high MR immunoreactivity has been demonstrated in most columnar epithelial cells (29). On the other hand, MR immunoreactivity in the human stomach was detected only in parietal cells, which represent a small proportion of the total cells lining the stomach. Therefore, the lower number of gastric binding sites could be due to the lower relative abundance of MR positive cells.

Pressley and Funder (8) reported that specific [3H]aldosterone binding sites could not be detected in the antral mucosa of rat stomach, consistent with the absence of MR in the antrum of the human stomach. However, they did not examine the body of stomach in which MR expression was detected in our study. In another report Fuller et al. (34) demonstrated the absence of MR gene expression in rat stomach, employing Northern blot analysis. The discrepancy in Northern blot findings may be due to species differences and the mucosal area examined.

We also examined the human gastric mucosa for the presence of 11ß-HSD2. Weak HUH23 immunoreactivity and the detection of low amounts of 11ß-HSD2 mRNA in the human gastric fundic mucosa RT-PCR suggest the presence of low amounts of activity. Smith et al. (35) also demonstrated the expression of low levels of 11ß-HSD2 protein in the rat stomach by Western blot analysis. The present study thus provides evidence of the colocalization of MR and 11ß-HSD2 in the parietal cells of human gastric mucosa, and suggests that the parietal cell is a mineralocorticoid target. Although the level of 11ß-HSD2 in parietal cells appeared to be low, these findings could be affected by medication or be modulated by the degree of stomach emptying. It should be emphasized that the only cell type in the stomach to exhibit colocalization was the parietal cell, suggesting that mineralocorticoid activity may be important for acid production.

The most important biological function of parietal cells is gastric acid secretion. Acute injection of aldosterone to Heidenhain pouch dogs decreased the Na+ content of gastric juice, consistent with the known actions of aldosterone (36, 37). In addition, continuous daily injections of aldosterone induced significant increases in the Na+/K+ ratio of gastric juice reminiscent of the escape phenomenon observed in the kidney (36, 37). Reduced gastric acid secretion has also been reported in adrenalectomized animal models, including rat (38, 39) and dog (40). Licorice and its congener, carbenoxolone sodium, have been employed as anti-ulcer agents but have been discontinued in clinical practice because of side effects resembling pseudohyperaldosteronism (41, 42). The concurrent administration of carbenoxolone, with either the mineralocorticoid antagonist spironolactone (43, 44) or the epithelial sodium channel inhibitor amiloride (45) has been reported to reduce both pseudohyperaldosteronism-like symptoms and anti-ulcer effects. Other studies revealed that licorice derivatives and carbenoxolone had a low affinity for the renal MR (46, 47) and inhibited 11ß-HSD2 activity strongly (48, 49, 50). Carbenoxolone leads to severe hypokalemia causing the inhibition of gastric acid secretion; this may explain the mechanism by which carbenoxolone might modulate gastric acid secretion (51). Subsequent investigations reported that carbenoxolone might also act by increasing prostaglandin production in the gastric mucosa (52).

In conclusion, our study supports previous findings implicating aldosterone action on human stomach via MR. However, the physiological significance of this action remains unknown, and further studies are required to clarify the role of aldosterone in gastric secretion in health and disease.

Received December 17, 1998.

Revised March 9, 1999.

Accepted April 9, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Edmonds CJ, Marriott J. 1970 Sodium transport and short circuit current in rat colon in vivo and the effect of aldosterone. J Physiol (London). 210(4):1021–1039.
  2. Levitan R, Ingelfinger FJ. 1965 Effect of d-aldosterone on salt and water absorption from the intact human colon. J Clin Invest. 44:801–808.
  3. Efstratopoulos AD, Peart WS, Wilson GA. 1974 The effect of aldosterone on colonic potential difference and renal electrolyte excretion in normal man. Clin Sci Mol Med. 46:489–499.[Medline]
  4. Feldman DJ, Funder JW, Edelman IS. 1972 Subcellular mechanisms in the action of adrenal steroids. Am J Med. 53:545–560.[CrossRef][Medline]
  5. Edelman IS. 1981 Receptors and effectors in hormone action on the kidney. Am J Physiol. 241:F333–339.
  6. Evans RM. 1988 The steroid and thyroid hormone receptor superfamily. Science. 240:889–895.[Abstract/Free Full Text]
  7. Swaneck GE, Highland E, Edelman IS. 1969 Stereospecific nuclear and cytosol aldosterone-binding proteins of various tissues. Nephron. 6:297–316.[Medline]
  8. Pressley L, Funder JW. 1975 Glucocorticoid and mineralocorticoid receptors in gut mucosa. Endocrinology97 :588–596.
  9. Rafestin-Oblin ME, Lombes M, Michiel JB, Michaud A, Claire M. 1984 Mineralocorticoid receptors in the epithelial cells of human colon and ileum. J Steroid Biochem. 20:311–315.[CrossRef][Medline]
  10. Schulman G, Miller-Diener A, Litwack G, Bastl CP. 1986 Characterization of the rat colonic aldosterone receptor and its activation process. J Biol Chem. 261:12102–12108.[Abstract/Free Full Text]
  11. Binder HJ, White A, Whiting D, et al. 1986 Demonstration of specific high affinity receptors for aldosterone in cytosol of rat colon. Endocrinology. 118:628–631.[Abstract]
  12. Edwards CR, Stewart PM, Burt D, Hayslett J. 1988 Localization of 11ß-hydroxysteroid dehydrogenase–tissue specific protector of the mineralocorticoid receptor. Lancet 2:986–989.
  13. Funder JW, Pearce PT, Smith RE, Smith AI. 1988 Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science. 242:583–585.[Abstract/Free Full Text]
  14. Monder C, White PC. 1993 11ß-hydroxysteroid dehydrogenase. Vitam Horm. 47:187–271.[Medline]
  15. Rusvai E, Naay-Fejes-Toh A. 1993 A new isoform of 11ß-hydroxysteroid dehydrogenase in aldosterone target cells. J Biol Chem. 268:10717–10720.[Abstract/Free Full Text]
  16. Hirasawa G, Sasano H, Takahashi K, et al. 1997 Colocalization of 11ß-hydroxysteroid dehydrogenase type II and mineralocorticoid receptor in human epithelia. J Clin Endocrinol Metab. 82:3859–3863.[Abstract/Free Full Text]
  17. Muallem S, Blissard D, Crago Jr EJ, Sachs G. 1988 Activation of Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell. J Biol Chem. 263:14703–14711.[Abstract/Free Full Text]
  18. Horie S, Yano S, Watanabe K. 1992 Effects of drugs acting on Cl--HCO3- and Na+-H+ exchangers on acid secretion in the rat gastric mucosa sheet preparation. Eur J Pharmacol. 229:15–19.[CrossRef][Medline]
  19. Paradiso AM, Tsien RY, Demarest JR, Machen TE. 1987 Na+-H+ and Cl--HCO3- exchange in rabbit oxyntic cells using fluorescence microscopy. Am J Physiol. 253:C30–36.
  20. Krozowski ZS, Funder JF. 1983 Renal mineralocorticoid receptors and hippocampal corticosterone-binding species have identical intrinsic steroid specificity. Proc Natl Acad Sci USA 80:6056–6060.
  21. Bradford MM. 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem. 72:248–254.[CrossRef][Medline]
  22. Armanini D, Strasser T, Weber PC. 1985 Characterization of aldosterone binding sites in circulating human mononuclear leukocytes. Am J Physiol. 248:E388–390.
  23. Shimada T, Yasuda K, Mori A, et al. 1993 Aldosterone binding to mineralocorticoid receptors of mononuclear leukocytes in diabetic subjects. Acta Endocrinol (Copenh). 128:529–535.[Medline]
  24. Doyle D, Smith R, Krozowski ZS, Funder JW. 1989 Mineralocorticoid specificity of renal type I receptors: binding and metabolism of corticosterone. J Steroid Biochem. 33:165–170.[CrossRef][Medline]
  25. Arriza JL, Weinberger C, Cerelli G, et al. 1987 Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science. 237:268–275.[Abstract/Free Full Text]
  26. Albiston AL, Obeyesekere VR, Smith RE, et al. 1994 Cloning and tissue distribution of the human 11ß-hydroxysteroid dehydrogenase type 2 enzyme. Mol Cell Endocrinol. 105:R11–17.
  27. Slight SH, Ganjam VK, Guez Snchez CE, Krozowski ZS. 1997 High Affinity NAD+-dependent 11ß-hydroxysteroid dehydrogenase in the Human Heart. J Mol Cell Cardiol. 28:781–787.
  28. Krozowski ZS, Rundle SE, Wallace C, et al. 1989 Immunolocalization of mineralocorticoid receptors with an antiserum against a peptide deduced from the complementary deoxyribonucleic acid sequence. Endocrinology. 125:192–198.[Abstract]
  29. Fukushima K, Sasano H, Nagura H, et al. 1991 Immunohistochemical localization of mineralocorticoid receptor in human gut. Tohoku J Exp Med. 165:155–163.[Medline]
  30. Sasano H, Fukushima K, Sasaki I, Matsuno S, Krozowski ZS. 1992 Immunolocalization of mineralocorticoid receptor in human kidney, pancreas, salivary, mammary and sweat glands: a light and electron microscopic immunohistochemical study. J Endocrinol. 132:305–310.[Abstract]
  31. Krozowski ZS, MaGuire JA, Stein Oakley AN, Dowling J, Smith RE, Andrews RK. 1995 Immunohistochemical localization of the 11ß-hydroxysteroid dehydrogenase type II enzyme in human kidney and placenta. J Clin Endocrinol Metab. 80:2203–2209.[Abstract]
  32. Hogan DL, Smith GH. 1982 Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues. J Histochem Cytochem. 30:1301–1306.[Abstract]
  33. Williams PL, Warwick R. 1980 Gray’s Anatomy. 36th ed. Edinburgh: Churchill Livingstone; 1336–1337.
  34. Fuller PJ, Verity K. 1990 Mineralocorticoid receptor gene expression in the gastrointestinal tract: distribution and ontogeny. J Steroid Biochem. 36:263–267.[CrossRef][Medline]
  35. Smith ER, Li KX, Andrews RK, Krozowski ZS. 1997 Immunohistochemical and molecular characterization of the rat 11ß-hydroxysteroid dehydrogenase type II enzyme. Endocrinology. 138:540–547.[Abstract/Free Full Text]
  36. Baddeley RM, Evans J, Griffin JA. 1969 Influence of hyperaldosteronism upon gastric secretion. Gut 10:143–145.
  37. August JT, Nelson DH, Thorn GW. 1958 Response of normal subjects to large amounts of aldosterone. J Clin Invest. 37:1549–1555.
  38. Tseng CC, Schmidt KL, Johnson LR. 1987 Hormonal effects on development of the secretary apparatus of parietal cells. Am J Physiol. 253:G284–289.
  39. Matsiuk IaR. 1987 A morphofunctional analysis in the parietal cells of the stomach after adrenalectomy. Arkh Anat Gistol Embriol. 92:61–66.[Medline]
  40. Krivova NA, Gridneva VI. 1982 Stomach secretion and excretion in mineralocorticoid deficiency. Biull Eksp Biol Med. 93:39–41.
  41. Conn JW, Rovner DP, Cohen EL. 1968 Licorice-induced pseudoaldosteronism. Hypertension, hypokalemia, aldosteropenia and suppressed plasma rennin activity. JAMA. 205:492–496.[CrossRef][Medline]
  42. Stewart PM, Corrie JE, Shackleton CH, Edwards CR. 1988 Syndrome of apparent mineralocorticoid excess. A defect in the cortisol-cortisone shuttle. J Clin Invest. 82:340–349.
  43. Davies GJ, Rhodes J, Calcraft BJ. 1974 Complications of carbenoxolone therapy. Br Med J. 3:400–402.
  44. Doll R, Langman MJ, Shawdon HH. 1968 Treatment of gastric ulcer with carbenoxolone: antagonistic effect of spironolactone. Gut. 9:42–45.[Free Full Text]
  45. Reed PI, Lewis SI, Vincent-Brown A, et al. 1980 The influence of amiloride on the therapeutic and metabolic effects of carbenoxolone in patients with gastric ulcer. A double-blind controlled trial. Scand J Gastroenterol Suppl. 60:51–57.
  46. Armanini D, Karbowiak I, Krozowski Z, Funder JW, Adam WR. 1982 The mechanism of mineralocorticoid action of carbenoxolone. Endocrinology. 111:1683–1686.[Abstract]
  47. Armanini D, Karbowiak I, Funder JW. 1983 Affinity of liquorice derivatives for mineralocorticoid and glucocorticoid receptors. Clin Endocrinol (Oxf). 19:609–612.[Medline]
  48. Monder C, Stewart PM, Lakshmi V, Valentino R, Burt D, Edwards CR. 1989 Licorice inhibits corticosteroid 11ß-dehydrogenase of rat kidney and liver: in vivo and in vitro studies. Endocrinology. 125:1046–1053.[Abstract]
  49. Funder JW. 1990 11ß-hydroxysteroid dehydrogenase and the meaning of life. Mol Cell Endocrinol. 68:C3–5.
  50. McKenzie MA, Hoefnagels WH, Jansen RW, Benraad TJ, Kloppenborg PW. 1990 The influence of glycyrrhetinic acid on plasma cortisol and cortisone in healthy young volunteers. J Clin Endocrinol Metab. 70:1637–1643.[Abstract]
  51. Baron JH. 1977 Effect of carbenoxolone sodium on human gastric acid secretion. Gut. 18:721–722.[Abstract/Free Full Text]
  52. Martin A, Gurrieri G, Sturniolo GC, Naccarato R. 1983 Effect of carbenoxolone on gastric prostaglandin E release and cytoprotection in the rat. Adv Prostaglandin Thromboxane Leukot Res12 :409–410.



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