This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shimojo, M. Articles by Stewart, P. M. Search for Related Content PubMed PubMed Citation Articles by Shimojo, M. Articles by Stewart, P. M.

Immunodetection of 11ß-Hydroxysteroid Dehydrogenase Type 2 in Human Mineralocorticoid Target Tissues: Evidence for Nuclear Localization¹

Departments of Medicine, Immunology (G.D.J., A.R.B.) and Pathology (A.J.H.), University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom B15 2TH

Address all correspondence and requests for reprints to: Prof. Paul M. Stewart, Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom B15 2TH.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
11ß-Hydroxysteroid dehydrogenase (11ßHSD) is an enzyme complex responsible for the conversion of hormonally active cortisol to inactive cortisone; two isoforms of the enzyme have been cloned and characterized. Clinical observations from patients with the hypertensive syndrome apparent mineralocorticoid excess, recently explained on the basis of mutations in the human 11ßHSD2 gene, suggest that it is the 11ßHSD2 isoform that serves a vital role in dictating specificity upon the mineralocorticoid receptor (MR). We have raised a novel antibody in sheep against human 11ßHSD2 using synthetic multiantigenic peptides and have examined the localization and subcellular distribution of 11ßHSD2 in mineralocorticoid target tissues.

The immunopurified antibody recognized a single band of approximately 44 kDa in placenta, trophoblast, and distal colon. In kidney tissue, two bands of approximately 44 and 48 kDa were consistently observed. No signal was seen in decidua, adrenal, or liver. Immunoperoxidase studies on the mineralocorticoid target tissues, kidney, colon, and parotid gland indicated positive staining in epithelial cells known to express the MR: respectively, renal collecting ducts, surface and crypt colonic epithelial cells, and parotid duct epithelial cells. No staining was seen in these tissues in other sites. The intracellular localization of 11ßHSD2 in kidney and colon epithelial cells was addressed using confocal laser microscopy. Parallel measurements of 11ßHSD2 and nuclear propidium iodide fluorescence on sections scanned through an optical section of approximately 0.1 µm indicated significant 11ßHSD2 immunofluorescence in the nucleus.

In human kidney, colon, and salivary gland, 11ßHSD2 protects the MR from glucocorticoid excess in an autocrine fashion. Furthermore, within these tissues, 11ßHSD2, which had been considered to be a microsomal enzyme, is also found in the nucleus, suggesting that the interaction between the MR and aldosterone or cortisol is in part a nuclear event.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE ENZYME COMPLEX 11ß-HYDROXYSTEROID dehydrogenase (11ßHSD) is responsible for the interconversion of cortisol to hormonally inactive cortisone. Two isoforms of 11ßHSD have been cloned and characterized in human tissues (1, 2, 3), but it is the 11ßHSD2 isozyme that dictates specificity upon the mineralocorticoid receptor (MR). In vitro the MR has equal affinity for aldosterone and cortisol (4), but the inactivation of cortisol to cortisone in the kidney, colon, and salivary gland ensures that normal in vivo specificity for the MR is maintained with the binding of aldosterone (5, 6). Apparent mineralocorticoid excess is a form of human hypertension due to mutation in the gene encoding 11ßHSD2 (7, 8, 9); a defect in this cortisol-cortisone shuttle mechanism results, and cortisol acts as a potent mineralocorticoid (10, 11). Inhibition of 11ßHSD2 also explains the mineralocorticoid excess state seen after licorice ingestion (12), and the enzyme is overwhelmed by substrate excess in the ectopic ACTH syndrome (13, 14). Thus, 11ßHSD2 serves an important role in normal physiology, and alterations in its activity are involved in the pathogenesis of human hypertension.

11ßHSD2 messenger RNA and activity have been demonstrated in human kidney and colon (3, 15). In the colon, activity studies have suggested that 11ßHSD2 is expressed in the surface epithelial cells (16), the site of expression of the MR. Furthermore, in situ hybridization (15) and immunohistochemistry studies (17, 18) have confirmed the site of expression of 11ßHSD2 within the kidney to be the MR-expressing collecting ducts. However, there remains some debate as to whether other structures within the kidney express 11ßHSD2, such as the vasculature. In addition, although 11ßHSD2 has been considered to be a microsomal enzyme, earlier activity studies in kidney tissue did suggest the presence of 11ßHSD activity within a crude nuclear fraction (19, 20).

Using a sheep antiserum raised against human 11ßHSD2, we carried out immunohistochemical studies, including confocal microscopy and Western blot analyses, to characterize and more clearly define the localization of 11ßHSD2 within human mineralocorticoid target tissues.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tissues were obtained from the Department of Pathology, University of Birmingham. In every case tissues were histologically normal, derived from kidney, colon, and parotid gland. In each case a minimum of four separate normal tissues were studied, and the results shown to be consistent. Formalin-fixed, paraffin-wax embedded sections (5 µm) were mounted onto charged glass slides.

Synthesis of a human 11ßHSD2 antiserum
Using hydrophilicity profiles, two regions were selected from the published amino acid sequence of the type 2 isoform of human 11ßHSD, amino acids 137–160 and 334–358. These sequences were synthesized as eight-branched multiantigenic peptides and the two multiantigenic peptides mixed with Freund’s complete adjuvant and used to immunize a single sheep. An IgG fraction was prepared from the immune serum by ammonium sulfate precipitation and ion exchange chromatography.

Immunoperoxidase method
Sections were dewaxed in xylene, washed, and then boiled in 0.01 M citrate buffer, pH 6.0, for 2 min. After cooling at room temperature, endogenous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 15 min. The sections were rinsed in 0.05 M Tris-buffered saline, pH 7.6 (TBS), and incubated with human 11ßHSD2 antiserum [1:100 dilution in 10% normal swine serum in TBS (NSS/TBS)] for 60 min at room temperature. After washing in TBS, a 1:25 dilution of an antidonkey sheep horseradish peroxidase conjugate (Binding Site, Birmingham, UK) was applied to the sections for 30 min. Sections were rinsed in TBS covered with 10 mg 3,3'-diaminobenzidine tetrahydrochloride in 20 ml 0.1 M Tris buffer, pH 8.2, with 20 µl hydrogen peroxide for 10 min. The sections were washed in tap water, counterstained with hematoxylin, dehydrated, and mounted.

Immunofluorescence and confocal laser microscopy
This was carried out on human kidney tissues. Paraffin sections were processed as described above, incubated with a 1:25 dilution of the 11ßHSD2 antiserum in 10% NSS/TBS for 60 min, washed in PBS for 10 min, and then incubated with a 1:25 dilution of an antidonkey sheep fluorescent conjugate (Binding Site) for 30 min. Sections were washed in PBS for 60 min and then counterstained with the nuclear-specific propidium iodide (0.5 µg/ml) (21) and rinsed in PBS. Sections were mounted in glycerol containing 1,4-diazabicyclo-(2.2.2) octane and initially examined under a Zeiss Universal fluorescent microscope as previously reported (22). A Bio-Rad 500 laser scanning confocal system (Bio-Rad, Richmond, CA) attached to a Leitz SM-Lux microscope (Leitz, Rockleigh, NJ) was used to analyze the sections through an optical section of approximately 0.1 µm (in contrast to the immunoperoxidase studies analyzed on the light microscope on 5-µm sections) (23). The determination of nuclear vs. cytoplasmic 11ßHSD2 immunofluorescence within human kidney and colon was achieved by simultaneous multichannel analysis on the dual labeled sections (11ßHSD2 and propidium iodide) by merging and displacing the paired images collected in the dual fluorescence mode, again through an optical section of 0.1 µm. From a mean of 10 epithelial cells in any given field, quantification of the 2 fluorescent labels was measured, from which the relative nuclear area and nuclear fluorescent signal could be calculated. Finally, by optical scanning of the section in the z or vertical axis, determination of whether nuclear staining was membrane or internal was made.

Controls
In each case negative controls were carried out with the primary antiserum omitted, but in the presence of 10% NSS. Further extensive control experiments were carried out using the immunizing 11ßHSD2 peptides. Peptide 1 (representing amino acids 137–160 of 11ßHSD2; 11.0 mg/ml) and peptide 2 (amino acids 334–358; 4.1 mg/ml) were diluted with 10% NSS/TBS from 1:2 to 1:128,000 dilution and incubated with the 11ßHSD2 antiserum (1:100 dilution with the diluted peptide) at 4 C overnight. After centrifugation, the supernatants were taken and used as the primary antiserum as described above.

Western blot analysis
Western analysis of proteins, prepared from homogenates of intact term placenta, trophoblast, and decidua; adrenal; liver; colon; and kidney, was performed by SDS-PAGE on discontinuous acrylamide gels. After a series of preliminary experiments to optimize conditions, 15 µg sample protein were denatured at 95 C in 2% SDS, 10% glycerol, 62.5 mM Tris (pH 6.8), and 0.1% dithiothreitol and electrophoresed at 200 mV through 4.5% stacking and 10% resolving gels using a Mini-Protean II Western apparatus (Bio-Rad). After electrophoresis, proteins were transferred to Immobilon P membranes (Millipore Corp., Bedford, MA), the membranes were blocked with 20% nonfat milk in PBS-0.1% Tween-20 (PBS-T) for 1 h, washed three times in PBS-T, and incubated with a 1:1000 dilution of the human 11ßHSD2 antiserum in 0.05% PBS-T overnight at 4 C. After washing in 0.1% PBS-T, membranes were incubated with a 1:50,000 dilution of an antisheep IgG peroxidase conjugate for 90 min at room temperature. Protein bands were analyzed using the ECL detection system (Amersham International, Aylesbury, UK) and exposed immediately to DuPont Cronex x-ray film (Wilmington, DE) for 10 min. Western blot analysis was also carried out on kidney and colon using antisera preincubated with a 1:64,000 dilution of peptide 1 mixed with a 1:16 dilution of peptide 2 (see below).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Localization of 11ßHSD2 within mineralocorticoid target tissues
Kidney. Intense immunoreactivity was seen only in cortical and medullary collecting ducts. No staining was observed over vascular structures, glomeruli, proximal tubules, thin limbs of the loop of Henle, or thick limbs of the loop of Henle, identified by their positions in the outer medulla, in medullary rays in the cortex, and at the macula densa adjacent to glomeruli. In the collecting ducts, immunoreactivity for 11ßHSD2 was much stronger in some cells than others; these have the morphological characteristics of principal cells, although specific markers for these cells were not employed (Fig. 1a).

View larger version (137K): [in this window] [in a new window]   Figure 1. Immunolocalization of 11ßHSD2 in normal kidney cortex (a). Intense positivity of cortical collecting ducts (brown staining) is shown. Other tubular structures, including proximal tubules and thick ascending loop of Henle (identified by their position adjacent to glomeruli) are negative, as are vascular endothelial cells within the glomerular tuft. b, Negative control using antiserum incubated with the immunizing 11ßHSD2 peptide. Magnification, x150. c, Immunolocalization of 11ßHSD2 to normal human distal colonic mucosa, indicating positive staining in surface and, to a lesser extent, crypt epithelial cells. Nonepithelial structures are negative (magnification, x100). d, Normal parotid gland indicating 11ßHSD2 localization to ductular epithelial cells. Adjacent acini and adipocytes are negative (magnification, x100).

Salivary gland. Immunoreactivity was seen in the epithelium of ducts in the parotid gland, with no detectable staining of glandular acini, adipocytes, or connective tissues (Fig. 1d).

The specificity of the antisera was confirmed by demonstrating the absence of staining with omission of the antiserum in the presence of normal sheep serum and also from the control studies with the immunizing peptide. Thus, with peptide 1, dilutions of 1:2 to 1:64,000 added to the antisera showed no staining, whereas a 1:128,000 showed some positivity over renal collecting ducts. With peptide 2, the absence of staining was seen with dilutions of 1:2 to 1:16, but positivity was restored when dilutions of 1:32 and less were used. The negative controls depicted here used a 1:64,000 dilution of peptide 1 mixed with a 1:16 dilution of peptide 2 incubated with the antisera as described in Materials and Methods (Fig. 1b). Further negative controls were carried out on human liver tissue that was devoid of any immunoreactivity (data not shown). The antibody was further characterized by Western blot analysis.

Western blot analysis of 11ßHSD2 in human mineralocorticoid target tissues
A single band of approximately 44 kDa was observed in term placenta and trophoblast and colon, but not in decidua, adrenal, or liver. In renal tissue, a second band was seen of approximately 48 kDa (Fig. 2a). The significance of this is uncertain, but has been a consistent finding in our Western blot analysis of human renal tissue (both cortex and medulla). Both the 44- and 48-kDa bands were eliminated in the presence of the absorbed antiserum (Fig. 2b). No additional bands were found in placenta or colon.

View larger version (41K): [in this window] [in a new window]   Figure 2. Western blot analysis of 11ßHSD2 in human tissues. a, A single band of approximately 44 kDa is seen in term placenta and trophoblast and colon. In kidney tissue, two bands of approximately 44 and 48 kDa are seen. Decidua, adrenal, and liver are negative. These protein bands are eliminated after incubation of the antiserum with the immunizing peptide (depicted in the + lanes in b), as described in Materials and Methods.

View larger version (54K): [in this window] [in a new window]   Figure 3. Confocal laser microscopy of 11ßHSD2 immunofluoresence in kidney and colon. The images depict the distribution of immunofluorescence signal as scanned through an optical section of approximately 0.1 µm. The merged image of epithelial cells in renal collecting ducts (a) and colon (b) stained with 11ßHSD2 antiserum (green signal) and propidium iodide (red signal) to highlight nuclei is shown. A nuclear localization of 11ßHSD2 is indicated by the yellow signal (magnification, x400). This is demonstrated on a higher magnification (x1200) of 11ßHSD2 immunofluorescence in epithelial cells within a renal collecting duct (c). In the left panel, total cellular 11ßHSD2 immunofluorescence is shown. For comparison, the right panel shows 11ßHSD2 immunofluorescence after subtraction of the nuclear area (as established from the propidium iodide immunofluorescence). With an optical section of approximately 0.1 µm through the center of most of the nuclei, signal over the nuclear area is clearly demonstrated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study we have addressed the immunolocalization of 11ßHSD2 in human mineralocorticoid target tissues, kidney, colon, and salivary gland (24, 25, 30) using a novel antihuman 11ßHSD2 antiserum raised in sheep. The antibody recognizes a protein of approximately 44 kDa in human kidney, colon, and placenta, but not in human adrenal, liver, or decidua. This finding is in keeping with a predicted M_r of 44.14 kDa for 11ßHSD2 and earlier reports on the expression of 11ßHSD2 messenger RNA in human tissues (3, 15). 11ßHSD2 immunoreactivity was observed only in cells known to express the MR, i.e. cells within renal collecting ducts (24, 31), surface and crypt epithelial cells within the colon (25), and ductal epithelial cells within the parotid gland (30), in keeping with the conclusion that 11ßHSD2 protects the MR in an autocrine fashion. Two recent reports, using differing 11ßHSD2 antisera, have described the immunolocalization of 11ßHSD2 within human kidney and colon (but not salivary gland) (17, 18), and there are differences from our data. Kro-zowski et al. (17) report immunostaining to vascular endothelial cells within the kidney (although not apparently the placenta), a finding that we are unable to reproduce. Although vascular staining was not seen in the study by Kyossev et al. (18), immunoreactivity was observed in the cortical ascending limb of the loop of Henle. We are confident that these tubular structures are negative in our study. The reasons for these discrepancies remain uncertain; whereas preimmune serum was used as a control in all three immunohistochemical studies, only our study used the more rigorous control of neutralizing the antiserum with the immunizing peptide.

The confocal laser microscopy presented here indicates that approximately 40% of the total immunostaining seen in renal collecting ducts and colonic epithelial cells is nuclear in origin. Furthermore, our data suggest that the enzyme is not associated with the nuclear membrane but has an intranuclear localization. 11ßHSD1 has been considered to be a microsomal enzyme (32), with data also suggesting that it is found on the lumenal side of the endoplasmic reticulum (33). There is, however, some evidence for nuclear 11ßHSD in kidney tissue. One of the first studies describing renal 11ßHSD activity indicated significant nuclear activity (19), a finding that has been confirmed subsequently (20), albeit on crude subcellular preparations and before the description of the two distinct isoforms of 11ßHSD. While this work was in progress, a fluorescent microscopy study reported that 11ßHSD2 is exclusively localized to the cytoplasmic surface of the endoplasmic reticulum (34). This study, however, used a chimeric rabbit 11ßHSD2/green fluorescent protein fusion gene transfected into a nonmineralocorticoid target tissue, Chinese hamster ovary cells, and the significance of this work to our work on the human 11ßHSD2 in classical mineralocorticoid target tissues remains uncertain. Indeed, in view of the established role for 11ßHSD in protecting the MR, these data raise questions concerning the mechanisms underlying MR specificity and the intracellular trafficking of the MR. For other members of the steroid hormone receptor superfamily, such as the glucocorticoid and progesterone receptors, this is well characterized; after the binding of ligand to the receptor in the cytosol and the dissociation of heat shock proteins, the activated receptor complex is translocated to the nucleus (reviewed in Ref.35). The intracellular localization of the MR, however, is still in some doubt. Although a predominantly cytosolic MR has been shown to translocate to the nucleus upon exposure to aldosterone in baculovirus-infected Sf9 cells (36), data from rabbit (37) and rat kidney (24) and pituitary cells (38) suggest both a nuclear and a cytosolic localization for the MR, independent of ligand, suggesting that the interaction of the MR with its ligand may in part be a nuclear event. The data presented here on human kidney would be in keeping with these data; in a given mineralocorticoid-responsive epithelial cell, 11ßHSD2 is ideally placed both inside and outside the nucleus to confer specificity upon the MR. It remains to be seen whether this pattern of subcellular localization of the enzyme extends to other tissues expressing the MR and 11ßHSD2, for example in the central nervous system (39), and if so, what factors in the structure of 11ßHSD determine this pattern of distribution. On this note, our Western blot analysis did reveal a second band of approximately 48 kDa in human kidney tissue homogenate, a band that was not observed in the study of Kyossev et al. (18), who carried out Western analysis on microsomal fractions. It seems unlikely, however, that this second band reflects a modified nuclear 11ßHSD2 protein, because it was absent in the colon, where our confocal laser studies also confirmed nuclear localization of 11ßHSD2.

In summary, we have developed a novel human 11ßHSD2 antiserum and have used this to confirm that MR specificity is effected at an autocrine level within human kidney, colon, and parotid. The observation that a significant proportion of cellular 11ßHSD2 is localized to the nuclear compartment suggests that the interaction between the MR and its ligand, be it cortisol or aldosterone, is a nuclear event and as such is more akin to the thyroid hormone receptor than to its closer relatives in the steroid-thyroid hormone receptor superfamily, the glucocorticoid and progesterone receptors.

	Acknowledgments

 
We thank Graham Mead for assistance in raising the antisera.

	Footnotes

 
¹ This work was supported by the Medical Research Council.

² Medical Research Council Senior Clinical Fellow.

Received August 19, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Tannin GM, Agarwal AK, Monder C, New MI, White PC 1991 The human gene for 11ß-hydroxysteroid dehydrogenase. Structure, tissue distribution, and chromosomal localization. J Biol Chem 266:16653–16658[Abstract/Free Full Text]
2. Stewart PM, Murry BA, Mason JI 1994 Human kidney 11ß-hydroxysteroid dehydrogenase is a high affinity NAD+-dependent enzyme and differs from the cloned "type I" isoform. J Clin Endocrinol Metab 79:480–484[Abstract]
3. Albiston AL, Obeyesekere VR, Smith RE, Krozowski ZS 1994 Cloning and tissue distribution of the human 11ß-hydroxysteroid dehydrogenase type 2 enzyme. Mol Cell Endocrinol 105:R11–R17
4. Arriza JL, Weinberger C, Cerelli G, Glaser TM, Handelin BL, Houseman DE, Evans RM 1998 Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor. Science 237:268–275
5. Edwards CRW, Stewart PM, Burt D, Brett L, McIntyre MA, Sutanto WS, DeKloet ER, Monder C 1988 Localisation of 11ß-hydroxysteroid dehydrogenase-tissue specific protector of the mineralocorticoid receptor. Lancet 2:986–989[CrossRef][Medline]
6. Funder JW, Pearce PT, Smith R, Smith AI 1988 Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated. Science 242:583–585[Abstract/Free Full Text]
7. Wilson RC, Krozowski ZS, Li K, Obeyesekere VR, Razzaghy-Azar M, Harbison MD, Wei JQ, Shackleton CHL, Funder JW, New MI 1995 A mutation in the HSD11B2 gene in a family with apparent mineralocorticoid excess. J Clin Endocrinol Metab 80:2263–2266[Abstract]
8. Mune T, Rogerson FM, Nikkila H, Agarwal AK, White PC 1995 Human hypertension caused by mutations in the kidney isozyme of 11ß-hydroxy-steroid dehydrogenase. Nat Genet 10:394–399[CrossRef][Medline]
9. Stewart PM, Krozowski ZS, Gupta A, Milford DV, Howie AJ, Sheppard MC, Whorwood CB 1996 Hypertension in the syndrome of apparent mineralocorticoid excess due to mutation of the 11ß-hydroxysteroid dehydrogenase type 2 gene. Lancet 347:88–91[CrossRef][Medline]
10. Dimartino-Nardi J, Stoner E, Martin K, Balfe JW, Jose PA, New MI 1987 New findings in apparent mineralocorticoid excess. Clin Endocrinol (Oxf) 27:49–62[Medline]
11. Stewart PM, Corrie JET, Shackleton CHL, Edwards CRW 1988 Syndrome of apparent mineralocorticoid excess: a defect in the cortisol-cortisone shuttle. J Clin Invest 82:340–349
12. Stewart PM, Wallace AM, Valentino R, Burt D, Shackleton CHL, Edwards CRW 1987 Mineralocorticoid activity of liquorice: 11ß-hydroxysteroid dehydrogenase deficiency comes of age. Lancet 2:821–824[Medline]
13. Ulick S, Wang JZ, Blumenfeld JD, Pickering TG 1992 Cortisol inactivation overload: a mechanism of mineralocorticoid hypertension in the ectopic adrenocorticotropin syndrome. J Clin Endocrinol Metab 74:963–967[Abstract]
14. Stewart PM, Walker BR, Holder G, O’Halloran D, Shackleton CHL 1995 11ß-Hydroxysteroid dehydrogenase activity in Cushing’s syndrome: explaining the mineralocorticoid excess state of the ectopic ACTH syndrome. J Clin Endocrinol Metab 80:3617–3620[Abstract]
15. Whorwood CB, Mason JI, Ricketts ML, Howie AJ, Stewart PM 1995 Detection of human 11ß-hydroxysteroid dehydrogenase isoforms using reverse transcriptase-polymerase chain reaction and localization of the type 2 isoform to renal collecting ducts. Mol Cell Endocrinol 110:R7–R12
16. Whorwood CB, Ricketts ML, Stewart PM 1994 Epithelial cell localization of type 2 11ß-hydroxysteroid dehydrogenase in rat and human colon. Endocrinology 135:2533–2541[Abstract]
17. Krozowski Z, 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]
18. Kyossev Z, Walker PD, Reeves WB 1996 Immunolocalization of NAD-dependent 11ß-hydroxysteroid dehydrogenase in human kidney and colon. Kidney Int 49:271–281[Medline]
19. Mahesh VB, Ulrich F 1960 Metabolism of cortisol and cortisone by various tissues and subcellular particles. J Biol Chem 235:356–360[Free Full Text]
20. Kobayashi N, Schulz W, Hierholzer K 1987 Corticosteroid metabolism in rat kidney in vitro. IV. Subcellular sites of 11ß-hydroxysteroid dehydrogenase activity. Pflugers Arch 408:46–53[CrossRef][Medline]
21. Ockleford CD, Bae-Li H, Wakely J, Badley RA, Whyte A, Page Faulk W 1981 Propidium iodide as a nuclear marker in immunofluorescence. I. Use with tissue and cytoskeleton studies. J Immunol Methods 43:261–267[CrossRef][Medline]
22. Howie AJ, Johnson GD 1992 Confocal microscopic and other observations on the distal end of the thick limb of the loop of Henle. Cell Tissue Res 267:11–16[CrossRef][Medline]
23. White JG, Amos WB, Fordham M 1987 An evaluation of confocal vs. conventional imaging of biological structures by fluorescence microscopy. J Cell Biol 105:41–48[Abstract/Free Full Text]
24. Krozowski ZS, Rundle SE, Wallace C, Castell MJ, Shen JH, Dowling J, Funder JW, Smith AI 1989 Immunolocalization of renal mineralocorticoid receptors with an antiserum against a peptide deduced from the complementary deoxyribonucleic acid sequence. Endocrinology 123:192–198[Abstract]
25. Fukushima K, Sasano H, Nagura H, Sasaki I, Matsuno S, Krozowski Z 1991 Immunohistochemical localization of mineralocorticoid receptor in the human gut. Tohoku J Exp Med 165:155–163[Medline]
26. Whorwood CB, Barber PC, Gregory J, Sheppard MC, Stewart PM 1993 11ß-Hydroxysteroid dehydrogenase and corticosteroid hormone action in the rat colon. Am J Physiol 264:E951–E957
27. Rusvai E, Fejes-Toth AN 1993 A new isoform of 11ß-hydroxysteroid dehydrogenase in aldosterone target cells. J Biol Chem 268:10717–10720[Abstract/Free Full Text]
28. Brown RW, Chapman KE, Edwards CRW, Seckl JR 1993 Human placental 11ß-hydroxysteroid dehydrogenase: evidence for and partial purification of a distinct NAD-dependent isoform. Endocrinology 132:2614–2621[Abstract]
29. Agarwal AK, Mune T, Monder C, White PC NAD-dependent isoform of 11ß-hydroxysteroid dehydrogenase. Cloning and characterization of cDNA from sheep kidney. J Biol Chem 269:25959–25962
30. Sheppard K, Funder JW 1987 Type 1 receptors in parotid, colon and pituitary are aldosterone selective in vitro. Am J Physiol 253:E467–E471
31. Fejes-Toth AN, Rusvai E, Fejes-Toth G 1994 Mineralocorticoid receptors and 11ß-steroid dehydrogenase activity in renal principal and intercalating cells. Am J Physiol 266:F76–F80
32. Monder C, White PC 1993 11ß-Hydroxysteroid dehydrogenase. Vitam Horm 47:187–271[Medline]
33. Ozols J 1995 Lumenal orientation and post-translational modifications of the liver microsomal 11ß-hydroxysteroid dehydrogenase. J Biol Chem 270:2305–2312[Abstract/Free Full Text]
34. Naray-Fejes-Toth A, Fejes-Toth G 1996 Subcellular localization of the type 2 11ß-hydroxysteroid dehydrogenase. J Biol Chem 271:15436–15442[Abstract/Free Full Text]
35. Carson-Jurica MA, Schrader WT, O’Malley BW 1990 Steroid receptor family: structure and functions. Endocr Rev 11:201–220[Abstract]
36. Robertson NM, Schulman G, Karnik S, Alnemri E, Litwack G 1993 Demonstration of nuclear translocation of the mineralocorticoid receptor (MR) using an anti-MR antibody and confocal laser scanning microscopy. Mol Endocrinol 7:1226–1239[Abstract]
37. Lombes M, Farman N, Oblin ME, Baulieu EE, Bonvalet JP, Erlanger BF, Gasc JM 1990 Immunohistochemical localization of renal mineralocorticoid receptor by using an anti-idiotypic antibody that is an internal image of aldosterone. Proc Natl Acad Sci USA 87:1086–1088[Abstract/Free Full Text]
38. Pearce PT, McNally M, Funder JW 1986 Nuclear localization of type 1 aldosterone binding sites in steroid-unexposed GH3 cells. Clin Exp Pharmacol Physiol 13:647–654[Medline]
39. Roland BL, Li KX, Funder JW 1995 Hybridization-histochemical localization of 11ß-hydroxysteroid dehydrogenase type 2 in rat brain. Endocrinology 136:4697–4700[Abstract]

This article has been cited by other articles:

				J. Kim, K. A. Temple, S. A. Jones, K. N. Meredith, J. L. Basko, and M. J. Brady Differential Modulation of 3T3-L1 Adipogenesis Mediated by 11beta-Hydroxysteroid Dehydrogenase-1 Levels J. Biol. Chem., April 13, 2007; 282(15): 11038 - 11046. [Abstract] [Full Text] [PDF]

				C. U Onyimba, N. Vijapurapu, S J. Curnow, P. Khosla, P. M Stewart, P. I Murray, E. A Walker, and S. Rauz Characterisation of the prereceptor regulation of glucocorticoids in the anterior segment of the rabbit eye. J. Endocrinol., August 1, 2006; 190(2): 483 - 493. [Abstract] [Full Text] [PDF]

				M. Quinkler, D. Zehnder, J. Lepenies, M. D Petrelli, J. S Moore, S. V Hughes, P. Cockwell, M. Hewison, and P. M Stewart Expression of renal 11{beta}-hydroxysteroid dehydrogenase type 2 is decreased in patients with impaired renal function Eur. J. Endocrinol., August 1, 2005; 153(2): 291 - 299. [Abstract] [Full Text] [PDF]

				H. Chisaka, J. F. Johnstone, M. Premyslova, Z. Manduch, and J. R.G. Challis Effect of Pro-inflammatory Cytokines on Expression and Activity of 11{beta}-Hydroxysteroid Dehydrogenase Type 2 in Cultured Human Term Placental Trophoblast and Human Choriocarcinoma JEG-3 Cells Reproductive Sciences, July 1, 2005; 12(5): 303 - 309. [Abstract] [PDF]

				C. A. Wagner, K. E. Finberg, S. Breton, V. Marshansky, D. Brown, and J. P. Geibel Renal Vacuolar H+-ATPase Physiol Rev, October 1, 2004; 84(4): 1263 - 1314. [Abstract] [Full Text] [PDF]

				R. Norregaard, T. R. Uhrenholt, C. Bistrup, O. Skott, and B. L. Jensen Stimulation of 11-{beta}-hydroxysteroid dehydrogenase type 2 in rat colon but not in kidney by low dietary NaCl intake Am J Physiol Renal Physiol, August 1, 2003; 285(2): F348 - F358. [Abstract] [Full Text] [PDF]

				N. Alfaidy, S. Gupta, C. DeMarco, I. Caniggia, and J. R. G. Challis Oxygen Regulation of Placental 11{beta}-Hydroxysteroid Dehydrogenase 2: Physiological and Pathological Implications J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4797 - 4805. [Abstract] [Full Text] [PDF]

				S. Kataoka, A. Kudo, H. Hirano, H. Kawakami, T. Kawano, E. Higashihara, H. Tanaka, F. Delarue, J.-D. Sraer, T. Mune, et al. 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Is Expressed in the Human Kidney Glomerulus J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 877 - 882. [Abstract] [Full Text] [PDF]

				M. Y. H. Zhang, X. Wang, J. T. Wang, N. A. Compagnone, S. H. Mellon, J. L. Olson, H. S. Tenenhouse, W. L. Miller, and A. A. Portale Dietary Phosphorus Transcriptionally Regulates 25-Hydroxyvitamin D-1{alpha}-Hydroxylase Gene Expression in the Proximal Renal Tubule Endocrinology, February 1, 2002; 143(2): 587 - 595. [Abstract] [Full Text] [PDF]

				S. Rauz, E. A. Walker, C. H. L. Shackleton, M. Hewison, P. I. Murray, and P. M. Stewart Expression and Putative Role of 11{beta}-Hydroxysteroid Dehydrogenase Isozymes within the Human Eye Invest. Ophthalmol. Vis. Sci., August 1, 2001; 42(9): 2037 - 2042. [Abstract] [Full Text] [PDF]

				M. Korbonits, I. Bujalska, M. Shimojo, J. Nobes, S. Jordan, A. B. Grossman, and P. M. Stewart Expression of 11{beta}-Hydroxysteroid Dehydrogenase Isoenzymes in the Human Pituitary: Induction of the Type 2 Enzyme in Corticotropinomas and Other Pituitary Tumors J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2728 - 2733. [Abstract] [Full Text] [PDF]

				P.M. Driver, M.D. Kilby, I. Bujalska, E.A. Walker, M. Hewison, and P.M. Stewart Expression of 11{beta}-hydroxysteroid dehydrogenase isozymes and corticosteroid hormone receptors in primary cultures of human trophoblast and placental bed biopsies Mol. Hum. Reprod., April 1, 2001; 7(4): 357 - 363. [Abstract] [Full Text] [PDF]

				G. J. Pepe, M. G. Burch, and E. D. Albrecht Localization and Developmental Regulation of 11{beta}-Hydroxysteroid Dehydrogenase-1 and -2 in the Baboon Syncytiotrophoblast Endocrinology, January 1, 2001; 142(1): 68 - 80. [Abstract] [Full Text] [PDF]

				D. ZEHNDER, R. BLAND, E. A. WALKER, A. R. BRADWELL, A. J. HOWIE, M. HEWISON, and P. M. STEWART Expression of 25-Hydroxyvitamin D3-1{alpha}-Hydroxylase in the Human Kidney J. Am. Soc. Nephrol., December 1, 1999; 10(12): 2465 - 2473. [Abstract] [Full Text]

				A. Odermatt, P. Arnold, A. Stauffer, B. M. Frey, and F. J. Frey The N-terminal Anchor Sequences of 11beta -Hydroxysteroid Dehydrogenases Determine Their Orientation in the Endoplasmic Reticulum Membrane J. Biol. Chem., October 1, 1999; 274(40): 28762 - 28770. [Abstract] [Full Text] [PDF]

				M. Tetsuka, M. Milne, G.E. Simpson, and S.G. Hillier Expression of 11ß-Hydroxysteroid Dehydrogenase, Glucocorticoid Receptor, and Mineralocorticoid Receptor Genes in Rat Ovary Biol Reprod, February 1, 1999; 60(2): 330 - 335. [Abstract] [Full Text] [PDF]

				J. Condon, C. Gosden, D. Gardener, P. Nickson, M. Hewison, A. J. Howie, and P. M. Stewart Expression of Type 2 11{beta}-Hydroxysteroid Dehydrogenase and Corticosteroid Hormone Receptors in Early Human Fetal Life J. Clin. Endocrinol. Metab., December 1, 1998; 83(12): 4490 - 4497. [Abstract] [Full Text]

				G. Mazzocchi, G. P. Rossi, G. Neri, L. K. Malendowicz, G. Albertin, and G. G. Nussdorfer 11ß-Hydroxysteroid dehydrogenase expression and activity in the human adrenal cortex FASEB J, November 1, 1998; 12(14): 1533 - 1539. [Abstract] [Full Text]

				K. E. Sheppard, K. Khoo, Z. S. Krozowski, and K. X. Z. Li Steroid specificity of the putative DHB receptor: evidence that the receptor is not 11beta HSD Am J Physiol Endocrinol Metab, July 1, 1998; 275(1): E124 - E131. [Abstract] [Full Text] [PDF]

				A. Naray-Fejes-Toth and G. Fejes-Toth Extranuclear Localization of Endogenous 11{beta}-Hydroxysteroid Dehydrogenase-2 in Aldosterone Target Cells Endocrinology, June 1, 1998; 139(6): 2955 - 2959. [Abstract] [Full Text] [PDF]

				M. L. Ricketts, J. M. Verhaeg, I. Bujalska, A. J. Howie, W. E. Rainey, and P. M. Stewart Immunohistochemical Localization of Type 1 11{beta}-Hydroxysteroid Dehydrogenase in Human Tissues J. Clin. Endocrinol. Metab., April 1, 1998; 83(4): 1325 - 1335. [Abstract] [Full Text]

				S. H.M. van Uum, A. R.M.M. Hermus, P. Smits, T. Thien, and J. W.M. Lenders The role of 11{beta}-hydroxysteroid dehydrogenase in the pathogenesis of hypertension Cardiovasc Res, April 1, 1998; 38(1): 16 - 24. [Abstract] [Full Text] [PDF]

				R. E. Smith, L. A. Salamonsen, P. A. Komesaroff, K. X. Z. Li, K. M. Myles, M. Lawrence, and Z. Krozowski 11{beta}-Hydroxysteroid Dehydrogenase Type II in the Human Endometrium: Localization and Activity during the Menstrual Cycle J. Clin. Endocrinol. Metab., December 1, 1997; 82(12): 4252 - 4257. [Abstract] [Full Text] [PDF]

				G. Hirasawa, H. Sasano, K.-i. Takahashi, K. Fukushima, T. Suzuki, N. Hiwatashi, T. Toyota, Z. S. Krozowski, and H. Nagura Colocalization of 11{beta}-Hydroxysteroid Dehydrogenase Type II and Mineralocorticoid Receptor in Human Epithelia J. Clin. Endocrinol. Metab., November 1, 1997; 82(11): 3859 - 3863. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shimojo, M.