A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy

David J. Morris1, Syed A. Latif1, Michael D. Rokaw2, Charles O. Watlington3, and John P. Johnson2

1 Department of Pathology and Laboratory Medicine, The Miriam Hospital, Lifespan, and Brown University School of Medicine, Providence, Rhode Island 02903; 2 Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213; and 3 Department of Medicine, Division of Endocrinology, Medical College of Virginia, Richmond, Virginia 23298

    ABSTRACT
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Abstract
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Methods
Results
Discussion
References

We have confirmed that A6 cells (derived from kidney of Xenopus laevis), which contain both mineralocorticoid and glucocorticoid receptors, do not normally possess 11beta -hydroxysteroid dehydroxgenase (11beta -HSD1 or 11beta -HSD2) enzymatic activity and so are without apparent "protective" enzymes. A6 cells do not convert the glucocorticoid corticosterone to 11-dehydrocorticosterone but do, however, possess steroid 6beta -hydroxylase that transforms corticosterone to 6beta -hydroxycorticosterone. This hydroxylase is cytochrome P-450 3A (CYP3A). We have now determined the effects of 3alpha ,5beta -tetrahydroprogesterone and chenodeoxycholic acid (both inhibitors of 11beta -HSD1) and 11-dehydrocorticosterone and 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone (inhibitors of 11beta -HSD2) and carbenoxalone, which inhibits both 11beta -HSD1 and 11beta -HSD2, on the actions and metabolism of corticosterone and active Na+ transport [short-circuit current (Isc)] in A6 cells. All of these 11beta -HSD inhibitory substances induced a significant increment in corticosterone-induced Isc, which was detectable within 2 h. However, none of these agents caused an increase in Isc when incubated by themselves with A6 cells. In all cases, the additional Isc was inhibited by the mineralocorticoid receptor (MR) antagonist, RU-28318, whereas the original Isc elicited by corticosterone alone was inhibited by the glucocorticoid receptor antagonist, RU-38486. In separate experiments, each agent was shown to significantly inhibit metabolism of corticosterone to 6beta -hydroxycorticosterone in A6 cells, and a linear relationship existed between 6beta -hydroxylase inhibition and the MR-mediated increase in Isc in the one inhibitor tested. Troleandomycin, a selective inhibitor of CYP3A, inhibited 6beta -hydroxylase and also significantly enhanced corticosterone-induced Isc at 2 h. These experiments indicate that the enhanced MR-mediated Isc in A6 cells may be related to inhibition of 6beta -hydroxylase activity in these cells and that this 6beta -hydroxylase (CYP3A) may be protecting the expression of corticosterone-induced active Na+ transport in A6 cells by MR-mediated mechanism(s).

steroid 6beta -hydroxylase; sodium transport

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

IN RECENT YEARS it has become abundantly clear that mineralocorticoid receptors (MR) in mineralocorticoid target cells such as the kidney and parotid gland are protected from the effects of endogenous glucorticoids. Experiments have shown that the glucocorticoids corticosterone and cortisol and the mineralocorticoid aldosterone have equal binding affinities for MR in vitro (1, 35). However, glucocorticoids do not bind to MR in vivo even though the endogenous circulating levels of glucocorticoids (in humans and in rats) are ~500 times greater than that of aldosterone. Because under normal conditions glucocorticoids do not cause mineralocorticoid-like actions (particularly Na+ retention), it is believed that protective and specificity-conferring mechanisms operate that prevent them from gaining access to renal MR in vivo. Edwards et al. (9) and Funder et al. (11) proposed that, in vivo, renal MR remains aldosterone specific because the enzyme, 11beta -hydroxysteroid dehydrogenase (11beta -HSD), metabolized glucocorticoids to their respective 11-dehydro products (5), which have low binding affinities for MR, do not elicit mineralocorticoid-like effects, and are considered inactive (9, 11, 19, 35).

Several experiments have offered additional support for the hypothesis that the enzyme 11beta -HSD acts as a guardian, conferring specificity on MR-mediated actions on Na+ (17, 25-27, 32, 40, 41, 44). Experiments from our laboratories have shown that the glucocorticoids, which normally do not elicit the usual Na+-retaining response (as does the mineralocorticoid aldosterone), do, however, display a potent Na+ retention and amplification of K+ excretion (36) in adrenalectomized rats pretreated with the 11beta -HSD inhibitor carbenoxolone (a succinate of glycyrrhetinic acid). The "mineralocorticoid-like" effects on Na+ retention conferred on glucocorticoids by carbenoxalone are inhibited by the specific MR antagonist RU-28318 but not by the glucocorticoid receptor (GR) antagonist RU-38486, indicating that these effects are mediated by occupation of MR (38). In other experiments using the isolated toad bladder preparation, which also possesses 11beta -HSD2 enzymatic activity, the short-circuit current (Isc, active Na+ transport) caused by glucocorticoids is enhanced when carbenoxalone is added to the incubation medium (3, 12).

There are at least two isoforms of 11beta -HSD: 1) 11beta -HSD1 in liver and proximal portions of the renal tubule of rats, which is bidirectional and NADP+ dependent, and 2) the NAD+-dependent 11beta -HSD2, which is unidirectional, possesses a much lower Michaelis-Menten constant for corticosterone, and is present in the cortical collecting duct segment of the renal tubule (22, 32). 3alpha ,5beta -Tetrahydroprogesterone and the bile acid chenodeoxycholic acid both inhibit 11beta -HSD1 (20, 29) and confer significant Na+ retention on the glucocorticoid corticosterone in adrenalectomized rats (20). 11-Dehydrocorticosterone (32) and 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone are strong inhibitors of 11beta -HSD2 (21). These agents all confer Na+ retention on glucocorticoids in adrenalectomized rat (20, 21).

Although there is abundant evidence to support the hypothesis that 11beta -HSD functions as a "protective" enzyme for MR, it is by no means clear that this hypothesis is sufficient to explain all aspects of receptor specificity in tissues that express both MR and GR. A number of observations suggest that the current paradigm, 11beta -HSD2 protection, is not sufficient to explain all observed phenomena and that other protective mechanisms may be involved. For example, it does not explain the observations that selective inhibitors of either isoform of 11beta -HSD can induce glucocorticoid-mediated Na+ reabsorption even though type 1 is characteristically found in tissues lacking MR. Moreover, 11beta -HSD inhibitors amplify the antinaturietic activity of aldosterone and deoxycorticosterone, which are not substrates for the enzyme (24), and Na+ retention may be induced by several "inactive" steroids (11-deoxycortisol and 11-dehydrocorticosterone), which neither are substrates for the enzyme nor bind to receptors (20, 37). In addition, the function of the enzyme is not clear in colon, where GR and MR apparently regulate differing pathways of Na+ (2). Finally, GR and MR are expressed in central nervous system tissues, which appear to be functionally unprotected by 11beta -HSD, further suggesting that other specificity-enhancing mechanisms for receptor activation may exist (10, 39).

The active Na+-transporting epithelial cell line, A6 (derived from toad kidney), possesses both MR and GR receptors (6, 43); however, active Na+ transport stimulation induced by both mineralocorticoids and glucocorticoids is thought to be mediated via GR (43). This transport stimulation is not reminiscent of that described for GR in the Na+-retaining segments of colon (2) but is in every way typical of MR-induced activation of the hormone response element (HRE) expressed as increase in amiloride-sensitive Na+ channels and Na+-K+-ATPase activity seen in MR "protected" tissues (16, 42). The major pathway of metabolism in A6 cells for both mineralocorticoids and glucocorticoids has been reported to be a steroid 6beta -hydroxylase enzyme activity that converts corticosterone and aldosterone to their 6beta -hydroxylated products (8, 14, 23). The enzyme is a cytochrome P-450 3A (CYP3A), as demonstrated by enzyme methodology (15) and mammalian probes (33). Immunohistochemistry in rat kidney localizes the CYP3A to the collecting duct (33). It is not known whether inhibition of steroid metabolism affects glucocorticoid-mediated transport events, although this has been proposed in the absence of 11beta -HSD (7). The availability of a cell line expressing both MR and GR without apparent protective enzyme but with an easily measured physiological response to steroids provides a model system to assess the role of other metabolic pathways on the regulation of access of glucocorticoids to MR. Experiments were therefore undertaken to reexamine the pathways of glucocorticoid metabolism and to determine the effects of the above 11beta -HSD-inhibiting steroidal substances on actions and metabolism of corticosterone on Isc in A6 cells. These experiments might shed further light on the protective mechanisms governing MR- and possibly GR-mediated Na+ transport in epithelial cells.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

A6 cells. All studies were performed on A6 cells grown on semipermeable supports. Cells were grown as described (43) in amphibian media (BioWhittaker, Walkersville, MD) with 10% fetal bovine serum (Sigma, St. Louis, MO) in an atmosphere of humidified air-4% CO2 at 28°C. Cells were grown on Millicell-HA inserts (Millipore, Bedford, MA). Transepithelial potential difference and Isc were measured using a sterile in-hood short-circuiting device as previously described (43).

Chemicals. 3alpha ,5beta -Tetrahydroprogesterone, 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone, 3alpha ,5beta -tetrahydropregnane, 11-dehydrocorticosterone, corticosterone, and chenodeoxycholic acid and cholic acid were obtained from Steraloids (Wilton, NH) and maintained as stock solutions at 10-3 M in absolute ethanol. All experiments were performed in serum-free media, and equivalent amounts of vehicle were added to control preparations.

11beta -HSD assays. Assays of 11beta -HSD isoforms 1 and 2 (11beta -HSD1 and 11beta -HSD2, respectively) were performed as previously described (21). For the 11beta -HSD1 assay, 50 µg of cell lysates were incubated at 37°C for 10 min with 5 µM corticosterone containing 0.5 µCi [3H]corticosterone in 50 mM Tris · HCl, pH 8.4, containing 3.4 mM NADP+ and 5 mM MgCl2 in a total volume of 250 µl. The enzymatic reaction was terminated by freezing in a dry ice-ethanol slurry. For the 11beta -HSD2 assay, 50 µg of cell lysate were incubated at 37°C for 1 h with 50 nM corticosterone containing 0.5 µCi [3H]corticosterone, 200 µM NAD+, and 5 mM MgCl2 in 50 mM Tris · HCl, pH 7.4, in a final incubation volume of 250 µl.

6beta -Hydroxylase assays. For assay of 6beta -hydroxylase catalytic activity, A6 cells were scraped from filters in PBS, centrifuged, and resuspended in 0.5 ml of 0.1 M K2PO4 buffer, pH 7.4. Cells were disrupted by sonication on ice and centrifuged at 100,000 g at 4°C for 30 min in a Sorvall (Wilmington, DE) ultracentrifuge. The resulting pellet was used as a microsomal preparation for assay as previously described (23). The pellet was resuspended in 0.4 ml of 0.1 M K2PO4 containing 2 mM EDTA and 25% glycerol, adjusted to pH 7.4 (buffer A). An aliquot was removed for protein determination, and 110 µl of the microsomal preparation were diluted to 400 µl with 5 mM K2PO4 buffer (pH 7.4) containing 1 mM NADPH, 50 mM sucrose, 3 mM MgCl2, and 10 nM [3H]corticosterone. The preparation was incubated for 45 min at 28°C, and the reaction was terminated by freezing.

In separate experiments, 10 nM [3H]corticosterone was added to the cells from the apical side in medium, and the cells were incubated for 1 h. Medium from the apical side was discarded, and the basal medium was collected. The cells were washed, scraped from the filters, and pelleted. Frozen media and pellets were then analyzed by HPLC.

HPLC. Aliquots of methanol extracts from incubation medium in the above experiments were diluted with water to 45% methanol (HPLC grade; Fisher Scientific, Medford, MA) and chromatographed using HPLC on a DuPont Zorbax C8 reversed-phase column at 44°C with 62% aqueous methanol. Radioactive metabolite peaks were detected by an on-line detection system (radiomatic model FLO-ONE/Beta, radiochromatography detector, Packard Instrument, Meriden, CT). Nonradioactive corticosterone, 11-dehydrocorticosterone, and 6beta -hydroxycorticosterone were used as HPLC standards, employing a photodiode array detector (Packard Instrument).

Immunoblot analysis. Immunoblot analysis of electrophoretically separated microsomal proteins was performed as previously described (23). Confluent A6 cells were scraped from filters and disrupted by sonication. A crude microsomal pellet was obtained by centrifugation at 100,000 g for 30 min, and proteins were subjected to electrophoresis on 15% SDS-PAGE, transferred to nitrocellulose, and reacted with anti-cytochrome P-450 IgG (kindly provided by Dr. Erin Schuetz, St. Jude Children's Research Hospital, Memphis, TN). Samples were then exposed to peroxidase-conjugated second antibody (rabbit anti-goat IgG, Sigma), and reaction was visualized by enhanced chemiluminescence technique.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects on Isc elicited by corticosterone in A6 cells. Initial experiments were designed to determine whether agents that inhibit 11beta -HSD in other systems had any agonist effect on Na+ transport in A6 cells and whether they enhanced the effect of corticosterone on Isc. The substances chosen for these experiments were carbenoxolone, two bile acids (chenodeoxycholic acid and cholic acid), two progesterone metabolites (3alpha ,5beta -tetrahydroprogesterone and 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone), and 11-dehydrocorticosterone, the end product of 11beta -HSD. In addition, 3alpha ,5beta -tetrahydropregnane, which is not a known substrate for or inhibitor of the enzyme, was employed. A similar protocol was used for all experiments. After overnight incubation in steroid-free medium, A6 cells were exposed to the agents or vehicle and Isc was measured hourly for 2 h. As shown in Table 1, none of the agents employed produced any significant increase in Isc, suggesting that they are not, in themselves, agonists for either GR- or MR-mediated Na+ transport. After this initial incubation, corticosterone was added to all cells (final concentration of 10 nM) and Isc measurements followed for an additional 3 h. The results in Table 1 demonstrate that all the 11beta -HSD inhibitors induced a significant increment in corticosterone-induced Isc that was detectable within 2 h. 3alpha ,5beta -Tetrahydropregnane, which does not inhibit either isoform of 11beta -HSD, partially antagonized the effect of corticosterone.

                              
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Table 1.   Effect of 11beta -HSD inhibitors on basal and corticosterone-induced Isc

As an additional control, the effects of two of the inhibitors on Isc were followed for the entire 5-h period to ensure that no late agonist effect of the agents was missed. 11beta -Hydroxy-3alpha ,5beta -tetrahydroprogesterone and chenodeoxycholic acid (both at 10-6 M) were added to A6 cells, and Isc was measured at 3 and 5 h following addition. Ratios of experimental to control Isc at 3 and 5 h were 0.99 ± 0.01 and 1.07 ± 0.10 for 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone, and 0.96 ± 0.04 and 1.05 ± 0.08 for chenodeoxycholic acid. There was no significant difference between Isc in control and inhibitor-treated cells over this time course.

Measurements for 11beta -HSD1 and 11beta -HSD2 enzyme activity in A6 cells. Although previous studies of corticosterone metabolism in A6 cells have not demonstrated any significant 11beta -HSD activity (7, 8, 13, 14), the findings with the above inhibitors suggested that this enzyme might be active in the cells. Cell lysates were examined for 11beta -HSD activity under conditions favoring either type 1 or type 2 isoforms as describe in METHODS. There was no evidence of metabolism of corticosterone to 11-dehydrocorticosterone in these cells under these conditions, although activity was readily seen in the toad urinary bladder cell lysates using these methods (Fig. 1). When whole cells were similarly incubated with isotopically labeled corticosterone for 2 h and whole cells and media were sampled, 11-dehydrocorticosterone was not seen, although a more polar peak consistent with 6beta -hydroxycorticosterone was detected (Fig. 2).


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Fig. 1.   Attempt to demonstrate 11beta -hydroxysteroid dehydroxgenase (11beta -HSD) (via HPLC) activity in A6 cells. Cell lysates were incubated with [3H]corticosterone (10-8 M), and lysates were examined under conditions favoring either 11beta -HSD1 or 11beta -HSD2 (see METHODS). No evidence of either isoform activity was seen. In contrast, toad urinary bladder cells readily metabolized [3H]corticosterone (compound B) to its 11-dehydro derivative (compound A). cpm, Counts per minute.


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Fig. 2.   HPLC showing synthesis of 6beta -hydroxycorticosterone (6beta -OH-B) from [3H]corticosterone (compound B; 10-8 M) in A6 cells (control) and inhibition when coincubated with 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone (11beta -OH-3alpha ,5alpha -THProg; 10-6 M).

Measurement of 6beta -hydroxylase activity in A6 cells. These findings tended to confirm the previous observation (8, 14) that 6beta -hydroxycorticosterone was the major metabolite of corticosterone in A6 cells. The next experiments examined whether the agents that accentuated/enhanced the action of corticosterone on Isc had any effect on the metabolism of corticosterone to its 6beta -hydroxy derivative. Similar to the example shown in Fig. 2, all agents examined significantly inhibited 6beta -hydroxylase activity at the concentrations that led to an enhancement of the corticosterone-induced current (Table 1). 3alpha ,5beta -Tetrahydropregnane, which did not enhance the effect of corticosterone on Isc, had no effect on 6beta -hydroxylase activity. Because there appeared to be some variability between percent enzyme inhibition produced by each agent and relative increase in Isc, we examined this relationship directly over a wide concentration range for a single inhibitor. A dose-response comparison of the effect of 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone on inhibition of 6beta -hydroxylase activity and enhancement of corticosterone-induced Isc is shown in Fig. 3. The degree of inhibition of 6beta -hydroxylase activity correlated with the stimulation of Na+ transport.


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Fig. 3.   Relationship between enzyme inhibition and transport stimulation by 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone. A6 cells were incubated with corticosterone (10-8 M) in the presence or absence of 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone at concentration ranging from 10-6 to 10-9 M (log dilutions). Increment of short-circuit current (Isc) measurements compared with that observed with corticosterone (corti) alone is plotted on the ordinate, and % inhibition of metabolism of corticosterone to its 6beta -hydroxy derivative is plotted on the abscissa. Correlation by linear regression analysis gives r value of 0.99, P < 0.01; n = 4-6 for each concentration of inhibitor.

Presence of CYP3A in A6 cells. To determine if the CYP3A present in liver and kidney (7, 23) and thought to mediate 6beta -hydroxylase activity was also present in A6 cells, immunoblot analysis of microsomal fractions of A6 was carried out, with a sample of rat hepatic microsomes examined as the control. Figure 4 demonstrates that the antibody to mammalian CYP3A recognizes a protein of the same molecular mass in A6 microsomes.


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Fig. 4.   Western blot analysis of A6 membranes. Fifty micrograms of A6 microsomes (lane 1) and 5 µg of rat hepatocyte microsomes from a dexamethasone-treated rat (lane 2) were resolved by 15% SDS-PAGE, transferred to nitrocellulose, and reacted with antibody to cytochrome P-450 3A (CYP3A) as described in METHODS. Molecular mass markers are shown at left.

Effects of troleandromycin on corticosterone-induced Isc in A6 cells. The effects of troleandromycin, a selective inhibitor of the CYP3A enzyme, were also examined. This agent inhibits steroid 6beta -hydroxylase (34). Incubation for 2 h with 10-6 M troleandromycin alone had no effect on basal Isc. However, troleandromycin significantly enhanced corticosterone-induced Isc at 2 h following addition of 10-8 M corticosterone (corticosterone increased Isc from 14.2 ± 0.9 to 25.5 ± 3.8 µA/cm2; corticosterone in the presence of troleandromycin increased Isc from 14.7 ± 1.3 to 41 ± 1.8 µA/cm2). This concentration of troleandromycin virtually completely inhibited 6beta -hydroxylase activity in our cells (data not shown).

Effects of MR and GR antagonists on corticosterone-induced Isc. The simplest explanation for these findings would be that unmetabolized corticosterone acted through its cognate receptor to produce the enhanced transport response when metabolism was inhibited. To examine this possibility, studies were then carried out with specific antagonists of GR and MR. As shown in Table 2, all Isc induced by corticosterone under the conditions of this study are mediated by GR, as it is specifically inhibited by excess RU-28486, a GR antagonist. RU-28318, a specific MR antagonist, had no effect on either basal or corticosterone-induced Na+ transport. The MR and GR antagonists were then employed to probe the additional effects of 10-8 M corticosterone on Isc caused by either of 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone, 3alpha ,5beta -tetrahydroprogesterone, or chenodeoxycholic acid. The results for each agent were qualitatively similar and are shown in Fig. 5. RU-28318, the MR antagonist, blocked the enhanced Isc induced by each agent so that the current observed in combination with corticosterone was not different from that seen with corticosterone alone. The GR antagonist RU-28486 reduced Isc but not to a level equal to control cells. These results indicate that the increment in Isc conferred on corticosterone by each of these substances is mediated through MR, whereas Isc induced by corticosterone alone is mediated through GR.

                              
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Table 2.   Effect of glucocorticoid receptor antagonist (RU-28486) and mineralocorticoid receptor antagonist (RU-28318) on corticosterone-induced Isc


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Fig. 5.   Top: effect of specific glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) antagonists on the increment in Isc induced by 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone in the presence of corticosterone. black-square, Corticosterone (10-8 M) alone; black-triangle, compound B + 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone (10-7 M); black-down-triangle , compound B + 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone + MR antagonist RU-28318 (10-6 M); black-lozenge , compound B + 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone + GR antagonist RU-28486 (10-6 M); bullet , control cells. 11beta -hydroxy-3alpha ,5beta -tetrahydroprogesterone was added at time 0. At 2 h, compound B and MR and GR antagonists were added. * Isc significantly greater than that seen with compound B alone; ** Isc significantly greater than control; n = 6 for each observation. Middle: similar experiment with chenodeoxycholic acid (10-6 M). Bottom: similar experiment with 3alpha ,5beta -tetrahydroprogesterone (10-6 M). For middle and bottom: bullet , controls; black-square, compound B alone; black-triangle, compound B + inhibitor; black-down-triangle , compound B + inhibitor + RU-28318; black-lozenge , compound B + inhibitor + RU-28486.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The A6 cell line has been widely used to study steroid regulation of Na+ transport in model epithelia (reviewed in Refs. 16 and 42). Although the cell line possesses both MR and GR, with GR in greater abundance (6, 43), activation of the transport response appears to be mediated primarily via GR and correlates well with occupancy of GR (34, 43). It is not clear what the function of MR is in this cell line. Unlike many mammalian or anuran tissues that express 11-dehydrocorticosterone activity in tissues that possess both MR and GR (9, 17, 21, 25, 28), A6 cells do not appear to express this protective enzyme under normal culture conditions (8, 14). The main pathway of steroid metabolism in A6 cells appears to be via steroid 6beta -hydroxylation (8, 14, 15).

Because results from mammalian studies suggested that the specificity of 11beta -HSD2 inhibition does not always correlate with the ability of inactive steroids to induce MR-mediated Na+ retention (20-22, 29), we sought to examine the effects of known inhibitors of both 11beta -HSD isoforms in a cell line that expressed both MR and GR but not 11-dehydrocorticosterone. Any effects on steroid action under such conditions would suggest that more than one "MR-protective" mechanism might exist. Our results confirm earlier studies that neither 11beta -HSD1 nor 11beta -HSD2 enzymatic activity is detectable, that the major pathway of glucocorticoid metabolism is by 6beta -hydroxylation, and that stimulation of Na+ transport under usual culture conditions is exclusively via GR (14, 18, 43).

We examined a variety of specific inhibitors of either 11beta -HSD1 or 11beta -HSD2 or inhibitors of both isoforms (20-22, 29, 37), all of which are known to confer MR activity on glucocorticoids in mammalian systems, for effects on basal or glucocorticoid-stimulated Na+ transport in A6 cells. None of the agents appears to have any agonist activity at the concentrations employed, yet all enhance the Na+ transport response induced by corticosterone. This enhancement occurs over a period of several hours, during which considerable metabolism of corticosterone to 6beta -hydroxycorticosterone was normally observed. Each of the agents examined inhibits 6beta -hydroxylase activity at the concentrations that enhance Na+ transport, and there is a close correlation between inhibition of enzyme activity and magnitude of the enhanced transport stimulation for the one inhibitor studied. This enzyme is really identifiable in A6 by an antibody to mammalian CYP3A, which has also been employed to identify the enzyme in the steroid- responsive collecting ducts of mammalian kidney (7). Finally, troleandromycin, an inhibitor of CYP3A activity with no known effects on 11beta -HSD, also confers enhanced transport stimulation on corticosterone.

Studies with specific antagonists of GR and MR indicate that the stimulation of Na+ transport induced by corticosterone alone is mediated exclusively via GR, as previously described (18, 43). However, the increment in transport seen with inhibition of 6beta -hydroxylase activity appears to be mediated via MR. The GR-induced transport response is not affected, suggesting that metabolism of corticosterone to 6beta -hydroxycorticosterone does not affect activation of GR under the conditions of these experiments. Indeed, 6beta -hydroxycorticosterone has not been described to have any activity at concentrations below 10-8 M (14). The metabolite has been described to have agonist activity at concentrations of 10-6 M that are not mediated via either MR or GR (14). Because corticosterone concentrations did not exceed 10-8 M, it is unlikely that metabolite concentrations could exceed these under the present conditions.

The simplest hypothesis to explain our findings would be that the 6beta -hydroxy metabolite of corticosterone serves to protect MR from corticosterone binding. In other words, corticosterone is normally metabolized to 6beta -hydroxycorticosterone, which acts as an antagonist of MR, leading to the sole occupancy of GR. In the presence of inhibitors of 6beta -hydroxylase, corticosterone could bind to both GR and MR and enhance transport. This hypothesis would require studies of specific binding of corticosterone to MR in the presence and absence of metabolism or, alternatively, studies of transcriptional activation under those conditions to be verified. The current results suggest that this enzyme may serve as a "guardian" mechanism protecting MR in A6 cells from excessive stimulation by the glucocorticoid corticosterone. The enzyme 6beta -hydroxylase is present mainly in liver of mammals (4) but is also expressed, albeit to a lesser degree, in human and rat kidneys (33). Because the same agents inhibit both 11beta -HSD and 6beta -hydroxylase activity, it is possible that the MR-mediated mineralocorticoid-like Na+ retention conferred by corticosterone in vivo in adrenalectomized rats by 3alpha ,5beta -tetrahydroprogesterone, chenodeoxycholic acid, and even carbenoxylone (20, 38) may be caused in part by 6beta -hydroxylase inhibition.

Further investigations are necessary to help better understand the respective role(s) and function(s) of MR and GR and 6beta -hydroxylase in A6 cells. The unusual aspect of A6 cells is that the transport response, whether initiated by mineralocorticoid or glucocorticoid, is mediated under standard culture conditions via GR. Eaton and colleagues (D. Eaton, personal communication) have demonstrated that 11beta -HSD activity may be induced by preincubation with glucocorticoid and under these conditions transport stimulation is mediated by MR. This would be consistent with the notion that 11beta -HSD "protects" both MR and GR in mineralocorticoid target tissues (10). In our experiments, in the absence of 11beta -HSD, stimulation of the physiological response is via GR but is enhanced by an MR-mediated component when 6beta -hydroxylase is inhibited. This synergism between MR and GR is intriguing, especially since both are thought to bind to consensus HRE (30, 31). Activated MR displacing GR from such a site might be expected to downregulate the response (43). In fact, the physiological response is amplified. This could represent a physiological expression of heterodimerization between GR and MR as has been described for central nervous system tissues, which, like A6 cells, possess both MR and GR.

The present studies indicate that A6 cells will provide a good steroid-responsive target epithelial cell model to explore and determine other enzyme or specific protein-containing mechanisms/processes that govern the magnitude of the MR-signaling Na+ transport mechanism. These mechanisms may be distinct from the 11beta -HSD guardian mechanism and may also be present and play a role in other mineralocorticoid target tissues, including mammalian kidney. In fact, these findings that 11beta -HSD inhibitors also inhibit 6beta -hydroxylase may offer an explanation for the inconsistencies in experimental tests of the "MR protective hypothesis" in mammals described above.

    ACKNOWLEDGEMENTS

We thank Michael West for excellent technical assistance and Elizabeth Gifford for excellent secretarial assistance.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-21404 and DK-47874, by the Miriam Hospital Research Foundation, and by a National Kidney Foundation Young Investigator Grant (to M. D. Rokaw).

Address for reprint requests: J. P. Johnson, Dept. of Medicine/Renal-Electrolyte Division, The Univ. of Pittsburgh Medical Center, A935 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15213.

Received 23 May 1997; accepted in final form 23 January 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

1.   Armanini, D., I. Karobowiak, J. W. Funder, and W. R. Adam. The mechanism of mineralocorticoid action of carbenoxolone. Endocrinology 111: 1683-1686, 1982[Abstract].

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3.   Brem, A. S., K. L. Matheson, T. Conca, and D. J. Morris. Effect of carbenoxolone on glucocorticoid metabolism and Na transport in toad bladder. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F700-F704, 1989[Abstract/Free Full Text].

4.   Brem, A. S., K. L. Matheson, S. A. Latif, and D. J. Morris. Activity of 11beta -hydroxysteroid dehydrogenase in toad bladder: effects of 11-dehydrocorticosterone. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F854-F858, 1993[Abstract/Free Full Text].

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