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A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy DAVID J. MORRIS,1 SYED A. LATIF,1 MICHAEL D. ROKAW,2 CHARLES O. WATLINGTON,3 AND JOHN P. JOHNSON2 1Department of Pathology and Laboratory Medicine, The Miriam Hospital, Lifespan, and Brown University School of Medicine, Providence, Rhode Island 02903; 2Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213; and 3Department of Medicine, Division of Endocrinology, Medical College of Virginia, Richmond, Virginia 23298 steroid 6b-hydroxylase; sodium transport 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, IN RECENT YEARS 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 Na1 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, 11bhydroxysteroid dehydrogenase (11b-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 11b-HSD acts as a guardian, conferring specificity on MR-mediated actions on Na1 (17, 25–27, 32, 40, 41, 44). Experiments from our laboratories have shown that the glucocorticoids, which normally do not elicit the usual Na1retaining response (as does the mineralocorticoid aldosterone), do, however, display a potent Na1 retention and amplification of K1 excretion (36) in adrenalectomized rats pretreated with the 11b-HSD inhibitor carbenoxolone (a succinate of glycyrrhetinic acid). The ‘‘mineralocorticoid-like’’ effects on Na1 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 11bHSD2 enzymatic activity, the short-circuit current (Isc, active Na1 transport) caused by glucocorticoids is enhanced when carbenoxalone is added to the incubation medium (3, 12). There are at least two isoforms of 11b-HSD: 1) 11b-HSD1 in liver and proximal portions of the renal tubule of rats, which is bidirectional and NADP1 dependent, and 2) the NAD1-dependent 11b-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). 3a,5b-Tetrahydroprogesterone and the bile acid chenodeoxycholic acid both inhibit 11b-HSD1 (20, 29) and confer significant Na1 retention on the 0363-6143/98 $5.00 Copyright r 1998 the American Physiological Society C1245 Downloaded from http://ajpcell.physiology.org/ by 10.220.33.6 on May 8, 2017 Morris, David J., Syed A. Latif, Michael D. Rokaw, Charles O. Watlington, and John P. Johnson. A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy. Am. J. Physiol. 274 (Cell Physiol. 43): C1245–C1252, 1998.—We have confirmed that A6 cells (derived from kidney of Xenopus laevis), which contain both mineralocorticoid and glucocorticoid receptors, do not normally possess 11b-hydroxysteroid dehydroxgenase (11bHSD1 or 11b-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 6b-hydroxylase that transforms corticosterone to 6b-hydroxycorticosterone. This hydroxylase is cytochrome P-450 3A (CYP3A). We have now determined the effects of 3a,5b-tetrahydroprogesterone and chenodeoxycholic acid (both inhibitors of 11b-HSD1) and 11-dehydrocorticosterone and 11b-hydroxy-3a,5b-tetrahydroprogesterone (inhibitors of 11b-HSD2) and carbenoxalone, which inhibits both 11b-HSD1 and 11b-HSD2, on the actions and metabolism of corticosterone and active Na1 transport [short-circuit current (Isc )] in A6 cells. All of these 11b-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 6bhydroxycorticosterone in A6 cells, and a linear relationship existed between 6b-hydroxylase inhibition and the MRmediated increase in Isc in the one inhibitor tested. Troleandomycin, a selective inhibitor of CYP3A, inhibited 6b-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 6b-hydroxylase activity in these cells and that this 6bhydroxylase (CYP3A) may be protecting the expression of corticosterone-induced active Na1 transport in A6 cells by MR-mediated mechanism(s). C1246 STEROID METABOLISM IN A6 CELLS reexamine the pathways of glucocorticoid metabolism and to determine the effects of the above 11b-HSDinhibiting 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 Na1 transport in epithelial cells. METHODS 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. 3a,5b-Tetrahydroprogesterone, 11b-hydroxy3a,5b-tetrahydroprogesterone, 3a,5b-tetrahydropregnane, 11dehydrocorticosterone, corticosterone, and chenodeoxycholic acid and cholic acid were obtained from Steraloids (Wilton, NH) and maintained as stock solutions at 1023 M in absolute ethanol. All experiments were performed in serum-free media, and equivalent amounts of vehicle were added to control preparations. 11b-HSD assays. Assays of 11b-HSD isoforms 1 and 2 (11b-HSD1 and 11b-HSD2, respectively) were performed as previously described (21). For the 11b-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 NADP1 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 11b-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 NAD1, and 5 mM MgCl2 in 50 mM Tris · HCl, pH 7.4, in a final incubation volume of 250 µl. Table 1. Effect of 11b-HSD inhibitors on basal and corticosterone-induced Isc Ratio of Isc Agent Cholic acid (1025 M) 11 Ketoprogesterone (1027 M) 11b-OH-3a,5a-tetrahydroprogesterone (1027 M) Chenodeoxycholic acid (1026 M) 3a,5b-Tetraprogesterone (1026 M) 11-Dehydrocorticosterone (1025 M) Carbenoxylone (1025 M) 3a,5b-Tetrahydropregnane (1026 M) 2h 1 h1B 2 h1B 3 h1B 6b-Hydroxylase Inhibition, % 0.99 1.17* 1.29* 1.25* ND 1.1 1.27* 1.21* 1.27* 84.4 1.06 1.17* 1.25* 1.54* 48.9 1.05 1.21* 1.21* 1.20* 78.1 1.03 1.07 1.10* 1.15* 70 0.98 1.36* 1.15* 1.32* ND 1.02 1.52* 1.26* 1.22* ND 0.76 0.64 0.56 0 Values are the ratios of short-circuit current (Isc ) using various agents compared with control for the first 2-h period. Subsequently, 1028 M corticosterone (B) was added to all cells and values are the ratio of B 1 agent to B alone; n 5 4–6 for each observation. Inhibition of 6b-hydroxylase activity was measured separately as described in METHODS. * Isc of B 1 agent was significantly greater than B alone, P , 0.05 by t-test of independent means. ND, not done. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.6 on May 8, 2017 glucocorticoid corticosterone in adrenalectomized rats (20). 11-Dehydrocorticosterone (32) and 11b-hydroxy3a,5b-tetrahydroprogesterone are strong inhibitors of 11b-HSD2 (21). These agents all confer Na1 retention on glucocorticoids in adrenalectomized rat (20, 21). Although there is abundant evidence to support the hypothesis that 11b-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, 11b-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 11b-HSD can induce glucocorticoidmediated Na1 reabsorption even though type 1 is characteristically found in tissues lacking MR. Moreover, 11b-HSD inhibitors amplify the antinaturietic activity of aldosterone and deoxycorticosterone, which are not substrates for the enzyme (24), and Na1 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 Na1 (2). Finally, GR and MR are expressed in central nervous system tissues, which appear to be functionally unprotected by 11b-HSD, further suggesting that other specificity-enhancing mechanisms for receptor activation may exist (10, 39). The active Na1-transporting epithelial cell line, A6 (derived from toad kidney), possesses both MR and GR receptors (6, 43); however, active Na1 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 Na1-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 Na1 channels and Na1K1-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 6b-hydroxylase enzyme activity that converts corticosterone and aldosterone to their 6b-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 11b-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 STEROID METABOLISM IN A6 CELLS 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 6b-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 Fig. 1. Attempt to demonstrate 11b-hydroxysteroid dehydroxgenase (11b-HSD) (via HPLC) activity in A6 cells. Cell lysates were incubated with [3H]corticosterone (1028 M), and lysates were examined under conditions favoring either 11b-HSD1 or 11b-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. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.6 on May 8, 2017 6b-Hydroxylase assays. For assay of 6b-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. C1247 C1248 STEROID METABOLISM IN A6 CELLS second antibody (rabbit anti-goat IgG, Sigma), and reaction was visualized by enhanced chemiluminescence technique. RESULTS Fig. 2. HPLC showing synthesis of 6b-hydroxycorticosterone (6b-OH-B) from [3H]corticosterone (compound B; 1028 M) in A6 cells (control) and inhibition when coincubated with 11b-hydroxy-3a,5b-tetrahydroprogesterone (11b-OH-3a,5a-THProg; 1026 M). Downloaded from http://ajpcell.physiology.org/ by 10.220.33.6 on May 8, 2017 Effects on Isc elicited by corticosterone in A6 cells. Initial experiments were designed to determine whether agents that inhibit 11b-HSD in other systems had any agonist effect on Na1 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 (3a,5btetrahydroprogesterone and 11b-hydroxy-3a,5b-tetrahydroprogesterone), and 11-dehydrocorticosterone, the end product of 11b-HSD. In addition, 3a,5b-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 Na1 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 11b-HSD inhibitors induced a significant increment in corticosterone-induced Isc that was detectable within 2 h. 3a,5b-Tetrahydropregnane, which does not inhibit either isoform of 11b-HSD, partially antagonized the effect of corticosterone. 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. 11b-Hydroxy-3a,5b-tetrahydroprogesterone and chenodeoxycholic acid (both at 1026 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 6 0.01 and 1.07 6 0.10 for 11b-hydroxy3a,5b-tetrahydroprogesterone, and 0.96 6 0.04 and 1.05 6 0.08 for chenodeoxycholic acid. There was no significant difference between Isc in control and inhibitor-treated cells over this time course. Measurements for 11b-HSD1 and 11b-HSD2 enzyme activity in A6 cells. Although previous studies of corticosterone metabolism in A6 cells have not demonstrated any significant 11b-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 11b-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, 11dehydrocorticosterone was not seen, although a more STEROID METABOLISM IN A6 CELLS polar peak consistent with 6b-hydroxycorticosterone was detected (Fig. 2). Measurement of 6b-hydroxylase activity in A6 cells. These findings tended to confirm the previous observation (8, 14) that 6b-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 6b-hydroxy derivative. Similar to the example shown in Fig. 2, all agents examined significantly inhibited 6b-hydroxylase activity at the concentrations that led to an enhancement of the corticosterone-induced current (Table 1). 3a,5b-Tetrahydropregnane, which did not enhance the effect of corticosterone on Isc, had no effect on 6b-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 11b-hydroxy3a,5b-tetrahydroprogesterone on inhibition of 6bhydroxylase activity and enhancement of corticosteroneinduced Isc is shown in Fig. 3. The degree of inhibition of 6b-hydroxylase activity correlated with the stimulation of Na1 transport. Presence of CYP3A in A6 cells. To determine if the CYP3A present in liver and kidney (7, 23) and thought to mediate 6b-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. 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 6b-hydroxylase (34). Incubation for 2 h with 1026 M troleandromycin alone had no effect on basal Isc. However, troleandromycin significantly enhanced corticosterone-induced Isc at 2 h following addition of 1028 M corticosterone (corticosterone increased Isc from 14.2 6 0.9 to 25.5 6 3.8 µA/cm2; corticosterone in the presence of troleandromycin increased Isc from 14.7 6 1.3 to 41 6 1.8 µA/cm2 ). This concentration of troleandromycin virtually completely inhibited 6b-hydroxylase activity in our cells (data not shown). Effects of MR and GR antagonists on corticosteroneinduced 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 Na1 transport. The MR and GR antagonists were then employed to probe the additional effects of 1028 M corticosterone on Isc caused by either of 11b-hydroxy-3a,5b-tetrahydroprogesterone, 3a,5b-tetrahydroprogesterone, or chenodeoxycholic acid. The results for each agent were qualitatively similar and are shown in Fig. 5. RU28318, 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 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% SDSPAGE, transferred to nitrocellulose, and reacted with antibody to cytochrome P-450 3A (CYP3A) as described in METHODS. Molecular mass markers are shown at left. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.6 on May 8, 2017 Fig. 3. Relationship between enzyme inhibition and transport stimulation by 11b-hydroxy-3a,5b-tetrahydroprogesterone. A6 cells were incubated with corticosterone (1028 M) in the presence or absence of 11b-hydroxy-3a,5b-tetrahydroprogesterone at concentration ranging from 1026 to 1029 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 6b-hydroxy derivative is plotted on the abscissa. Correlation by linear regression analysis gives r value of 0.99, P , 0.01; n 5 4–6 for each concentration of inhibitor. C1249 C1250 STEROID METABOLISM IN A6 CELLS Table 2. Effect of glucocorticoid receptor antagonist (RU-28486) and mineralocorticoid receptor antagonist (RU-28318) on corticosterone-induced Isc Isc , µA/cm2 Control B (1028 M) B (1028 M) 1 RU-28486 (1026 M) B (1028 M) 1 RU-28318 (1026 M) Basal 3h 6 6 0.7 5.25 6 1 6 6 0.7 5.1 6 0.6 6.1 6 1.0 15 6 1* 5.25 6 1.2 15.1 6 0.7* Values are means 6 SE. Cells were incubated with B, B 1 RU-28486, B 1 RU-28318, or diluent for 3 h, and Isc was measured. * Significantly different from control, P , 0.05. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.6 on May 8, 2017 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. DISCUSSION The A6 cell line has been widely used to study steroid regulation of Na1 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 6b-hydroxylation (8, 14, 15). Because results from mammalian studies suggested that the specificity of 11b-HSD2 inhibition does not always correlate with the ability of inactive steroids to induce MR-mediated Na1 retention (20–22, 29), we sought to examine the effects of known inhibitors of both 11b-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 11b-HSD1 nor 11b-HSD2 enzymatic activity is detectable, that the major pathway of glucocorticoid metabolism is by 6b-hydroxylation, and that stimulation of Na1 transport under usual culture conditions is exclusively via GR (14, 18, 43). We examined a variety of specific inhibitors of either 11b-HSD1 or 11b-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 Na1 transport in A6 cells. None of the agents appears to have any agonist activity at the concentrations employed, yet all enhance the Na1 transport response induced by corticosterone. This enhancement occurs over a period of several hours, during which considerable metabolism Fig. 5. Top: effect of specific glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) antagonists on the increment in Isc induced by 11b-hydroxy-3a,5b-tetrahydroprogesterone in the presence of corticosterone. j, Corticosterone (1028 M) alone; l, compound B 1 11bhydroxy-3a,5b-tetrahydroprogesterone (1027 M); m, compound B 1 11b-hydroxy-3a,5b-tetrahydroprogesterone 1 MR antagonist RU-28318 (1026 M); p, compound B 1 11b-hydroxy-3a,5b-tetrahydroprogesterone 1 GR antagonist RU-28486 (1026 M); r, control cells. 11b-hydroxy-3a,5b-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 5 6 for each observation. Middle: similar experiment with chenodeoxycholic acid (1026 M). Bottom: similar experiment with 3a,5b-tetrahydroprogesterone (1026 M). For middle and bottom: r, controls; j, compound B alone; l, compound B 1 inhibitor; m, compound B 1 inhibitor 1 RU-28318; p, compound B 1 inhibitor 1 RU-28486. STEROID METABOLISM IN A6 CELLS GR. Eaton and colleagues (D. Eaton, personal communication) have demonstrated that 11b-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 11b-HSD ‘‘protects’’ both MR and GR in mineralocorticoid target tissues (10). In our experiments, in the absence of 11b-HSD, stimulation of the physiological response is via GR but is enhanced by an MR-mediated component when 6b-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 Na1 transport mechanism. These mechanisms may be distinct from the 11b-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 11b-HSD inhibitors also inhibit 6b-hydroxylase may offer an explanation for the inconsistencies in experimental tests of the ‘‘MR protective hypothesis’’ in mammals described above. We thank Michael West for excellent technical assistance and Elizabeth Gifford for excellent secretarial assistance. 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 1. Armanini, D., I. Karobowiak, J. W. Funder, and W. R. Adam. The mechanism of mineralocorticoid action of carbenoxolone. Endocrinology 111: 1683–1686, 1982. 2. Bastl, C. P., and J. P. Hayslett. The cellular action of aldosterone in target epithelia. Kidney Int. 42: 250–264, 1992. 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. 4. Brem, A. S., K. L. Matheson, S. A. Latif, and D. J. Morris. Activity of 11b-hydroxysteroid dehydrogenase in toad bladder: effects of 11-dehydrocorticosterone. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F854–F858, 1993. 5. Bush, I. E., S. A. Hunter, and R. A. Meigs. Metabolism of 11-oxygenated steroids. Biochem. J. 107: 239–257, 1968. 6. Claire, M., B. Mackard, M. Lombes, M. Oblin, J. Bonvalet, and N. Farman. Aldosterone receptors in A6 cells: physicochemical characterization and autoradiographic study. Am. J. Physiol. 257 (Cell. Physiol. 26): C665–C677, 1989. 7. Clore, J., A. Schoolwerth, and C. O. Watlington. When is cortisol a mineralocorticoid? Kidney Int. 42: 1297–1308, 1992. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.6 on May 8, 2017 of corticosterone to 6b-hydroxycorticosterone was normally observed. Each of the agents examined inhibits 6b-hydroxylase activity at the concentrations that enhance Na1 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 steroidresponsive collecting ducts of mammalian kidney (7). Finally, troleandromycin, an inhibitor of CYP3A activity with no known effects on 11b-HSD, also confers enhanced transport stimulation on corticosterone. Studies with specific antagonists of GR and MR indicate that the stimulation of Na1 transport induced by corticosterone alone is mediated exclusively via GR, as previously described (18, 43). However, the increment in transport seen with inhibition of 6b-hydroxylase activity appears to be mediated via MR. The GR-induced transport response is not affected, suggesting that metabolism of corticosterone to 6b-hydroxycorticosterone does not affect activation of GR under the conditions of these experiments. Indeed, 6b-hydroxycorticosterone has not been described to have any activity at concentrations below 1028 M (14). The metabolite has been described to have agonist activity at concentrations of 1026 M that are not mediated via either MR or GR (14). Because corticosterone concentrations did not exceed 1028 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 6b-hydroxy metabolite of corticosterone serves to protect MR from corticosterone binding. In other words, corticosterone is normally metabolized to 6b-hydroxycorticosterone, which acts as an antagonist of MR, leading to the sole occupancy of GR. In the presence of inhibitors of 6b-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 6b-hydroxylase is present mainly in liver of mammals (4) but is also expressed, albeit to a lesser degree, in human and rat kidneys (33). 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