Download A second enzyme protecting mineralocorticoid receptors from

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
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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).
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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.
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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.
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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.
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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).
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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.
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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.
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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.
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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.
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