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
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ABSTRACT |
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We have confirmed that A6 cells (derived from
kidney of Xenopus laevis), which
contain both mineralocorticoid and glucocorticoid receptors, do not
normally possess 11-hydroxysteroid dehydroxgenase (11
-HSD1 or
11
-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 6
-hydroxylase that transforms corticosterone to
6
-hydroxycorticosterone. This hydroxylase is cytochrome
P-450 3A (CYP3A). We have now
determined the effects of 3
,5
-tetrahydroprogesterone and
chenodeoxycholic acid (both inhibitors of 11
-HSD1) and
11-dehydrocorticosterone and
11
-hydroxy-3
,5
-tetrahydroprogesterone (inhibitors of
11
-HSD2) and carbenoxalone, which inhibits both 11
-HSD1 and
11
-HSD2, on the actions and metabolism of corticosterone and active
Na+ transport [short-circuit
current
(Isc)] in
A6 cells. All of these 11
-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
6
-hydroxycorticosterone in A6 cells, and a linear relationship
existed between 6
-hydroxylase inhibition and the MR-mediated
increase in Isc
in the one inhibitor tested. Troleandomycin, a selective inhibitor of
CYP3A, inhibited 6
-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 6
-hydroxylase activity in these
cells and that this 6
-hydroxylase (CYP3A) may be protecting the
expression of corticosterone-induced active Na+ transport in A6 cells by
MR-mediated mechanism(s).
steroid 6-hydroxylase; sodium transport
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INTRODUCTION |
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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,
11-hydroxysteroid dehydrogenase (11
-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 11-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 11
-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 11
-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 11-HSD:
1) 11
-HSD1 in liver and proximal
portions of the renal tubule of rats, which is bidirectional and
NADP+ dependent, and
2) the
NAD+-dependent 11
-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). 3
,5
-Tetrahydroprogesterone and the
bile acid chenodeoxycholic acid both inhibit 11
-HSD1 (20, 29) and
confer significant Na+ retention
on the glucocorticoid corticosterone in adrenalectomized rats (20).
11-Dehydrocorticosterone (32) and
11
-hydroxy-3
,5
-tetrahydroprogesterone are strong inhibitors of
11
-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
11-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, 11
-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 11
-HSD can induce glucocorticoid-mediated Na+ reabsorption even though type
1 is characteristically found in tissues lacking MR. Moreover,
11
-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 11
-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 6-hydroxylase enzyme activity that converts corticosterone and aldosterone to their
6
-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 11
-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
11
-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.
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METHODS |
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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.
3,5
-Tetrahydroprogesterone,
11
-hydroxy-3
,5
-tetrahydroprogesterone,
3
,5
-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.
11-HSD assays.
Assays of 11
-HSD isoforms 1 and 2 (11
-HSD1 and 11
-HSD2,
respectively) were performed as previously described (21). For the
11
-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 11
-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.
6-Hydroxylase assays.
For assay of 6
-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.
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
6-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.
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RESULTS |
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Effects on Isc elicited
by corticosterone in A6 cells.
Initial experiments were designed to determine whether agents that
inhibit 11-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 (3
,5
-tetrahydroprogesterone and
11
-hydroxy-3
,5
-tetrahydroprogesterone), and
11-dehydrocorticosterone, the end product of 11
-HSD. In addition,
3
,5
-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 11
-HSD inhibitors induced a significant increment in
corticosterone-induced
Isc that was
detectable within 2 h. 3
,5
-Tetrahydropregnane, which does not
inhibit either isoform of 11
-HSD, partially antagonized the effect
of corticosterone.
|
Measurements for 11-HSD1 and 11
-HSD2
enzyme activity in A6 cells.
Although previous studies of corticosterone metabolism in A6 cells have
not demonstrated any significant 11
-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 11
-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 6
-hydroxycorticosterone was detected (Fig.
2).
|
|
Measurement of 6-hydroxylase activity in A6 cells.
These findings tended to confirm the previous observation (8, 14) that
6
-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 6
-hydroxy
derivative. Similar to the example shown in Fig. 2, all agents examined
significantly inhibited 6
-hydroxylase activity at the concentrations
that led to an enhancement of the corticosterone-induced current (Table
1). 3
,5
-Tetrahydropregnane, which did not enhance the effect of
corticosterone on
Isc, had no
effect on 6
-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
11
-hydroxy-3
,5
-tetrahydroprogesterone on inhibition of
6
-hydroxylase activity and enhancement of corticosterone-induced Isc is shown in
Fig. 3. The degree of inhibition of
6
-hydroxylase activity correlated with the stimulation of
Na+ transport.
|
Presence of CYP3A in A6 cells.
To determine if the CYP3A present in liver and kidney (7, 23) and
thought to mediate 6-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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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 6-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 6
-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 108 M
corticosterone on
Isc caused by
either of 11
-hydroxy-3
,5
-tetrahydroprogesterone, 3
,5
-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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DISCUSSION |
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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 6-hydroxylation (8, 14, 15).
Because results from mammalian studies suggested that the specificity
of 11-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 11
-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
11
-HSD1 nor 11
-HSD2 enzymatic activity is detectable, that the
major pathway of glucocorticoid metabolism is by 6
-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 11-HSD1 or
11
-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 6
-hydroxycorticosterone was normally observed.
Each of the agents examined inhibits 6
-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 11
-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 6-hydroxylase activity appears to be mediated via
MR. The GR-induced transport response is not affected, suggesting that
metabolism of corticosterone to 6
-hydroxycorticosterone does not
affect activation of GR under the conditions of these experiments.
Indeed, 6
-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
6-hydroxy metabolite of corticosterone serves to protect MR from
corticosterone binding. In other words, corticosterone is normally
metabolized to 6
-hydroxycorticosterone, which acts as an antagonist
of MR, leading to the sole occupancy of GR. In the presence of
inhibitors of 6
-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 6
-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
11
-HSD and 6
-hydroxylase activity, it is possible that the
MR-mediated mineralocorticoid-like
Na+ retention conferred by
corticosterone in vivo in adrenalectomized rats by
3
,5
-tetrahydroprogesterone, chenodeoxycholic acid, and even
carbenoxylone (20, 38) may be caused in part by 6
-hydroxylase inhibition.
Further investigations are necessary to help better understand the
respective role(s) and function(s) of MR and GR and 6-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 11
-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 11
-HSD "protects" both MR and
GR in mineralocorticoid target tissues (10). In our experiments, in the
absence of 11
-HSD, stimulation of the physiological response is via
GR but is enhanced by an MR-mediated component when
6
-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 11-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
11
-HSD inhibitors also inhibit 6
-hydroxylase may offer an
explanation for the inconsistencies in experimental tests of the "MR
protective hypothesis" in mammals described above.
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ACKNOWLEDGEMENTS |
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We thank Michael West for excellent technical assistance and Elizabeth Gifford for excellent secretarial assistance.
![]() |
FOOTNOTES |
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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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