© 1995 American Heart Association, Inc.
Articles |
Corticosterone Metabolism and Effects on Angiotensin II Receptors in Vascular Smooth Muscle
From the Division of Nephrology, Department of Medicine, Medical University of South Carolina, Charleston.
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Abstract It has been postulated that mineralocorticoids can bind to corticosteroid receptors in the kidney, because glucocorticoids are metabolized to inactive compounds. The present study was performed to delineate glucocorticoid metabolism by rat vascular tissue and to determine the activity of these metabolites. Vascular segments converted 25% to 30% of corticosterone (compound B), the major glucocorticoid in the rat, to 11-dehydrocorticosterone (compound A) but not to aldosterone or 6ß-hydroxycorticosterone. In cultured vascular smooth muscle cells, 10% of compound B was converted to compound A, whereas >60% of compound A was converted to compound B. The 11ß-hydroxysteroid dehydrogenase inhibitor carbenoxolone (1 µmol/L) completely blocked conversion in both directions. Whereas 6ß-hydroxycorticosterone did not upregulate angiotensin II receptor binding (a marker for corticosteroid action in vascular smooth muscle), compound A caused concentration-dependent upregulation. Compound A was almost (75%) as effective and as potent as compound B in upregulating angiotensin II binding. Upregulation elicited by exposure to compound A persisted in the presence of 1 µmol/L carbenoxolone, which completely prevented the conversion of compound A to compound B. Compound A, even in the presence of carbenoxolone, effected other glucocorticoid actions by inhibiting cell growth and potentiating angiotensin II–stimulated inositol phosphate formation. In summary, compound B and compound A are interconverted in vascular tissue, and the latter displays significant glucocorticoid action. The concentration excess of compound B in the circulation and the activity of its metabolite compound A will make it difficult for mineralocorticoids to gain access to corticosteroid receptors in the vasculature.
Key Words: angiotensin II • vascular smooth muscle • corticosterone • 11-dehydrocorticosterone
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Introduction |
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Glucocorticoids are essential for the maintenance of blood pressure and, in excess, cause hypertension. Although the mechanisms of the blood pressure–elevating effects of glucocorticoids are not completely worked out, increases in peripheral vascular resistance contribute substantially.1 2 3 Glucocorticoids (and mineralocorticoids as well) increase vascular tone by potentiating the vasoconstrictor action of a number of pressor hormones, including
-adrenergic agonists4 5 6 7 and angiotensin
II (Ang II),4 8 and the first step in this potentiation is
upregulation of the receptors in vascular smooth muscle for these
pressor hormones. Ang II receptor number is increased in resistance
vessels from animals made hypertensive with
corticosteroids9 10 11 and in vascular smooth
muscle cells (VSMCs) exposed in culture to a number of natural and
synthetic glucocorticoids and mineralocorticoids.11 12 13 It
is most likely that corticosteroids upregulate Ang II
receptors by inducing the gene for the Ang II AT1 receptor,
the predominant Ang II receptor subtype in vascular smooth muscle. The
presence of specific receptors for glucocorticoids and
mineralocorticoids in vascular smooth muscle has been
documented,14 15 16 and incubation of cultured VSMCs with the
synthetic glucocorticoid dexamethasone resulted in
increases in steady state levels of AT1 receptor
mRNA.17 18 Although it is well established that glucocorticoids potentiate Ang II receptor function, it is unclear whether glucocorticoid metabolites do likewise. An obvious homeostatic role for metabolism is limitation of hormone action by hormone inactivation. Most of our knowledge of metabolism of glucocorticoids (cortisol in humans and corticosterone [compound B] in rats) and of activity of metabolites of compound B derives from studies in renal and hepatic tissue. The 6ß-hydroxylase metabolite of compound B, 6ß-hydroxycorticosterone, stimulates sodium transport in certain renal preparations.19 More attention has been paid to the 11ß-hydroxysteroid dehydrogenase (11ßOHSD) product of compound B, 11-dehydrocorticosterone (compound A). Funder et al20 have suggested that the abundance of 11ßOHSD in the kidney explains how the kidney can be a mineralocorticoid target tissue despite the facts that circulating compound B concentrations exceed circulating mineralocorticoid (ie, aldosterone) concentrations by 1000-fold and that compound B and aldosterone bind with comparable affinities to isolated preparations of mineralocorticoid receptors. This theory assumes that 11ßOHSD converts the active compound B to an inactive product, compound A. However, recent studies suggest that compound A may not be inactive in renal tissue.21 22
Patterns of metabolism of compound B by vascular smooth muscle and activity of these metabolites in the blood vessel have not been investigated in detail. Several studies over the past few years have demonstrated 11ßOHSD activity in vascular smooth muscle,23 24 25 suggesting that the vasculature may be a mineralocorticoid target tissue like the kidney. Compound A would be generated within VSMCs upon metabolism of compound B by 11ßOHSD. Whether VSMCs contain 6ß-hydroxylase is unknown. It is also unknown whether compound A or 6ß-hydroxycorticosterone, like compound B, potentiates Ang II action in VSMCs.
The purpose of these studies, therefore, was to test the hypothesis that metabolites of compound B potentiate Ang II action in vascular smooth muscle. Only metabolites previously demonstrated to possess activity in nonvascular tissue were studied in vascular tissue. After delineating compound B metabolism in intact blood vessels and cultured VSMCs, we determined how these metabolites affect Ang II receptor binding, Ang II–stimulated signal transduction, and Ang II–stimulated vasoconstriction.
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Materials and Methods |
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General Methods
Unless otherwise indicated, all experiments were performed at 37°C. Control treatment was exposure to equal concentrations (<1%) of the steroid vehicle ethanol. Data are presented as mean±SEM.
Preparation of [3H]Compound A
Four kidneys were removed from 250- to 300-g
Sprague-Dawley rats and homogenized with a
polytron (Kinematica) for 30 seconds in 5 mL Krebs-Henseleit buffer.
[3H]Compound B was converted to
[3H]compound A in identical reactions in 12 test tubes
with polytetrafluoroethylene
(Teflon)-coated tops, each of which contained 275 µL renal
homogenate, 3 µL ethanol, 3 µL 20% bovine serum
albumin, 3 µL 20 mmol/L NADP, and 0.1 nmol
[3H]compound B. The reaction proceeded for 4 hours and
was stopped with enough chloroform/methanol (1:2) to achieve a single
phase. After a 24-hour extraction period, two phases were created by
the addition of H2O. The organic phase was isolated, dried
with N2, dissolved in 250 µL ethanol, and spotted
on silica G thin-layer chromatography plates (250
µm thick) in 12 separate lanes. Unlabeled compound A and compound B
also were spotted. Migration solution consisted of chloroform/ethanol
(92:8). Silica corresponding to compound A was scraped from all 12
lanes and combined (Rf for compound B is 0.57, and
Rf for compound A is 0.71). Lipids were extracted from the
silica with 2 mL chloroform/methanol (1:2) for 24 hours, and
thin-layer chromatography was repeated to further
purify compound A. Comparison of radioactivity in silica corresponding
to compounds A and B after the second thin-layer
chromatography demonstrated that >95% of putative
compound A was indeed compound A.
Corticosteroid Metabolism
Confluent VSMCs in six-well plates or 30-mm segments of
aorta, which were free of fat and adventitia, from 250- to 300-g
Sprague-Dawley rats were incubated with 0.015 nmol
[3H]compound A or [3H]compound B (200 000
cpm) in 2 mL MEM for 24 hours. Metabolism was stopped by
adding tissue and medium to chloroform/methanol (1:2) in sufficient
quantity to achieve a single phase. After a 24-hour period of organic
extraction, two phases were created with H2O. The organic
phase was isolated, dried, dissolved in ethanol, and spotted on Silica
G thin-layer chromatography plates (250 µm
thick). Unlabeled standards of corticosteroids and
metabolites also were spotted. Migration solution consisted of
chloroform/ethanol (92:8). For each sample spotted, the silica
corresponding to the standards was scraped and counted in 10 mL
scintillation fluid. Blanks contained no vascular tissue but otherwise
were handled similarly.
Mass Spectrometry
Liquid secondary ion mass spectrometry ionization was carried
out on a JEOL HX110/HX110 high-performance tandem mass
spectrometer operating at a resolution of 1500.
Corticosteroids were dissolved in ethanol (1 mmol/L)
and mixed with glycerol (1:1 [vol/vol]) on the sample probe.
Samples were bombarded with 15-keV Cs+ ions to achieve
ionization. Typically, three to five scans were summed to produce a
mass spectrum.
VSMC Isolation, Maintenance, and
Characterization
Aortas from Sprague-Dawley rats weighing 125 to 300 g
were cleaned of endothelium, fat, and adventitia.
Smooth muscle strips were incubated in collagenase (2 mg/2
mL) for 2 hours, cut into 2-mm2 pieces, and allowed to
adhere to a culture flask. Then a covering layer of growth medium (10%
[vol/vol] newborn calf serum and 1% [vol/vol] nonessential amino
acids, 100 U/mL penicillin, and 100 µg/mL streptomycin in MEM) was
added. Cells were incubated in humidified 5%
CO2/95% air atmosphere until confluent. Medium was
changed every 5 days. Cells were passaged every 7 to 10 days by
harvesting with trypsin-EDTA and seeded at a ratio of 1:4. Cells
exhibited characteristic stellate VSMC morphology and stained
positively for smooth muscle -actin.26
Ang II Binding
Binding studies were performed in duplicate wells of 24-well
plates. Binding buffer consisted of 50 mmol/L Tris, 100 mmol/L NaCl, 5
mmol/L KCl, 5 mmol/L MgCl2, 0.25% bovine serum
albumin, and 0.5 mg/mL bacitracin, pH 7.4. Incubation volume
was 300 µL. Single-concentration Ang II receptor binding studies
were performed by adding 125I–Ang II (50 fmol) to all
wells and 1 µmol/L unlabeled Ang II to certain wells (for the
determination of nonspecific binding, <15% of total binding). Full
competition binding studies for Scatchard analysis were
performed by adding 125I–Ang II (50 fmol) to all wells and
10 concentrations of unlabeled Ang II (0.5 nmol/L to 10 µmol/L) to
various wells. Studies were performed at 4°C for 90 minutes to obtain
surface binding equilibrium only.27 Free hormone was
removed by washing monolayers three times with ice-cold saline.
Cells were solubilized with 0.1% SDS/0.1N NaOH, and gamma
radioactivity was counted. In previous studies we have found that Ang
II receptors on cultured VSMCs, both basal and upregulated, are of the
AT1 subtype and that Ang II receptor binding
parameters are not affected by cell passage (passages 3 to
10) or the body weight of the animals at the time of study (125 to 300
g).13
Phospholipid Labeling and Measurement of Inositol
Phosphates
Cells in six-well plates were incubated with 5 to 10 µCi
myo-[2-3H]inositol/2 mL per well in
inositol-deficient growth medium for 24 hours. Preliminary studies
revealed that steady state uptake of [3H]myoinositol
occurred after 24 hours and ranged from 100 000 to 500 000 cpm per
well. After exposure to effectors, reactions were terminated by the
addition of 1 mL ice-cold 20% trichloroacetic acid. Protein
precipitates were discarded, and supernatants were extracted three
times with equal volumes of diethyl ether. The upper ether phase was
discarded. Samples were adjusted to pH 7 with 50 mmol/L Tris base and
transferred to 20-mm columns of AG1-X8 anion exchange resin at room
temperature. Radioactivity elutable with water and borax (5 mmol/L
sodium borate and 60 mmol/L sodium formate) was discarded. Total
inositol phosphates were eluted with 1.0 mol/L ammonium formate in 0.1
mol/L formic acid. Fractions were counted in a scintillation counter.
Basal values for total inositol phosphates ranged from 3000 to 6000 cpm
per well (15 000 to 25 000 cpm/mg protein).
Thymidine Incorporation
This assay was performed as a measure of DNA synthesis. After
the effector treatment period, VSMCs in duplicate wells of 24-well
plates were incubated with [3H]thymidine (1 µCi/1 mL
per well) for 5 hours. After two saline washes, 0.5 mL of 0.3 mol/L
perchloric acid was added for 30 seconds. One saline wash was followed
by solubilization in 1 mL of 0.1% SDS/0.1N NaOH. Cells were scraped
into vials containing 5 mL of scintillation fluid, and radioactivity
was determined in a scintillation counter. In preliminary studies,
VSMCs that were serum deprived for 24 hours and then incubated with
[3H]thymidine and varying concentrations (0.1% to 10%)
of serum incorporated thymidine in direct proportion to the
concentration of serum. Basal thymidine incorporation ranged from
10 000 to 50 000 cpm per well.
Protein Determination
Cell protein content was determined by a minor alteration of the
method of Lowry et al,28 in that absorbance was read at
660 nm.
Aortic Ring Contractions
Thoracic aortas from 250- to 300-g Sprague-Dawley rats
were cleaned of adventitia and cut into rings 5 mm in length. Rings
were exposed to steroids or vehicle for 24 hours in 50 mL of
Krebs-Henseleit bicarbonate buffer bubbled with 95%
O2/5% CO2 at pH 7.4 at room
temperature. Then rings were attached to an isometric
force-displacement transducer under 2 g of tension and equilibrated
for 1 hour in the same buffer system at 37°C. Effectors of
contraction (Ang II and KCl) in 10-µL volumes were added
simultaneously to control and experimental rings in
side-by-side 10-mL organ chambers to achieve the desired final
concentrations. Ang II (100 nmol/L) elicited transient contractions
that peaked in 4 to 6 minutes and spontaneously resolved (even in the
continued presence of Ang II) in 8 to 10 minutes. KCl (120 mmol/L)
elicited a prolonged contraction. Intensity of contraction (ie, the
peak of the contraction curve) was expressed as grams tension per
milligram dry weight of aorta. Comparisons between treatments were made
in a paired manner between rings derived from the same aorta, since
contractions varied considerably from aorta to aorta.
Materials
Compound B, compound A, carbenoxolone, bovine serum
albumin, nonessential amino acids, penicillin, streptomycin,
bacitracin, and trypsin-EDTA were from Sigma Chemical Co; Ang II, from
Peninsula Laboratories; radioisotopes, from New England Nuclear; rats,
from Harlan Sprague Dawley, Indianapolis, Ind; MEM, from Mediatech;
collagenase, from Worthington Biochemical;
6ß-hydroxycorticosterone, from Steraloids, Inc; newborn calf
serum, from Hyclone Laboratories; thin-layer
chromatography plates, from Alltech Associates, Inc;
and anion exchange resin, from Bio-Rad Laboratories. RU38486 was kindly
provided by Dr D. Philibert, Roussel Uclaf, Romainville, France.
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Corticosteroid Metabolism
Initial studies were performed to determine the metabolic products of compound B by use of intact rat aorta. Rat aorta was used because cultured VSMCs were explanted from rat aorta. Conversion to compound A, aldosterone, and 6ß-hydroxycorticosterone were evaluated because compound B can be converted in the adrenal gland and possibly in VSMCs29 to aldosterone, which is a known upregulator of Ang II receptor number and stimulated signal transduction,12 13 and compound A and 6ß-hydroxycorticosterone may be active in renal tissue.19 21 22 Incubation of these vessels for 24 hours with compound B resulted in 25% to 30% conversion to compound A but no conversion to aldosterone or 6ß-hydroxycorticosterone (Fig 1
).
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Additional studies were performed in cultured VSMCs to determine if
compound B can be converted to compound A and vice versa. Incubation
with compound B resulted in moderate (10%) conversion to compound A
after 24 hours, whereas incubation with compound A resulted in
significantly more conversion (>60%) to compound B after 24 hours
(Fig 2). The 11ßOHSD inhibitor
carbenoxolone completely blocked conversion in both directions.
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Effects of Metabolites on Ang II Receptor Binding
Relative abilities of metabolites of compound B to regulate Ang II
receptor binding were investigated (Fig 3). Incubation
of VSMCs for 24 hours with compound B resulted in
concentration-dependent increases in Ang II binding, as
demonstrated by us previously.13
6ß-Hydroxycorticosterone, in concentrations ranging from 0.1 nmol/L
to 1 µmol/L, did not alter Ang II binding, whereas compound A caused
concentration-dependent upregulation of Ang II binding. The
threshold for the upregulating effect was between 0.1 and 1 nmol/L for
both compounds B and A, but the maximal effect at 1 µmol/L was 50%
greater with compound B than with compound A. Values of maximal
upregulation of Ang II binding with 1 µmol/L compound A and with 1
µmol/L of the mineralocorticoid aldosterone were similar:
163±2% of control (aldosterone) versus 168±10% of
control (compound A) (n=4). To determine if upregulation of Ang II
receptors on exposure to compound B or compound A is mediated through
glucocorticoid receptors, studies were performed in the presence of
RU38486, a specific antagonist for glucocorticoid
receptors.30 Upregulation of Ang II binding by compound A
or compound B was completely inhibited by RU38486 (Fig 3). It has been
demonstrated previously in VSMCs that RU38486 alone does not alter Ang
II binding.13
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Although commercially obtained compound A was used in the studies
detailed above, compound A itself may not have mediated the
upregulation in Ang II binding. Studies were performed to examine this
possibility. The commercially obtained stock of compound A was studied
for cross contamination with compound B. Samples of 1-mmol/L stocks of
compounds A and B in ethanol were spotted in separate lanes on
thin-layer chromatography plates, and iodine
staining revealed that compound A was not contaminated with compound B
(data not shown). Mass spectrometric analysis of these stocks
confirmed this result (data not shown). In additional studies (Fig 4), compound A was added to VSMCs in the presence of
carbenoxolone to prevent completely the conversion of compound A to
compound B. Compound A upregulated Ang II binding as effectively in the
presence of carbenoxolone as in its absence. Compound B was added to
VSMCs in the presence of carbenoxolone to prevent the small amount of
conversion of compound B to compound A (Fig 4). Upregulation was
similar in the presence and absence of carbenoxolone. Carbenoxolone
alone (1 µmol/L) did not alter Ang II binding (97±3% of control,
n=4). Scatchard analysis of complete competition binding data
from control cells and cells treated for 24 hours with 1 µmol/L
carbenoxolone and 1 µmol/L compound A revealed parallel lines (Fig 5), suggesting that compound A increased Ang II surface
receptor number without altering binding affinity. Finally, 100
µmol/L losartan was as effective as 100 nmol/L unlabeled Ang
II in displacing 125I–Ang II from VSMCs treated for 24
hours with 1 µmol/L carbenoxolone and 1 µmol/L compound A
(7523±411 cpm per well [no competitors] versus 2043±123 cpm per
well [Ang II] versus 2185±277 cpm per well [losartan],
n=16). These data, which are similar to those involving VSMCs treated
with aldosterone,13 suggest that Ang II
receptors upregulated by compound A (as well as basal Ang II receptors)
are of the AT1 subtype.
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Effects of Metabolites on Ang II Action
Since compound A, like unmetabolized glucocorticoids, upregulates
Ang II receptors in VSMCs (Figs 3
and 4), studies were performed to
determine if compound A simulates glucocorticoids in other ways, ie, by
inhibition of VSMC growth31 32 and potentiation of Ang
II–stimulated action. Fig 6 demonstrates that
incubation of VSMCs for 24 hours with compound B or A results in
concentration-dependent decreases in thymidine incorporation. In
separate experiments, we demonstrated that these effects are mediated
through the glucocorticoid receptor, in that reductions in thymidine
incorporation by 100 nmol/L compound A (to 72±6% of control) or 100
nmol/L compound B (to 46±8% of control) were prevented by 1 µmol/L
RU38486 (90±5% of control and 98±4% of control, respectively)
(n=4). When 1 µmol/L carbenoxolone was included with compound A,
inhibition was reduced but still significant (Fig 6). Carbenoxolone did
not alter compound B–mediated decreases in thymidine incorporation
(Fig 6).
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In previous studies, we observed that incubation of VSMCs with the
mineralocorticoid aldosterone resulted in upregulation of
Ang II receptor number and proportional enhancement of Ang
II–stimulated inositol phosphate formation.12 13 In the
present study, enhancement of Ang II–stimulated inositol phosphate
formation by compounds B and A, corticosteroids that
also upregulate Ang II receptors (Fig 3), was compared. Fig 7 demonstrates that Ang II–stimulated inositol
phosphate formation was more than doubled if 1 µmol/L compound B was
added to the cells for 24 hours before Ang II stimulation. Compound A
also enhanced Ang II–stimulated inositol phosphate formation, but the
enhancement was less than that effected by compound B. Enhancement by
compound A persisted in the presence of carbenoxolone, and
carbenoxolone did not further enhance the compound B effect.
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Finally, the effects of compounds B and A on Ang II–stimulated
vasoconstriction were determined (Fig 8). Aortic rings
were incubated ex vivo with 500 nmol/L compound B, 500 nmol/L compound
A, or vehicle for 24 hours, and then contraction was stimulated with
Ang II or KCl. The technique of maintaining blood vessels ex vivo has
been described previously.33 34 Peak stimulated tension in
response to KCl was not different between any of the groups (data not
shown). However, peak tension in response to Ang II was significantly
greater in compound B–treated rings (0.74±0.06 g/mg dry wt) than in
control rings (0.55±0.06 g/mg dry wt). Ang II contractions in compound
A–treated rings were numerically greater than those in control rings
(0.66±0.05 g/mg dry wt [compound A] versus 0.59±0.05 g/mg dry wt
[vehicle]), but this difference did not reach statistical
significance.
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Glucocorticoids are metabolized predominantly in the liver. The majority of urinary metabolites of compound B are formed on reduction of the double bond between carbons 4 and 5 (by 5ß-reductase) and reduction of the double bond in the keto group at carbon 3 (by 3
-hydroxysteroid dehydrogenase). The resulting compounds are
inactive. The 6ß-hydroxylase product of compound B,
6ß-hydroxycorticosterone, though a minor hepatic metabolite, is
of interest, since it is the agonist for type IV
corticosteroid receptors.19 This receptor
subtype, recently described in a renal epithelial cell line, is
distinct from other corticosteroid receptors and
mediates sodium transport. The results of the present study
demonstrate that vascular tissue does not convert compound B to
6ß-hydroxycorticosterone (Fig 1). In states of glucocorticoid
excess, the major pathways of compound B metabolism may
become saturated, and 6ß-hydroxycorticosterone formation may
rise. Although some of the compound may reach the vasculature, our
results demonstrate that VSMCs do not respond to
6ß-hydroxycorticosterone with upregulation of Ang II receptors
(Fig 3). Although steroid binding analysis was not performed,
it is most likely that VSMCs do not contain type IV
corticosteroid receptors. Fig 1 also demonstrates that
compound B was not converted to aldosterone. Although it is
generally felt that aldosterone biosynthesis occurs only in
the adrenal cortex, a recent report has demonstrated the presence of
the mRNA for P-450c11, the key mitochondrial enzyme
responsible for aldosterone biosynthesis, in cultured
VSMCs.29 Production of aldosterone by
vascular tissue may be below the levels of detection by thin-layer
chromatography.
11ßOHSD is responsible for extrahepatic metabolism of
compound B; more 11ßOHSD is found in the kidney than in the liver.
11ßOHSD, in fact, contains two enzyme activities: a dehydrogenase
that converts compound B to compound A and an oxoreductase that
converts compound A to compound B. Activity in the kidney appears to
favor the dehydrogenase for glucocorticoid inactivation, as discussed
by Funder et al.20 Similarly, neither isolated toad
bladders22 nor freshly isolated rabbit cortical collecting
duct cells35 were able to convert compound A to compound
B. The nearly complete conversion of compound B to compound A by the
kidney allowed us to isolate [3H]compound A, which is not
available commercially, in our laboratory. A number of recent studies
have demonstrated the presence of 11ßOHSD in vascular tissue as
well.23 24 25 The dominant enzyme activity in vascular smooth
muscle, however, is controversial. Homogenates of rat aorta
and mesenteric artery converted 20% to 30% of compound B to compound
A, whereas activity in the reverse direction was nil.23 It
should be noted that the 20% to 30% conversion of compound B to
compound A is of a magnitude similar to the conversion in the
present study (Fig 1). In contrast, we (Fig 2) and
others25 report greater conversion of compound A to
compound B than of compound B to compound A by freshly isolated aorta
and cultured VSMCs. Reasons for this discrepancy are unclear but may
relate to differences between intact tissues and broken cell
preparations.
In a seminal observation by Funder et al,20 it was suggested that compound A is inactive in renal tissue, since compound A bound to glucocorticoid and mineralocorticoid receptors with 1/300 the affinity of dexamethasone and aldosterone, respectively. It was postulated that inactivation of vast concentrations of glucocorticoids by 11ßOHSD allows smaller concentrations of mineralocorticoids to access mineralocorticoid receptors. However, compound A may not be inactive in renal tissue. In adrenalectomized rats, compound A was found to be a more potent stimulator of sodium reabsorption than compound B,21 and compound A was a more potent inhibitor of aldosterone-stimulated short-circuit currents than compound B.22
The present study extends this controversy to vascular tissue, and
the results suggest that compound A possesses significant
glucocorticoid agonist properties. Exposure of VSMCs to compound A
resulted in concentration-dependent upregulation of Ang II binding
(Fig 3). The results from the competition studies and Scatchard
analysis imply that compound A upregulates Ang II receptor
number without change in binding affinity (Fig 5). Compound A appears
to bind to type II (glucocorticoid) receptors, since the specific type
II corticoid receptor antagonist RU38486 completely
inhibited Ang II receptor upregulation by compound A (Fig 3), by the
natural glucocorticoid compound B (Fig 3), and by the selective
synthetic glucocorticoid dexamethasone.13 The
enhancement of Ang II–stimulated inositol phosphate formation (Fig 7)
suggests that the upregulated Ang II receptors are coupled to more
distal aspects of the Ang II signal transduction pathway. Ang II
receptor upregulation and proportional increases in Ang II–stimulated
inositol phosphate formation after exposure of VSMCs to the
mineralocorticoid aldosterone have been demonstrated
previously.12 13 An additional glucocorticoid property,
inhibition of VSMC growth,31 32 was displayed by compound
A (Fig 6).
Because conversion of compound A to compound B by cultured VSMCs is
substantial (Fig 2) and because compound B is a potent upregulator of
Ang II receptor binding in its own right (Fig 3), compound A itself may
not have caused the glucocorticoid actions. The addition of the
11ßOHSD inhibitor carbenoxolone, which blocked all
interconversion (Fig 2), allowed direct comparison of the
glucocorticoid effects of compound A and compound B. Upregulation of
Ang II binding, enhancement of Ang II–stimulated inositol phosphate
formation, and inhibition of VSMC growth by compound A persisted even
in the presence of carbenoxolone (Figs 3, 6, and 7). The use of
carbenoxolone allowed us to determine that compound A was 40% to 50%
weaker in all glucocorticoid effects compared with compound B. We also
considered the possibility that the addition of carbenoxolone to
compound B might potentiate its glucocorticoid properties by preventing
the small amount of conversion to compound A (a somewhat weaker
glucocorticoid). However, this was not the case, because upregulation
of Ang II binding, enhancement of Ang II–stimulated inositol phosphate
formation, and inhibition of VSMC growth by compound B were not
affected by the addition of carbenoxolone. It is likely that the small
(10%) reduction in the compound B content and the intrinsic activity
of compound A explain this lack of effect.
Exposure of aortic rings ex vivo to compound B for 24 hours resulted in
greater contraction to 1 µmol/L Ang II than did exposure to vehicle,
whereas exposure to compound A elicited contractions to 1 µmol/L Ang
II that were numerically but not statistically greater than those in
rings exposed to vehicle (Fig 8). Exposure to
corticosteroids was performed in this isolated setting
to study their direct effects and to avoid the influence of systemic
perturbations. Enhanced vasoconstriction to -adrenergic agonists
and Ang II has been demonstrated in vessels removed from animals after
glucocorticoid or mineralocorticoid treatment in vivo.4 5 6 7 8
It is possible that compound A would have increased the potency of Ang
II contractions (ie, lower EC50) more significantly than it
increased contractions at maximal (1 µmol/L) Ang II concentrations.
However, such studies are based on cumulative
contractions—contractions to a set of increasing concentrations of
vasoconstrictor, each added when the contraction to the previous
concentration has plateaued. Such studies are impossible with Ang II,
whose contractions do not plateau but abate spontaneously and become
desensitized to subsequent Ang II exposure.36 We did not
examine the effects of compound A per se by including carbenoxolone
with compound A in the overnight incubation, because it has been
suggested that carbenoxolone is vasoactive in its own
right.37
Results from the present study argue against the role of blood vessels as a mineralocorticoid target tissue, despite the presence of 11ßOHSD in vascular smooth muscle. Since only 25% to 30% of circulating compound B is metabolized by vascular smooth muscle, the large excess of glucocorticoid over mineralocorticoid would not be overcome. This is in contrast to the kidney, which contains much greater amounts of 11ßOHSD and can metabolize almost all glucocorticoids. In addition, compound A, the product of compound B metabolism by 11ßOHSD, is not inactive in vascular smooth muscle but is as potent in upregulating Ang II receptor function as aldosterone. Furthermore, much of the compound A gaining access to the circulation after genesis in the liver and kidney would be converted within vascular smooth muscle to compound B as a result of the predominant oxoreductase moiety of 11ßOHSD. Only if a disease state were characterized by a massive excess of circulating mineralocorticoid would mineralocorticoids be able to compete with glucocorticoids for access to mineralocorticoid receptors in vascular smooth muscle. Such a scenario may occur in animal models of mineralocorticoid hypertension, whereas plasma aldosterone levels are only elevated 5- to 10-fold in human primary or secondary hyperaldosteronism.
Although carbenoxolone did not enhance the effects of compound B in our
in vitro experiments (Figs 3, 6, and 7), carbenoxolone may enhance
glucocorticoid action in vivo. Treatment of normal humans for 7 days
with carbenoxolone resulted in greater pressor responses to
intravenous norepinephrine and greater forearm
blood flow reduction to intra-arterial
norepinephrine than did control treatment.38
In the absence of carbenoxolone, the moderate degree (25% to 30%) of
conversion of compound B to the less active compound A by 11ßOHSD in
vascular tissue may limit potentiation of pressor hormone action. In
the presence of carbenoxolone, which maintains compound B in the
unmetabolized state, potentiation of pressor hormone action can be
full. Although the anti–peptic ulcer drug carbenoxolone is no
longer in common usage, licorice and chewing tobacco contain related
11ßOHSD inhibitors such as glycyrrhizic acid.
Hypertension from such exposure continues to be seen in clinical
practice.
|
Acknowledgments |
|---|
This study was supported by the American Heart Association, South Carolina Affiliate, Inc, and by biomedical research support grants from the Medical University of South Carolina. Expert technical assistance was provided by Jana J. Fine. The authors thank Lois B. McKamey for secretarial support. Mass spectrometric data were kindly obtained by Dr Kevin L. Schey at the Medical University of South Carolina Mass Spectrometry Research Resource Facility.
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Footnotes |
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Reprint requests to Dr Michael E. Ullian, Division of Nephrology, Department of Medicine, Medical University of South Carolina, Clinical Sciences Bldg 829, 171 Ashley Ave, Charleston, SC 29425.
Previously presented in part in abstract form at the annual meeting of the American Society of Nephrology, Orlando, Fla, October 26-29, 1994.
Received January 12, 1995; accepted June 6, 1995.
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