The llfl=OHSD inhibitor, carbenoxolone, enhances Na retention by aldosterone and II-deoxycorticosterone DAVID
J. MORRIS
AND
GRAHAM
W. SOUNESS
Department of Pathology and Laboratory Medicine, The Miriam Hospital, and the Division of Biology and Medicine, Brown University, Providence, Rhode Island 02906 MORRIS, DAVID J., AND GRAHAM W. SOUNESS. The llpOHSD inhibitor, carbenoxolone, enhances Na retention by aldosterone and 11 -deoxycorticosterone. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F756-F759, 1990.-Carbenoxolone sodium, CS, a liquorice derivative associated with hypertension and sodium retention, has been demonstrated to inhibit 11/3-hydroxysteroid dehydrogenase? an enzyme that metabolizes cortisol and corticosterone to their respective inactive 11-dehydro products (cortisone and 11-dehydrocorticosterone). It has been proposed that the increased bioavailability of unmetabolized corticosterone and cortisol following llP-OHSD inhibition allows these steroids to act on renal mineralocorticoid receptors to elicit the mineralocorticoid action. Here we describe how CS amplifies the antinatriuretic activity of aldosterone and deoxycorticosterone; the latter steroid is of particular importance in that it does not possess a hydroxyl group at the C-11 position in the steroid ring, indicating that another mechanism(s) in addition to lib-OHSD inhibition is responsible for the amplification of the action of deoxycorticosterone.
liquorice;
sodium retention;
adrenalectomized
rats
11@-hydroxysteroid dehydrogenase (lipthe glucocorticoid hormones cortisol and corticosterone to cortisone and ll-dehydrocorticosterone, their putative inactive products (2,6,7). Renewed interest in this enzyme was sparked by the experiments of Ulick, New, and co-workers (10, 14, 15, 23, 24), who demonstrated that hypertensive children with the syndrome of apparent mineralocorticoid excess (AME) lack 11/3-OHSD enzyme activity, a condition that results in altered peripheral metabolism of cortisol. These patients exhibit Na+ retention, K+ wasting, and increased blood pressure without measurable increases in circulating aldosterone (Aldo). A pharmacological equivalent of this congenital condition results from the ingestion of glycyrrhetinic acid (GA), which is present as glycoside derivatives in liquorice [Stewart et al. (20, 21)]. GA has now been shown to be a potent inhibitor of both hepatic and renal ll@-OHSD (11). These discoveries taken together led several investigators (4,522) to suggest that lowered llfl-OHSD enzyme activity results in higher peripheral and intrarenal concentrations of corticosterone in experimental animals and cortisol in humans, which may then interact with mineralocorticoid (MC) receptors and promote Na+ reabsorption. We recently reported that acute pretreatment of ad-
THE
ENZYME
OHSD) metabolizes
F756
0363-6127/90
$1.50 Copyright
renalectomized (ADX) male rats with carbenoxolone sodium (CS), a succinate derivative of GA, caused both the glucocorticoids corticosterone and cortisol to display MC-like activity (19); neither corticosterone nor cortisol when administered at these dosages alone possessed intrinsic antinatriuretic activity. Using the same sensitive bioassay (modified from Kagawa) that measures changes in urinary electrolyte excretion (13), we have now examined the effects of acute administration of CS on the MC activity of both deoxycorticosterone (DOC) and Aldo in ADX male rats. Both steroids are key adrenal MCs; DOC is -5% as active as Aldo (8), and, importantly, DOC does not possess a C-11 hydroxyl group. MATERIALS
AND METHODS
Chemicals. CS was obtained from Biorex Laboratories (London, UK). DOC was obtained from Sigma Chemical (St. Louis, MO) and Aldo from Andard Mount (London, UK). Rat bioassay. Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were bilaterally adrenalectomized under ether anesthesia at 6-8 wk of age. Thereafter, rats were allowed free access to 0.9% NaCl as drinking water at all times and to Purina rat chow (Ralston Purina, St. Louis, MO) until 16 h before experimentation. Rats (160-200 g body wt) were maintained in a temperatureand light-controlled room and were used 7-12 days postadrenalectomy. On the morning of the test, rats were injected subcutaneously with 2.5 mg CS (time zero minus 30 min) dissolved in 0.2 ml of 0.154 M NaCl; control animals received the saline alone. Thirty minutes later rats were made to urinate with a whiff of ether and were then injected subcutaneously (time zero) with 3.0 ml of 0.154 M NaCl and various dosages of DOC or Aldo dissolved in a mixture of 0.154 M NaCl (90%) ethanol (10%); control rats received this vehicle alone. Urine was collected (again induced with ether) from the time periods O-l, l-3, and 3-4 h postinjection of either steroid and analyzed for Na+, K+, and creatinine, as previously described (16). Comparisons (using analysis of variance and the Bonferroni t test) were made between 1) animals that received the same dosages of DOC or Aldo with and without CS pretreatment, 2) animals that received DOC or Aldo alone and animals that received vehicle only, and 3) animals that received CS alone and animals that received vehicle only (see RESULTS and legends to Figs. l-4).
0 1990 the American
Physiological
Society
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LIQUORICE
ENHANCES
NA+
RETENTION
OF
ALDOSTERONE
AND
F757
DOC
excretion of Na’, K+ (Fig. 1, A and B), or creatinine. In addition, CS caused DOC (5.0 and 10.0 ,ug/rat) to be DOC. DOC at a dosage of 2.5 pg/rat had no effect but significantly antinatriuretic in the latent period (O-l h) at 5.0 pg/rat it had a small effect on urinary Na+ excre(Fig. 2A) and also prolonged urinary Na+ retention into tion in the I- to 3-h urine collection period compared with animals given vehicle alone (Fig. IA). However, at the 3-4-h urine collection period (Fig. 2B). CS did not affect the K+ excretion caused by DOC in either urine a dosage of 10 pg/rat, DOC caused a significant antinacollection period, O-l h or 3-4 h. triuresis on its own; Na+-to-creatinine ratios (means t Aldo. Aldo at dosages of 0.05 and 0.1 pg/rat caused SE) fell to 307 t 27 compared with 530 t 58 mmol/g (P significant Na+ retention compared with vehicle alone in < 0.05) for the control group (Fig. IA). DOC had no the l-3-h urine collection period (Fig. 3A). Na+-to-creeffect on urinary K+ excretion at 2.5 or 5.0 lug/rat during the l-3-h urine collection period (Fig. 1B) but caused a atinine ratios (means t SE) for the control group were significant kaliuresis at 10 ,ug/rat. K+-to-creatinine ratios 530 t 58 compared with 384 t 35 mmol/g for 0.05 ,ug (means t SE) were increased to 270 t 20 compared with Aldo/rat (P < 0.05) and 311 t 18 mmol/g for 0.1 pg 139 t 14 mmol/g for the control group (Fig. 1B). At all Aldo/rat (P < 0.01). Aldo also caused a significant kaliuresis at these dosages; urinary K+-to-creatinine ratios dosagesDOC did not affect urinary creatinine output. (means t SE) for controls were 139 t 14 compared with When the ADX rats were pretreated (subcutaneously) for 30 min with 2.5 mg CS/rat, the Na+-retaining effects 238 t 19 mmol/g (P < 0.01) for 0.05 pg Aldo/rat, and 247 t 20 mmol/g (P < 0.001) for 0.1 pg Aldo/rat (Fig. of both dosages of DOC (5 and 10 pg/rat) were significantly enhanced when compared with the same dosages 3B) CS (2.5 mg/rat) also significantly enhanced the Na+ of DOC alone in the l-3-h urine collection period (Fig. retention caused by 0.05 and 0.1 pg Aldo/rat in the 1-3IA). CS did not alter the urinary K+ excretion caused by h urine collection period (Fig. 3A). CS had no effect on any dosage of DOC used (Fig. IB), whereas CS alone the kaliuresis induced by both the 0.05- and O.l-pg/rat (2.5 mg/rat) had no intrinsic effects on the urinary dosages of Aldo (Fig. 3B). Urinary creatinine excretion was not significantly altered by either Aldo or CS. As A URINARY NA+/cRwTININE (mm&s/g) with DOC, CS enhanced the antinatriuretic effects of 1 - 3 HOURS Aldo in the O-l-h (0.05 pg Aldo/rat) and 3-4-h (0.05 and RESULTS
600
T
t
A
URINARY 8001
200--
*.*-
. 0'
(mmoles/g)
NS
P(O.001
q - ....* . . .
100
NA+/CREATlNlNE 0 -1HOUR
51.0
. . . . -....
ib
P(O.001
1 o!o
DOC(pg/rat)
URINARY
B
SHAM DOC
K+/CREATININE(mmoles/g) 1 - 3 HOURS
300T
I
= SHAM
2.5/q
DOC
5.0
/q
DOC
lO.Opg
I
CS
DOC
= 2.5mgCS
T
250
B
t
URINARY
200
(mw ioles/g)
NA+/CREATlNINE 3 -4 HOUR
700 150
A
T
600
I loo--IINS 50 --
I
P(O.01
0 O!
I 2.5
5!0
lO!O
D WPg/rat)
FIG. 1. Mineralocorticoid activity of 2.5, 5.0, and 10.0 pg/rat DOC with (W---H) and without (o----@ CS pretreatment (2.5 mg/rat). CS was given SC 30 min before DOC in male ADX rats, and urine was collected l-3 h after injection of the steroid. Antinatriuretic activity (A) is expressed as a decrease in the Na+-to-creatinine ratio and kaliuretic activity (B) by an increase in the K+-to-creatinine ratio. Values shown are means f. SE (n = 13-16 rats). A, animals given CS vehicle followed by DOC vehicle; A, mean ratios for animals treated with CS (2.5 mg/rat) alone. Groups were statistically compared using ANOVA followed by the Bonferroni t test.
I FIG. periods analyses dosages shown
SHAM DOC
2.5j.q
= SHAM
CS
DOC
5.0
pg
DOC
I
P(O.01
lO.Opq
DOC
= 2.5mgCS
2. Antinatriuretic activity of DOC or vehicle in urine collection O-l h (A) and 3-4 h (B) after injection of steroid. Statistical (see Fig. 1) were made between animals that received the same of DOC (or vehicle) with and without CS pretreatment. Values are means & SE (n = 13-17 rats).
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F758
LIQUORICE
ENHANCES
A URINARY NA+/CREATlNlNE
NA+
RETENTION
OF
(mmoles/g)
ALDOSTERONE
AND
A URINARY NA’/CREATININE (mmoles/g)
1 - 3HOUR
O800
600 -I-
T
DOC
hlC
700--
f
NS
1 HOUR T
600-500--
3004
400-300-200 -lOO--
0 SHAM ALDO
=
= SHAM
0.05pg
ALDO
CS
0.1~
0
ALDO
SHAM ALDO
= 2.5mgCS
-
= SHAM
0.05p-g
ALDO
CS
O.lpg
U
ALDO
= 2.5mgCS
B URINARY NA’/CREATlNlNE (mmoles/g) 3-
4HOUR
0 SHAM AID0
-
= SHAM
CS
FIG. 3. Antinatriuretic (A) and kaliuretic 0.1 lug/rat Aldo with and without 2.5 mg/rat shown are means t SE (n = 6-8 rats).
I
= 2.5mgCS (B) activities of 0.05 and CS pretreatment. Values
0.1 pugAldo/rat) postinjection periods (Fig. 4, A and B). The above results describing the effects of CS on DOC and Aldo were expressed with Na+-to-creatinine and K+to-creatinine ratios; in addition, Table 1 shows the absolute excretory rates for Na+, K+, and creatinine for sham experiments, 5.0 pugDOC (with or without CS) and 0.1 ,ug Aldo (with or without CS) in the l-3-h urine collection period. DISCUSSION
We had earlier shown that carbenoxolone sodium causes the glucocorticoids corticosterone and cortisol which contain an lip-hydroxyl grouping, to display MClike activity (19). The present studies show that the Na+retaining effects of two other adrenal MCs, Aldo and DOC, can be enhanced by the liquorice derivative CS. DOC does not possess a C-11 hydroxyl grouping; thus the increased Na+-retaining properties conferred by CS upon DOC must occur by a mechanism(s) in addition to that of llfl-OHSD inhibition. In aqueous solutions a small proportion of Aldo exists as its ILhydroxyl form (3), thus a role for lip-OHSD inhibition in the enhanced Na+-retaining properties of Aldo by CS cannot be ruled out. It should be noted that others have shown enhancement of the MC actions of Aldo, although with much greater dosagesof CS than used in these experiments (1, 16); in contrast, one study showed that CS did not affect
-
= SHAM
0.05pg
ALDO
CS
FIG. 4. Antinatriuretic activity CS (2.5 mg CS/rat) pretreatment (A) and 3-4 h (B) after injection SE (n = 6-8 rats).
0.1/q
I
ALDO
I = 2.5mgCS
of 0.05 and 0.1 pg Aldo/rat following in the urine collection periods O-l h of the steroid. Values are means t
1. Composition of urine from CS-treated and DOC- and Aldo-treated (HZ’S) ADX rats
TABLE
Na’, pmol Control Sham CS Sham Steroid 2.5 mg CS Sham Steroid DOC Sham CS 5.0 pg DOC 2.5 mg CS 5.0 pg DOC Aldo Sham CS 0.1 Pg Aldo 2.5 mg CS 0.1 Pg Aldo
K+, pmol
Creatinine,
169.7 t20.3 155.7 NS +13.4
44.1 rt4.4 39.3 NS t4.2
327.5 t23.8 356.2 NS t25.6
146.0 t18.7 60.5 P < 0.001 t6.6
61.2 t6.6 56.0 NS -1-4.0
377.8 t13.1 364.1 NS t16.5
68.6 t7.6 66.3 NS t5.6
278.3 k21.5 280.9 NS t19.4
86.1 t8.3 46.9 t8.9
P < 0.01
pug
Values are means k SE; n = 8-12 rats. Urine from male ADX rats (160-200 g body wt) was collected l-3 h after injection of either steroid or vehicle (see METHODS). Statistical comparisons were made between groups of animals that received DO@ or Aldo with and without CS. The control set of experiments shows the effects of CS alone.
the MC activity of Aldo at all (17). At the present time, the mechanisms by which CS causes this apparent amplification of the Na’-retaining effects of DOC and Aldo, and even on which portions of the nephron CS is working, are all unknown. An increased bioavailability of the
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LIQUORICE
ENHANCES
NA+
RETENTION
unmetabolized parent MCs may possibly account for these observations. In addition to demonstrating inhibition of llfl-OHSD (ll), we have recently shown by in vitro experiments with both DOC (S. A. Latif, personal communication) and Aldo (9) as substrates, that CS inhibits hepatic A*-5P-reductase and also 3P-hydroxysteroid dehydrogenase, but not A*-5cu-reductase. Thus the metabolism of Aldo, DOC, and possibly other glucocorticoids in the rat may be rerouted in the presence of CS, resulting in the accumulation of unmetabolized hormones and their corresponding 5a-dihydro derivatives. Both 5a-dihydro-Aldo and 5cr-dihydro-DOC are also biologically active compounds (13, 18). It is interesting that, as shown in our earlier studies with corticosterone and cortisol, the enhanced Na+ retention observed with both Aldo and DOC in rats pretreated with CS was also visible earlier as well as being extended longer into the 3-4-h urine collection period. This does not usually occur with physiological dosages of Aldo where a longer latent period is observed (12). In addition, the previous studies showed the large antinatriuretic responses elicited by corticosterone and cortisol in the presence of CS were disproportionate to the kaliuretic effects observed (19). It is noteworthy that the kaliuretic properties of DOC and Aldo were not affected by CS in the present studies. These observations offer clues in designing the further experiments needed to 1) elucidate how CS may alter the interactions of these MCs with steroid receptors in the kidney, and 2) determine which segments of the renal tubule and which biochemical processes are affected by CS. Thus liquorice derivatives may enhance or amplify the MC activity of a variety of other adrenal steroids (besides corticosterone and cortisol), including some that do not possess an lip-hydroxyl group. Hence a variety of secretory products of the adrenal gland (from both the fascicular and glomerular zones) may be affected by liquorice ingestion or treatment with foodstuffs and drugs containing chemical derivatives of glycyrrhetinic acid.
OF
9.
10
11.
12.
13.
14.
15.
16.
17.
18. The authors thank Dr. M. Sheff, Miriam Hospital, Providence, RI, for helpful discussions concerning this work. This work was supported by National Institutes of Health Grant DK-21404 and The Miriam Hospital Research Foundation. Address for reprint requests: D. J. Morris, The Miriam Hospital, Dept. of Laboratory Medicine, 164 Summit Ave., Providence, RI 02906. Received
30 August
1989; accepted
in final
form
17 November
1989.
19.
20.
REFERENCES 1. ARMANINI, D., I. KARBOWIAK, 2. KROZOWSKI, J. W. FUNDER, AND W. R. ADAM. The mechanism of mineralocorticoid action of carbenoxolone. Endocrinology 111: 1683-1686, 1982. 2. BUSH, I. E., S. A. HUNTER, AND R. A. MEIGS. Metabolism of lloxygenated steroids. Biochem. J. 107: 239-257, 1968. 3. CARTER, B. G., D. N. KIRK, AND P. J. BURKE. 18-Substituted steroids, Part 14. The high-field lH and 13C nuclear magnetic resonance spectra of aldosterone; full assignments for the main equilibrating forms in solution. J. Chem. Sot. 31, Perhin Trans. II: 1247-1252,1987. 4. EDWARDS, C. R. W., P. M. STEWART, D. BURT, L. BRETT, M. A. MCINTYRE, W. S. SUTANTO, E. R. DEKLOET, AND C. MONDER. Localization of II/?-hydroxysteroid dehydrogenase; tissue specific protector of the mineralocorticoid receptor. Lancet 2: 986-989, 1988. 5. FUNDER, J. W., P. T. PEARCE, R. SMITH, AND A. I. SMITH.
21,
22.
23.
24.
ALDOSTERONE
AND
DOC
F759
Mineralocorticoid action: Target tissue specificity is enzyme, not receptor, mediated. Science Wash. DC 242: 583-585, 1988. HELLMAN, L., K. NAKADA, G. B. ZUMOFF, D. FUKUSHIMA, H. L. BRADLOW, AND T. F. GALLACHER. Renal capture and oxidation of cortisol in man. J. Clin. Endocrinol. Metab. 33: 52-62, 1971. JENKINS, J. S. The metabolism of cortisol by human extrahepatic tissues. J. Endocrinol. 34: 51-56, 1966. KAGAWA, C. M., F. M. STURTEVANT, AND C. G. VAN ARMAN. Pharmacology of a new steroid that blocks salt activity of aldosterone and desoxycorticosterone. J. Pharmacol. Exp. Ther. 126: 123-130,1959. LATIF, S. A., T. J. CONCA, AND D. J. MORRIS. The effects of the liquorice derivatives, glycyrrhetinic acid, on hepatic ECU- and 3@hydroxysteroid dehydrogenases and ECU- and 5P-reductase pathways of metabolism of aldosterone in male rats. Steroids. 55: 52-58, 1990. MONDER, C., C. H. L. SHACKLETON, H. L. BRADLOW, M. I. NEW, E. STONER, F. IOHAN, AND V. LAKSHMI. The syndrome of apparent mineralocorticoid excess: its association with llp-dehydrogenase and 5fl-reductase deficiency and some consequences for corticosteroid metabolism. J. Clin. Endocrinol. Metab. 63: 550-557, 1986. MONDER, C., P. M. STEWART, V. LAKSHMI, R. VALENTINO, D. BURT, AND C. R. W. EDWARDS. Liquorice inhibits corticosteroid ll/?-dehydrogenase of rat kidney and liver: In vivo and in vitro studies. Endocrinology 125: 1046-1053, 1989. MORRIS, D. J., AND A. S. BREM. Metabolic derivatives of aldosterone. Am. J. Physiol. 252 (Renal Fluid Electrolyte Physiol. 21): F365F373,1987. MORRIS, D. J., C. J. KENYON, S. A. LATIF, M. MCDERMOTT, AND T. GOODFRIEND. The possible biological significance of aldosterone metabolites. Hypertension Dallas, 5 Suppl. 1: 1-35-I-40, 1983. NEW, M. I., S. E. OBERFIELD, R. CAREY, F. GREIG, S. ULICK, AND L. S. LEVINE. A genetic defect in cortisol metabolism as the basis for the syndrome of apparent mineralocorticoid excess. In: Endocrinology of Hypertension, edited by F. Mantero, C. Biglieri, and C. R. W. Edwards. New York: Academic, 1982, p. 85-101. OBERFIELD, S. E., L. S. LEVINE, R. M. CAREY, F. GREIG, S. ULICK, AND M. I. NEW. Metabolic and blood pressure responses to hydrocortisone in the syndrome of apparent mineralocorticoid excess. J. Clin. Endocrinol. Metab. 56: 332-339, 1983. PORTER, G. A. Synergistic effects of carbenoxolone sodium on aldosterone-enhanced active sodium transport in toad skin. In: Carbenoxolone Sodium, edited by J. H. Baron, and F. M. Sullivan. London: Butterworths, 1970, p. 33. PORTER, G. A., C. RHODES, AND P. SACRA. Comparative studies on the mineralocorticoid action of aldosterone and carbenoxolone sodium in the adrenalectomized rat. Pharmacology 12: 224-229, 1974. SEKIHARA, H., D. P. ISLAND, AND G. W. LIDDLE. New mineralocorticoids: 5&-dihydroaldosterone and 5cu-dihydro-ll-deoxycorticosterone. Endocrinology 103: 1450-1453, 1978. SOUNESS, G. W., AND D. J. MORRIS. The antinatriuretic and kaliuretic effects of glucocorticoids corticosterone and cortisol following pretreatment with carbenoxolone sodium (a liquorice derivative) in the adrenalectomized rat. Endocrinology 124: 1588-1590, 1989. STEWART, P. M., C. H. L. SHACKLETON, AND C. R. W. EDWARDS. The cortisol-cortisone shuttle and the genesis of hypertension. In: Corticosteroids and Peptide Hormones in Hypertension, edited by F. Mantero and P. Vecscei. New York: Raven, 1987, vol. 39, p. 163-178. STEWART, P. M., R. VALENTINO, A. M. WALLACE, D. BURT, C. H. L. SHACKLETON, AND C. R. W. EDWARDS. Mineralocorticoid activity of liquorice: ll-P-hydroxysteroid dehydrogenase deficiency comes of age. Lancet 2: 821-823, 1987. ULICK, S., C. CHAN, K. N. RAO, J. EDASSERY, AND F. MANTERO. A new form of the syndrome of apparent mineralocorticoid excess. J. Steroid Biochem. 32: 209-212, 1989. ULICK, S., L. S. LEVINE, P. GUNCZLER, G. ZANCONATO, L. C. RAMIREZ, W. RAUH, A. ROSLER, H. L. BRADLOW, AND M. I. NEW. A syndrome of apparent mineralocorticoid excess associated with defects in peripheral metabolism of cortisol. J. Clin. Endocrinol. Metab. 49: 757-764, 1979. ULICK, S., L. C. RAMIREZ, AND M. I. NEW. An abnormality in steroid reductive metabolism in a hypertensive syndrome. J. Clin. Endocrinol. Metab. 44: 799-802, 1977.
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J. MORRIS
AND
GRAHAM
W. SOUNESS
Department of Pathology and Laboratory Medicine, The Miriam Hospital, and the Division of Biology and Medicine, Brown University, Providence, Rhode Island 02906 MORRIS, DAVID J., AND GRAHAM W. SOUNESS. The llpOHSD inhibitor, carbenoxolone, enhances Na retention by aldosterone and 11 -deoxycorticosterone. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F756-F759, 1990.-Carbenoxolone sodium, CS, a liquorice derivative associated with hypertension and sodium retention, has been demonstrated to inhibit 11/3-hydroxysteroid dehydrogenase? an enzyme that metabolizes cortisol and corticosterone to their respective inactive 11-dehydro products (cortisone and 11-dehydrocorticosterone). It has been proposed that the increased bioavailability of unmetabolized corticosterone and cortisol following llP-OHSD inhibition allows these steroids to act on renal mineralocorticoid receptors to elicit the mineralocorticoid action. Here we describe how CS amplifies the antinatriuretic activity of aldosterone and deoxycorticosterone; the latter steroid is of particular importance in that it does not possess a hydroxyl group at the C-11 position in the steroid ring, indicating that another mechanism(s) in addition to lib-OHSD inhibition is responsible for the amplification of the action of deoxycorticosterone.
liquorice;
sodium retention;
adrenalectomized
rats
11@-hydroxysteroid dehydrogenase (lipthe glucocorticoid hormones cortisol and corticosterone to cortisone and ll-dehydrocorticosterone, their putative inactive products (2,6,7). Renewed interest in this enzyme was sparked by the experiments of Ulick, New, and co-workers (10, 14, 15, 23, 24), who demonstrated that hypertensive children with the syndrome of apparent mineralocorticoid excess (AME) lack 11/3-OHSD enzyme activity, a condition that results in altered peripheral metabolism of cortisol. These patients exhibit Na+ retention, K+ wasting, and increased blood pressure without measurable increases in circulating aldosterone (Aldo). A pharmacological equivalent of this congenital condition results from the ingestion of glycyrrhetinic acid (GA), which is present as glycoside derivatives in liquorice [Stewart et al. (20, 21)]. GA has now been shown to be a potent inhibitor of both hepatic and renal ll@-OHSD (11). These discoveries taken together led several investigators (4,522) to suggest that lowered llfl-OHSD enzyme activity results in higher peripheral and intrarenal concentrations of corticosterone in experimental animals and cortisol in humans, which may then interact with mineralocorticoid (MC) receptors and promote Na+ reabsorption. We recently reported that acute pretreatment of ad-
THE
ENZYME
OHSD) metabolizes
F756
0363-6127/90
$1.50 Copyright
renalectomized (ADX) male rats with carbenoxolone sodium (CS), a succinate derivative of GA, caused both the glucocorticoids corticosterone and cortisol to display MC-like activity (19); neither corticosterone nor cortisol when administered at these dosages alone possessed intrinsic antinatriuretic activity. Using the same sensitive bioassay (modified from Kagawa) that measures changes in urinary electrolyte excretion (13), we have now examined the effects of acute administration of CS on the MC activity of both deoxycorticosterone (DOC) and Aldo in ADX male rats. Both steroids are key adrenal MCs; DOC is -5% as active as Aldo (8), and, importantly, DOC does not possess a C-11 hydroxyl group. MATERIALS
AND METHODS
Chemicals. CS was obtained from Biorex Laboratories (London, UK). DOC was obtained from Sigma Chemical (St. Louis, MO) and Aldo from Andard Mount (London, UK). Rat bioassay. Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were bilaterally adrenalectomized under ether anesthesia at 6-8 wk of age. Thereafter, rats were allowed free access to 0.9% NaCl as drinking water at all times and to Purina rat chow (Ralston Purina, St. Louis, MO) until 16 h before experimentation. Rats (160-200 g body wt) were maintained in a temperatureand light-controlled room and were used 7-12 days postadrenalectomy. On the morning of the test, rats were injected subcutaneously with 2.5 mg CS (time zero minus 30 min) dissolved in 0.2 ml of 0.154 M NaCl; control animals received the saline alone. Thirty minutes later rats were made to urinate with a whiff of ether and were then injected subcutaneously (time zero) with 3.0 ml of 0.154 M NaCl and various dosages of DOC or Aldo dissolved in a mixture of 0.154 M NaCl (90%) ethanol (10%); control rats received this vehicle alone. Urine was collected (again induced with ether) from the time periods O-l, l-3, and 3-4 h postinjection of either steroid and analyzed for Na+, K+, and creatinine, as previously described (16). Comparisons (using analysis of variance and the Bonferroni t test) were made between 1) animals that received the same dosages of DOC or Aldo with and without CS pretreatment, 2) animals that received DOC or Aldo alone and animals that received vehicle only, and 3) animals that received CS alone and animals that received vehicle only (see RESULTS and legends to Figs. l-4).
0 1990 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 16, 2019.
LIQUORICE
ENHANCES
NA+
RETENTION
OF
ALDOSTERONE
AND
F757
DOC
excretion of Na’, K+ (Fig. 1, A and B), or creatinine. In addition, CS caused DOC (5.0 and 10.0 ,ug/rat) to be DOC. DOC at a dosage of 2.5 pg/rat had no effect but significantly antinatriuretic in the latent period (O-l h) at 5.0 pg/rat it had a small effect on urinary Na+ excre(Fig. 2A) and also prolonged urinary Na+ retention into tion in the I- to 3-h urine collection period compared with animals given vehicle alone (Fig. IA). However, at the 3-4-h urine collection period (Fig. 2B). CS did not affect the K+ excretion caused by DOC in either urine a dosage of 10 pg/rat, DOC caused a significant antinacollection period, O-l h or 3-4 h. triuresis on its own; Na+-to-creatinine ratios (means t Aldo. Aldo at dosages of 0.05 and 0.1 pg/rat caused SE) fell to 307 t 27 compared with 530 t 58 mmol/g (P significant Na+ retention compared with vehicle alone in < 0.05) for the control group (Fig. IA). DOC had no the l-3-h urine collection period (Fig. 3A). Na+-to-creeffect on urinary K+ excretion at 2.5 or 5.0 lug/rat during the l-3-h urine collection period (Fig. 1B) but caused a atinine ratios (means t SE) for the control group were significant kaliuresis at 10 ,ug/rat. K+-to-creatinine ratios 530 t 58 compared with 384 t 35 mmol/g for 0.05 ,ug (means t SE) were increased to 270 t 20 compared with Aldo/rat (P < 0.05) and 311 t 18 mmol/g for 0.1 pg 139 t 14 mmol/g for the control group (Fig. 1B). At all Aldo/rat (P < 0.01). Aldo also caused a significant kaliuresis at these dosages; urinary K+-to-creatinine ratios dosagesDOC did not affect urinary creatinine output. (means t SE) for controls were 139 t 14 compared with When the ADX rats were pretreated (subcutaneously) for 30 min with 2.5 mg CS/rat, the Na+-retaining effects 238 t 19 mmol/g (P < 0.01) for 0.05 pg Aldo/rat, and 247 t 20 mmol/g (P < 0.001) for 0.1 pg Aldo/rat (Fig. of both dosages of DOC (5 and 10 pg/rat) were significantly enhanced when compared with the same dosages 3B) CS (2.5 mg/rat) also significantly enhanced the Na+ of DOC alone in the l-3-h urine collection period (Fig. retention caused by 0.05 and 0.1 pg Aldo/rat in the 1-3IA). CS did not alter the urinary K+ excretion caused by h urine collection period (Fig. 3A). CS had no effect on any dosage of DOC used (Fig. IB), whereas CS alone the kaliuresis induced by both the 0.05- and O.l-pg/rat (2.5 mg/rat) had no intrinsic effects on the urinary dosages of Aldo (Fig. 3B). Urinary creatinine excretion was not significantly altered by either Aldo or CS. As A URINARY NA+/cRwTININE (mm&s/g) with DOC, CS enhanced the antinatriuretic effects of 1 - 3 HOURS Aldo in the O-l-h (0.05 pg Aldo/rat) and 3-4-h (0.05 and RESULTS
600
T
t
A
URINARY 8001
200--
*.*-
. 0'
(mmoles/g)
NS
P(O.001
q - ....* . . .
100
NA+/CREATlNlNE 0 -1HOUR
51.0
. . . . -....
ib
P(O.001
1 o!o
DOC(pg/rat)
URINARY
B
SHAM DOC
K+/CREATININE(mmoles/g) 1 - 3 HOURS
300T
I
= SHAM
2.5/q
DOC
5.0
/q
DOC
lO.Opg
I
CS
DOC
= 2.5mgCS
T
250
B
t
URINARY
200
(mw ioles/g)
NA+/CREATlNINE 3 -4 HOUR
700 150
A
T
600
I loo--IINS 50 --
I
P(O.01
0 O!
I 2.5
5!0
lO!O
D WPg/rat)
FIG. 1. Mineralocorticoid activity of 2.5, 5.0, and 10.0 pg/rat DOC with (W---H) and without (o----@ CS pretreatment (2.5 mg/rat). CS was given SC 30 min before DOC in male ADX rats, and urine was collected l-3 h after injection of the steroid. Antinatriuretic activity (A) is expressed as a decrease in the Na+-to-creatinine ratio and kaliuretic activity (B) by an increase in the K+-to-creatinine ratio. Values shown are means f. SE (n = 13-16 rats). A, animals given CS vehicle followed by DOC vehicle; A, mean ratios for animals treated with CS (2.5 mg/rat) alone. Groups were statistically compared using ANOVA followed by the Bonferroni t test.
I FIG. periods analyses dosages shown
SHAM DOC
2.5j.q
= SHAM
CS
DOC
5.0
pg
DOC
I
P(O.01
lO.Opq
DOC
= 2.5mgCS
2. Antinatriuretic activity of DOC or vehicle in urine collection O-l h (A) and 3-4 h (B) after injection of steroid. Statistical (see Fig. 1) were made between animals that received the same of DOC (or vehicle) with and without CS pretreatment. Values are means & SE (n = 13-17 rats).
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F758
LIQUORICE
ENHANCES
A URINARY NA+/CREATlNlNE
NA+
RETENTION
OF
(mmoles/g)
ALDOSTERONE
AND
A URINARY NA’/CREATININE (mmoles/g)
1 - 3HOUR
O800
600 -I-
T
DOC
hlC
700--
f
NS
1 HOUR T
600-500--
3004
400-300-200 -lOO--
0 SHAM ALDO
=
= SHAM
0.05pg
ALDO
CS
0.1~
0
ALDO
SHAM ALDO
= 2.5mgCS
-
= SHAM
0.05p-g
ALDO
CS
O.lpg
U
ALDO
= 2.5mgCS
B URINARY NA’/CREATlNlNE (mmoles/g) 3-
4HOUR
0 SHAM AID0
-
= SHAM
CS
FIG. 3. Antinatriuretic (A) and kaliuretic 0.1 lug/rat Aldo with and without 2.5 mg/rat shown are means t SE (n = 6-8 rats).
I
= 2.5mgCS (B) activities of 0.05 and CS pretreatment. Values
0.1 pugAldo/rat) postinjection periods (Fig. 4, A and B). The above results describing the effects of CS on DOC and Aldo were expressed with Na+-to-creatinine and K+to-creatinine ratios; in addition, Table 1 shows the absolute excretory rates for Na+, K+, and creatinine for sham experiments, 5.0 pugDOC (with or without CS) and 0.1 ,ug Aldo (with or without CS) in the l-3-h urine collection period. DISCUSSION
We had earlier shown that carbenoxolone sodium causes the glucocorticoids corticosterone and cortisol which contain an lip-hydroxyl grouping, to display MClike activity (19). The present studies show that the Na+retaining effects of two other adrenal MCs, Aldo and DOC, can be enhanced by the liquorice derivative CS. DOC does not possess a C-11 hydroxyl grouping; thus the increased Na+-retaining properties conferred by CS upon DOC must occur by a mechanism(s) in addition to that of llfl-OHSD inhibition. In aqueous solutions a small proportion of Aldo exists as its ILhydroxyl form (3), thus a role for lip-OHSD inhibition in the enhanced Na+-retaining properties of Aldo by CS cannot be ruled out. It should be noted that others have shown enhancement of the MC actions of Aldo, although with much greater dosagesof CS than used in these experiments (1, 16); in contrast, one study showed that CS did not affect
-
= SHAM
0.05pg
ALDO
CS
FIG. 4. Antinatriuretic activity CS (2.5 mg CS/rat) pretreatment (A) and 3-4 h (B) after injection SE (n = 6-8 rats).
0.1/q
I
ALDO
I = 2.5mgCS
of 0.05 and 0.1 pg Aldo/rat following in the urine collection periods O-l h of the steroid. Values are means t
1. Composition of urine from CS-treated and DOC- and Aldo-treated (HZ’S) ADX rats
TABLE
Na’, pmol Control Sham CS Sham Steroid 2.5 mg CS Sham Steroid DOC Sham CS 5.0 pg DOC 2.5 mg CS 5.0 pg DOC Aldo Sham CS 0.1 Pg Aldo 2.5 mg CS 0.1 Pg Aldo
K+, pmol
Creatinine,
169.7 t20.3 155.7 NS +13.4
44.1 rt4.4 39.3 NS t4.2
327.5 t23.8 356.2 NS t25.6
146.0 t18.7 60.5 P < 0.001 t6.6
61.2 t6.6 56.0 NS -1-4.0
377.8 t13.1 364.1 NS t16.5
68.6 t7.6 66.3 NS t5.6
278.3 k21.5 280.9 NS t19.4
86.1 t8.3 46.9 t8.9
P < 0.01
pug
Values are means k SE; n = 8-12 rats. Urine from male ADX rats (160-200 g body wt) was collected l-3 h after injection of either steroid or vehicle (see METHODS). Statistical comparisons were made between groups of animals that received DO@ or Aldo with and without CS. The control set of experiments shows the effects of CS alone.
the MC activity of Aldo at all (17). At the present time, the mechanisms by which CS causes this apparent amplification of the Na’-retaining effects of DOC and Aldo, and even on which portions of the nephron CS is working, are all unknown. An increased bioavailability of the
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LIQUORICE
ENHANCES
NA+
RETENTION
unmetabolized parent MCs may possibly account for these observations. In addition to demonstrating inhibition of llfl-OHSD (ll), we have recently shown by in vitro experiments with both DOC (S. A. Latif, personal communication) and Aldo (9) as substrates, that CS inhibits hepatic A*-5P-reductase and also 3P-hydroxysteroid dehydrogenase, but not A*-5cu-reductase. Thus the metabolism of Aldo, DOC, and possibly other glucocorticoids in the rat may be rerouted in the presence of CS, resulting in the accumulation of unmetabolized hormones and their corresponding 5a-dihydro derivatives. Both 5a-dihydro-Aldo and 5cr-dihydro-DOC are also biologically active compounds (13, 18). It is interesting that, as shown in our earlier studies with corticosterone and cortisol, the enhanced Na+ retention observed with both Aldo and DOC in rats pretreated with CS was also visible earlier as well as being extended longer into the 3-4-h urine collection period. This does not usually occur with physiological dosages of Aldo where a longer latent period is observed (12). In addition, the previous studies showed the large antinatriuretic responses elicited by corticosterone and cortisol in the presence of CS were disproportionate to the kaliuretic effects observed (19). It is noteworthy that the kaliuretic properties of DOC and Aldo were not affected by CS in the present studies. These observations offer clues in designing the further experiments needed to 1) elucidate how CS may alter the interactions of these MCs with steroid receptors in the kidney, and 2) determine which segments of the renal tubule and which biochemical processes are affected by CS. Thus liquorice derivatives may enhance or amplify the MC activity of a variety of other adrenal steroids (besides corticosterone and cortisol), including some that do not possess an lip-hydroxyl group. Hence a variety of secretory products of the adrenal gland (from both the fascicular and glomerular zones) may be affected by liquorice ingestion or treatment with foodstuffs and drugs containing chemical derivatives of glycyrrhetinic acid.
OF
9.
10
11.
12.
13.
14.
15.
16.
17.
18. The authors thank Dr. M. Sheff, Miriam Hospital, Providence, RI, for helpful discussions concerning this work. This work was supported by National Institutes of Health Grant DK-21404 and The Miriam Hospital Research Foundation. Address for reprint requests: D. J. Morris, The Miriam Hospital, Dept. of Laboratory Medicine, 164 Summit Ave., Providence, RI 02906. Received
30 August
1989; accepted
in final
form
17 November
1989.
19.
20.
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