J. Steroid Biochem. Molec. Biol. Vol. 45, No. 4, pp. 235-238, 1993 0960-0760/93 $6.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1993 Pergamon Press Ltd

5 ~ - D I H Y D R O - 1 1 - D E O X Y C O R T I C O S T E R O N E A S A

M I N E R A L O C O R T I C O I D A G O N I S T A N D A N T A G O N I S T :

E V I D E N C E F O R A W E A K M I N E R A L O C O R T I C O I D A S A N

A N T A G O N I S T O F P O T E N T M I N E R A L O C O R T I C O I D S

HISAHIKO SEKIHARA* and YOSHIO YAZAKI

The Third Department of Internal Medicine, University of Tokyo Faculty of Medicine, Hongo, Tokyo 113, Japan

(Received 11 August 1992; accepted 16 November 1992)

Summary--To evaluate the possibility that 5g-dihydro-1 l-deoxycorticosterone (5ct-DH-DOC), a weak mineralocorticoid, is an antagonist of a more potent mineralocorticoid, aldosterone, 0.25 #g aldosterone was injected into adrenalectomized rats simultaneously with 200-800 #g 5a-DH-DOC and urinary Na/K ratio and Na and K excretion were evaluated. Urinary Na/K ratio and Na excretion were significantly lower than those of control rats regardless of whether rats were treated with 0.25/zg aldosterone alone or 400-800 #g 5at-DH-DOC alone. Urinary Na/K ratio and Na excretion of rats given a combination of 0.25 #g aldosterone plus 400- 800 # g 5ct-DH-DOC were significantly higher than those of rats given 0.25/~ g aldosterone alone. None of the treatment caused significant changes in urinary K excretion. The results demonstrate that 5~-DH-DOC, a weak mineralocorticoid, is an antagonist of the sodium-retaining action of a more potent mineralocorticoid, aldosterone. Progesterone which has weak mineralocorticoid activity is also known as an antagonist of more potent mineralocorticoids. The results of the present study demonstrate further evidence that weak mineralocorticoids may work as antagonists of more potent mineralocorticoids.

INTRODUCTION

Among 5g-dihydrosteroids, 5~-dihydroaldo- sterone (5g-DH-ALDO) and 5g-dihydro- 1 l-de- oxycorticosterone (5g-DH-DOC) are mineralo- corticoids [1, 2]. The mineralocorticoid potency of 5a-DH-ALDO is 1.8% of aldosterone and almost the same as l l-deoxycorticosterone (DOC) and that of 5~-DH-DOC is 2.5% of DOC and 0.04% of aldosterone.

As progesterone, which has weak mineralo- corticoid activity, is known as an antagonist of mineralocorticoids [3-5], in the present study we evaluated the possibility that 5~-DH-DOC, a weak mineralocorticoid, is an antagonist of more potent mineralocorticoids and found that 5~-DH-DOC antagonizes the sodium-retaining action of aldosterone. The present paper demon- strates further evidence that weak mineralo- corticoids work as antagonists of more potent mineralocorticoids.

EXPERIMENTAL

Materials

Aldosterone was obtained from Sigma Chemical Co. (St Louis, U.S.A.). 5~-DH-DOC

*To whom correspondence should be addressed.

(5~-pregnan-21-ol-3, 20-dione) was purchased from Makor Chemicals Ltd (Jerusalem, Israel). Male Sprague-Dawley rats weighing 120-150 g were obtained from Clea Japan Inc. (Tokyo, Japan).

Mineralocorticoid bioassay

Mineralocorticoid bioassay was performed as described previously[l, 6-8]. The method, in brief, is as follows: male rats were bilaterally adrenalectomized under sodium pentobarbital anesthesia and then fasted overnight. The following day, 3 ml saline (154mM sodium chloride) along with 0.25 #g aldosterone alone, graded doses (200-800#g) of 5~-DH-DOC alone and a combination of 0.25 #g aidosterone plus graded doses (200-800 #g) of 5~-DH-DOC dissolved in 0.5 ml of 10% ethanol were injected intraperitoneally. The rats were sacrificed 3 h later and the urine was aspirated from the bladders. The concentration of Na and K of the urine was measured with a flame photometer. Urinary Na/K ratio was calculated from the concentration of Na and K and was used as the index of mineralocorticoid activity. Urinary Na and K excretion was calculated from the concentration of Na and K and urine volume.

235

236 HISAHIKO SEKIHARA and Y o s m o YAZAKI

Table I. Comparison of urine volume, Na and K concentration, Na/K ratio and Na and K excretion of rats given 0.25 #g aldosterone with those of control rats

Na K Na K Urine volume concentration concentration excretion excretion

(ml/3 h) (mEq/I ) (mEq/I) Na/K (mEq/3 h) (mEq/3 h)

Control 0.98 ___ 0.17 207 + 20 81 + 8 2.36 + 0.15 0.193 _+ 0.026 0.084 + 0.014 Aldosterone 0.80 __. 0.13 142 ___ 11 111 _+ 4 1.27 + 0.10 0.096 + 0.034 0.087 + 0.012

0.25/~g P NS <0.05 <0.0l <0.001 <0.05 NS

The mean + SE (n = 6) of each value is shown.

Dose-response curves of mineralocorticoid activity of aldosterone, DOC, 5~-DH-ALDO and 5~t-DH-DOC were described previously [1]. On the dose-response curve of aldosterone, 0.25#g aldosterone showed the maximum mineralocorticoid activity. The changes in urine volume, Na and K concentration, Na/K ratio and Na and K excretion after the administration of 0.25 #g aldosterone are shown in Table 1. The administration of 0.25#g aldosterone caused a significant decrease in urinary Na/K ratio and Na excretion, but did not cause any significant change in urinary K excretion. Relative potency of mineralocorticoid activity (and 95% confidence limits) of DOC, 5~-DH- ALDO and 5g-DH-DOC is as follows: aldo- sterone 100, DOC 1.6 (0.6-3.7), 5g-DH-ALDO 1.8 (0.7-4.7), 5ct-DH-DOC 0.04 (0.0096-0.059). The index of precision of the bioassay ranged between 0.2 and 0.3.

Urinary Na/K ratio and Na and K excretion of each group were compared by the multiple comparison method of Duncan.

R E S U L T S

To examine the antagonistic effect of 5~-DH- DOC on aldosterone, 0.25 #g aldosterone alone, 200-800/~g 5~-DH-DOC alone and a combin- ation of 0.25#g aldosterone plus 200-800#g 5~-DH-DOC were injected into rats and urinary Na/K ratio (Fig. 1) and Na and K excretion (Fig. 2) were evaluated.

Urinary Na/K ratio was significantly lower than that of control rats regardless of whether rats were treated with 0,25#g aldosterone alone or 200, 400 or 800 #g 5~-DH-DOC alone. Urinary Na excretion was also significantly lower than that of control rats regardless of whether rats were treated with 0.25/~g aldo- sterone alone or 400 or 800#g 5~-DH-DOC alone. Urinary Na/K ratio of rats given a combination of 0.25#g aldosterone plus 400 or 800/~g 5~-DH-DOC was significantly higher than that of rats given 0.25gg aldosterone alone. Urinary Na excretion of rats given a combination of 0.25 #g aldosterone plus 400 or 800#g 5~-DH-DOC was also significantly

_

3- Ig Z

. [ 2-

el-

_T..

Control Aldo 0.25

P<0.01 I I I

P<0.01 [ ]1

N$ P<0.01 I I I

N$

145

0.25 0.25 0.25 200 400 800t, g + . + +

~I-00C I~I.D0C I~.I~C 2OO 4O0 8O0

Fig. I. Ur ina ry N a / K ra t io (the m e a n + SE) of con t ro l ra ts (n = 13) and rats given 0.25 p g a ldos te rone (Aldo) a lone (n = 10), 200-800 # g 5 ,~-DH-DOC ( D H - D O C ) a lone (n = 8) and a c o m b i n a t i o n of 0 . 25 / l g aldosterone plus 2 0 0 - 8 0 0 # g 5 ~ - D H - D O C (n = 10). Black bars are s ignif icantly lower than cont ro l

(P < 0.01).

Interaction of mineralocorticoids 237

O.

C 0 ==;o.

z

= 0"2 t . 0 ~

" 0

P<O.O1 NS w[ P<O.01 NS

I [ I

Control Aldo AMo Aldo Aldo DH.DOC DH.DOC DH.DOC 0.25 0.25 0.25 0.25 200 400 8~g

+ + + DH.DOC DH.DOC DH-DOC

200 400 800

N Control

f] f]n A f] f]N Aldo Aldo Aide Aldo DH.DOC I~I.IX~ I~1.1~ 0.25 0.25 0.25 0.25 200 400 8~g

4- + + DH.DOC DH.DOC DH.DOC

200 400 800 Fig. 2. Urinary Na and K excretion (the mean + SE) of control rats (n = 13) and rats given 0.25 #g aldosteron¢ (Aldo) alone (n = 10), 200-800 #g 5~t-DH-DOC (DH-DOC) alone (n = 8) and a combination of 0.25/zg aldostcrone plus 200400 #g 5~t-DH-DOC (n = 10). Black bars are significantly lower than

control (P < 0.01). White bars are not significantly different from control.

higher than that of rats given 0.25 #g aldo- sterone alone. In contrast, urinary Na/K ratio and Na excretion of rats given a combination of 0.25 #g aldosterone plus 400 or 800/~g 50t- DH-DOC were not significantly different from those of rats given 400 or 800/lg 5~-DH-DOC alone. None of the treatments caused significant changes in urinary K excretion.

DISCUSSION

In the present study, the sodium-retaining action of aldosterone was antagonized by the simultaneous administration of a 1600-fold ex- cess of 5~t-DH-DOC. Therefore, 5~t-DH-DOC, which was reported to be a weak mineralo- corticoid [1, 2], is an antagonist of the action of a more potent mineralocorticoid, aldosterone.

Progesterone which has weak mineralo- corticoid activity is also known as an antagonist of mineralocorticoids. Progesterone decreased urinary Na/K ratio when it was administered alone. However, progesterone increased low urinary Na/K ratio caused by more potent mineralocorticoids when it was administered simultaneously with the latter compounds [3-5].

The results of the present study show that 5~t-DH-DOC has the activities similar to pro- gesterone. Therefore, weak mineralocorticoids are considered to work as antagonists of more potent mineralocorticoids.

The antagonistic effect of 5~t-DH-DOC on aldosterone, however, is considered only to the level of intrinsic activity of 5~t-DH-DOC, because no significant difference of mineralo- corticoid activity was obtained between 5~t-DH- DOC and a combination of aldosterone plus 5~t-DH-DOC even though a 3200-fold excess of 5at-DH-DOC was administered simultaneously.

According to current concepts of the mechan- ism of physiological action of mineralocorticoids, the initial event in the intracellular action is considered to involve binding of the steroid to stereospecific cytoplasmic receptors in renal tubular cells. The steroid-receptor complex is then activated and transferred to the nucleus where it attaches to chromatin acceptor sites and initiates DNA-dependent RNA synthesis [10]. Progesterone[9] was demonstrated to inhibit the binding of aidosterone to the renal cytoplasmic receptors and to diminish the generation of nuclear aldosterone complexes

SBMB 45/~--D

238 HISAHIKO SEKIHARA and YOSHIO YAZAKI

capable of a t taching to chromat in . Therefore, 5 ~ - D H - D O C is considered to antagonize the sodium-retaining action of aldosterone probably inhibi t ing the b inding of aldosterone to renal cytoplasmic receptors and diminishing the generat ion of nuclear aldosterone complexes. The details should be evaluated further.

Cort isol /cort isone shuttle in the renal tubular cells is now considered to be impor tan t in the

regulat ion of blood pressure and the genesis of hypertension [11]. According to this shuttle theory, cortisol which escapes conversion to

cortisone by 1 l fl-hydroxysteroid dehydrogenase binds to mineralocort icoid receptors and works as a potent mineralocort icoid [12]. Al though the role of cortisone in this shuttle is not taken into considerat ion at the present time, cortisone has mineralocort icoid activity, the potency of which is 0.17% of aldosterone[13, 14]. Therefore, cort isone is a weak mineralocort icoid similar to 5~ -DH-DOC. According to the results of the present study, it is inferred that cort isone is working as an antagonis t of aldosterone and cortisol in the shuttle to regulate the action of both potent mineralocort icoids. The effects of cortisone on the act ion of aldosterone and cortisol will be evaluated in the following

studies.

Acknowledgements--This work was supported in part by Grants-in-Aid for Scientific Research from The Ministry of Education, Science, and Culture, Japan (61570540 and 62570503).

REFERENCES

1. Sckihara H., Island D. P. and Liddle G. W.: New mineralocorticoids: 5~-dihydroaldosterone and 5~-dihydro-ll-deoxycorticosterone. Endocrinology 103 (1978) 1450-1452.

2. Morris D. J.: The metabolism and mechanism of action of aldosterone. Endocrine Rev. 2 (1981) 234-247.

3. Kagawa C. M.: Blocking urinary electrolyte effects of desoxycorticosterone with progesterone in rats. Proc. Soc. Exp. Biol. Med. 99 (1958) 705-707.

4. Rosemberg E. and Engel I.: The effect ofhydrocortisone and progesterone on the electrolyte action exhibited by desoxycorticosterone in the adrenalectomized rat. Endocrinology 69 (1961) 496-503.

5. Uete T. and Venning E. H.: Effects of 16c(-OH-A a- and AS-pregnene compounds with respect to electrolyte excretion of aldosterone. Endocrinology 72 (1963) 397-402.

6. Sekihara H., Hollifield J. W., Island D. P., Slaton P. E. and Liddle G. W.: Evidence for heterogeneity of mineralocorticoids in urine of patients with low renin essential hypertension. J. Clin. Endocr. Metab. 48 (I 979) 143 147.

7. Kagawa C. M., Shipley E. G. and Meyer R. K.: A biological method for determining small quantities of sodium retaining substances. Proc. Soc. Exp. Biol. Meal 80 (1952) 281-285.

8. Sennett J. A., Brown R. D., Island D. P., Yarbro L. R., Watson J. T., Slaton P. E., Hollifield J. W. and Liddle G. W.: Evidence for a new mineralocorticoid in patients with low-renin essential hypertension. Circ. Res. 36/37 (Suppl. I) (1975) I-2-I-9.

9. Wambach G. and Higgins J. R.: Antimineralocorticoid action of progesterone in the rat: correlation of the effect on electrolyte excretion and interaction with renal mineralocorticoid receptors. Endocrinology 102 (1978) 1686--1693.

10. Marver D., Goodman D. and Edelman I. S.: Relationships between renal cytoplasmic and nuclear aldosterone-receptors. Kidney Int. 1 (1972) 210 223.

11. Ulick S., Levine L. S., Gunczler P., Zanconato G., Ramirez L. C., Rauh W., Rrsler A., Bradlow H. L. and New M. I.: A syndrome of apparent mineralocorticoid excess associated with defects in the peripheral metab- olism of cortisol. J. Clin. Endocr. Metab. 49 (1979) 757-764.

12. Arriza J. L., Weinberger C., Cerelli G., Glaser T. M., Handelin B. L., Housman D. E. and Evans R. M.: Cloning of human mineralocorticoid receptor comple- mentary DNA: structural and functional kinship with glucocorticoid receptor. Science 237 (1987) 268-275.

13. Liddle G. W.: The adrenals. In Textbook of Endo- crinology (Edited by R. H. Williams). W. B. Saunders, Philadelphia, 6th Edn (1981) pp. 249-292.

14. Roberts K. E. and Pitts R. F.: The influence of cortisone on renal function and electrolyte excretion in the adrenalectomized dog. Endocrinology 50 (1952) 51 60