Specific inhibition of the last steps of aldosterone biosynthesis by 18-vinylprogesterone in bovine adrenocortical cells

J. Steroid Biochem. Molec. Biol. Vol. 57, No. 1/2, pp. 141-147, 1996

Pergamon

0960-0760(95)00252-9

Copyright © 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-0760/96 $15.00 + 0.00

Specific Inhibition of the Last Steps of Aldosterone Biosynthesis by 18..Vinylprogesterone in Bovine Adrenocortical Cells G. D e f a y e , 1. A . P i f f e t e a u , 2 C. D e l o r m e

2 and A. Marquet 2

ICEA, I N S E R M U244, Laboratoire de Biochimie des R~gulations Cellulaires Endocrines, Departement de Biologie Moldculaire et Structurale, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France and 2Laboratoire de Chimie Organique Biologique, URA C N R S 493, Universitd Pierre et Marie Curie, 4 Place Jussieu, 75252 Paris Cedex 05, France

1 8 - V i n y l p r o g e s t e r o n e (18-VP), d e s i g n e d f o r m e c h a n i s m - b a s e d specific i n h i b i t i o n o f t he last steps o f t h e a l d o s t e r o n e biosynthesis, was us ed to c h a r a c t e r i z e t he m e c h a n i s m o f t he 11- a n d 1 8 - h y d r o x y l a s e activities o f b o v i n e c y t o c h r o m e P4501tp. In t h e p r e s e n t work, its a c t i o n was s t u d i e d by o b s e r v a t i o n s on a p r i m a r y c u l t u r e o f b o v i n e a d r e n o c o r t i c a l cells. First, we i n v e s t i g a t e d t he effects o f 18-VP on t h e d i f f e r e n t e n z y m a t i c steps o f t he b i o s y n t h e s i s o f cort i sol a n d a l d o s t e r o n e . T h e p r o d u c t i o n o f cortisol, b a s e l i n e or h o r m o n e - s t i m u l a t e d ( A C T H or AID, was i n h i b i t e d by 18-VP in a dosed e p e n d e n t m a n n e r with a m a x i m a l i n h i b i t i o n at 5/iM. S u p p l y o f d i f f e r e n t e x o g e n o u s s u b s t r a t e s to s u p p o r t s t e r o i d o g e n e s i s r e v e a l e d an i n h i b i t i o n o f t he last step o f cort i sol or c o r t i c o s t e r o n e biosynthesis. We t h e n us ed specific b l o c k e r s to m e a s u r e i n d i v i d u a l activities a n d c o n c l u d e t h a t 111~-hydroxylation was t he only e n z y m a t i c a c t i v i t y affected. A l d o s t e r o n e , as well as 1 8 - h y d r o x y c o r t i c o s t e r o n e , was also m e a s u r e d following a d d i t i o n o f c o r t i c o s t e r o n e . T h e 1 8 - h y d r o x y l a t i o n o f c o r t i c o s t e r o n e was i n h i b i t e d by 18-VP, with 50% i n h i b i t i o n o c c u r r i n g at 0.04 p M c o m p a r e d w ith the 50% i n h i b i t i o n value o f 0.3/~ M o b t a i n e d f o r 1 1 - h y d r o x y l a t i o n . S u r p r i s i n g l y , 1 8 - e t h y n y l - p r o g e s t e r o n e (18-EP), w h i c h has ~, s t r u c t u r e v e r y s i m i l a r to 18-VP, only weakly i nhi bi t s 1 1 f l - h y d r o x y l a t i o n . T h e i n h i b i t i o n o f a l d o s t e r o n e f o r m a t i o n was also m u c h l o w e r with 18-EP t h a n with 18-VP. T h e s e studies d e m o n s t r a t e t h a t 18-VP i nhi bi t s only t he l a t e r steps o f a l d o s t e r o n e b i o s y n t h e s i s a n d m o r e specifically 18- t h a n 1 1 - h y d r o x y l a t i o n activity. C o p y r i g h t © 1996 E l s e v i e r Science Ltd.

j . Steroid Biochem. Molec. Biol., Vol. 57, No. 1/2, pp. 141-147, 1996

substrate analogs modified at the 18-methyl group. These specifically targeted compounds were designed as mechanism-based inhibitors of the enzyme that catalyses the formation of aldosterone from corticosterone [2, 3]. T he two steps of the biosynthesis of aldosterone from corticosterone, namely the 18-hydroxylation of corticosterone and the transformation of 18-hydroxycorticosterone to aldosterone, are both catalysed in the bovine [4, 5] or the porcine [5] by cytochrome P450H~ (EC 1.14.15.4). This cytochrome is also responsible for the l lfl-hydroxylation of deoxycorticosterone and deoxycortisol which leads, respectively, to corticosterone and cortisol. In the bovine adrenal cortex [6], two distinct cytochromes P450H~ encoded by two different genes have been isolated [7]. T he two proteins are found in both the glomerulosa and the fasciculata

INTRODUCTION Aldosterone, the most potent mineralocorticoid in mammals, is produced in the zona glomerulosa of the adrenal cortex. It regulates electrolyte balance by promoting potassium elimination and sodium retention [1]. In pathological stal:es, overproduction of aldosterone leads to some forms of arterial hypertension. Among many other therapeutic options, these disorders may be clinically treated with spironolactone, which acts mainly at the receptor level [1] but which can also produce severe side ettects. We have developed a different approach, namely enzymatic inhibition of aldosterone biosynthesis by *Correspondence to G. Def-~cye. Received 25 Mar. 1995; accepted 18 Sep. 1995 141

142

G. Defaye et al.

and have the same catalytic properties [5, 8]. T w o cDNAs, 11/3-2 and 11/3-3, corresponding to these forms have been cloned [9] and expressed in COS-7 cells [10]. At the same time, Mathew et al. [11] have established that a e D N A clone, equivalent to 11/3-3, encodes the major form of P450ua found in bovine adrenal cortex, and is able to catalyse l l/3-hydroxylation of D O C as well as aldosterone synthesis from D O C in COS-1 cells. Among the 18-methyl substituted derivatives of progesterone, developed to bind covalently to the prosthetic heme group of the cytochrome [2, 3], the most potent inhibitors of aldosterone biosynthesis by rat adrenal cell-free extracts are 18-vinylprogesterone (18-VP) and 18-ethynyl-progesterone (18-EP) (Fig. 1). T h e inhibition, in calf adrenal glomerulosa cells, by the closely related compound 18ethynyldeoxycorticosterone (18-EDOC), has also been studied recently by both Yamakita et al. [12] and Gomez-Sanchez et al. [13]. In addition, the biochemical and pharmacological effects of mainly ethynyl inhibitors of aromatase and aldosterone synthase have been published [14]. In this work, we present results concerning the action of these compounds on cortisol and aldosterone production by primary cultures of bovine adrenocortical cells. T h e y inhibit 11/3-18 hydroxylase activity, as expected, but more specifically 18- than 11-hydroxylation. MATERIALS AND M E T H O D S

Table 1. Effect of 18-VP on hormone activated and steroid supported cortisol production Cortisol production (pmol/h/106 cells) 18-VP (/aM) C o m p o u n d added No A C T H (10 nM) A I I (30 nM) 25-Hydroxycholesterol Pregnenolone (15 # M ) Progesterone (15 # M ) 17OH-Pregnenolone (15 # M ) 17OH-Progesterone (15 p M )

0

10

23__+4 347 + 38 247 + 11 202 _ 15 205 _ 8 273 _ 9 1851 _ 33 2738 _ 138

14+3 219 + 8 139 + 14 67 __+3 75 + 4 120 _+ 14 522 __+24 752 _ 29

On day 5, bovine adrenocortical cells were incubated for 2 h in F-12 medium containing the indicated concentrations of hormones or steroids in the presence or absence of 18-VP (10#M). After 2 h at 37°C the medium was collected and its cortisol content was determined by radioimmunoassay. T h e values represent the mean + SD of three experiments.

extraction of the medium with dichloromethane and evaporation of the solvent. Cortisol anti-serum was raised in the rabbit in the laboratory; aldosterone monoclonal anti-serum was a generous gift from N. Cittanova [15]. Adrenocortical cells

Bovine adrenals were obtained in a local slaughterhouse. T h e glands were cut, the medulla zone was discarded and the cortex was sliced on a Stadie-Riggs

Materials

All chemicals were purchased from Boehringer Mannheim and Sigma. Silica gel plates 60F 254 were from Merck (Darmstadt, Germany). Synthetic A C T H (1-24) (Synacten) was provided by Ciba (Basel, Switzerland). [1,2,6,7- 3H]dehydro-epiandrosterone (64.5 Ci/mmol), [7-3H-(N)]pregnenolone (25 Ci/mmol), aldosterone and [1,2-3H-(N)]ll-deoxycortisol (41.7 Ci/ mmol) were from New England Nuclear; [1,2,6,73H]corticosterone (60Ci/mmol), [1,2,6,7-3H]cortisol (100Ci/mmol) and [1,2,6,7-3H]17-hydroxyprogester one (89Ci]-mmol) were from the Radiochemical Centre (Amersham, U.K.). Cortisol and aldosterone were measured in cell culture medium by radioimmunoassay. Cortisol was measured directly in the medium and aldosterone after

A 1,0

o ~o = •" o ~

0,s

o o

" 0,0 0

0.05

0.1

le-vp o

18-VP

o

18-EP

Fig. 1. S t r u c t u r e of 1 8 - V P a n d 18-EP

0.5

1

5

10

(I~M)

Fig. 2. P r o d u c t i o n o f c o r t i s o l by cells in t h e p r e s e n c e o f 1 8 - V P . O n d a y 5, b o v i n e a d r e n o c o r t i c a l c e l l s w e r e i n c u b a t e d for 2 h in F-12 m e d i u m c o n t a i n i n g A C T H (10 n M ) a n d t h e c o n c e n trations indicated of inhibitor. Cortisol content was determ i n e d b y r a d i o i m m u n o a s s a y . E a c h a c t i v i t y is t h e a v e r a g e ( + S D ) o f t r i p l i c a t e m e a s u r e m e n t s in t h e s a m e e x p e r i m e n t , e x p r e s s e d p e r 10 6 c e l l s p e r h o u r . S h o w n is a r e p r e s e n t a t i v e sample of three independent experiments.

Inhibition of Aldosterone Biosynthesis

143

Table 2. Inhibition by 18-VP of the different enzymatic steps nmol produced/106 cells/h 18-VP (#M) Enzymatic step

0

1

5

10

0.8±0.1

--

1.30±0.2

1.40±0.05

1.12±0.15

1.28±0.1

2.1±0.1

2.2±0.2

1.9±0.2

1.8±0.2

13.8±0.7

--

--

12.3±0.5

Side chain cleavage Pregnenolone*** (from 25-OH cholesterol 25 #M)

0.85±0.2

--

17ot-hydroxylase [3H]17-OH-pregnenolone* (from [7-3H]pregnenolone 5 0 # M )

21-hydroxylase [3H]l 1-deoxycortisol** (from [1,2,6,7-3H] 17-OH-progesterone 100 pM) 3 ~-Hydroxysteroid -dehydrog~ nase [3H]androstenedione** (from [1,2,6,7-3H]DHEA 100 #M)

*in the presence of trilostane (2 #M). **in the presence of metyrapone (2/~M). ***in the presence of trilostane (2ltM) and SU 10603 (0.5 #M). On day 5, bovine adrenocortical cells were incubated for 2 h in F-12 medium containing the indicated concentrations of 18-VP and the different steroids. After 2 h ~t 37°C the medium was collected and the products separated by T L C as described in Materials and Methods. T h e values are the average -: SD of 3 measurements in the same experiment. They are representative of three independent experiments.

microtome. Adrenocortical cells were then prepared by successive digestion [16]. Cells were grown in Hams' F12 medium supplemented with insulin (5#g/ml), transferrin (5/~g/ml) and selenous acid (5 ng/ml) ( I T S medium) and seeded in 12-well plates.

Inhibition of cortisol production Cells were used on day 5 of culture. For stimulation of steroidogenesis concentrations of 10 n M of A C T H and 0.3 p M of AII were used. Steroidogenesis was studied with or without addition of exogenous substrates, (25-hydrox,.ccholesterol, pregnenolone, 17hydroxypregnenolone, progesterone or 17-hydroxyprogesterone). In each case, incubations were performed at 37°C for 2 h in the presence of 18-VP (0-10 pM). Cortisol production was measured in the medium by RIA.

Enzymatic activities Side chain cleavage activity. Side chain cleavage activity was measured by incubation of the cells for 2 h at 37°C with 25-hydroxycholesterol (25 # M ) in the presence of trilostane (2: # M ) (gift of Winthrop Laboratories, France) and SU 10603 (5 ktM) as blockers of 3/%hydroxysteroid dehydrogenase/isomerase and 17hydroxylase activity, respectively. After extraction of the steroids into dichloromethane, the pregnenolone content was quantified by RIA. 17o~-Hydroxylase activity. 17~-Hydroxylase activity was assayed by measuring the transformation of [7- 3H-(N)]pregnenolone to [3H]17~-hydroxypregnenolone. T h e cells were incubated for 2 h at 37°C in Ham's F12 medium (Gibco) containing 5 0 m M of [3H]pregnenolone (New England Nuclear) and 2 # M of trilostane (kind gift from Winthrop Laboratories, France) as a blocker of' 5-en-3fl-hydroxysteroid dehydrogenase/isomerase. T h e steroids were extracted from

the medium with chloroform. T h e organic extract was analysed by thin layer chromatography on silica gel plates (F-254, Merck) with chloroform-ethyl acetate (1:1, v/v). T h e spots corresponding to the substrate and the product of the reaction were scraped off the plates and counted for radioactivity in a liquid scintillation spectrophotometer (Kontron, Paris, France). T h e amounts of steroid produced were calculated by the formula A x B/C, where A is the initial amount of substrate, B is the total 3H-counts of the zones corresponding to products and C the sum of the counts corresponding to residual substrate and products.

3~ -Hydroxysteroid-dehydrogenase /isomerase. 3~ Hydroxysteroid-dehydrogenase/isomerase activity was measured by incubation of [1,2,6,7-3H]dehydro epiandrosterone ( D H E A ) ( 1 0 0 # M ) in the presence of metyrapone ( 2 # M ) ; dehydroepiandrosterone and androstenedione were separated on T L C using chloroform-acetone (9: 1, v/v). 21-Hydroxylase. 21-Hydroxylase activity was measured with [1,2,6,7-3H]17-hydroxyprogesterone ( 5 0 # M ) as substrate in the presence of metyrapone (2 #M); [3H]17-hydroxyprogesterone and [3H]deoxycortisol were separated on T L C using chloroform-ethyl acetate (1:1, v/v). Ii ~-Hydroxylase. 11/~-Hydroxylase activity was measured with [1,2-3H-(N)]deoxycortisol (100/~M) as substrate; [3H]cortisol and [3H]deoxycortisol were separated on T L C using chloroform-ethyl acetate (1 : 1,

v/v). 18-Hydroxylation. 18-Hydroxylation of corticosterone was measured after incubation of the cells with [1,2,6,7-3H]corticosterone ( 1 0 p M ) during 2h. T h e products corticosterone, 18-hydroxycorticosterone and aldosterone were separated by H P L C (silica RP-18 (5 mm) column and a mixture of methanol/water (53 %)

G. Defaye et al.

144

at 1 m l / m n flow rate). T h e eluted fractions were collected and the radioactivity corresponding to each peak was measured. [ 3 H ] 1 8 - H y d r o x y c o r t i c o s t e r o n e formation was established by the ratio of the radio40 A It 6 O tD O

RESULTS

30

m O

E i-

20

v

It

10 O O

0.05

0.1

0.5

18-VP

1

5

10

(~tM)

(B) I 8-hydroxylation

400

(corticosterone-18-OHcorticosterone)

O ~0 O

300 Q O

E a. 200

m

o.

100

0,05

0.1 18-VP

0.5

1

5

(gM)

2O

aldosterone synthase corticosterone-aldosterone)

O v-

We then explored each step of the biosynthetic pathway to ascertain which steps were inhibited by 18-VP. Exogenous substrates, intermediaries in the biosynthesis of cortisol, were added to the cells and cortisol production was measured (Table 1). T h e production of cortisol was inhibited for all the substrates added. T h i s result suggests that, as expected, there was no influence of 18-VP on the first steps of the biosynthesis of cortisol. T h i s was confirmed by measuring individual enzyme activities. As shown in T a b l e 2, the different steps were not affected by 18-VP at concentrations of 1, 5 and 1 0 # M . T a k e n together, these results substantiate the hypothesis that 18-VP inhibits cortisol production by selectively decreasing the last step of its biosynthesis, namely, the 11/~-hydroxylation.

Inhibition by 18-VP of the cytochrome P450,~ supported activities As previously described [4] in the bovine adrenal cortex, both l l-hydroxylation of deoxycortisol and deoxycorticosterone and the transformation of corticosterone to 18-hydroxycorticosterone and aldosterone are accomplished by cytochrome P45011~. Cells were

(c)

A m m m O O

T h e main steroid produced by BAC cells is cortisol and production is strongly stimulated by A C T H or A I I . As shown on T a b l e 1, at a concentration of 10/zM of 18-VP, inhibition of the baseline as well as the hormone-stimulated production of cortisol was observed. Cortisol secretion stimulated by A C T H was measured for various concentrations of 18-VP (0-10/zM). T h i s secretion is inhibited by 18-VP in a dose-dependent manner with a m a x i m u m inhibition at 5 ~tM and an ICs0 of 0.06 + 0.02 # M (Fig. 2).

Determination of the step(s) inhibited by I 8 - V P

500

A m D

activity contained in the corresponding peak over the total radioactivity measured. Aldosterone formation. Aldosterone formation was studied under the same conditions with corticosterone (10 p M ) . T h e aldosterone produced was measured by radioimmunoassay, a more sensitive method than the H P L C method described above.

J¢

_o O

E.

10'

6 C

P o It O "D m m

0.05

0.1

0.5

18-vP

(rtM)

1

5

Fig. 3. I n h i b i t i o n of t h e activities s u p p o r t e d by c y t o c h o m e P45011 p. On day 5, cells were i n c u b a t e d w i t h [3H]deoxycortisol (100 IIM) (A), [3H]corticosterone (10 pM) (B) o r c o r t i c o s t e r o n e (10ptM) (C) in t h e p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s of 18-VP as described in Materials and Methods. [3H]deoxycortisol a n d [3H]cortisol were s e p a r a t e d by TLC. [3H]corticosterone a n d [3H]18hydroxycorticosterone were s e p a r a t e d by HPLC. A l d o s t e r o n e f o r m a t i o n w a s d e t e r m i n e d by r a d i o i m m u n o a s s a y . E a c h activity is t h e m e a n of t r i p l i c a t e m e a s u r e m e n t s i n t h e s a m e e x p e r i m e n t , e x p r e s s e d p e r 106 cells p e r h o u r . S h o w n is a r e p r e s e n t a t i v e s a m p l e of t h r e e independent experiments.

Inhibition of Aldosterone Biosynthesis

145

Table 3. Inhibition of cortisol production and of 1lfl-hydroxylation activity by 18-VP or 18-EP Inhibitor (/~M) 0 Cortisol production* 11/~-Hydroxylation**

18-EP 18-VP 18-EP 18-VP

28.3 28.3 35.6 35.3

+ + + +

1 0.9 0.9 0.6 0.6

24.3 9.2 34.6 6.4

+ 0.4 + 0.5 _+ 1.8 + 0.8

5 11.0 8.8 28.3 4.1

_ + + +

10 0.6 0.4 0.1 0.6

8.7 _+ 0.4 8.5 _+ 0.2

*praol/h/106 cells. **~mol/h/106 cells. Production of cortisol by B A C cells incubated during 2 h in the presence of various concentrations of the two inhibitors 18-EP or 18-VP. Cortisol content was determined by radioimmunoassay. Each value is the average production ( + SD) of 3 wells. T h r e e independent experiments were performed. l l/]-hydoxylase activity was measured by the production of cortisol by B A C cells incubated during 2 h with [3H]deoxycortisol (100 ~ M ) in the presence of the inhibitors (18.-VP or 18-EP) at the concentrations indicated. [3H]deoxycortisol and [3H]cortisol were separated by T L C as described in Materials and Methods. Each value is the average production ( + SD) of 3 wells. T h r e e independent experiments were performed.

thus incubated with deoxycortisol to measure 1 lfl-hydroxylase activity by the production of cortisol, and with corticosterone to measure 18-hydroxylase activity by the production of 18-hydroxycorticosterone. Similarly, aldosterone synthase activity was measured by the production of aldosterone. T h e dose-dependence of the inhibition by 18-VP is shown in Fig. 3. Cortisol production was inhibited with an ICs0 of 0.3 ___0.1 # M whereas the aldosterone and 18-hydroxycorticosterone production was inhibited with 0.07 __+0.02 and 0.04 ___0.01/~M, respectively. For example, a concentration of 0.1 #/vl of 18-VP inhibited the cortisol 120

100

A

0 C 0

h.

60

0 W 0

"D

40

20

0.0

0.1 Inhlbilor

0.5 (pM)

Fig. 4. I n h i b i t i o n o f t h e t r a n s f o r m a t i o n o f c o r t i c o s t e r o n e to aldosterone by 18-VP or 18-EP. Production of aldosterone f r o m c o r t i c o s t e r o n e in t h e p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f 1 8 - V P a n d 18-]EP a s d e s c r i b e d f o r Fig. 3. 100% is t h e p r o d u c t i o n w i t h o u t i n h i b i t o r . E a c h v a l u e is t h e a v e r a g e p r o d u c t i o n o f 3 wells. S h o w n is a r e p r e s e n t a t i v e s a m p l e o f three indepe:adent measurements.

formation from deoxycortisol by 35 %, 18-hydroxycorticosterone formation from corticosterone by 60~o and aldosterone formation from corticosterone by 90%. Comparison of the action of 1 8 - V P and 18-EP on B A C cells First, the inhibition of cortisol production by 18-VP and 18-EP was examined (Table 3). In contrast with 18-VP, the inhibition by 18-EP was very weak for concentrations below 5/~M. T h e effects of 18-VP and 18-EP on the 1 lfl-hydroxylation step were then compared. T h e transformation of deoxycortisol to cortisol was measured at different inhibitor concentrations. As shown in T a b l e 3, for a concentration of 1 # M , there is a marked decrease in cortisol production with 18-VP (only 20% of the total activity remaining), but no inhibition in the presence of 18-EP. A concentration of 5/~M of 18-EP was needed to obtain a significant decrease in cortisol formation (20%). T h e formation of aldosterone from corticosterone was also measured in the presence of the two inhibitors. Figure 4 shows that the inhibition of aldosterone production is m u c h lower with 18-EP than with 18-VP. T h u s , aldosterone formation is not inhibited by 18-EP at a concentration of 0.1/~M and by only about 50% at a concentration of 0.5 # M . DISCUSSION A m o n g several 18-substituted progesterone derivatives 18-VP and 18-EP are the most potent inhibitors of aldosterone biosynthesis when tested with rat adrenal cell-free extracts [2]. In this study, our goal was to determine the influence of these inhibitors on the steroidogenesis in bovine adrenocortical cells where the enzyme is inserted into the native mitochondrial

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m e m b r a n e . A d i s a d v a n t a g e is that the s i t u a t i o n is m o r e c o m p l e x i n cells because the i n h i b i t o r s m a y also act as s u b s t r a t e s (or i n h i b i t o r s ) for e n z y m a t i c steps o t h e r t h a n 11- or 1 8 - h y d r o x y l a t i o n . 18-VP a n d 18-EP were d e s i g n e d as m e c h a n i s m based i n h i b i t o r s to be c o v a l e n t l y b o u n d to the p r o s t h e t i c h e m e g r o u p of the P45011~ [17, 18]. I n d e e d , w h e n the cells were i n c u b a t e d w i t h tritiated 18-VP (1 # M ) , 2 5 % of the radioactivity a p p e a r e d to be covalently associated with the m i t o c h o n d r i a l m e m b r a n e s after T C A p r e c i p i t a t i o n (data n o t shown). T h e r e are two different c y t o c h r o m e P45011/~ h y d r o x ylases (CYP11B1 a n d C Y P 1 1 B 2 ) in the h u m a n , rat a n d m o u s e a d r e n a l g l a n d [6], one r e s p o n s i b l e for the 1 l f l h y d r o x y l a t i o n a n d the other for the a l d o s t e r o n e s y n thesis. I n the b o v i n e a d r e n a l gland, the two i s o e n z y m e s P45011~ are able to c o n v e r t corticosterone to aldostero n e as well as d e o x y c o r t i c o s t e r o n e to corticosterone a n d b o t h show similar activities [7]. F u r t h e r m o r e , they are f o u n d i n almost equal a m o u n t s in all the zones of the a d r e n a l cortex. H e n c e the zona g l o m e r u l o s a specificity of aldosterone p r o d u c t i o n is e x p l a i n e d n e i t h e r b y different catalytic p r o p e r t i e s n o r b y the zonal distrib u t i o n of the two forms [5, 7]. W e have s h o w n that the selectivity of 18-VP t o w a r d the 18-position c a n n o t certainly be e x p l a i n e d b y a p r e f e r e n t i a l i n t e r a c t i o n with one of the two different isoforms p r e s e n t in o u r p r e p a r a t i o n [19]. T h e differences i n e n z y m e kinetics m i g h t well reflect c o n f o r m a t i o n a l change of c y t o c h r o m e P450H~, in that l l f l - h y d r o x y l a t i o n m a y occur i n the native site whereas 1 8 - h y d r o x y l a t i o n m a y i n v o l v e a different o r i e n t a t i o n of the steroid w i t h respect to the h e m e i r o n atom. T h i s w o u l d suggest that the i n h i b i t o r shows a h i g h e r affinity for the c o n f o r m a t i o n in w h i c h 1 8 - h y d r o x y l a t i o n is t a k i n g place [19]. T h o u g h 18-EP is a less p o t e n t i n h i b i t o r t h a n 18-VP, a r o u g h e s t i m a t i o n of the ICs0 values for the 11- a n d 1 8 - h y d r o x y l a t i o n s (18 a n d 0.4 # M ) indicates that it is also m o r e selective for the 18-position. T h e i n h i b i t i o n b y the 1 8 - e t h y n y l derivative of D O C was r e c e n t l y s t u d i e d b y Y a m a k i t a et al. [12] a n d G o m e z - S a n c h e z et al. [13] with c u l t u r e d calf zona g l o m e r u l o s a cells a n d with freshly isolated cells. T h e ICs0 o b t a i n e d was 0.25pM for a l d o s t e r o n e p r o d u c t i o n a n d 4 8 p M for the cortisol p r o d u c t i o n , d e m o n s t r a t i n g similar selectivity. T h u s , 18-VP a n d 1 8 - E P c o u l d be useful i n h i b i t o r s of a l d o s t e r o n e biosynthesis. W e have also s h o w n that they have a n excellent affinity for the h u m a n m i n e r a l o c o r t i coid receptor [20] a n d behave as a n t a g o n i s t s of a l d o s t e r o n e h u m a n receptor [21; M . E . R a f e s t i n - O b l i n , p e r s o n a l c o m m u n i c a t i o n ] . H o w e v e r , this class of m o l e c u l e s has n o t yet b e e n tested w i t h the h u m a n e n z y m e . I f they are also effective i n h i b i t o r s of a l d o s t e r o n e b i o s y n t h e s i s i n h u m a n s , they could lead to interesting new antimineralocorticoids. Some c o m p l e m e n t a r y i n v e s t i g a t i o n s are o b v i o u s l y necessary in o r d e r to evaluate their p h a r m a c e u t i c a l potential.

Acknowledgements--We are indebted to C. Blanc-Brude for her

assistance with the primary culture preparations. This work was supported by the Institut National de la Sant6 et de la Recherche M6dicale (Unit6 244), the Centre de l'Energie Atomique (DSV/DBMS/BRCE) and the Centre National de la Recherche Scientifique (URA 493).

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