0013-7227/91/1295-2361$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 5 Printed in U.S.A.
Inhibition of Aldosterone Biosynthesis by 18-Ethynyl Deoxycorticosterone* NORIYOSHI YAMAKITAf, SHIRLEY CHIOU, AND CELSO E. GOMEZ-SANCHEZ Department of Internal Medicine, University of South Florida Health Sciences Center, and the James A. Haley Veterans Hospital, Tampa, Florida 33612; and the Third Department of Internal Medicine, Gifu University School of Medicine (N. Y.), Gifu, Japan
ABSTRACT. The inhibiting effects of 18-ethynyl-deoxycorticosterone (18-E-DOC) as a mechanism-based inhibitor on the late steps of aldosterone biosynthetic pathway were examined in calf adrenal zona glomerulosa cells placed in the primary culture. Baseline and ACTH (10'9 M)-, angiotensin-II (10'8 M)-, and potassium (12 mM)-stimulated production of aldosterone and 18-hydroxycorticosterone were inhibited in a dose- and timedependent manner. At 1 /xM, 18-E-DOC produced a 73% inhibition, and at 10 nM, it produced a 94.6% inhibition of aldosterone secretion. Preincubation with 10 nM 18-E-DOC for 5 min followed by washing resulted in 75% inhibition of aldosterone secretion. The maximal degree of inhibition was reached after 60 min of preincubation. The degree of the inhibition of 18-
A
LDOSTERONE is produced in the adrenal zona glomerulosa by a series of enzymatic reactions that follow the sequence cholesterol —> pregnenolone —» progesterone —> deoxycorticosterone —*• corticosterone —> 18-hydroxycorticosterone (18-OH-B) —> aldosterone (1). The two regulatory steps in the synthesis of aldosterone occur in the mitochondria and have been called the early pathway (conversion of cholesterol to pregnenolone) and the late pathway (conversion of corticosterone to aldosterone) (1). In the mitochondria, deoxycorticosterone (DOC) is hydroxylated to corticosterone by the cytochrome P-450 lljS/18-hydroxylase, which is further hydroxylated to 18-OH-B and aldosterone by the cytochrome P-450 methyl oxidase-I and -II (1, 2). Corticosterone exhibits a high rate of conversion to aldosterone, but the putative intermediate 18-OH-B shows lower conversion rates to aldosterone (3). Ulick (2) has suggested that the true intermediate product of the action of the methyl oxidases is a reactive monooxygenated derivative Received May 6,1991. Address all correspondence and requests for reprints to: Celso E. Gomez-Sanchez, M.D., James A. Haley Veterans Hospital, 13000 Bruce B. Downs Boulevard, VAH 111 M, Tampa, Florida 33612. * This work was supported by Medical Research Funds from the Department of Veterans Affairs and NIH Grant HL-27255. t On sabbatical leave from Gifu University and supported by a grant from the Ministry of Education, Culture, and Science of Japan.
hydroxycorticosterone production was almost same as that of aldosterone. Preincubation with 10 fiU 18-E-DOC for 60 min, followed by extensive washing and reincubation with medium for 24 h, resulted in recovery to more than half the production of the control cells. Minimal changes occurred in the production of corticosterone (slight increase), 18-hydroxydeoxycorticosterone (slight increase) in zona glomerulosa cells, and cortisol (no changes) in zona fasciculata cells. These studies show that 18-E-DOC is a specific inhibitor of the late pathway of aldosterone biosynthesis. 18-E-DOC could be valuable as a therapeutic agent in those conditions associated with increased aldosterone production where a specific inhibitor would be useful. (.Endocrinology 129: 2361-2366,1991)
complex with the enzyme, which is then further hydroxylated to a germinal diol, which spontaneously dehydrates to aldosterone or leaves the enzyme complex to spontaneously rearrange to the stable form of 18-OH-B. Two distinct cytochrome P-450 proteins have been isolated from adrenocortical mitochondria: the cytochrome P-450 side-chain cleavage and the 11/3/18-hydroxylase (4). The latter enzyme catalyzes the 11/3-, 18-, and 19-hydroxylation of DOC and other steroid substrates (4, 5). Isolated mitochondria from the zona fasciculata do not convert corticosterone to aldosterone as would be expected (6). However, detergent-extracted mitochondria and purified preparations of the 11/?hydroxylase from bovine or porcine zona fasciculata have been shown to convert corticosterone into 18-OH-B and aldosterone (7, 8). On the basis of these observations, it has been postulated that the hypothetical enzymes P450 methyl oxidase-I and -II [called aldosterone synthase by others (9, 10)] are the P-450 11/3-hydroxylase, and that for as yet unknown reasons there is a zonal distribution of the various activities of the same enzyme (7, 8). There are significant differences between rat and bovine 11/3-hydroxylases in their ability to catalyze the formation of aldosterone. In contrast with bovine or porcine mitochondria, detergent-extracted rat zona fasciculata mitochondria synthesize almost no aldosterone;
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SUICIDE INHIBITION OF ALDOSTERONE SYNTHESIS
as expected, zona glomerulosa does it very effectively (6). Recently, 3 groups have reported the cloning of enzymes from human or rat adrenals that are believed to be aldosterone synthases (9-11). Two 11/3-hydroxylases have been isolated from bovine adrenals that have aldosterone synthase activity (12), although 1 of them (form 2) was more efficient at generating aldosterone from DOC. Four bovine 110-hydroxylase cDNAs were recently cloned, 2 of which correspond to pseudogenes (13). Expression of the cDNAs P-45O(110)-3 and P-45O(110)2 in COS-7 cells revealed that both had 110/18/19hydroxylase activity and converted corticosterone to aldosterone, but convesion by P-450(ll/?)-3 was greater than that by P-450(ll/3)-2 (14). Expression of the 2 kinds of P-450(ll#) mRNAs in adrenal cortex from 10 bovines showed that most individuals expressed both mRNAs, but in 4 of 10 animals, only 1 was expressed (14). Aldosterone generation in the adrenal of most species is in the zona glomerulosa and is regulated in a different fashion from that of cortisol/corticosterone from the zona fasciculata. In the bovine adrenal it is hard to explain this selectivity in view of the lack of specificity of the isolated 11/3-hydroxylase or its cDNA product. We are presenting studies using a mechanism-based (kcat) inhibitor, 18-ethynyl-deoxycorticosterone [18-E-DOC; 21-hydroxy-13-(2-propynyl)18-nor-preg-4-ene-3,20dione], of the hypothetical enzymes P-450 methyl oxidase-I and -II (aldosterone synthase) on the production of corticosterone, 18-hydroxy-ll-deoxycorticosterone (18-OH-DOC), 18-OH-B, and aldosterone by cultured calf zona glomerulosa cells. The studies demonstrate that functionally, the 11/3-hydroxylase is distinct from P-450 methyl oxidase-I and -II.
Materials and Methods Materials
Endo • 1991 Voll29'No5
dispersed mechanically by aspiration through a latex tube fitted to a 60-ml syringe. The cells were collected by centrifugation at 150 x g for 8 min, washed twice with medium, and finally resuspended in medium containing 12 mM potassium, 10% serum mixture (10% fetal calf-horse with 5% bovine embryonic fluid), antibiotic mixture, and an antioxidant mixture (5 nM metyrapone, 50 nM selenous acid, 100 nM butylated hydroxyanisole, and 100 mM dimethylsulfoxide). as described by Crivello et al. (15). The cells were then plated in Primaria 24-well plates (Falcon Plastics, Becton Dickinson Co., Lincoln Park, NJ; 200,000/well) and incubated at 370 C in a 5% CO2 air environment. An extra 1 ml medium was added 24 h later, and the cells were cultured for 3-5 days before being used. All experiments were performed in triplicate and repeated at least twice. Dose response to 18-E-DOC inhibition On the day of the experiments, plates were washed with Hanks' Balanced Salt Solution twice, and the cells were incubated with medium containing 0.5% BSA for 2 h to remove the intracellularly trapped metyrapone. The cells were washed again and incubated in the presence of 0.01-100 fiM 18-E-DOC for 2 h. The final concentration of dimethylsulfoxide (DMSO) used to dissolve 18-E-DOC was 0.5% and did not affect steroid secretion. Cells in 24-well plates were washed and preincubated with 10 MM 18-E-DOC in DMSO or DMSO alone. At the end of the pretreatment time, the cells were washed again with HBBS twice and incubated for 2 h in medium containing vehicle (control), ACTH (109 M), angiotensin-II (A-II; 10 8 M), or potassium (12 mM). The medium was then removed and stored at -20 C. Time course of inhibition The dose of 18-E-DOC necessary to produce maximal inhibition of aldosterone secretion after 2 h of preincubation was 10 /iM, and all subsequent experiments were performed with this concentration. Cells were preincubated, as described above, for various times, and the supernatant was collected as described above. Recovery of aldosterone production after washing 18-E-DOC
Tissue culture medium and serum were obtained from Sigma Chemical Co. (St. Louis, MO). All steroids were obtained from Sigma or Andard Mount Co. (London, England). The 18ethynyl-DOC was kindly provided by Dr. J. O'Neil Johnston from Marion Merrell Dow Research Institute (Cincinatti, OH). Culture of adrenal zona glomerulosa cells Mixed breed calf adrenal glands were obtained from a local abattoir and trimmed clean of fat and adhering tissue under sterile conditions. The outer 500 nm was sliced off using a Stadie-Riggs Microtome. Slices were washed using Hanks' Balanced Salt Solution and suspended in modified Ham's F-12 medium (137 mM NaCl, 2.1 mM CaCl2, 5 mM KC1, and 25 mM HEPES) containing 2 mg/ml collagenase (Worthington Biochemicals, Freehold, NJ), 0.4 mg/ml deoxyribonuclease-I, 0.3 mg/ml neutral protease-IX, 0.5% BSA (Sigma), and antibiotic mixture (100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2.5 mg/ml amphotericin-B). After incubation for 1 h, cells were
After pretreatment with 10 nM 18-E-DOC for 60 min on the last day of the culture, cells were intensively washed and cultured in the complete medium for an additional 24 h. The following day the cells were stimulated with 10'9 M ACTH, 10'8 M A-II, or 12 mM potassium, as described above. Controls received pretreatment with vehicle alone and were handled as described above. Inhibition of cortisol secretion A study similar to that described above was performed using cultures of the second 500-/im layer (zona fasciculata) to determine whether 18-E-DOC inhibited cortisol production. Steroid measurements Aldosterone was measured by direct RIA, using a monoclonal antibody (16). Corticosterone and cortisol were also measured by direct RIA (17). 18-OH-B and 18-OH-DOC were measured by RIA after extracting with dichloromethane (17).
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SUICIDE INHIBITION OF ALDOSTERONE SYNTHESIS
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300
Statistical analysis Statistical comparisons were made by analysis of variance, comparison between treatment means were made using Fishers PLSD test and Dunnett's t test, using Statview 512+ computer program (Brain Power, Calabazas, CA).
Control A-I110 nM ACTH nM Potassium 12 mM
200
"o S a.
Results Aldosterone secretion was inhibited in a dose-dependent fashion, with inhibition becoming significant at a 1fiM concentration of 18-E-DOC (Fig. 1). Inhibition of ACTH-stimulated aldosterone secretion was 73% with 1 AiM, 94.6% with 10 ixM, and 97.5% with 100 nM 18-EDOC. The percentage of 18-E-DOC inhibition of aldosterone secretion by cells stimulated by A-II or potassium and that by control unstimulated cells were significantly less than that by cells stimulated by ACTH, but the absolute secretion of aldosterone at the higher doses of inhibitor was not significantly different. Even at the highest dose of 18-E-DOC, there was significant production of aldosterone. Preincubation with 18-E-DOC for as little as 5 min accomplished significant inhibition of aldosterone production, but 60 min were required for maximal inhibition (Fig. 2). The inhibition of 18-OH-B production was similar to that of aldosterone and followed a similar time course. The lack of effect of 18-E-DOC on corticosterone and 18-OH-DOC in the zona glomerulosa and of cortisol in the zona fasciculata indicated that the inhibition did not affect the production of these steroids (Fig. 3). The inhibition of aldosterone secretion could be partially recovered by incubating the treated cells overnight in new medium, suggesting that new enzyme must be synthesized (Fig. 4). In cells stimulated with ACTH, 1-h treatment with 10 /xM 18-E-DOC resulted in a 90 ± 0.8%
Control ACTH nM
100"
40 PREINCUBATION
TIME
60 (MIN)
"o S Q. UJ
z o
QC •
I00
X
o cc o
0
20 40 PREINCUBATION TIME (MIN)
60
FIG. 2. Top panel, Effect of preincubation time with 18-E-DOC (106 M) on aldosterone secretion in unstimulated and ACTH (109 M)-, A-II (10'8 M)-, or potassium (12 mM)-stimulated calf zona glomerulosa cells in culture. Bottom panel, Effect of preincubation time with 18-E-DOC (106 M) on 18-OH-B secretion in unstimulated and ACTH (109 M)-, A-II (10'8 M)-, or potassium (12 mM)-stimulated calf zona glomerulosa cells in culture.
A-II 10 nM
inhibition of aldosterone secretion and 91 ± 2% inhibition of 18-OH-B secretion. Cells washed and reincubated for 24 h in complete culture medium exhibited an increase in aldosterone to 58 ± 0.5% and in 18-OH-B secretion to 31.5 ± 4% of the level in the uninhibited control cells.
K + 12mM
20 -
oJ
Discussion r— 0
.01
.1
1
10
100
1000
18-Ethynyl-DOC concentration (|iM)
FIG. 1. Dose response of 18-E-DOC on the inhibition of aldosterone secretion in unstimulated and ACTH (109 M)-, A-II (10 8 M)-, or potassium (12 mMJ-stimulated calf zona glomerulosa cells in culture.
There are three classes of mechanism-based inhibitors of cytochrome P-450 monooxygenases: those that either covalently bind to the protein (class I) or to the prosthetic heme group (class III), and those that coordinate quasiirreversibly with the iron atom (class II) (18, 19). Several mechanism-based inhibitors of the cytochrome
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SUICIDE INHIBITION OF ALDOSTERONE SYNTHESIS
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Endo• 1991 Voll29«No5
300
800
P |
1 Bm. • 0
1
10
18-ETHYNYLDEOXYCORTICOSTERONE CONCENTRATION friM)
ill H
1 i
raKg
Ha
1 1
0 1 10 18-ETHYNYL-DEOXYCORTICOSTERONE CONCENTRATION (pM)
FiG. 3. Effect of 18-E-DOC (0,1, and 10 JUM) on corticosterone, 18-OH-DOC, and 18-OH-B secretion in unstimulated and ACTH (109 M)-, A-II (10'8 M)-, or potassium (12 mM)-stimulated calf zona glomerulosa cells and of cortisol secretion in zona fasciculata cells in culture. *, P < 0.05; • • , P < 0.01; • * * , P < 0.001.
P-450s have been described (18-21). Viger et al. (20, 22) reported that the class III compounds are the most potent inhibitors of aldosterone. They reported that 0.8 nM 18ethynyl-progesterone produce a 63% inhibition and 8 nM completely inhibited aldosterone biosynthesis. The structure of 18-E-DOC used in this study is very similar to that of 18-ethynyl-progesterone, and the degree of inhibition of aldosterone production was almost the same as that of 18-ethynyl-progesterone (20, 22). The inhibition of aldosterone secretion is likely to be by covalent binding of the inhibitor to the cytochrome P-450, since exposure of the cells to even a short period of time and extensive washing of the cells resulted in profound inhibition of aldosterone secretion. In the studies of Viger et al. (20, 22), the inhibitor was incubated simultaneously with precursors, and inactivation of the enzyme could not be addressed. Reincubation of the cells for 24 h resulted in a significant recovery of the enzymatic activity, although at 24 h, activity had not returned to control values. It is very likely that new synthesis of the aldosterone synthase is required, although this was not studied
directly. The production of aldosterone and 18-OH-B was inhibited to the same degree after pretreatment with 18E-DOC, whereas the production of corticosterone and 18-OH-DOC in zona glomerulosa cells slightly increased, and that of cortisol in zona fasciculata cells did not change. The recovery of 18-OH-B synthesis was significantly less than that of aldosterone 24 h after exposure to 18-E-DOC; the explanation for this finding is unclear. 18-OH-B is believed by some to be an intermediate in the synthesis of aldosterone (3), atthough Ulick (2) postulated that 18-OH-B is a terminal product from an intermediate on the way to aldosterone synthesis by the cytochrome P-450 enzyme. The adrenal gland shows a distinct zonation regulated by common and independent regulatory factors. In vitro incubations of tissue slices or isolated mitochondria have indicated that aldosterone is produced in the zona glomerulosa of the adrenal (6, 17, 23). However, isolated cytochrome P-450 ll/?-hydroxylase from the zona glomerulosa or fasciculata of porcine or bovine adrenals can
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SUICIDE INHIBITION OF ALDOSTERONE SYNTHESIS 200
ALDOSTERONE 18-HVDROXYCORTICOSTERONE
f o D C o o
o= =
100-
a
ol
5 o
*/'
2
B
C
TREATMENT
FIG. 4. Recovery of aldosterone and 18-OH-B production in cultured zona glomerulosa cells after preincubation with 18-E-DOC (10 fiM) for 60 min. A, Cells were preincubated with 18-E-DOC, washed, and stimulated with ACTH (10'9 M) for 2 h. The degree of stimulation was significantly less that for than untreated cells (*, P < 0.001). B, Cells were preincubated with medium without 18-E-DOC, washed, incubated for 24 h with complete culture medium, washed, and stimulated with ACTH as described above. C, Cells were preincubated with medium with 18-E-DOC, washed, incubated for 24 h with complete culture medium, washed, and stimulated with ACTH, as described above. The degree of stimulation was significantly less than that for untreated cells (*, P < 0.001), but it was significantly greater than for cells treated and incubated immediately (**, P < 0.001).
convert corticosterone to aldosterone quite effectively (7, 8, 24), leading to the suggestion that the aldosterone synthase or corticosterone methyl oxidase activity resides within the 11/3-hydroxylase. Corticosterone, 18OH-DOC, and cortisol were not inhibited by 18-E-DOC, indicating that the enzyme that was inactivated was not responsible for the ll/?/18-hydroxylase activity. It is of interest to note that for unknown reasons the 18-hydroxylation of DOC by the 11/3-hydroxylase was not affected by 18-E-DOC. It is possible that the presence of a bulky group at position 13 inhibited binding to cytochrome P-450 110/18-hydroxylase, but not that to aldosterone synthase. The cloning of aldosterone synthase cDNAs from the rat (9, 10) and a human aldosteroneproducing adrenal adenoma (11) and of bovine 11/3hydroxylase cDNAs (12, 25) has been described. The expression products of these cDNAs have aldosteronegenerating ability. Aldosterone synthase is likely to be a variant of the lljS-hydroxylase with specialized activity (12, 25). This distinct gene is probably located in the chromosome very near the gene for the lljS-hydroxylase (26). These studies of enzyme function show that P-450 methyl oxidase-I and -II exist in bovine adrenal, with no evidence for 11/3-hydroxylase activity. It is possible that the isolated bovine 11/3-hydroxylases and cDNAs are not the responsible for the generation of aldosterone from corticosterone and that the gene responsible remains to be identified.
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In conclusion., in the calf adrenal cortex, 18-E-DOC inhibited the last steps of the aldosterone biosynthetic pathway without affecting the production of intermediates that correspond to the most important glucocorticoids produced by the adrenal gland. This drug, if it has the proper phamacokinetic properties, could be useful in the treatment of those disorders associated with increased aldosterone production because of its specificity, in contrast to currently used spironolactone drugs, which antagonize aldosterone as well as other adrenal steroids at the receptor level (27).
Acknowledgments The 18-E-DOC was kindly provided by Dr. J. O'Neil Johnston from Marion Merrell Dow Research Institute, a division of Marion Merrell Dow, Inc. The technical help of Mark F. Foecking is gratefully acknowledged.
References 1. Muller J 1988 Pathway of aldosterone biosynthesis. In: Muller J (ed) Regulation of Aldosterone Biosynthesis. Physiological and Clinical Aspects. Springer-Verlag, Berlin, pp 5-15 2. Ulick S 1976 Diagnosis and nomenclature of the disorders of the terminal portions of the aldosterone biosynthetic pathway. J Clin Endocrinol Metab 43:92-96 3. Fattah DI, Whitehouse BJ, Vinson GP 1977 Biosynthesis of aldosterone from 18-hydroxylated precursors by rat adrenal tissue in vitro. J Endocrinol 75:187-195 4. Sato H, Ashida N, Suhara K 1978 Properties of an adrenal cytochrome P-450 (P-450-llb) for the hydroxylations of corticosteroids. Arch Biochem Biophys 190:307-314 5. Watanuki M, Tilley BE, Hall PF 1978 Cytochrome P-450 for lib and 18-hydroxylase activities of bovine adrenocortical mitochondria: one enzyme or two. Biochemistry 17:127-130 6. Ohnishi T, Wada A, Lauber M, Yamano T, Okamoto M 1988 Aldosterone biosynthesis in mitochondria of isolated zones of adrenal cortex. J Steroid Biochem 31:73-81 7. Wada A, Ohnishi T, Nonaka Y, Okamoto M, Yamano T 1985 Synthesis of aldosterone by a reconstituted system of cytochrome P-450-llb from bovine adrenocortical mitochondria. J Biochem 98:245-256 8. Yanagibashi K, Haniu M, Shively JE, Shem WH, Hall P 1986 The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glom«rulosa) possess one enzyme for lib, 18-hydroxylation, and aldehyde synthesis. J Biol Chem 261:3556-3562 9. Matsukawa N, Nonaka Y, Ying Z, Higaki J, Ogihara T, Okamoto M 1990 Molecular cloning and expression of cDNAs encoding rat aldosterone synthase: variants of cytochrome P-450 lib. Biochem Biophys Res Commun 169:245-252 10. Imai M, Shimada H , Okada Y, Matsuhima-Hibiya Y, Ogishima T, Ishimura Y 1990 Molecular cloning of cDNA encoding aldosterone synthase cytochrome P-450 in rat adrenal cortex. FEBS Lett 263:299-302 11. Kawamoto T, Mitsuuchi Y, Ohnishi T, Ichikawa Y, Yokoyama Y, Sumimoto H, Toda K, Miyahara K, Kuribayashi I 1991 Cloning and expression of a cDNA for human cytochrome P-450 aldo as related to primary aldosteronism. Biochem Biophys Res Commun 173:309-316 12. Ogishima T, Mitani F, Ishimura Y 1989 Isolation of two distinct cytochromes P-450 lib with aldosterone synthase activity from bovine adrenocortical mitochondria. J Biochem 105:497-499 13. Hashimoto T, Morohashi K-I, Omura T 1989 Cloning and characterization of bovine cytochrome P-450(llb) genes. J Biochem 105:676-679
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14. Morohashi K-I, Nonaka Y, Kirita S, Hatano 0, Takakusu A, Okamoto M, Omura T 1990 Enzymatic activities of P-450(llb)s expressed by two cDNAs in COS-7 cells. J Biochem 107:635-640 15. Crivello JF, Hornsby PJ, Gill GN 1982 Metyrapone and antioxidants are required to maintain aldosterone synthesis by cultured bovine adrenocortical zona glomerulosa cells. Endocrinology 111:469-479 16. Gomez-Sanchez CE, Foecking MF, Ferris MW, Chavarri MR, Uribe L, Gomez-Sanchez EP 1987 The production of monoclonal antibodies against aldosterone. Steroids 49:581-587 17. Gomez-Sanchez CE, Ferris MW, Foecking MF, Gomez-Sanchez EP 1989 Synthesis of 18-hydroxycortisol and 18-oxocortisol in bovine adrenal slices. J Steroid Biochem 33:595-598 18. Ortiz de Montellano PR 1984 The inactivation of cytochrome P450. Annu Rep Med Chem 19:201-211 19. Johnston JO, Wright CL, Metcalf BW 1984 Biochemical and endocrine properties of a mechanism-based inhibitor of aromatase. Endocrinology 115:776-785 20. Viger A, Coustal S, Perard S, Chappe B, Marquet A 1988 Synthesis and activity of new inhibitors of aldosterone biosynthesis. J Steroid Biochem 30:469-472
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21. Nagahisa A, Spencer RW, Orme-Johnson WH 1983 Acetylenic mechanism-based inhibitors of cholesterol side chain cleavage by cytochrome P450 sec. J Biol Chem 258:6721-6723 22. Viger A, Coustal S, Perard S, Piffeteau A, Marquet A 1989. 18Sustituted progesterone derivatives as inhibitors of aldosterone biosynthesis. J Steroid Biochem 33:119-124 23. Giroud CJP, Stachenko J, Venning EH 1956 Secretion of aldosterone by the zona glomerulosa of rat adrenal glands incubated in vitro. Proc Soc Exp Biol Med 92:154-158 24. Ohnishi T, Wada A, Nonaka Y, Okamoto M, Yamano T 1984 Effect of phospholipid on aldosterone biosynthesis by a cytochrome P-450-llb-reconstituted system. Biochem Int 9:715-723 25. Mathew PA, Mason JI, Trant JM, Sanders D, Waterman MR 1990 Amino acid substitutions Phe^-Lew and Ser126-Pro abolish cortisol and aldosterone synthesis by bovine cytochrome P-450 lib. J Biol Chem 265:20228-20233 26. Globerman H, Rosier A, Theodor R, New MI, White PC 1988 An inherited defect in aldosterone biosynthesis caused by a mutation in or near the gene for steroid 11-hydroxylase. N Engl J Med 319:1193-1197 27. Fanestil DD 1988 Mechanism of action of aldosterone blockers. Semin Nephrol 8:249-263
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Vol. 129, No. 5 Printed in U.S.A.
Inhibition of Aldosterone Biosynthesis by 18-Ethynyl Deoxycorticosterone* NORIYOSHI YAMAKITAf, SHIRLEY CHIOU, AND CELSO E. GOMEZ-SANCHEZ Department of Internal Medicine, University of South Florida Health Sciences Center, and the James A. Haley Veterans Hospital, Tampa, Florida 33612; and the Third Department of Internal Medicine, Gifu University School of Medicine (N. Y.), Gifu, Japan
ABSTRACT. The inhibiting effects of 18-ethynyl-deoxycorticosterone (18-E-DOC) as a mechanism-based inhibitor on the late steps of aldosterone biosynthetic pathway were examined in calf adrenal zona glomerulosa cells placed in the primary culture. Baseline and ACTH (10'9 M)-, angiotensin-II (10'8 M)-, and potassium (12 mM)-stimulated production of aldosterone and 18-hydroxycorticosterone were inhibited in a dose- and timedependent manner. At 1 /xM, 18-E-DOC produced a 73% inhibition, and at 10 nM, it produced a 94.6% inhibition of aldosterone secretion. Preincubation with 10 nM 18-E-DOC for 5 min followed by washing resulted in 75% inhibition of aldosterone secretion. The maximal degree of inhibition was reached after 60 min of preincubation. The degree of the inhibition of 18-
A
LDOSTERONE is produced in the adrenal zona glomerulosa by a series of enzymatic reactions that follow the sequence cholesterol —> pregnenolone —» progesterone —> deoxycorticosterone —*• corticosterone —> 18-hydroxycorticosterone (18-OH-B) —> aldosterone (1). The two regulatory steps in the synthesis of aldosterone occur in the mitochondria and have been called the early pathway (conversion of cholesterol to pregnenolone) and the late pathway (conversion of corticosterone to aldosterone) (1). In the mitochondria, deoxycorticosterone (DOC) is hydroxylated to corticosterone by the cytochrome P-450 lljS/18-hydroxylase, which is further hydroxylated to 18-OH-B and aldosterone by the cytochrome P-450 methyl oxidase-I and -II (1, 2). Corticosterone exhibits a high rate of conversion to aldosterone, but the putative intermediate 18-OH-B shows lower conversion rates to aldosterone (3). Ulick (2) has suggested that the true intermediate product of the action of the methyl oxidases is a reactive monooxygenated derivative Received May 6,1991. Address all correspondence and requests for reprints to: Celso E. Gomez-Sanchez, M.D., James A. Haley Veterans Hospital, 13000 Bruce B. Downs Boulevard, VAH 111 M, Tampa, Florida 33612. * This work was supported by Medical Research Funds from the Department of Veterans Affairs and NIH Grant HL-27255. t On sabbatical leave from Gifu University and supported by a grant from the Ministry of Education, Culture, and Science of Japan.
hydroxycorticosterone production was almost same as that of aldosterone. Preincubation with 10 fiU 18-E-DOC for 60 min, followed by extensive washing and reincubation with medium for 24 h, resulted in recovery to more than half the production of the control cells. Minimal changes occurred in the production of corticosterone (slight increase), 18-hydroxydeoxycorticosterone (slight increase) in zona glomerulosa cells, and cortisol (no changes) in zona fasciculata cells. These studies show that 18-E-DOC is a specific inhibitor of the late pathway of aldosterone biosynthesis. 18-E-DOC could be valuable as a therapeutic agent in those conditions associated with increased aldosterone production where a specific inhibitor would be useful. (.Endocrinology 129: 2361-2366,1991)
complex with the enzyme, which is then further hydroxylated to a germinal diol, which spontaneously dehydrates to aldosterone or leaves the enzyme complex to spontaneously rearrange to the stable form of 18-OH-B. Two distinct cytochrome P-450 proteins have been isolated from adrenocortical mitochondria: the cytochrome P-450 side-chain cleavage and the 11/3/18-hydroxylase (4). The latter enzyme catalyzes the 11/3-, 18-, and 19-hydroxylation of DOC and other steroid substrates (4, 5). Isolated mitochondria from the zona fasciculata do not convert corticosterone to aldosterone as would be expected (6). However, detergent-extracted mitochondria and purified preparations of the 11/?hydroxylase from bovine or porcine zona fasciculata have been shown to convert corticosterone into 18-OH-B and aldosterone (7, 8). On the basis of these observations, it has been postulated that the hypothetical enzymes P450 methyl oxidase-I and -II [called aldosterone synthase by others (9, 10)] are the P-450 11/3-hydroxylase, and that for as yet unknown reasons there is a zonal distribution of the various activities of the same enzyme (7, 8). There are significant differences between rat and bovine 11/3-hydroxylases in their ability to catalyze the formation of aldosterone. In contrast with bovine or porcine mitochondria, detergent-extracted rat zona fasciculata mitochondria synthesize almost no aldosterone;
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SUICIDE INHIBITION OF ALDOSTERONE SYNTHESIS
as expected, zona glomerulosa does it very effectively (6). Recently, 3 groups have reported the cloning of enzymes from human or rat adrenals that are believed to be aldosterone synthases (9-11). Two 11/3-hydroxylases have been isolated from bovine adrenals that have aldosterone synthase activity (12), although 1 of them (form 2) was more efficient at generating aldosterone from DOC. Four bovine 110-hydroxylase cDNAs were recently cloned, 2 of which correspond to pseudogenes (13). Expression of the cDNAs P-45O(110)-3 and P-45O(110)2 in COS-7 cells revealed that both had 110/18/19hydroxylase activity and converted corticosterone to aldosterone, but convesion by P-450(ll/?)-3 was greater than that by P-450(ll/3)-2 (14). Expression of the 2 kinds of P-450(ll#) mRNAs in adrenal cortex from 10 bovines showed that most individuals expressed both mRNAs, but in 4 of 10 animals, only 1 was expressed (14). Aldosterone generation in the adrenal of most species is in the zona glomerulosa and is regulated in a different fashion from that of cortisol/corticosterone from the zona fasciculata. In the bovine adrenal it is hard to explain this selectivity in view of the lack of specificity of the isolated 11/3-hydroxylase or its cDNA product. We are presenting studies using a mechanism-based (kcat) inhibitor, 18-ethynyl-deoxycorticosterone [18-E-DOC; 21-hydroxy-13-(2-propynyl)18-nor-preg-4-ene-3,20dione], of the hypothetical enzymes P-450 methyl oxidase-I and -II (aldosterone synthase) on the production of corticosterone, 18-hydroxy-ll-deoxycorticosterone (18-OH-DOC), 18-OH-B, and aldosterone by cultured calf zona glomerulosa cells. The studies demonstrate that functionally, the 11/3-hydroxylase is distinct from P-450 methyl oxidase-I and -II.
Materials and Methods Materials
Endo • 1991 Voll29'No5
dispersed mechanically by aspiration through a latex tube fitted to a 60-ml syringe. The cells were collected by centrifugation at 150 x g for 8 min, washed twice with medium, and finally resuspended in medium containing 12 mM potassium, 10% serum mixture (10% fetal calf-horse with 5% bovine embryonic fluid), antibiotic mixture, and an antioxidant mixture (5 nM metyrapone, 50 nM selenous acid, 100 nM butylated hydroxyanisole, and 100 mM dimethylsulfoxide). as described by Crivello et al. (15). The cells were then plated in Primaria 24-well plates (Falcon Plastics, Becton Dickinson Co., Lincoln Park, NJ; 200,000/well) and incubated at 370 C in a 5% CO2 air environment. An extra 1 ml medium was added 24 h later, and the cells were cultured for 3-5 days before being used. All experiments were performed in triplicate and repeated at least twice. Dose response to 18-E-DOC inhibition On the day of the experiments, plates were washed with Hanks' Balanced Salt Solution twice, and the cells were incubated with medium containing 0.5% BSA for 2 h to remove the intracellularly trapped metyrapone. The cells were washed again and incubated in the presence of 0.01-100 fiM 18-E-DOC for 2 h. The final concentration of dimethylsulfoxide (DMSO) used to dissolve 18-E-DOC was 0.5% and did not affect steroid secretion. Cells in 24-well plates were washed and preincubated with 10 MM 18-E-DOC in DMSO or DMSO alone. At the end of the pretreatment time, the cells were washed again with HBBS twice and incubated for 2 h in medium containing vehicle (control), ACTH (109 M), angiotensin-II (A-II; 10 8 M), or potassium (12 mM). The medium was then removed and stored at -20 C. Time course of inhibition The dose of 18-E-DOC necessary to produce maximal inhibition of aldosterone secretion after 2 h of preincubation was 10 /iM, and all subsequent experiments were performed with this concentration. Cells were preincubated, as described above, for various times, and the supernatant was collected as described above. Recovery of aldosterone production after washing 18-E-DOC
Tissue culture medium and serum were obtained from Sigma Chemical Co. (St. Louis, MO). All steroids were obtained from Sigma or Andard Mount Co. (London, England). The 18ethynyl-DOC was kindly provided by Dr. J. O'Neil Johnston from Marion Merrell Dow Research Institute (Cincinatti, OH). Culture of adrenal zona glomerulosa cells Mixed breed calf adrenal glands were obtained from a local abattoir and trimmed clean of fat and adhering tissue under sterile conditions. The outer 500 nm was sliced off using a Stadie-Riggs Microtome. Slices were washed using Hanks' Balanced Salt Solution and suspended in modified Ham's F-12 medium (137 mM NaCl, 2.1 mM CaCl2, 5 mM KC1, and 25 mM HEPES) containing 2 mg/ml collagenase (Worthington Biochemicals, Freehold, NJ), 0.4 mg/ml deoxyribonuclease-I, 0.3 mg/ml neutral protease-IX, 0.5% BSA (Sigma), and antibiotic mixture (100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2.5 mg/ml amphotericin-B). After incubation for 1 h, cells were
After pretreatment with 10 nM 18-E-DOC for 60 min on the last day of the culture, cells were intensively washed and cultured in the complete medium for an additional 24 h. The following day the cells were stimulated with 10'9 M ACTH, 10'8 M A-II, or 12 mM potassium, as described above. Controls received pretreatment with vehicle alone and were handled as described above. Inhibition of cortisol secretion A study similar to that described above was performed using cultures of the second 500-/im layer (zona fasciculata) to determine whether 18-E-DOC inhibited cortisol production. Steroid measurements Aldosterone was measured by direct RIA, using a monoclonal antibody (16). Corticosterone and cortisol were also measured by direct RIA (17). 18-OH-B and 18-OH-DOC were measured by RIA after extracting with dichloromethane (17).
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SUICIDE INHIBITION OF ALDOSTERONE SYNTHESIS
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300
Statistical analysis Statistical comparisons were made by analysis of variance, comparison between treatment means were made using Fishers PLSD test and Dunnett's t test, using Statview 512+ computer program (Brain Power, Calabazas, CA).
Control A-I110 nM ACTH nM Potassium 12 mM
200
"o S a.
Results Aldosterone secretion was inhibited in a dose-dependent fashion, with inhibition becoming significant at a 1fiM concentration of 18-E-DOC (Fig. 1). Inhibition of ACTH-stimulated aldosterone secretion was 73% with 1 AiM, 94.6% with 10 ixM, and 97.5% with 100 nM 18-EDOC. The percentage of 18-E-DOC inhibition of aldosterone secretion by cells stimulated by A-II or potassium and that by control unstimulated cells were significantly less than that by cells stimulated by ACTH, but the absolute secretion of aldosterone at the higher doses of inhibitor was not significantly different. Even at the highest dose of 18-E-DOC, there was significant production of aldosterone. Preincubation with 18-E-DOC for as little as 5 min accomplished significant inhibition of aldosterone production, but 60 min were required for maximal inhibition (Fig. 2). The inhibition of 18-OH-B production was similar to that of aldosterone and followed a similar time course. The lack of effect of 18-E-DOC on corticosterone and 18-OH-DOC in the zona glomerulosa and of cortisol in the zona fasciculata indicated that the inhibition did not affect the production of these steroids (Fig. 3). The inhibition of aldosterone secretion could be partially recovered by incubating the treated cells overnight in new medium, suggesting that new enzyme must be synthesized (Fig. 4). In cells stimulated with ACTH, 1-h treatment with 10 /xM 18-E-DOC resulted in a 90 ± 0.8%
Control ACTH nM
100"
40 PREINCUBATION
TIME
60 (MIN)
"o S Q. UJ
z o
QC •
I00
X
o cc o
0
20 40 PREINCUBATION TIME (MIN)
60
FIG. 2. Top panel, Effect of preincubation time with 18-E-DOC (106 M) on aldosterone secretion in unstimulated and ACTH (109 M)-, A-II (10'8 M)-, or potassium (12 mM)-stimulated calf zona glomerulosa cells in culture. Bottom panel, Effect of preincubation time with 18-E-DOC (106 M) on 18-OH-B secretion in unstimulated and ACTH (109 M)-, A-II (10'8 M)-, or potassium (12 mM)-stimulated calf zona glomerulosa cells in culture.
A-II 10 nM
inhibition of aldosterone secretion and 91 ± 2% inhibition of 18-OH-B secretion. Cells washed and reincubated for 24 h in complete culture medium exhibited an increase in aldosterone to 58 ± 0.5% and in 18-OH-B secretion to 31.5 ± 4% of the level in the uninhibited control cells.
K + 12mM
20 -
oJ
Discussion r— 0
.01
.1
1
10
100
1000
18-Ethynyl-DOC concentration (|iM)
FIG. 1. Dose response of 18-E-DOC on the inhibition of aldosterone secretion in unstimulated and ACTH (109 M)-, A-II (10 8 M)-, or potassium (12 mMJ-stimulated calf zona glomerulosa cells in culture.
There are three classes of mechanism-based inhibitors of cytochrome P-450 monooxygenases: those that either covalently bind to the protein (class I) or to the prosthetic heme group (class III), and those that coordinate quasiirreversibly with the iron atom (class II) (18, 19). Several mechanism-based inhibitors of the cytochrome
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SUICIDE INHIBITION OF ALDOSTERONE SYNTHESIS
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Endo• 1991 Voll29«No5
300
800
P |
1 Bm. • 0
1
10
18-ETHYNYLDEOXYCORTICOSTERONE CONCENTRATION friM)
ill H
1 i
raKg
Ha
1 1
0 1 10 18-ETHYNYL-DEOXYCORTICOSTERONE CONCENTRATION (pM)
FiG. 3. Effect of 18-E-DOC (0,1, and 10 JUM) on corticosterone, 18-OH-DOC, and 18-OH-B secretion in unstimulated and ACTH (109 M)-, A-II (10'8 M)-, or potassium (12 mM)-stimulated calf zona glomerulosa cells and of cortisol secretion in zona fasciculata cells in culture. *, P < 0.05; • • , P < 0.01; • * * , P < 0.001.
P-450s have been described (18-21). Viger et al. (20, 22) reported that the class III compounds are the most potent inhibitors of aldosterone. They reported that 0.8 nM 18ethynyl-progesterone produce a 63% inhibition and 8 nM completely inhibited aldosterone biosynthesis. The structure of 18-E-DOC used in this study is very similar to that of 18-ethynyl-progesterone, and the degree of inhibition of aldosterone production was almost the same as that of 18-ethynyl-progesterone (20, 22). The inhibition of aldosterone secretion is likely to be by covalent binding of the inhibitor to the cytochrome P-450, since exposure of the cells to even a short period of time and extensive washing of the cells resulted in profound inhibition of aldosterone secretion. In the studies of Viger et al. (20, 22), the inhibitor was incubated simultaneously with precursors, and inactivation of the enzyme could not be addressed. Reincubation of the cells for 24 h resulted in a significant recovery of the enzymatic activity, although at 24 h, activity had not returned to control values. It is very likely that new synthesis of the aldosterone synthase is required, although this was not studied
directly. The production of aldosterone and 18-OH-B was inhibited to the same degree after pretreatment with 18E-DOC, whereas the production of corticosterone and 18-OH-DOC in zona glomerulosa cells slightly increased, and that of cortisol in zona fasciculata cells did not change. The recovery of 18-OH-B synthesis was significantly less than that of aldosterone 24 h after exposure to 18-E-DOC; the explanation for this finding is unclear. 18-OH-B is believed by some to be an intermediate in the synthesis of aldosterone (3), atthough Ulick (2) postulated that 18-OH-B is a terminal product from an intermediate on the way to aldosterone synthesis by the cytochrome P-450 enzyme. The adrenal gland shows a distinct zonation regulated by common and independent regulatory factors. In vitro incubations of tissue slices or isolated mitochondria have indicated that aldosterone is produced in the zona glomerulosa of the adrenal (6, 17, 23). However, isolated cytochrome P-450 ll/?-hydroxylase from the zona glomerulosa or fasciculata of porcine or bovine adrenals can
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SUICIDE INHIBITION OF ALDOSTERONE SYNTHESIS 200
ALDOSTERONE 18-HVDROXYCORTICOSTERONE
f o D C o o
o= =
100-
a
ol
5 o
*/'
2
B
C
TREATMENT
FIG. 4. Recovery of aldosterone and 18-OH-B production in cultured zona glomerulosa cells after preincubation with 18-E-DOC (10 fiM) for 60 min. A, Cells were preincubated with 18-E-DOC, washed, and stimulated with ACTH (10'9 M) for 2 h. The degree of stimulation was significantly less that for than untreated cells (*, P < 0.001). B, Cells were preincubated with medium without 18-E-DOC, washed, incubated for 24 h with complete culture medium, washed, and stimulated with ACTH as described above. C, Cells were preincubated with medium with 18-E-DOC, washed, incubated for 24 h with complete culture medium, washed, and stimulated with ACTH, as described above. The degree of stimulation was significantly less than that for untreated cells (*, P < 0.001), but it was significantly greater than for cells treated and incubated immediately (**, P < 0.001).
convert corticosterone to aldosterone quite effectively (7, 8, 24), leading to the suggestion that the aldosterone synthase or corticosterone methyl oxidase activity resides within the 11/3-hydroxylase. Corticosterone, 18OH-DOC, and cortisol were not inhibited by 18-E-DOC, indicating that the enzyme that was inactivated was not responsible for the ll/?/18-hydroxylase activity. It is of interest to note that for unknown reasons the 18-hydroxylation of DOC by the 11/3-hydroxylase was not affected by 18-E-DOC. It is possible that the presence of a bulky group at position 13 inhibited binding to cytochrome P-450 110/18-hydroxylase, but not that to aldosterone synthase. The cloning of aldosterone synthase cDNAs from the rat (9, 10) and a human aldosteroneproducing adrenal adenoma (11) and of bovine 11/3hydroxylase cDNAs (12, 25) has been described. The expression products of these cDNAs have aldosteronegenerating ability. Aldosterone synthase is likely to be a variant of the lljS-hydroxylase with specialized activity (12, 25). This distinct gene is probably located in the chromosome very near the gene for the lljS-hydroxylase (26). These studies of enzyme function show that P-450 methyl oxidase-I and -II exist in bovine adrenal, with no evidence for 11/3-hydroxylase activity. It is possible that the isolated bovine 11/3-hydroxylases and cDNAs are not the responsible for the generation of aldosterone from corticosterone and that the gene responsible remains to be identified.
2365
In conclusion., in the calf adrenal cortex, 18-E-DOC inhibited the last steps of the aldosterone biosynthetic pathway without affecting the production of intermediates that correspond to the most important glucocorticoids produced by the adrenal gland. This drug, if it has the proper phamacokinetic properties, could be useful in the treatment of those disorders associated with increased aldosterone production because of its specificity, in contrast to currently used spironolactone drugs, which antagonize aldosterone as well as other adrenal steroids at the receptor level (27).
Acknowledgments The 18-E-DOC was kindly provided by Dr. J. O'Neil Johnston from Marion Merrell Dow Research Institute, a division of Marion Merrell Dow, Inc. The technical help of Mark F. Foecking is gratefully acknowledged.
References 1. Muller J 1988 Pathway of aldosterone biosynthesis. In: Muller J (ed) Regulation of Aldosterone Biosynthesis. Physiological and Clinical Aspects. Springer-Verlag, Berlin, pp 5-15 2. Ulick S 1976 Diagnosis and nomenclature of the disorders of the terminal portions of the aldosterone biosynthetic pathway. J Clin Endocrinol Metab 43:92-96 3. Fattah DI, Whitehouse BJ, Vinson GP 1977 Biosynthesis of aldosterone from 18-hydroxylated precursors by rat adrenal tissue in vitro. J Endocrinol 75:187-195 4. Sato H, Ashida N, Suhara K 1978 Properties of an adrenal cytochrome P-450 (P-450-llb) for the hydroxylations of corticosteroids. Arch Biochem Biophys 190:307-314 5. Watanuki M, Tilley BE, Hall PF 1978 Cytochrome P-450 for lib and 18-hydroxylase activities of bovine adrenocortical mitochondria: one enzyme or two. Biochemistry 17:127-130 6. Ohnishi T, Wada A, Lauber M, Yamano T, Okamoto M 1988 Aldosterone biosynthesis in mitochondria of isolated zones of adrenal cortex. J Steroid Biochem 31:73-81 7. Wada A, Ohnishi T, Nonaka Y, Okamoto M, Yamano T 1985 Synthesis of aldosterone by a reconstituted system of cytochrome P-450-llb from bovine adrenocortical mitochondria. J Biochem 98:245-256 8. Yanagibashi K, Haniu M, Shively JE, Shem WH, Hall P 1986 The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glom«rulosa) possess one enzyme for lib, 18-hydroxylation, and aldehyde synthesis. J Biol Chem 261:3556-3562 9. Matsukawa N, Nonaka Y, Ying Z, Higaki J, Ogihara T, Okamoto M 1990 Molecular cloning and expression of cDNAs encoding rat aldosterone synthase: variants of cytochrome P-450 lib. Biochem Biophys Res Commun 169:245-252 10. Imai M, Shimada H , Okada Y, Matsuhima-Hibiya Y, Ogishima T, Ishimura Y 1990 Molecular cloning of cDNA encoding aldosterone synthase cytochrome P-450 in rat adrenal cortex. FEBS Lett 263:299-302 11. Kawamoto T, Mitsuuchi Y, Ohnishi T, Ichikawa Y, Yokoyama Y, Sumimoto H, Toda K, Miyahara K, Kuribayashi I 1991 Cloning and expression of a cDNA for human cytochrome P-450 aldo as related to primary aldosteronism. Biochem Biophys Res Commun 173:309-316 12. Ogishima T, Mitani F, Ishimura Y 1989 Isolation of two distinct cytochromes P-450 lib with aldosterone synthase activity from bovine adrenocortical mitochondria. J Biochem 105:497-499 13. Hashimoto T, Morohashi K-I, Omura T 1989 Cloning and characterization of bovine cytochrome P-450(llb) genes. J Biochem 105:676-679
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SUICIDE INHIBITION OF ALDOSTERONE SYNTHESIS
14. Morohashi K-I, Nonaka Y, Kirita S, Hatano 0, Takakusu A, Okamoto M, Omura T 1990 Enzymatic activities of P-450(llb)s expressed by two cDNAs in COS-7 cells. J Biochem 107:635-640 15. Crivello JF, Hornsby PJ, Gill GN 1982 Metyrapone and antioxidants are required to maintain aldosterone synthesis by cultured bovine adrenocortical zona glomerulosa cells. Endocrinology 111:469-479 16. Gomez-Sanchez CE, Foecking MF, Ferris MW, Chavarri MR, Uribe L, Gomez-Sanchez EP 1987 The production of monoclonal antibodies against aldosterone. Steroids 49:581-587 17. Gomez-Sanchez CE, Ferris MW, Foecking MF, Gomez-Sanchez EP 1989 Synthesis of 18-hydroxycortisol and 18-oxocortisol in bovine adrenal slices. J Steroid Biochem 33:595-598 18. Ortiz de Montellano PR 1984 The inactivation of cytochrome P450. Annu Rep Med Chem 19:201-211 19. Johnston JO, Wright CL, Metcalf BW 1984 Biochemical and endocrine properties of a mechanism-based inhibitor of aromatase. Endocrinology 115:776-785 20. Viger A, Coustal S, Perard S, Chappe B, Marquet A 1988 Synthesis and activity of new inhibitors of aldosterone biosynthesis. J Steroid Biochem 30:469-472
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21. Nagahisa A, Spencer RW, Orme-Johnson WH 1983 Acetylenic mechanism-based inhibitors of cholesterol side chain cleavage by cytochrome P450 sec. J Biol Chem 258:6721-6723 22. Viger A, Coustal S, Perard S, Piffeteau A, Marquet A 1989. 18Sustituted progesterone derivatives as inhibitors of aldosterone biosynthesis. J Steroid Biochem 33:119-124 23. Giroud CJP, Stachenko J, Venning EH 1956 Secretion of aldosterone by the zona glomerulosa of rat adrenal glands incubated in vitro. Proc Soc Exp Biol Med 92:154-158 24. Ohnishi T, Wada A, Nonaka Y, Okamoto M, Yamano T 1984 Effect of phospholipid on aldosterone biosynthesis by a cytochrome P-450-llb-reconstituted system. Biochem Int 9:715-723 25. Mathew PA, Mason JI, Trant JM, Sanders D, Waterman MR 1990 Amino acid substitutions Phe^-Lew and Ser126-Pro abolish cortisol and aldosterone synthesis by bovine cytochrome P-450 lib. J Biol Chem 265:20228-20233 26. Globerman H, Rosier A, Theodor R, New MI, White PC 1988 An inherited defect in aldosterone biosynthesis caused by a mutation in or near the gene for steroid 11-hydroxylase. N Engl J Med 319:1193-1197 27. Fanestil DD 1988 Mechanism of action of aldosterone blockers. Semin Nephrol 8:249-263
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