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Medical Research Council Blood Pressure Group, Department of Medicine and Therapeutics (A.F., E.C.F., J.M.C.C., R.F., E.D.), Western Infirmary, Glasgow, Scotland G11 6NT; Department of Biochemistry, Universität des Saarlandes (R.B.), D-66041 Saarbrucken, Germany; and University of Mississippi Medical Center and the G. V. (Sonny) Montgomery Veterans Affairs Hospital (C.G.-S.), Jackson, Mississippi 39216
Address all correspondence and requests for reprints to: Prof. R. Fraser, Medical Research Council Blood Pressure Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow, Scotland G11 6NT. E-mail: rfraser{at}clinmed.gla.ac.uk
Abstract
In glucocorticoid-suppressible hyperaldosteronism, 11ß- hydroxylase activity is impaired. A chimeric enzyme formed from the control elements of 11ß-hydroxylase and the structural elements of aldosterone synthase is expressed ectopically in the zona fasciculata, thus exposing cortisol to aldosterone synthase. Increased quantities of 18-hydroxycortisol and 18-oxocortisol are synthesized, which, it has been suggested, might have a local inhibitory effect on the normal 11ß-hydroxylase. The effects of these compounds and also of 18-hydroxydeoxycorticosterone were tested in cells stably transfected with CYP11B1 and CYP11B2, the genes encoding 11ß-hydroxylase and aldosterone synthase, respectively. Neither 18-hydroxycortisol nor 18-oxocortisol affected the efficiency of use of 11-deoxycorticosterone or 11-deoxycortisol as substrates by the enzymes. 18-Hydroxydeoxycorticosterone significantly reduced the conversion rate of 11-deoxycorticosterone to corticosterone and that of 11-deoxycortisol to cortisol by both enzymes, but the production rate of 18- hydroxycorticosterone and aldosterone by aldosterone synthase increased. Aldosterone synthase was able to convert 18-hydroxydeoxycorticosterone to 18-hydroxycorticosterone and aldosterone, although its affinity for this substrate was lower (4.76 µmol/liter) than that for 11-deoxycorticosterone (0.11 µmol/liter). 11ß-Hydroxylase was unable to convert 18- hydroxydeoxycorticosterone to 18-hydroxycorticosterone. 18-Hydroxycortisol and 18-oxocortisol are not, therefore, the cause of lower 11ß-hydroxylase activity in glucocorticoid- suppressible hyperaldosteronism. 18-Hydroxydeoxycorticosterone can be converted to aldosterone, but its local concentration in man and its Km suggest that it is unlikely to be important.
ALDOSTERONE (Aldo) SYNTHASE is synthesized
and acts exclusively in the zona glomerulosa of the adrenal cortex. It
catalyzes the conversion of 11-deoxycorticosterone (DOC) to Aldo
(1). The zona glomerulosa does not express
17
-hydroxylase and therefore is unable to synthesize cortisol (F).
In the zona fasciculata, 11ß-hydroxylase catalyzes the conversion of
DOC and 11-deoxycortisol (S) to corticosterone (B) and F, respectively.
It can also 18-hydroxylate a variety of precursors, including
progesterone, DOC, S, B (2), and, to a limited extent, F
(3). It is unable to form an aldehyde group at
C18.
In two rare conditions, however, F is exposed to Aldo synthase. In these cases the rates of synthesis of 18-hydroxycortisol (18-OHF) and 18-oxocortisol (18OXOF) are markedly increased (4, 5). 18-Hydroxycorticosterone (18OHB) levels are also raised (5). In Conn’s syndrome, an adrenocortical tumor that secretes Aldo autonomously usually contains both Aldo-secreting and F-secreting cells (4). In glucocorticoid-suppressible hyperaldosteronism (GSH), a chimeric enzyme comprising the 11ß-hydroxylase control region and the catalytic domain of Aldo synthase is expressed ectopically in the zona fasciculata (6). In both conditions, but particularly in GSH, 11ß-hydroxylation is impaired (7, 8). It has been postulated that abnormally high concentrations of 18- hydroxysteroids may be responsible for this (8). This hypothesis was investigated using cells stably transfected with human CYP11B1 or CYP11B2, the genes encoding 11ß-hydroxylase and Aldo synthase, respectively.
Materials and Methods
Cells from a Chinese hamster ovary cell line (V79) were stably transfected with CYP11B1 or CYP11B2 (9, 10). They were maintained in DMEM supplemented with 2 mM L-glutamine, 5% FCS, 100 U/ml penicillin G, and 100 µg/ml streptomycin and amphotericin (Life Technologies, Inc., Paisley, UK). They were passaged at 90% confluence. Before use, they were washed (1 x 5 ml trypsin-EDTA solution; Life Technologies, Inc.) and suspended in 10 ml fresh culture medium (see above). Aliquots (4 ml) were transferred to each well of six-well culture plates and incubated for 24 h (37 C, 5% CO2). The medium was then replaced with medium containing the requisite steroid substrate concentrations and the cells were incubated for an additional 24 h. The medium was then removed and stored at -20 C until required for steroid analysis (11). The cell protein concentration was determined as previously described (Bio-Rad Laboratories, Inc. Hemel Hempstead, UK) (11). Derivation of the Km of Aldo synthase for DOC and 18-hydroxydeoxycorticosterone (18-hydroxyDOC) was carried out in whole cells incubated with substrate (0.1–10 µM) for 8 h. A low passage number was used in the kinetic study to obtain greater sensitivity at lower substrate concentrations. All incubations were carried out in quadruplicate.
Data were analyzed by ANOVA, followed by t test where appropriate.
Results
Effects of 18OHF and 18OXOF
Neither 18OHF nor 18OXOF at 10 µM (data not shown) or 20 µM affected the efficiency of B production from DOC (basal B, 10.4 ± 1.0; +18OXOF, 8.0 ± 1.2; basal, 7.4 ± 0.5; +18OHF, 8.2 ± 1.1 nmol/mg·24 h) or F production from S (basal F, 11.5 ± 2.4; +18OXOF, 13.0 ± 0.5; basal, 12.8 ± 2.6; +18OHF, 12.7 ± 0.9 nmol/mg·24 h) by 11ß-hydroxylase (CYP11B1-transfected cells). Similarly, the ability of Aldo synthase (CYP11B2-transfected cells) to convert S to F (basal F, 5.8 ± 0.5; +18OXOF, 6.2 ± 0.4; basal, 5.0 ± 0.5; +18OHF, 4.0 ± 0.3 nmol/mg·24 h) or DOC to B (basal B, 4.6 ± 0.2; +18OXOF, 4.2 ± 0.6; basal, 6.8 ± 0.9; +18OHF, 5.4 ± 1.2 nmol/mg·24 h), 18OHB (basal 18OHB, 9.3 ± 1.1; +18OXOF, 9.6 ± 1.6; basal, 10.0 ± 1.4; +18OHF, 9.9 ± 1.0 nmol/mg·24 h) or Aldo (basal Aldo, 5.2 ± 0.6; +18OXOF, 6.0 ± 0.7; basal, 6.4 ± 0.6; +18OHF, 6.4 ± 0.6 nmol/mg·24 h) was unaffected by either compound.
Effects of 18-hydroxyDOC
In cells transfected with CYP11B1, inhibition of B synthesis from
DOC by 10 µM 18-hydroxyDOC was significant at DOC
concentrations of 0.01 µM and above (Fig. 1
). B synthesis from a DOC concentration
of 0.001 µM was below the reliable sensitivity limits
of the assay. 18-HydroxyDOC similarly inhibited the conversion of S to
F (Fig. 1). In cells transfected with CYP11B2, 10 µM
18-hydroxyDOC significantly decreased the production of B and F from
DOC and S, respectively (Fig. 2).
However, in these cells the production rates of both 18OHB and Aldo
were enhanced by the presence of 18-hydroxyDOC (Fig. 3). Incubation of CYP11B2-transfected
cells with 18-hydroxyDOC shows a close positive dose- response
relationship with 18OHB and Aldo production (Fig. 4). Thus, 18-hydroxyDOC is a substrate
for human Aldo synthase, but the affinity is less
(Km, 4.76 µmol/liter) than that for DOC
(Km, 0.11 µmol/liter; data not shown). Human
11ß-hydroxylase (CYP11B1-transfected cells) could convert DOC to
18OHB (0.5 µmol DOC, 2.1 ± 0.2 nmol/mg·24 h; 10 µmol DOC,
6.5 ± 1.3 nmol/mg·24 h; P < 0.0001), but not
18-hydroxyDOC (0.5 µmol 18-hydroxyDOC, 0.25 ± 0.02
nmol/mg·24 h; 10 µmol 18-hydroxyDOC, 0.7 ± 0.05
nmol/mg·24 h; P > 0.05).
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It has been suggested that 18OHF and 18OXOF account for apparently subnormal 11ß-hydroxylase activity in GSH and Conn’s syndrome (8). In this study, 18OHF, 18OXOF, and also 18-hydroxyDOC were assessed in vitro for effects on 11ß-hydroxylation by human Aldo synthase and 11ß-hydroxylase. Urinary levels of 18OHF are some 10-fold greater than normal in GSH patients. Comparable concentrations of 18OHF or 18OXOF did not affect 11ß-hydroxylation of S or DOC by either 11ß-hydroxylase or Aldo synthase. In addition, neither steroid affected DOC conversion to 18OHB or Aldo by Aldo synthase. Local tissue concentrations of 18OHF or 18OXOF may be higher than those observed in plasma or urine, although it is generally accepted that hormonal steroids are not stored at high concentrations (see, however, Ref. 12). Other possible explanations might account for the defective 11ß-hydroxylation observed in GSH. The high rate of expression of the chimeric gene (13) must indirectly down-regulate zona glomerulosa CYP11B2 expression. DOC to B and S to F conversion must be due to the sum of the activities of the chimeric enzyme and 11ß-hydroxylase. Levels of chimeric mRNA in the zona fasciculata are greater than levels of CYP11B1 mRNA (13). High Aldo concentrations in the zona fasciculata may inhibit the conversion of DOC by the chimeric Aldo synthase. Aldo has been shown to inhibit the formation of B in cultured adrenocortical cells (14). F inhibits the conversion of B, 18OHB, or 18-hydroxyDOC to Aldo by rat mitochondria, but has no effect on the conversion of DOC to 18-hydroxyDOC or B (15). However, neither Aldo nor F at the levels attained here would be expected to affect the conversion of S to F.
In contrast, 18-hydroxyDOC significantly inhibited 11ß-hydroxylation of S and DOC by Aldo synthase and 11ß-hydroxylase. The mechanism is probably competitive inhibition. 18-HydroxyDOC can act as a substrate for Aldo synthase. 11ß-Hydroxylase can convert it to 18OHB, but produces only small quantities. However, it is unlikely that 18-hydroxyDOC accounts for the 11ß-hydroxylase deficiency in GSH; it is a poor substrate and unlikely to compete with DOC or S.
These experiments show 18-hydroxyDOC to be a substrate for human Aldo synthase and, to a lesser extent, 11ß- hydroxylase in vitro. Whether it is an important substrate in vivo remains to be established. In the rat, 18-hydroxyDOC is produced by 11ß-hydroxylase. It may be sequestered within the adrenal cortex and made available as an alternative substrate for Aldo biosynthesis (see below). Based on their respective plasma and urinary concentrations in man, the local tissue 18-hydroxyDOC concentration may be some 10-fold higher than that of DOC. DOC has a much lower Km for Aldo synthase than does 18-hydroxyDOC. It seems unlikely that 18-hydroxyDOC competes effectively with DOC for the Aldo synthase active site. The Km of 11ß-hydroxylase for 18- hydroxyDOC was not assessed, but the yield of 18OHB was much lower than that of DOC. This suggests that 18- hydroxyDOC is probably also not an important substrate for 11ß-hydroxylase in vivo. The efficiency of 18-hydroxyDOC conversion to Aldo by cloned rat Aldo synthase in vitro is also poor (16), and it is not a good substrate for bovine 11ß-hydroxylase, which synthesizes Aldo in this species (17).
The situation may be different in the rat (12, 18). The route by which zona fasciculata-produced 18-hydroxyDOC finds its way to the zona glomerulosa is not known. That it is made by human or rat Aldo synthase in situ in the zona glomerulosa in useful quantities is also unlikely (19).
In summary, neither 18OHF nor 18OXOF can account for the subnormal 11ß-hydroxylase activity in Conn’s syndrome and GSH. 18-HydroxyDOC inhibits DOC to B and S to F conversion in vitro and can be converted to Aldo and 18OHB by human Aldo synthase and 11ß-hydroxylase, respectively. However, the affinity of these enzymes for 18-hydroxyDOC is much lower than that of DOC, questioning its importance as an in vivo substrate in man.
Acknowledgments
Footnotes
This work was supported by Medical Research Council Program Grant G93/17119 (to J.M.C.C., R.F., and E.D.) and grants from the Deutsche Forschungsgemeinschaft (Be 1343/2-5) and the Fonds der Chemischen Industrie (to R.B.).
Abbreviations: Aldo, Aldosterone; B, corticosterone; F, cortisol; S, 11-deoxycortisol; DOC, 11-deoxycorticosterone; 18-hydroxyDOC, 18-hydroxydeoxycorticosterone; GSH, glucocorticoid-suppressible hyperaldosteronism; 18OHB, 18-hydroxycorticosterone; 18OHF, 18-hydroxycortisol; 18OXOF, 18-oxocortisol.
Received February 23, 2001.
Accepted May 17, 2001.
References
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