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Experimental Studies |
Department of Endocrinology, Klinikum Benjamin Franklin, Freie Universität Berlin, Germany
Address all correspondence and requests for reprints to: Sven Diederich, Department of Endocrinology, Klinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.
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Abstract |
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Oxidation of F to E: NAD was the exclusively used cosubstrate; the affinity [Michael’s constant (Km) for F = 25.5 nmol/L] and the maximum velocity (Vmax = 22.9 nmol/mg/min) were high. Reduction of E to F: Without the NADH/NADPH-regenerating system, this reaction was very slow. With this system, the Km value for E was in the nanomolar range (80.6 nmol/L) and the Vmax value was very low (0.88 nmol/mg/min). The reaction was clearly NADH-preferring. For the steroid pair F/E, the quotient Vmaxoxidation/Vmaxreduction (=26) demonstrates an equilibrium far on the 11-keto side. Oxidation of D to DH-D: With NAD as the only used cosubstrate, the kinetic analysis is compatible with the existence of two different NAD-dependent isoenzymes: Km for D = 327 nmol/L, Vmax = 53.5 nmol/mg/min and Km for D = 81.2 nmol/L; Vmax = 20.4 nmol/mg/min. Reduction of DH-D to D: The maximum velocity was higher than that of all other reactions tested: Vmax = 226.0 nmol/mg/min. The reaction was exclusively NADH-dependent; the Km value for DH-D was 68.4 nmol/L. For D/DH-D, the ratio Vmaxoxidation/Vmaxreduction was 0.24, demonstrating a shift to reductase activity with the reaction equilibrium far on the 11-hydroxy side. The reaction F to E was inhibited by E, DH-D, and D in a concentration-dependent manner.
In conclusion, the cosubstrate dependence, the Km value of the oxidation of F and the product inhibition are in good correspondence with data for the cloned human 11ß-HSD-II. The NADH-dependent 11ß-reduction of E and especially of DH-D are inconsistent with the dogma of an unidirectional 11ß-HSD-II. The preference of D for the reductase reaction in human kidney slices is probably caused by the fluor atom in position 9, is catalyzed by 11ß-HSD-II, and leads to an activation of 11-DH-D to D in the human kidney.
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Introduction |
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2 µmol/L) that is above the
physiological range of circulating free F (10 nmol/L) in man (9).
The isoenzyme 11ß-HSD-II seems to exclusively oxidize physiological
glucocorticoids, uses NAD as cosubstrate, is only found in MC target
tissues and the placenta, and has a Km value in the
nanomolar range that is closer to physiologically relevant amounts of
free F (10). This isoenzyme is optimized for the protection of MC
receptors from F. In this way, it provides MC selectivity to
aldosterone, which binds with the same affinity as F to the MC receptor
(11) but is not an 11ß-HSD-substrate (6).
9
-Halogenated 11-hydroxysteroids are metabolized in a manner
different from the unsubstituted molecules by 11ß-HSD. Bush et
al. (12, 13) found that rat liver homogenates (11ß-HSD-I
activity) reduced 9-fluorocortisone (9FE) more rapidly at the
11-position than cortisone (E), whereas 9-fluorocortisol (9FF)
was not oxidized at all, in contrast to F. Our group demonstrated
similar effects of 9-fluorination on 11ß-HSD-activity in human
kidney slices and in vivo (14). Kidney slices converted
F much faster to E than 9FF to 9FE, whereas the reduction of
9FE to 9FF was much more effective than that of E to F. In normal
males who took F by the oral route, 70% of the free steroid fraction
in urine was E and 30% was F. In contrast, after the oral
administration of 9-FF, 90% of the free steroid fraction was
9-FF and 10% was 9FE. Orally given 9FE was very effectively
reduced, and 90% was excreted as 9FF (14).
Siebe et al. (15) described similar in
vitro results with the 9-fluorinated steroid dexamethasone
(D) (9-fluoro-16-methyl-
1-dehydro-cortisol). In human kidney
slices, 11ß-oxidation of D was diminished and 11ß-reduction of
dehydro-D (DH-D) was increased compared with the steroid pair F/E. In
contrast to 9FF, which binds with the same affinity as F and
aldosterone to the MC receptor (14), D has very low affinity to the MC
receptor (6) but high affinity to the GC receptor (5).
In the present study, we attempted to find out which
11ß-HSD-isoenzyme is responsible for the 9-fluor-induced shift to
reductase activity observed in human kidney slices. We focused on the
steroid pairs F/E and D/DH-D and characterized the 11ß-HSD-isoenzymes
by performing enzyme kinetic analyses and defining the cosubstrate
dependence.
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Material and Methods |
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Unlabeled F, E, an D, the cofactors NAD/NADH and NADP/NADPH, D-glucose-6-phosphate-monosodium-salt (G6P), and sucrose were purchased from Sigma Chemical Co. (St. Louis, MO). Unlabeled DH-D was a gift from Dr. H. Laurent and Dr. H.J. Zentel, Schering AG (Berlin, Germany). [1,2,6,7-3H]F (specific activity: 70.0 Ci/mmol) was obtained from DuPont de Nemours GmbH (Bad Homburg, Germany). [1,2-3H]E (specific activity: 41 Ci/mmol) and [1,2,4,6,7-3H]D (specific activity: 70 Ci/mmol) were purchased from Amersham International plc (Buckinghamshire, England). [1,2,4,6,7-3H]DH-D was synthesized in our laboratory from tritiated D by oxidation with chromium-6-oxide (16). The identity of the tritiated DH-D was proved by three different methods. 1) Comparison of the retention time of the radioactive high performance liquid chromatography (HPLC) peak with that of the UV peak of the unlabeled reference steroid. 2) Mass spectometric analyses (Department of Pharmacology, Freie Universität Berlin) (15). 3) The synthesized [3H]DH-D was reduced to [3H]D by human kidney microsomes. All tritiated steroids were purified by HPLC before use.
Glucose-6-phosphate dehydrogenase (G6PDH) (from Leuconostoc mesenteroides) was purchased from Boehringer (Mannheim, Germany); sodium-hydrogenphosphate-dihydrate and sodium-hydrogenphosphate-monohydrate were obtained from Merck Ltd. (Darmstadt, Germany). The materials and solvents for extraction with Sep-Pak C18 cartridges (Waters Millipore GmbH, Eschborn, Germany) and for separation with HPLC were the same as described previously (17).
For thin-layer chromatography (TLC) that was used alternatively to HPLC, we also chose the extraction with Sep-Pak C18 cartridges. TLC plates coated with silicagel were obtained from Merck (Darmstadt, Germany); dichlormethane-methanol (75 + 5), used as mobile phase, was Lichrosolv from Merck. The scintilat Instagel Plus was from Packard (Frankfurt, Germany).
Preparation of microsomes
Human kidney tissue was taken from unaffected parts of kidneys removed because of renal cell carcinoma (14, 17). The microsomes were prepared from kidney cortex of six different organs (three male, and three female patients, age: 47–81 yr; mean, 60), no patient was treated with steroids or other hormones before this study. The whole tissue (total amount: 60 g) was homogenized in 0.01 M sodium phosphate buffer, containing 0.25 M sucrose. All subsequent steps were performed at 0–4 C. Microsomes were extracted by differential centrifugation using the method of Lakshmi and Monder (18). The homogenate was centrifuged at 750 x g for 30 min and at 20,000 x g for 30 min, saving the supernatant at each step. The last supernatant was recentrifuged at 105,000 x g for 60 min, the pellet was resuspended in the homogenizing buffer, and centrifuged again at 105,000 x g for 60 min. The washed microsomal pellets were resuspended in 14 mL 0.1 M phosphate-buffer and stored in portions of 200 µL in liquid nitrogen. The microsomes were used within 2 months, and no loss in enzyme activity was seen during this period. Because we used the same microsome pool for all reactions, the kinetic parameters are well comparable.
11ß-HSD activity determination
Protein quantification was done before every incubation by the method of Lowry et al. (19), and the microsomes were diluted to concentrations required. Incubations were carried out in wells of a plastic 24 multi-well incubation plate located in a steel chamber that had been preheated to 37 C in a shaking water bath.
We chose the following fixed conditions for all incubations: The total
incubate volume was 1 mL, the cosubstrate concentration was always
10-3 mol/L. In preliminary studies, we found the optimal
pH value for oxidation between 8.0 and 8.5 and for reduction between
6.0 and 6.5. Using a 0.1 mol/L sodium phosphate buffer, we chose pH 8.5
for all dehydrogenase reactions and pH 6.0 for all oxoreductase
reactions. The amount of labeled steroids was constant in every
experiment (100.000 cpm). The incubation was started by the addition
of microsomes to 10-min preincubated wells containing all the assay
components except the enzyme preparation. In all assays, blanks were
included containing all the assay components except that buffer
replaced the enzyme preparation. Incubations were terminated by
addition of 2 mL cold methanol and by rapid transfer of the multi-well
incubation plate on ice. Each experiment was at least done in
triplicate.
For each reaction tested, we did preliminary studies to determine optimal conditions: by varying the amount of enzyme preparation and the reaction time, the initial velocity was always in the linear range. For kinetic analyses we added increasing amounts of the corresponding unlabeled steroid. For the oxidation of F to E, the protein concentration was 0.025 mg/mL, and the incubation time was 40 min (preliminary time kinetic analyses had shown linearity up to a fractional conversion of 80% after 50 min!). Five different concentrations of F (between 9.5 and 150 nmol/L, n = 5) were used. The conditions for other reactions tested are described in the legends of the figures. For the reduction of E to F, no linear time kinetic could be obtained by changing the protein concentration or the incubation time. Using the NADH/NADPH-regenerating system (10-2 mol/L G6P and 10 U G6PDH for each incubation) (20), we could perform clean kinetic analyses for this reaction. Incubations of E and G6PDH without enzyme preparation showed no conversion of E to F. Because preliminary studies had not shown any effect of the NADH-regenerating system on the reduction of DH-D to D, this reduction was run without the cosubstrate-regenerating system.
Analytical procedure
The first analytical procedure for each reaction was done with the HPLC method, described by our group previously (17). Briefly, it involves the steroid extraction by Sep-Pak C18 cartridges, the steroid separation by HPLC, and radioactive detection and quantitation. Using this steroid analysis, we were unable to detect any other metabolites than the 11-oxo- or 11-hydroxy-steroids after incubation.
For the numerous measurements of the enzyme kinetics, we developed a faster and cheaper TLC method: steroids were extracted with Sep-Pak cartridges and dissolved in 50 µL methanol containing a mixture of the corresponding unlabeled steroids (F and E or D and DH-D). This solution was applied to TLC plates and developed with dichlormethane-methanol as the mobile phase. The spots were identified under UV light, cut out, and transferred to scintillation vials containing 1 mL H2O and 15 mL Instagel Plus. After incubation for 12 h, the recovery for all steroids tested was between 99 and 100%.
Measurement of percentage conversion rates allowed the determination of the initial velocity (nanomoles per milligrams per minute). We chose the Hanes-Woolf transformation for the calculations of Km and Vmax values (21).
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Results |
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, A-D). All measurements were in the linear part
of the time kinetic of the individual reaction, so that the initial
velocity (Vo) was measured. Therefore, incubation times and
protein concentrations were different for each steroid substrate. For
each reaction, two substrate concentrations were tested. Because even
at high substrate concentration, being closer to the Km
value of 11ß-HSD I, no NADP(H)-preferring activity was found,
11ß-HSD II seems to be the exclusive or dominant enzyme in human
kidney microsomes.
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, A and
B). In the Hanes-Woolf transformation, the negative Km
value corresponds to the intersection with the horizontal axis. The
slope of the line is the reciprocal value of the maximum velocity
(Vmax). For the isoenzyme with the lower Km
value (Fig. 2B), we only used the Vo values in the range of
10–50% of Vmax, because at higher substrate
concentrations the velocity rapidly increased because of additional
activity of the presumed second isoenzyme (Fig. 2A). For all other
kinetic estimations, we chose the substrate concentrations in such a
way, that the Vo values were in the ideal range of 10–90%
of Vmax.
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). The reduction
of DH-D is much more effective than that of E (compare the
Vmax values and consider that we did not add the
NADH-regenerating system for the reduction DH-D to D) (Fig. 3B). The
Km values for all four reactions are in the nanomolar order
of magnitude (Table 1). The Vmax values show
that the preferred reaction for F is the oxidation, whereas for D it is
the reduction (see the ratio
Vmaxoxidation/Vmaxreduction).
This contrast is most convincingly demonstrated by calculating the
intrinsic clearance values (ICV = Vmax/Km)
(22) of the different reactions (Table 1).
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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) are
similar to those of the cloned human 11ß-HSD-II [Km for
F: 47 nM (8)] and those of human fetal kidney microsomes
[Km for F: 60 nM (10)]. Moreover, the product
inhibition that we found in our experiments (Fig. 4), is characteristic
of 11ß-HSD-II, and not of 11ß-HSD-I (8). These similarities seem to
indicate that our microsomes predominantly or exclusively contained
11ß-HSD-II. This is also supported by the observation of Whorwood
et al. (23), who detected 11ß-HSD-II messenger RNA in
human kidney cortex and medulla, whereas small amounts of 11ß-HSD-I
messenger RNA were only found in kidney medulla.
The 9-fluorinated D is handled in a way markedly different from F by
human kidney 11ß-HSD. The oxidation of D (Fig. 2) was only
demonstrated with 11ß-HSD-II (8, 24) and seems to be an additional
characteristic of this isoenzyme. For this reaction, we found two
different NAD-dependent enzyme kinetics in human kidney cortex
microsomes (Fig. 2, A and B). Brown et al. (25) reported a
Km value of 119 nM in homogenized Chinese
hamster ovary cells transfected with human 11ß-HSD-II, whereas
Ferrari et al. (24) found a Km value of 890
nM in the same cell system. A careful view on the paper of
Ferrari et al. (24) shows that in their double-reciprocal
plot of the oxidation of D, a second kinetic with an additional
Km value may have been overlooked: the points of the lowest
D concentrations can be connected as an independent line. Thus, our
findings of two different Km values for the oxidation of D
may be an explanation for the diverging results of both groups (24, 25)
and may be because of a conformational perturbation of 11ß-HSD-II.
Apart from our suggestion of two kinetics in the plot of Ferrari
et al. (24), our own results are compatible with a second
NAD-dependent isoenzyme in human kidney cortex.
The oxidation of F is inhibited by the synthetic 11-ß-HSD-substrate D
(Fig. 4) suggesting competition for the binding domain of the enzyme.
In vivo, this competition probably plays no role, because
orally administered D totally suppresses adrenal secretion of
endogenous GCs.
For the first time, we could demonstrate a NADH-dependent reduction of E to F. Although the chosen conditions (NADH oversupply, NADH-regenerating system, and optimized pH value) are artificial and never found in vivo, this result is inconsistent with the dogma of an unidirectional 11ß-HSD-II. The NADH-regenerating system had to be used to be able to study the kinetics of E reduction at all. In addition to the cosubstrate dependence, the Km value (80.6 nmol/L) for the reduction of E is different from that described for 11ß-HSD-I (9). Thus, a contamination with 11ß-HSD-I as an explanation for our results is very unlikely.
The theory of unidirectionality of 11ß-HSD-II is based on studies
with human fetal kidney microsomes (10) and with homogenates of
11ß-HSD-II-transfected cells (8). The unexpected reductase activity
we found in human adult kidney microsomes may be explained by the
following facts. First, the time between surgical removal and storing
the tissue in liquid nitrogen ( -150 C) was relatively short, not
more than 1 h. Second, for the reduction of E we used high protein
concentrations and long incubation times (Fig. 1C). The NADH-support
system was never used before for testing human 11ß-HSD-II activity.
Furthermore, human adult kidney microsomes may be different from human
fetal microsomes and from 11ß-HSD-II-transfected cells.
The theory of a bidirectional 11-ß-HSD-II is clearly supported by our
results with the 9-fluorinated steroid DH-D as a substrate. For this
reaction, the NADH-regenerating system was not required, the amount of
microsomes was small, and the incubation times were short (Figs. 1D and 3B). These results prove that the reduction of 9-fluorinated
11-oxo-steroids found in human kidney slices (14, 15) is caused by
11ß-HSD-II. The apparent redox-equilibrium for D and DH-D is far on
the hydroxy side, whereas that for F and E is far on the keto side
(Table 1: ratios of Vmax or of intrinsic clearance values).
This shift seems to be induced by the 9-fluor substitution and leads to
biological activation of DH-D to the potent glucocorticoid D in the
human kidney (15). Thus, DH-D may be an interesting prodrug being
mainly activated to D in the kidney.
In summary, we have shown that human 11ß-HSD-II is not only an
oxidative enzyme, but can function as a reductase. For physiological
regulations, this function of 11ß-HSD-II seems to play no role,
because significant reduction of E could be demonstrated under
artificial in vitro conditions only (NADH-regenerating
system). For pharmacological applications, the very active reduction of
11-dehydro-D and of other 9-fluorinated steroids may be of interest
for renal drug targeting.
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Acknowledgments |
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Received October 24, 1996.
Revised January 3, 1997.
Accepted January 14, 1997.
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dehydro-D (DH-D), pH 8.5. Incubation time: 45 min.
Left, Concentration of D, 9.5 nmol/L. Protein
concentration: 0.075 mg/mL. Right, Concentration of D,
750 nmol/L. Protein concentration: 0.125 mg/mL. C, E 
