ENDOCRINE RESEARCH, 21(1&2), 471-475 (1995)

CORTISOL: A TOOL TO STUDY ALDOSTERONE BIOSYNTHESIS IN RATS

Laura Matkovid*, Celso E. Gomez-Sanchez#, Carlos P Lantos* and Eduardo N Cozza*

*Departamento de Quimica Biolbgica, Facultad de Cs. Exactas y Naturales-UBA and PRHOM-CONICET, Buenos Aires-1428, Argentina #Department of Internal Medicine, Harry S Truman Veterans' Hospital, University of Missouri, Columbia, MO 65201, USA

ABSTRACT.Corticosterone (B) and 18-hydroxy-11-deoxycorticosterone (180HDOC) but not 11-deoxycorticosterone (DOC) displaced cortisol (F) specifically bound to rat adrenal mitochondria. F competitively inhibited aldosterone formation from B, 180HB and l8OHDOC but did not inhibit conversions of DOC to B or 180HDOC. High concentrations of DOC increased its conversion to 180HDOC rather than B.

INTRODUCTION

Rat adrenal cortex possess two cytochromes P450 with 1113- hydroxylase activity each of which has 18-hydroxylase activity (1- 5). One, CYP11B1, is involved in the 18-hydroxylation of 11-

deoxycorticosterone (DOC) to 18-hydroxy-11-deoxycorticosterone (180HDOC) and a second one, CYP11B2, 18-hydroxylates corticosterone (B) to 18-hydroxycorticosterone (180HB) and aldosterone (Aldo).

Cortisol (F) is a foreign alternative substrate for rat adrenal cortex. We used cortisol to inhibit Aldo formation from different substrates.

MATERIALS AND METHODS

Steroids: Tritiated DOC, B, 180HB and 180HDOC were from Amersham Corp. (Arlington Heights, 11), and radioinert steroids were from Sigma Chem. (St. Louis, Mo). Animals: Male CHBB-Thom rats (150-200 grs) were used.

47 1

Copyright 0 1995 by Marcel Dekker, Inc.

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472 MATKOVIC ET AL.

Incubations: Mitochondria fractions (0.6 mg protein) from rat adrenal capsules were incubated in a final volume of 5OOpl in the presence of radioactive steroids as substrate, with or without other radioinert steroids. Incubations were started by the addition of 0.5 mM NADPH and stopped by placing the tubes in an ice-water bath followed by centrifugation. Quantification of products: Incubation supernatants were extracted with 5 vol. C12CH,, and 6 pg of the radioinert steroids to be measured were added to organic extracts to correct for losses. Steroids were then separated by TLC (silica gel plates, Mobile phase: Cl,CH,: CH,OH: CH$N (86:7:7)) followed by HPLC (Columm: pBondapak C-18, Flow rate 0.8 ml/min, Mobile phase: CH,OH + H20 (3+2)). Radioactivity associated to each steroid was measured by liquid scintillation and corrected for losses .

Purity of the radioactive steroid-fraction collected after HPLC was checked by recrystallization in acetone/water (6). Binding experiments: Specific binding of tritiated F was measured as already described (7). Miscellaneous: Proteins were determined by the method of Bradford (8). Apparent Km and Vmax were calculated by Lineaweaver-Burk plots which were obtained with six different substrate concentrations in duplicates.

RESULTS AND DISCUSSION

Cortisol as competitive inhibitor Figure 1 shows the effects of lOpM F on several reactions

It can be seen that while the reactions DOC to B and DOC to 180HDOC were not inhibited by F (cortisol insensitive -FIS- reactions), the other transformations were decreased to a different degree (cortisol sensitive -FS- reactions).

(substrate to products) occurring in rat adrenal mitochondria.

Taking into account the enzymes already identified by molecular biology studies, CYPllBl and CYPllB2 enzymes should be included in the FIS and FS categories respectively.

Lineaweaver-Burk plot for the transformation of B into Aldo indicated that F behaves as a competitive inhibitor with a Ki equal to 2 .5 f 0.5 pM.

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CORTISOL

SUBSTRATE. 1uM DOC

RATIO

1

0.5

n " B l 8 O H D O C A h 0

PRODUCTS

473

SUBSTRATE. 1pM B PRODUCT, ALDOSTERONE

UTlO

ALbO

PRODUCTS

UTIO

l8OHDOC lf3OHB

IwM SUBSTRATE

FIGURE 1

Effects of 10pM cortisol on different substrate to product reactions. a ~ ~ 0 . 0 5 , b ~ 0 . 0 2 , c ~ 0 . 0 1 , with respect to control with no cortisol (ANOVA). Plain bars, no cortisol. Diagonal bars, 10 pM cortisol. Ratio, transformation rate of substates into the indicated products in the presence of cortisol/same transformation rate in the absence of cortisol. Results are mean f SEM of 2 to 4 experiments performed in duplicates.

Surprisingly, Figure 1 also shows that the percentage of inhibition by F for the production of 180HB from B (79%) is higher than the one for Aldo production from B (57%). This result appears curious since proximal substrate inhibition exceeds the inhibition of Aldo production .

However, this is easily explained with apparent Km and Vmax values: 3.2 f 0.6 and 1.3 f 0.2 pM; and 1.37 f 0.09 and 0.69 f 0.09 runoles of product/min x mg prot, for the reactions B to 180HB and B to Aldo respectively. In effect, calculation of those percentages of inhibition using Michaelis-Menten equation for competitive inhibition yield the same pattern of inhibition: 76% for B to 180HB and 63% for B to Aldo.

Binding of Cortisol

Tritiated F specifically binds to mitochondria from rat adrenal capsules with an EC50 of 0.6 p M (Figure 2). The difference between this value and Ki for competitive inhibition by F of the

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474 MATKOVIC ET AL.

SPECIFIC BINDING OF CORTISOL

0 1 2 3 4 5 6 LOG(UNLABELED STEROID).nM

FIGURE 2

Specific binding of cortisol to rat adrenal mitochondria. Titriated cortisol was displaced by varying concentration of unlabeled cortisol(O), or by a constant concentration ( 1 0 p M ) of unlabeled B(+), 180HDOC(*) and DOC( ) . Data are mean 2 SEM of three experiments performed in duplicates.

transformation B to Aldo (2.5 pM) might be attributed to different incubation temperature (4OC vs. 37OC) and consequently to the absence of enzymatic metabolism of steroids at the low temperature of binding experiments.

On the other hand, 10 pM B and 180HDOC, but not DOC, significantly displaced the specific binding of tritiated F (Figure 2). These results confirm that B and 180HDOC are metabolized through FS reactions in rat adrenal mitochondria and that F behaves as a competitive inhibitor of those reactions.

B/180HDOC ratio Since CYPllBl is responsible for both, 1113- and 18-

hydroxylations of DOC, we searched for conditions of the reactions under which these hydroxylations could be regulated.

to B and to 180HDOC were 17.9 f 2.1 and 36.3 f 4.9 pM, and 16.5 f

2.9 and 53.2 f 4.2 nmoles of product/min x mg prot, respectively. These results suggest that at low concentrations of DOC its

metabolism could be preferentially directed to B (lower K m ) , whilst

Apparent Km and Vmax values for the transformations of DOC

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CORTISOL 475

l8OHDOC should be the preferred product at higher substrate concentrations (higher Vmax).

Such a suggestion was then placed under experimental verification. B/180HDOC ratios were 0.64, 0.58 and 0.51 for 0.1, 1 and 10 pM DOC respectively. Thus, B/180HDOC ratio decreased as substrate concentration increased.

CONCLUSIONS

1) Categorization into FS and FIS enzymes is compatible with the existence of the cloned CYPllBl and CYPllB2 enzymes. 2) F competitively inhibits B to Aldo and 180HDOC to Aldo transformations. 3) The substrate concentration-dependent regulation of the conversion of DOC into B or 180HDOC is due to apparent Km and Vmax values for those reactions.

ACKNOWLEDGEMENTS

This work was in part supported by grants from Fundacien Antorchas, Universidad de Buenos Aires, CONICET and NIH.

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