studies on isolated rat adrenal cells metabolism of hydroxylated sterols

Molecular and Cellular Endocrinology 3 (1975) 375-383. 0 North-Holland Pub]. Comp.

STUDIES ON ISOLATED RAT ADRENAL CELLS

METABOLISM OF HYDROXYLATED STEROLS*

H. E. FALKE, H. J. DEGENHART, G. J. A. ABELN and H. K. A. VISSER

Department of Pediatrics, Erasmus University and Academic Hospital Rotterdam and Sophia

Children’s Hospital and Neonatal Unit, Rotterdam, Netherlands

Received 9 April 1975 Accepted 20 June 1975

Corticosterone production by isolated rat adrenal cells in the absence of ACTH is stimulated by 20a-hydroxycholesterol, 22R-hydroxycholesterol and 25hydroxycholesterol. This effect is also seen at sub-maximal ACTH concentrations. Aminoglutethimide (AGJ) inhibits the stimulation caused by the three hydroxylated cholesterols. In the presence of ACTH, AGI also inhibits steroid production both under control conditions and in the presence of the sterols. The stimulation by 25hydroxycholesterol is dose-dependent, both in the presence and absence of a sub-maximal ACTH concentration. At maximal ACTH concentrations 25-hydroxycholesterol does not give additional stimulation. Cycloheximide has no effect on corticosterone production from 25-hydroxycholesterol whether ACTH is present or not. Our results indicate that 25-hydroxycholesterol is a good substrate for the study of the cholesterol side-chain cleaving system and the mechanism of action of ACTH at the level of the intact cell.

Keywords: isolated rat adrenal cells; cholesterol side-chain cleavage; ACTH mechanism of action; 25-hydroxycholesterol; 20a-hydroxycholesterol; 22R-hydroxycholesterol.

The adrenal cholesterol side-chain cleaving system is able to convert a wide

variety of sterols, including 5-cholesten-3P,20a-diol (20a-hydroxycholesterol),

5-cholesten-3b,22R-diol (22R-hydroxycholesterol) and 5-cholesten-3P,25-diol

(25-hydroxycholesterol), into steroids (Burstein and Gut, 1971). Usually the

metabolism of these sterols has been studied in vitro using isolated mito-

chondria or crude preparations of the cholesterol side-chain cleaving system

(Burstein and Gut, 1971; Jefcoate et al., 1974). Little is known about the

metabolism of these sterols in the intact adrenal cell. Hall and Young (1968)

*This work was supported by a grant from the Foundation for Medical Research, Sophia Children’s Hospital and Neonatal Unit, Rotterdam.

316 H. E. Falke et al.

observed a conversion of [7r~-~H]20a-hydroxycholesterol into cortisol in

bovine adrenal slices. Sharma (1973) found a production of deoxycorticosterone

and corticosterone from [7a-3H]20a-hydroxycholesterol in isolated rat adrenal

cells. In each case steroid production from 20a-hydroxycholesterol occurred

both in the absence and presence of ACTH.

This paper described the corticosterone production by isolated rat adrenal

cells incubated with 20a-hydroxycholesterol, 22R-hydroxycholesterol or 25

hydroxycholesterol and the effects of ACTH, aminoglutethimide (AGI) and

cycloheximide. Emphasis is put on 25-hydroxycholesterol in view of the fact

that side-chain cleavage of this sterol involves all the steps required for chol-

esterol side-chain cleavage. In the existing models for cholesterol side-chain

cleavage (Burstein and Gut, 1971; Kraaipoel et al., 1975) metabolism of 20a- hydroxycholesterol or 22R-hydroxycholesterol involves only part of the side-

chain cleaving system.

MATERIALS AND METHODS

Materials Incubations were carried out in a Krebs-Ringer bicarbonate buffer (pH 7.4)

containing 0.2% (w/v) glucose and 3 ‘A (w/v) albumin (KRBAG) (Bovine

serum albumin, Cohn Fraction V, Sigma Chemical Co.) or in a modified

Krebs-Ringer buffer with sodium bicarbonate replaced by 21.0 mM HEPES

(KRAHG) (Calbiochem). The use of HEPES buffers (Good et al., 1966) makes

it possible to do experiments in air and produces better pH control as compared

with bicarbonate buffers. Buffers were sterilized by filtration through Millipore

filters (HAWP 02500; pore size 0.45 uM).

Corticotrophin (ACTH) was a purified preparation of porcine ACTH

obtained from Organon Oss (CortrophineB). Aminoglutethimide phosphate

(AGI) was a generous gift from Ciba Co., Arnhem. Cycloheximide was obtained

from Boehringer. Cholesterol and 20a-hydroxycholesterol were obtained from

Sigma Chemical Co., 22R-hydroxycholesterol from Ikapharm and 25-hydroxy-

cholesterol from Steraloids. Solutions of these sterols were made up in ethanol.

All glass surfaces in contact with the cells or solutions containing ACTH

were siliconized (Siliclad, Clay Adams) and sterilized.

Incubations Incubations were carried out in round-bottomed glass centrifuge tubes

under 02-COZ (95-5 ‘A) for KRBAG or in air for KRHAG. The total incubation

volume was 1 ml. ACTH, AGI and cycloheximide were added in 0.1 ml buffer,

Adrenal metabolism of hydroxylated sterols 371

the sterols in 10 ~1 ethanol. After 2-h incubation at 37 “C. in a Dubnoff in-

cubator (100 rpm) the tubes were tightly capped, frozen rapidly and stored at

-20 “C. The number of cells per incubation was about 2 x 105.

Preparation of cell suspensions Suspensions of isolated rat adrenal cells were prepared as described pre-

viously (Falke et al., 1975). Adrenals of male Sprague-Dawley rats (200-250 g)

were used. No difference was observed between cells prepared in KRBAG or

KRHAG.

Analytic procedures Corticosterone was measured by fluorimetry as described previously (Falke

et al., 1975). The values obtained had to be corrected for the small amount of

fluorescence caused by 20a-hydroxycholesterol, 22R-hydroxycholesterol and

25hydroxycholesterol. The fluorescence of 1 ug of each of these sterols is

equivalent to 5.5 ng corticosterone at the maximal fluorescence wavelength of

the latter. The extent of the correction was based on the assumption that only a

small fraction of the sterols present was converted into steroids. That this was

a valid assumption was supported by measurement of the sterols by gas

chromatography. The method described by Kraaipoel et al. (1975) was used

with epicholesterol as an internal standard. Overall recovery of the sterols after

a 2-h incubation with isolated rat adrenal cells was 85-90x.

RESULTS

The production of corticosterone by isolated rat adrenal cells under several

conditions is shown in fig. 1. In the absence of ACTH, addition of 5ug/ml of

20a-hydroxycholesterol, 22R-hydroxycholesterol or 25-hydroxycholesterol caus-

ed a marked increase in corticosterone production (fig. 1, left side). 20a-

Hydroxycholesterol proved to be the best substrate, followed by 22R-hydroxy-

cholesterol and 25-hydroxycholesterol, respectively. Addition of cholesterol

failed to increase steroid production. AGI (70pM) inhibited corticosterone

production both in the control incubation and in the presence of each of the

sterols. Fig. 1 (right side) also shows the effect of the sterols on corticosterone

production in the presence of 1 mU ACTH/ml. Significant stimulation under

this condition was found with 5 ug/ml of 20a-hydroxycholesterol and 22R-

hydroxycholesterol. Very small effects were seen with cholesterol and 25-hydro-

xycholesterol. The effect of cholesterol surely is not significant and was not

found in other experiments, like the one shown in fig. 2. The effect of 25-


CORTICOSTERONE

(1g/2x105cells/2hrs. 1.75,

no ACTH

0 noAGl

70pM AGI

‘H (lmU/n

Fig. 1. Corticosterone production by isolated rat adrenal cells in the presence of cholesterol (B), 2ShydroxycholesteroI (C), 20a-hydroxycholesterol (D) and 22R-hydroxycholesterol (E). Sterols were present at a concentration of 5 pg/ml. A is the control. Vertical black bars

indicate the difference between duplicate incubations.

hydroxycholesterol also was not significant. Nevertheless a small effect of

5 ug 2Shydroxycholesterol/ml was found in all our experiments including

the experiment in fig. 2. Corticosterone production in the presence of ACTH

and the sterols was inhibited by AGI (fig. 1, right side). This effect was most

marked with 2Shydroxycholesterol. It should be mentioned that maximal

stimulation of corticosterone production in this series of experiments was

found with lo-100 mU ACTH/ml (see also fig. 3). The ACTH concentration

in the experiment shown in fig. 1 therefore was sub-maximal.

The increase in steroid production caused by 25-hydroxycholesterol depends

upon the concentration of the sterol (fig. 2). Higher concentrations of 25-

hydroxycholesterol resulted in an increase of the corticosterone production

both in the absence and presence of ACTH (1 mu/ml). Cholesterol, even at

25 ug/ml, had no effect on steroid production whether ACTH was present or


1,25

l&l I-

0,75 ,-

0,x l-

0,25 b-

.I _

0 -r 0

I_____ .-____:__--- # -----es_____

I I I 1 5 STEROL d:NCENTR::ION

20 25 pglml

Fig. 2. Corticosterone production by isolated rat adrenal cells in the presence of 25hydroxy- cholesterol (O---O), 2Shydroxycholesterol + 1 mU ACTH/ml (C--O), cholesterol (m- - -E) and cholesterol + 1 mU ACTH/ml ( q - - -3). Duplicate incubations are plotted.

not. Fig. 3 shows the effect of 25-hydroxycholesterol (25 pg/ml) in the presence

of increasing ACTH concentrations. The stimulation of corticosterone pro-

duction is clearly seen without ACTH and also at sub-maximal ACTH con-

centrations, but disappears when the stimulation of steroid production by

ACTH is maximal. The effect of cycloheximide (25 PM) on corticosterone pro-

duction in the presence of ACTH (1 FM/ml) and/or 25-hydroxycholesterol (25

pg/ml) is shown in fig. 4. Cycloheximide inhibited approximately 95% of the

effect of ACTH. The stimulation of corticosterone production caused by 25 ug

25-hydroxycholesterol/ml was not significantly altered by cycloheximide or

ACTH plus cycloheximide.


CORTICOSTERONE

~g/2x105cells12hrs. 1.751

Fig. 3. Corticosterone production by isolated rat adrenal cells in the presence of increasing ACTH concentrations without 25hydroxycholesterol (O--O) and with 25 ug 25-hydroxy-

cholesterol/ml (0- - -0). Duplicate incubations are plotted.

CORTICOSTERONE ~9/2x105cells/2hrs

ISO-

1.25-

lsm-

0.75-

0.50-

0.25-

0 CONTROL CH 25-OH 25PH ACTH ACTH ACTH ACTH

CH EH 25+OH 25+OH

CH

Fig. 4. Effect of 25 uM cycloheximide (CH), 1 mU ACTH/ml and 25 ug 25-hydroxycholesterol/ml (25OH) on corticosterone production by isolated rat adrenal cells. Vertical black

bars indicate the difference between duplicate incubations.


DISCUSSION

Corticosterone production by isolated rat adrenal cells is stimulated by

20a-hydroxycholesterol, 22R-hydroxycholesterol and 25-hydroxycholesterol.

In the presence of a sub-maximal ACTH concentration a similar stimulation

was found. This indicates that these sterols are good substrates for the cholester-

ol side-chain cleaving system in the intact adrenal cell. Hall and Young (1968)

and Sharma (1973), working with bovine adrenal slices and isolated rat adrenal

cells, respectively, previously recovered 3H-labeled C, i-steroids starting

with [7a- 3H]20a-hydroxycholesterol as a substrate. The synthesis of corti-

costerone from these sterols by isolated rat adrenal cells in the absence of

ACTH is compatible with the current hypothesis of the mechanism of action

of ACTH. According to this hypothesis ACTH exerts its effect prior to the

cholesterol side-chain cleaving system (Garren et al., 1971; Schulster, 1974).

The reason for the absence of a stimulating effect of cholesterol, a phenomenon

also reported by Sharma (1973), is not clear. Maybe, under our conditions,

exogeneous cholesterol cannot reach the side-chain cleaving system fast

enough to stimulate steroid synthesis. In addition Sharma (1973) found that

cholesterol inhibited the effect of ACTH on corticosterone production. The

absence of such an effect in our experiments may be due to the difference

in the cholesterol concentration used. The highest cholesterol concentration

in our experiments was 25 ug/ml (= 0.065 mM) while Sharma used 192 ug/ml

(= 0.5 mM). Formation of mono-molecular layers of cholesterol on the cell

surface might have been responsible for the inhibition caused by the high

cholesterol concentration used by Sharma.

In the classical scheme of cholesterol side-chain cleavage 20a-hydroxy-

cholesterol and 22R-hydroxycholesterol are considered as intermediates. In the

recent model proposed by Kraaipoel et al. (1975) however, these two sterols

are merely by-products. The calculations of Burstein and Gut (1971) also

indicate that the largest part of the steroid production from cholesterol does

not involve either sterol as an intermediate. Both in the model of Kraaipoel

et al. (1975) and in the model of Burstein and Gut (1971) the metabolism of

these two sterols involves only part of the cholesterol side-chain cleaving

system. Therefore they are of limited use for the study of the cholesterol side-

chain cleaving system. This is the reason why most of our work was carried out

only with 25-hydroxycholesterol. In our opinion 25-hydroxycholesterol is a

good substrate for the study of the cholesterol side-chain cleaving system.

The occurence of 25-hydroxycholesterol in mammals has been repeatedly

described. It is present among other sterols in human cord blood (Eberlein,

1965) human aorta (Van Lier and Smith, 1967) and brain (Van Lier and Smith,


1969). Formation of 25-hydroxycholesterol from cholesterol by a 25-hydroxy-

lase was found in rat liver (Bjiirkhem and Gustafsson, 1974). A 25-hydroxylase

for cholecalciferol is also present in mammalian liver (Norman and Henry,

1974). Conversion of 25-hydroxycholesterol to pregnenolone by the cholesterol

side-chain cleaving system was previously found by Burstein and Gut (1971)

in acetone powders of bovine adrenal mitochondria and by Degenhart et al.

(1974) and Jefcoate et al. (1974) in bovine and rat adrenal mitochondria.

Evidence exists that side-chain cleaving of cholesterol and 25-hydroxycholesterol

takes place by the same enzyme system. Metabolism of 25-hydroxycholesterol

is inhibited by AGI an inhibitor of the first step of cholesterol side-chain cleavage

(cholesterol+ 5,2@(22)-cholestadien-3P-01) (Degenhart et al., 1974). This also

indicates that all the steps of cholesterol side-chain cleaving are involved in the

metabolism of 25hydroxycholesterol. The competition between cholesterol

and 25-hydroxycholesterol (Jefccate et al., 1974) is further evidence for con-

version of both sterols by the same enzyme system. The inhibition of the meta-

bolism of 25-hydroxycholesterol by AGI (fig. 1) indicates that also in the

intact cell this sterol is converted by the cholesterol side-chain cleaving system.

The absence of additive stimulation by 25-hydroxycholesterol at maximal

ACTH concentrations points in the same direction. It seems that in isolated

rat adrenal cells at maximal ACTH concentrations the substrate flux to the

side-chain cleaving system matches the full capacity of this system; therefore

no extra effect of 25-hydroxy-cholesterol can be seen. The results on which

Jefcoate et al. (1974) base their assumption that the cytochrome P-450 for

side-chain cleavage of cholesterol is present in three different forms may just

reflect different binding properties of cholesterol and 25-hydroxycholesterol.

ACTH pretreatment in vivo has no effect on the total cholesterol side-chain

cleaving activity (Koritz and Kumar, 1970) nor on the K,, or I’,,,,, of the

enzyme system (Bell and Harding, 1974) in rat adrenal mitochondria. ACTH

therefore appears to have no direct effect on the cholesterol side-chain cleaving

system. Hcwever, Sharma (1973) found that ACTH, but not N6,02-dibutyryl-

adenosine 3’,5’-monophosphate stimulated the conversion of [7a-3H]20a-

hydroxycholesterol in isolated rat adrenal cells. This is in contrast with the

results of Hall and YotTng (1968) who found that ACTH s!ightly inhibited the

conversion of [7a-3H]20a-hydroxycholesterol in bovine adrenal slices. Neither

Hall and Young (1968) nor Sharma (1973) measured specific activities of the

compounds involved which makes exact interpretation of their results difficult.

Our results are consistent with the absence of a direct effect of ACTH on the

side-chain cleaving system. The metabolism of 25-hydroxycholesterol in the

absence of ACTH indicates that the side-chain cleaving system is invariably

present in an active form. At sub-maximal ACTH concentrations only part of


the activity is needed for side-chain cleavage of endogeneous cholesterol and

an additive effect of 25-hydroxycholesterol can be observed. Only at maximal

ACTH concentrations the full capacity of the side-chain cleaving system is

used for endogeneous cholesterol and 25-hydroxycholesterol cannot give

extra steroid production. Evidence that ACTH has no direct effect on the side-

chain cleaving system comes from our results with cycloheximide. Both in the

absence and in the presence of ACTH cycloheximide had no effect on the

amount of the stimulation caused by 25-hydroxycholesterol (fig. 4). The

failure of cycloheximide to influence the effect of 25-hydroxycholesterol is

further proof for the localization of the effect of ACTH on events prior to the

side-chain cleaving system.

ACKNOWLEDGEMENTS

Mrs. Mary Amesz-Heinrich, Mr. A. A. M. Kempers and Dr. D. A. Price are

gratefully acknowledged for their help with the preparation of the manuscript.

REFERENCES

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Visser, H. K. A. (1975) FEBS Lett. 50, 204. Koritz, S. B. and Kumar, A. M. (1970) J. Biol. Chem. 245, 152. Norman, A. W. and Henry, H. (1974) Recent Prog. Horm. Res. 30,431. Sharma, R. K. (1973) J. Biol. Chern. 248, 5473. Schulster, D. (1974) In: Advances in Steroid Biochemistry and Pharmacology, Vol. 4, Eds.:

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studies on isolated rat adrenal cells metabolism of hydroxylated sterols

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