Second

Department

University

of Pathology, of Helsinki, Helsinki

CORTICOSTERONE, 18-OH-DEOXYCORTICOSTERONE, DEOXYCORTICOSTERONE AND ALDOSTERONE SECRETION IN TISSUE CULTURE OF FOETAL RAT ADRENALS IN THE PRESENCE AND THE ABSENCE OF ACTH

By M.

Salmenper\l=a"\and

A. I. Kahri

ABSTRACT A method for simultaneous analysis of the main corticosteroids secreted by the rat adrenal is described. Purification is performed using previously established steps involving dichloromethane extraction, ethanol-cyclohexane partition and Sephadex-LH-20 column chromatography. Deoxycorticosterone and aldosterone are then quantitated with radioimmunoassay and corticosterone and 18-hydroxy-deoxycorticosterone with gas liquid chromatography as their 0-methyloxime-trimethylsilyl ethers. The coefficient of variation of the method for these steroids in concentrations found in the tissue culture of foetal rat adrenals varies between 3.9\p=n-\10.9% The secretion of these steroids in the tissue culture of foetal rat adrenals is studied. The steroid secretory pattern is correlated with a differentiation stage of the cultures as detected by electron microscopy. The initial secretory activity declines considerably after 15 days of cultivation to a low level. This is the case also with respect to aldosterone although the cortical cell population changed from the mixed population of the differentiated (zona fasciculata-like) and undifferentiated (zona glomerulosa-like) cells to homogenous undifferentiated growth. Addition of ACTH increased deoxycorticosterone secretion rapidly. Corticosterone, 18-hydroxy-deoxycorticosterone and aldosterone secretion was observed only after the differentiation of the cells (especially changes in their mitochondrial compartment) was evident morphologically.

Supported by

a

grant from The

Sigrid Jusélius

Foundation.

Dependence of adrenal cortical zonation on the ACTH has been described in vivo (Sabatini et al. 1962) and in tissue culture (Kahri 1966). The impor¬ tance of mitochondrial development during ACTH-induced differentiation of cortical cells was first suggested by Kahri (1968). Transformation of mito¬ chondrial inner membranes during ACTH stimulation to 600 Ä vesicles has been shown to be linked with elevated mitochondrial 11/3- and 18-hydroxy¬ lation (Kahri et al. 1970). An improvement in analytical methods was a pre-requisite for obtaining reliable data on the endogenous secretion of steroids during ACTH-induced differentiation, and for studying further the role of mitochondrial protein synthesis in cortical cells during their differentiation. One of the goals of the present study was to develop a method for simul¬ taneous analysis of corticosterone, 18-hydroxy-deoxycorticosterone, deoxycorticosterone and aldosterone in concentrations found in the tissue culture

of foetal by the rat adrenal cortex (Cortes et al. 1963; Baniukiewicz et al. 1968; Shapiro 8c Pérou 1973). The values obtained can be correlated with the stage of growth and differentiation of the cultures. rat adrenals.

These

are

the main steroids known to be secreted

MATERIALS AND METHODS

Chemicals and All solvents

glassware

of reagent grade and re-distilled before use. Dichloromethane, washed three times with distilled water, stored overnight over sodium hydroxide, distilled and stored in aliquots at 20°C. Pyridine was distilled and stored at room temperature in a brown bottle over potassium hydroxide flakes. Silylating reagents hexamethyldisilazane and trimethylchlorosilane were obtained from Fluka AG (Switzerland) and re-distilled. Methoxyamine hydrochloride was from East¬ man Organic chemicals (Rochester, N. Y., USA). Sephadex-LH-20 was purchased from Pharmacia (Uppsala, Sweden). Albumin (fraction 5) was obtained from Armour (East¬ bourne, England) and gamma-globulin (fraction 2) from Calbiochem AG (Löwen¬ graben, Switzerland). Norit A neutral charcoal and Dextran T-70 were from Amend (Irvington, England) and from Pharmacia, respectively. The non-disposable glassware was washed with hot detergent and chromic acid. Saturation analysis was performed in disposable ordinary laboratory glass tubes which were pre-rinsed with hot detergent.

reagent grade,

were was

Reference steroids [l,2-3H]deoxycorticosterone (42 Ci/mmol), [1,2-3H]cortico¬ (54 Ci/mmol), [4-HC]corticosterone (52 mCi/mmol), [1,2-3H] 18-OH-deoxycorticosterone (47 Ci/mmol) and [1,2-3H] aldosterone (48 Ci/mmol) were from the Radiochemical Centre (Amcrsham, United Kingdom). [l,2-3H]DOC was purified by thin-layer chromatography (Bergon et al. 1975) and other radioactive compounds on Sephadex-LH-20 columns (Shapiro Se Pérou 1972). About 107 dpm was purified at a Radioactive steroids.

sterone

-

time and used within 2 weeks.

Non-radioactive steroids. These were purchased mostly from Sigma Chemicals (St. Louis, USA). Other sources were: Steraloids (N.Y., USA): 18-OH-DOC, Ikapharm (Ramat-Gan, Israel): 18-OH-corticosterone, Koch-Light (Colnbrook, England): cholesteryl butyrate, and steroid reference collection: 11-dehydrocorticosterone, 11/J-OHandrostenedione, 20«-dihydroprogesterone. -

Antisera obtained from New England (Mass., USA). Antisera for the DOC assay in rabbit against deoxycorticosterone-21-succinyl-BSA, and that for aldo¬ sterone assay in sheep against aldosterone-18-21-dihemisuccinyl-BSA. These

is

were

prepared

Abbreviations and steroid nomenclature RIA: radioimmunoassay; GLC: gas-liquid chromatography; MO-TMS: O-methyloxime-trimethylsilyl ether; HMDS: hexamethyldisilazane; TMCS: trimethylchlorosilane; BSA: bovine serum albumin; LSC: liquid scintillation counting;

aldosterone: 11 /j,21 -dihydroxy-3,20-dioxo-4-pregnen-18-al ; androstenedione: 4-androstene-3,17-dione;

11/î-OH-androstenedione: ll/?-OH-4-androstene-3,17-dione; cholesteryl butyrate: 3/i-hydroxy-cholest-5-ene-3-n-butyrate; corticosterone: 1 l/?,21-dihydroxy-4-pregnene-3,20-dione; cortisol: ll/?,17,21-trihydroxy-4-pregnene-3,20-dione: cortisone: 17,21-dihydroxy-4-pregnene-3,ll,20-trione;

dehydroepiandrosterone: 3/?-hydroxy-5-androsten-17-one; 11-dehydrocorticosterone: 21-hydroxy-4-pregnene-3,20-trione; deoxycorticosterone (DOC): 21-hydroxy-4-pregnene-3,20-dione; 11-deoxycortisol: 17,21-dihydroxy-4-pregnene-3,20-dione; 18-OH-deoxycorticosterone (18-OH-DOC): 18,21-dihydroxy-4-pregnene-3,20-dione; 18-OH-corticosterone: ll/?,18,21-trihydroxy-4-pregnene-3,20-dione; pregnenolone: 3/S-hydroxy-5-pregnen-20-one; progesterone: 4-pregnene-3,20-dione;

20o-dihydro-progesterone: 20a-hydroxy-4-pregnen-3-one;

11/i-OH-progesterone: 1 l/?-hydroxy-4-pregnene-3,20-dione; 17/?-hydroxy-4-androsten-3-one.

testosterone:

Tissue culture The method of culturing foetal rat adrenals used by Kahri (1966) was used in this The medium was replaced every 5th day and consisted of: 50 % Melnick A, 25 % Eagle's Minimum Essential Medium and 25 % calf serum.

study.

Steroid extraction and About 1000 10 000 cpm of

purification

[l,2-3H]DOC, [1,2-3H]aldosterone, [4-UC]corticosterone and [1,2-3H] 18-OH-DOC in 50 pi of ethanol were added to the tissue

cpm

of

culture medium as recovery tracers. (The counts were obtained from the channel for 14C- and 3H-counting, respectively, see below). Extraction and purifica¬ tion were performed essentially as described by Shapiro Se Pérou (1973). The mediums were extracted with 10 vol. of dichlormethane. The extracts were washed with Vio vol. 0.1 n NaOH and twice with this volume distilled water. After drying in vacuo the samples were partitioned between 80% ethanol and cyclohexane 1:4 to remove the bulk of progesterone and to reduce the lipid load. After that the Sephadex-LH-20 column chromatography was performed. For the conditions and the collected fractions see Fig. 2.

optimized

Radioactivity

determinations

Bray's solution (5 g of Permaplend II (Packard, Zurich, Switzerland) and 120 g of naphthalene, Baker's (Denver, Holland) reagent grade, were dissolved in dioxane to make 1 1 of solution) was used for aqueous samples in RIA. Fifteen ml of this fluid

Fig.

1.

Gas Chromatographie tracings of the fraction II (Fig. 2) from the tissue culture me¬ dium. Peak 1 18-OH-DOC-MO-TMS (a-isomer, see text). Peak 2 corticosteroneMO-TMS (a-isomer) and about 5-7 % of the peak area is caused by b-isomer of 18-OH-DOC-MO-TMS. Peak 3 corticosterone-MO-TMS (b-isomer). Peak 4 internal standard, cholesteryl butyrate. The left chromatogram corresponds to the secretion found from a control culture (15th cultivation day, 5 days after medium change) internal standard 0.5 pg. Maximal useable sensitivity is used. In the right tracing from the same culture after 5 days of ACTH stimulation (100 mU/ml/day), internal standard 5/

Corticosterone, 18-OH-deoxycorticosterone, deoxycorticosterone and aldosterone secretion in tissue culture of foetal rat adrenals in the presence and the absence of ACTH.

Second Department University of Pathology, of Helsinki, Helsinki CORTICOSTERONE, 18-OH-DEOXYCORTICOSTERONE, DEOXYCORTICOSTERONE AND ALDOSTERONE SE...
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