STEROIDOGENIC PATHWAYS AND TROPHIC RESPONSE TO ADRENOCORTICOTROPHIN OF CULTURED ADRENOCORTICAL CELLS IN DIFFERENT STATES OF DIFFERENTIATION W. JULL AND N. AUERSPERG Research *Cancer Centre and Department of Zoology, British Columbia, Vancouver, Canada and ^Cancer Research Centre, University of University of British Columbia, Vancouver, Canada, V6T1W5 E. A.

SLAVINSKI, J.

(Received 20 April 1975) SUMMARY

Adrenocortical cells obtained from adult rats were propagated in monolayer culture. Depending on culture conditions, they grew either as lipid-containing epithelial-like cells with a high level of steroid production, or as fibroblast-like cells with a low level of steroid production. The major fluorogenic steroid secreted by both morphologic forms of adrenal cortical cell was corticosterone as determined by chromatography and acid fluorometry. Basal fluorogenic steroid production per 106 cells over 24 h was: epithelial-like cells, 5\m=.\0\g=m\g; fibroblast-like cells, 0\m=.\014\g=m\g. Stimulation with ACTH for 5 days increased fluorogenic steroid production and induced morphologic changes in both adrenal cell forms. ACTH stimulation of fluorogenic steroid production by both cell forms reached a maximum after 3 days, then dropped to a refractory state after 5 days. With maximal ACTH stimulation, production increased 25-fold in fibroblast-like cells and five-fold in epithelial-like cells. The latter rate of corticosterone production is similar, per cell, to ACTH-stimulated adrenal glands in vivo. Progressive morphologic changes were observed with ACTH stimulation : epithelial-like cells retracted from the substratum and lost lipid inclusions; fibroblast-like cells became more epithelial-like. Both adrenal cell types formed intermediates from [4-14C]pregnenolone including pregn-5-ene-3\g=b\,20\g=a\-dioland 20\g=a\-hydroxypregn-4-en-3-one. Control cultures of muscle fascia fibroblasts did not produce corticosterone or intermediates from [4-14C]pregnenolone and did not respond to ACTH functionally or

morphologically.

INTRODUCTION

Studies of steroid pathways and, particularly, of the trophic response of adrenocortical cells to ACTH have been limited by the in-vitro systems currently available. A tissue culture system offers considerable advantages over other in-vitro techniques, including longer cell survival which is required for studies of trophic stimulation, but until recently (Slavinski, Auersperg & Jull, 1974), the only available culture systems were a tumorigenic cell line (Buonassisi, Sato & Cohen, 1962) and normal adrenal cell populations maintained in primary culture (Kahri, 1966; Armato & Nussdorfer, 1972; O'Hare & Neville, 1973a). Although the tumour cell line provides large homogeneous populations of functional cells, it exhibits abnormal steroid biosynthetic pathways (Kowal & Fiedler, 1968). The steroid biosynthetic pathway of normal adrenal cells maintained in primary culture is Î Research Associate, National Cancer Institute of Canada.

more typical of adrenal cortical parenchyma in vivo (O'Hare & Neville, 1973a, b), but the presence of mixed cell populations makes biochemical analysis difficult. Furthermore, no corticosterone is detected once the cultures reach the steady baseline output after 10-14 days without ACTH (O'Hare & Neville, 1973 è), while adrenal cortical parenchyma continues to secrete corticosterone, although in reduced amounts, in the hypophysectomized animal (Shima, Matsuba & Pincus, 1968). As described in a recent report (Slavinski et al. 1974), we have been able to propagate in secondary culture, homogeneous populations of normal functional adrenocortical cells in what appear to be two distinct states of differentiation. Under one set of culture conditions, the cells expressed limited adrenocortical functions as suggested by a fibroblastic form, the presence of small amounts of lipid, a A5-3/?-hydroxysteroid dehydrogenase and a glucose6-phosphate dehydrogenase and, in the presence of ACTH, the production of small amounts of corticosteroids from [4-14C]pregnenolone. By altering culture conditions, adrenocortical cells were obtained which assumed an epithelial morphology. Their intense reaction for lipid and dehydrogenases, higher rate of [4-14C]pregnenolone metabolism and production, in the presence of ACTH, of the tissue-specific corticosterone from [4-14Qpregnenolone indicated a degree of differentiation higher than that attained by the adrenal cells in the fibroblast-like form. This report compares the endogenous steroid production of both morphologic forms of cultured adrenal cortical cells and characterizes in detail their morphologic and steroidogenic responses to trophic stimulation with ACTH.

MATERIALS AND METHODS

Cell culture Normal adrenocortical cells from 1-3 month old male Fischer rats were grown in primary culture as described previously (Slavinski et al. 1974). To obtain homogeneous secondary cultures of adrenal fibroblast-like cells, the primary monolayer outgrowths were dissociated with trypsin and subsequently grown in medium MB 752/1 with 10-25 % foetal calf serum. Homogeneous populations of adrenal epitheliallike cells were obtained by mechanically dissociating confluent primary outgrowths with a rubber policeman and scalpel and plating the resulting monolayer fragments into medium MB 752/1 supplemented with 3-10 % horse serum, depending on the serum batch. In both culture methods, cells were diluted 1:2 at the time of subculture. The stationary phase was reached by fibroblast-like cultures at a cell density of approximately 1 x 10e cells/culture dish and by epithelial-like cultures at a cell density of about 2 IO4 cells/culture dish. Monolayers were maintained for 20 to 24 days in culture before steroid production was determined. Cultures of connective tissue fibroblasts from muscle fascia were grown as controls in an identical manner. Steroids Non-radioactive steroids, pregnenolone, pregn-5-ene-3/?,20a-dioI, 20a-hydroxypregn-4-en3-one, deoxycorticosterone and corticosterone were obtained from Steraloids, U.S.A.

[4-14C]Pregnenolone (52-8 mCi/mmol), [4-14C]progesterone (57-3 mCi/mmol), [4-14C]corticosterone (57-3 mCi/mmol), [4-14C]deoxycorticosterone (54-3 mCi/mmol) and [1,2-3H(N)]20a-hydroxypregn-4-en-3-one (40 mCi/mmol) were purchased from New England Nuclear Corporation, Boston, Mass. Before use, they were chromatographed in the system PPC 1 (see below).

Liquid scintillation counting determined in toluene containing 2,5-diphenyloxazole (12g/l) and Radioactivity l,4-bis-[2-5(phenyloxazolyl)]-benzene (300 mg/1) using a Packard model 526 liquid scintil¬ lation spectrophotometer. Counting efficiency was 86 % for 14C and 35 % for 3H. was

Chromatography System PPC 1: Whatman No. 1 paper impregnated with propylene glycol and developed with light petroleum (b.p. 63-75 °C) for 24 h. System PPC 2: Whatman No. 1 paper impregnated with propylene glycol and developed with benzene : hexane (1:1, v/v) for 24 h.

Thin-layer chromatography systems (Silica Gel G plates) : (TLC 1) Benzene : ethyl acetate (3:1, v/v); (TLC 2) Chloroform : ethyl acetate (1:50, v/v); (TLC 3) Benzene : ethyl acetate (3:1, v/v). Inert carrier steroids were located either under u.v. light or by phosphomolybdate. Radioactive metabolites and standards were located using autoradiography as described by Jellinck & Goudy (1967). Isolation and identification of endogenous steroids Cultures were incubated with 2 ml of fresh medium for 24 h. The supernatant medium of each culture dish was then extracted with 10 ml dichloromethane. After removal of the aqueous phase and filtration through Whatman No. 1 filter paper, the extract was shaken with 2 ml of 65 % (v/v) ethanolic sulphuric acid. After 15 min the fluorescence of the sulphuric acid layer was determined with an Aminco-Bowman spectrophotofluorimeter using an excitation wavelength of 470 nm and an emission wavelength of 525 nm. Corti¬ costerone was used as the reference standard. To identify the major steroids produced endogenously by the two types of cells, the extracts of 8-10 incubates of each cell type were pooled. To each pool were added [4-14C]progesterone, [4-14C]pregnenolone, [4-14C]deoxycorticosterone and [l,2-3H(N)]20a-hydroxypregn-4-en-3-one (0-06/ig of each). Each mixture was chromatographed in the system PPC 1 and then in the system TLC 1. Radioactive areas, located by autoradiography, were eluted with ethanol. The final eluates were dried under N2, redissolved in dichloro¬ methane, extracted with 65 % ethanolic sulphuric acid and the fluorescence determined as above. Values were adjusted for loss of steroid during the chromatography and elution procedure as assessed by the loss of the radioactive standard.

Incubation, isolation and identification of the radiometabolites Stationary secondary cultures were incubated for 8 h with approximately 200000 d.p.m. of radioactive steroid per culture dish. The procedure for isolation of the radiometabolites has been described previously (Slavinski et al. 1974). Extracted radioactive metabolites were admixed with 100/¿g of unlabelled carrier and then sequentially chromatographed in the systems PPC 1, PPC 2, TLC 1, TLC 2 and, after acetylation, in TLC 3. The radiometabolites were finally identified by co-crystallization from methanol/water with 10-20 mg carrier steroid to constant specific activity. Since most of the metabolites were difficult to crystallize from solvent pairs other than methanol/water, this was used consistently. Axelrod, Matthijssen, Goldzieher & Pulliam (1965) have shown that the use of one solvent pair to obtain radiochemical purity is justifiable. Incubation with ACTH The effect of adrenocorticotrophin (ACTH) on the endogenous production of fluorogenic steroid and on [4-14C]pregnenolone metabolism by adrenal cells was examined by the

daily replacement of old medium with 2 ml of fresh medium containing 200 mu. ACTH (porcine, 88 i.u./mg, Sigma Chemicals, St. Louis, Mo.) for 1, 3 and 5 days before steroid determination. The stimulatory effect of ACTH on [4-14C]pregnenolone metabolism was statistically tested using a two-tailed r-test. Cultures were photographed during ACTH treatment using phase optics on a Wild inverted microscope. Cultures of connective tissue fibroblasts taken from muscle fascia identical manner and served as controls.

were

treated in

an

RESULTS

Endogenous production offluorogenic steroid The basal fluorogenic steroid production of 1 IO6 adrenal epithelial-like cells, as deter¬ mined by acid-induced fluorescence, was approximately 400 times more than that of the fibroblast-like cells. The addition of ACTH to both fibroblast-like and epithelial-like monolayers resulted in a characteristic rise in steroid production (Table 1). Endogenous production of steroid in both morphologic forms increased after 1 day of pre-incubation with the trophic hormone, reached a maximum after 3 days and dropped after 5 days. Steroid production increased approximately 25 times in maximally stimulated fibroblastlike cells and five times in maximally stimulated epithelial-like cells, a level that was approximately 70 times that of the maximally stimulated fibroblast-like cells. Table 1.

Effect of ACTH* on the secretion of endogenous fluorogenic steroide by adrenal epithelial-like cells, adrenal fibroblast-like cells and muscle fascia fibroblasts, expressed

per 1

10*

cells/24 h

(Means + s.e.m. of 8-10 cultures) Adrenal

Days of pre-

incubation with ACTH 0 1 3 5

epithelial-like cells

,-*-,

Fluorogenic steroid

(/tg)

Corticosteronet (%)

4-79 + 0-57 11-84 + 0-39

65 00

24-70+3-52

9312 —

500+1-30 —

Adrenal fibroblast-like cells

Muscle fascia fibroblasts

,-*-,

,-*-,

Fluorogenic steroid

(µ%) 0-014 + 0001 0-11 + 0-029 0-34 + 0037 010 + 0031

Corticosteronei (%) 71-43 79-40 —



Fluorogenic steroid

(µ%) 005 + 0-006 0-05 + 0-003 005 + 0-004 0-04 + 0003

Cortico-

steronet (%) 0

0





*

Every 24 h, culture medium in each dish was replaced with 2 ml of fresh medium containing 200 mu. of porcine ACTH. t Values have been adjusted for medium blanks. Î Chromatographie characterization of fluorogenic steroid secreted by cultures was determined for day 0

and 3. The dichloromethane extracts of the supernatant media from 8-10 cultures of each of the three cell types, together with 006/¿g [4-14C]corticosterone, as internal standard, were first chromatographed in ligroin for 24 h on Whatman No. 1 paper impregnated with propylene glycol, and then rechromatographed on Silica Gel G plates in benzene : ethyl acetate (3:1, v/v). Values were adjusted for loss of steroid during the isolation procedure, as assessed by loss of the radioactive standard, and expressed as a percentage for 1 x 106 cells.

Fluorometric analysis of the chromatographically isolated steroids secreted by adrenal cells in either the epithelial or fibroblastic form, indicated that corticosterone was the major end-product released into the surrounding medium. No fluorescence was detected in the Chromatographie areas of [4-14C]progesterone, [4-14C]pregnenolone, [4-14C]deoxycorticosterone, or [l,2-3H(N)]20a-hydroxypregn-4-en-3-one, although the last has been shown to fluoresce weakly (Kowal & Fiedler, 1968). Although cultures of muscle fascia fibroblasts did produce fluorescent material (Table 1), the monolayers did not respond to ACTH or produce corticosterone.

Metabolism of [4-uC]pregnenolone The conversion of [4-14C]pregnenolone to metabolites is given in Table 2. Adrenal epitheliallike cells not stimulated by ACTH metabolized approximately 65 % of the pregnenolone in 8 h, producing mainly pregn-5-ene-3/?,20a-diol and smaller amounts of 20a-hydroxypregn-4-en-3-one and deoxycorticosterone. The addition of trophic hormone did not significantly alter the amount of [4-14C]pregnenolone metabolized by adrenal epithelial-like cultures. However, after incubation with ACTH for 1 day, [4-14C]pregnenolone was metabolized to corticosterone. This response reached a maximum after 3 days of preincubation with the trophic hormone (P < 0-001) and appeared to drop after 5 days

(P

Steroidogenic pathways and trophic response to adrenocorticotrophin of cultured adrenocortical cells in different states of differentiation.

STEROIDOGENIC PATHWAYS AND TROPHIC RESPONSE TO ADRENOCORTICOTROPHIN OF CULTURED ADRENOCORTICAL CELLS IN DIFFERENT STATES OF DIFFERENTIATION W. JULL AN...
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