0021-972X/81/5206-1124$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1981 by The Endocrine Society

Vol. 52, No. 6 Printed in U.S.A.

The Role of Cyclic Adenosine 3',5'-Monophosphate in Cholesterol Metabolism and Steroidogenesis by the Human Fetal Adrenal Gland* BRUCE R. CARR, MASAO OHASHI, C. R. PARKER, JR., AND EVAN R. SIMPSON Cecil H. and Ida Green Center for Reproductive Biology Sciences, and the Departments of ObstetricsGynecology and Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas 75235

dbcAMP, or CT, there was a 2 to 3-fold increase of specific binding of [125I]iodo-LDL. In HFA tissue maintained in the presence of ACTH or CT, the rate of degradation of LDL was significantly increased compared to tissue maintained in the lipoprotein-poor serum alone. Finally, in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT there was a 6- to 10fold stimulation of the rate of incorporation of [14C]acetate into cholesterol. We conclude that steroidogenesis, LDL binding, and degradation, as well as de novo synthesis of cholesterol, are probably stimulated in HFA tissue via a cAMP-mediated pathway. ( J Clin Endocrinol Metab 52: 1124, 1981)

ABSTRACT. In the present investigation we studied the role of cAMP as a mediator of ACTH action in human fetal adrenal (HFA) tissue. We have characterized the response to ACTH, dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP), and cholera toxin (CT) with respect to steroidogenesis, low density lipoprotein (LDL) binding, degradation of LDL, and the rate of de novo synthesis of cholesterol. The rate of dehydroisoandrosterone sulfate secretion was similar in HFA tissue maintained in the presence of ACTH, dbcAMP, or CT. In contrast, cortisol secretion by HFA tissue was more sensitive to dbcAMP and CT than to ACTH. In membrane preparations obtained from HFA tissue maintained in the presence of ACTH,

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stimulates adenylate cyclase activity (6), with respect to steroidogenesis, LDL binding, degradation of LDL, and de novo synthesis of cholesterol.

N PREVIOUS investigations we found that plasma lipoproteins, and specifically low density lipoprotein (LDL), serve as a major source of the cholesterol that is required to maintain the high rate of steroid secretion by the human fetal adrenal (HFA) gland (1,2). Furthermore, we have demonstrated that ACTH causes an increase in the number of high affinity binding sites for LDL on HFA membrane preparations (Ohashi, M., B. R. Carr, and E. R. Simpson, unpublished observations). ACTH stimulates the metabolism of LDL (3) as well as the de novo synthesis of cholesterol (4) to supply optimal precursor for steroidogenesis in the HFA gland. The role of cAMP as a mediator or second messenger of ACTH action to stimulate steroidogenesis in adrenal cells of nonhuman species has been well characterized, and has been the topic of recent reviews (5, 6). In the present investigation, we sought to define the role of cAMP as a mediator of ACTH action in HFA tissue. Specifically, we sought to characterize the response of HFA tissue to dibutyryl adenosine 3',5'-cyclic monophosphoric acid (dbcAMP) and cholera toxin (CT), which

Materials and Methods Adrenal glands HFA glands were obtained from first and second trimester abortuses. Tissues were obtained in accordance with the Donors Anatomical Gift Act of the State of Texas after obtaining consent in writing from the woman to be aborted, using a consent form and protocol approved by the Human Research Review Committee of the University of Texas Health Science Center at Dallas, Texas. Abortion was accomplished by dilatation and extraction or else occurred spontaneously. The adrenal tissue was placed immediately in Waymouth-Gey's culture medium containing 1% antibiotics, and within one hour was minced into pieces of approximately 1 mm3. Tissue culture

Received September 29, 1980. Address correspondence and requests for reprints to: Bruce R. Carr, M.D., Cecil H. and Ida Green Center for Reproductive Biology Sciences 5323 Harry Hines Boulevard Dallas, Texas 75235. * This work was supported in part by USPHS Grant Nos. 2-R01HD-13234 and 5-P50-HD-11149.

Four fragments of fetal adrenal tissue were placed on lens paper that was supported by a stainless steel grid in each organ culture dish (60 X 15 mm; Falcon Plastics, Cockeysville, MD). To each dish was added 1 ml medium containing 60 vol Waymouth's MB 752/1 medium, 40 vol Gey's balanced salt solution (Grand Island Biological Company, Grand Island, NY) and 10 vol lipoprotein-poor serum (LPPS) or 10 vol pooled human

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ROLE OF cAMP IN HFA TISSUE serum (HS). Synthetic ACTH-(1-24) (Cortrosyn), obtained from Organon (West Orange, NJ), was added to the culture medium of some dishes to achieve a concentration of 1 /xg ml"1. dbcAMP and CT obtained from Sigma Chemical Co. (St. Louis, MO) were added to the culture medium of other dishes to achieve concentrations of 1 mg/ml and 10 /xg/ml, respectively. The tissue fragments were maintained at 37 C in a humidified atmosphere of 5% CO2-95% air. The medium was changed every 24 h. Steroid measurements In experiments designed to determine the rates of steroid secretion, tissue was incubated in medium containing 10% HS alone or 10% HS plus ACTH, dbcAMP, or CT. The medium was removed and replaced with fresh medium every 24 h. The media were frozen at —20 C until assayed. Cortisol and dehydroisoandrosterone sulfate (DS) were quantified by RIA (7, 8). At the end of the culture period, the tissue fragments were rinsed two times and homogenized in 1.0 ml NaCl (0.15 M). Aliquots of the homogenates were assayed for protein by the method of Lowry et al. (9). Each experimental condition was replicated in quintuplicate, and the results are expressed as the mean ± SE of the daily secretion rate of DS or cortisol (micrograms steroid mg"1 protein 24 h"1). Lipoproteins LDL (density, 1.019-1.063 g ml"1) and LPPS were prepared as described previously (2). [125I]Iodo-LDL was prepared according to the procedure of Bilheimer et al. (10) as adapted by Goldstein and Brown (11). The final specific activity of [125I]iodo-LDL ranged from 300-500 cpm ng"1 protein. Preparations of membrane fractions from HFA tissues On day 3 of culture, the tissue fragments from 5-10 dishes were pooled, suspended in 3 ml Tris-chloride (10 I M ) buffer, pH 7.4, containing NaCl (150 mM) and CaCl2 (1 mil), and centrifuged at 800 X g for 5 min at 4 C. The washed tissue pellet was suspended in 3 ml of the same buffer and homogenized with a tightly fitting Teflon glass homogenizer (15 strokes). The homogenate was centrifuged at 800 X g for 10 min at 4 C. The resultant pellet was suspended in 3 ml of the same buffer and centrifuged under the same conditions. The supernatant fractions were combined and centrifuged at 100,000 X g for 60 min at 4 C. The resulting pellet was stored in a liquid nitrogen freezer. 125

Binding of [

IJiodo-LDL to membrane fractions

Membrane-bound [125I]iodo-LDL was assayed by a modification of the method of Basu et al. (12). The binding assays were conducted in 1.5 ml polyethylene microfuge tubes (Brinkman Instruments, Palo Alto, CA) that had been precoated with 1% Siliclad (Clay-Adams, Inc., Parsippany, NJ). The usual binding assay was performed at a final pH of 7.5 in 100 /il Trischloride (50 mM) buffer containing NaCl (100 mM), CaCl2 (0.5 mM), bovine serum albumin (20 mg ml"1) and the indicated amounts of membrane protein, and [125I]iodo-LDL (10 /zg pro-

1125

tein ml '), with or without nonradiolabeled LDL (2 mg protein ml"1). Before use, the stock solution of [125I]iodo-LDL and nonradiolabeled LDL were passed through a Millipore filter (0.45 /xm; Millipore Corp., Bedford, MA). The binding reactions were initiated by the addition of [125I]iodo-LDL, and the reaction mixtures were incubated for 60 min at 0 C in an ice water bath. To quantify the amount of membrane-bound [125I]iodo-LDL, an aliquot (70 jul) of the reaction mixture was layered onto 150 /il 100% fetal calf serum in a thick cellulose proprionate tube (7.0 X 21.0 mm,; Beckman Instruments, Palo Alto, CA) that had been precoated with 1% Siliclad. The tubes were centrifuged at 100,000 X g for 20 min in a Beckman LP42Ti rotor. The supernatant fluid was discarded, and the surface of the pellet was washed by rinsing four times with 100 /il 100% fetal calf serum without suspending the pellet. After a final washing, the bottom portion of the tube was cut off with a hot razor blade and placed in a glass tube for radioactivity assay employing a Micromedic 4/600 series counter (Micromedic Systems, Niles, IL). The protein content of each membrane fraction was assayed by the method of Lowry et al. (9). Each value presented in the figure is the average of the results of triplicate assays. Degradation of lipoproteins by HFA tissue The tissue fragments were maintained in organ culture as described above. On the third day of culture the fragments were rinsed twice with 0.15 M NaCl. Fresh medium containing LPPS (10% vol/vol) and 10 jug [125I]iodo-LDL was added to each dish. The incubation was continued at 37 C for 24 h. The rate of degradation of [125I]iodo-LDL was determined as described (3). The results are expressed as nanograms [125I]iodo-LDL degraded mg"1 protein 24 h"1 and are the mean ± SE of quintuplicate replicates. Statistical analysis of the data was performed utilizing the Students' t test. Incorporation of [UCJacetate into cholesterol by HFA tissue The rate of incorporation of [HC]acetate into cholesterol was determined in adrenal tissues on the third day of culture as described (4). The results are expressed as nanomoles mg"1 protein h"1, and each value represents the average of duplicate measurements.

Results Steroid secretion rates The rate of secretion of DS by HFA tissue (maintained in culture medium containing 10% HS) as a function of days in culture is presented in Fig. 1. In the absence of ACTH, dbcAMP, or CT, the rate of DS secretion by HFA tissue fragments remained low. In the presence of ACTH, the secretion rate of DS was high on the first day in culture. Thereafter, DS secretion declined and subsequently was maintained at a steady rate, 5- to 8-fold greater than that obtained in HFA tissue maintained in the presence of HS alone. A similar pattern of DS secretion was observed in tissue maintained in the presence of

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CARR ET AL.

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Specific binding of LDL to membrane preparations of HFA tissue

10

E x CP

•ft

CO Q

1 2 3 4 5 10% HS

1 2 3 4 5 10% HS + ACTH

1 2 3 4 5 10% HS + dbcAMP

1 2 3 4 5 10% HS +C.T.

DAYS IN CULTURE

FIG. 1. Secretion rate of DS by HFA tissue as a function of days in culture. The tissue was maintained in the presence of HS (E), HS plus ACTH (ea), HS plus dbcAMP (eg) and HS plus CT M . The medium was changed daily and analyzed for steroids. The results presented are the mean ± SE offivereplicates and are expressed as jug DS mg"' tissue protein 24 h"1. Adrenal tissue as represented in Fig. 1 and 2 was obtained from a single abortus. -

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CM X

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JCE & M • 1981 Vol 52 • No 6

5

1 2 3 4 5 1 0 % HS + ACTH

1 2 3 4 5 10% HS +dbcAMP

Degradation of [nbI]iodo-LDL The rates of degradation of [125I]iodo-LDL by HFA maintained in the presence of 10% LPPS, and in the presence or absence of ACTH, dbcAMP, or CT, are presented in Fig. 4. In the presence of ACTH or CT there was a 2-fold greater rate of degradation of LDL than in the presence of LPPS alone (P < 0.01 and P < 0.05, respectively). In tissue maintained in the presence of dbcAMP there was a greater rate of degradation of LDL compared to that in tissue maintained in the presence of LPPS alone, but this difference was not statistically significant. Incorporation of [UC] acetate into cholesterol

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1 2 3 4 5 1 0 % HS

The specific binding of [125I]iodo-LDL (total binding minus binding when excess nonradiolabeled LDL was added) by membrane preparations of HFA prepared from tissue previously maintained in the presence of 10% LPPS and in the presence or absence of ACTH, dbcAMP, or CT are presented in Fig. 3. The specific binding of [125I]iodo-LDL by HFA tissues that were maintained in culture medium containing ACTH, dbcAMP, or CT was 2- to 3-fold greater than the specific binding by membranes prepared from nontreated tissues.

1 2 3 4 5 1 0 % HS +C.T.

The rates of incorporation of [14C]acetate into cholesterol by HFA tissue maintained in the presence of 10% LPPS and in the presence or absence of ACTH, dbcAMP, or CT are presented in Fig. 5. The rate of de novo, synthesis of cholesterol in HFA tissue maintained in the 300 r

DAYS IN CULTURE

FIG. 2. Secretion rate of cortisol by human fetal adrenal tissue as a function of days in culture. See Fig. 1 for details.

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§ 5 200

dbcAMP or CT. The rate of secretion of cortisol, as a function of days in culture, is presented in Fig. 2. Again, in the absence of ACTH, dbcAMP, and CT, cortisol secretion remained low. In the presence of ACTH, cortisol secretion increased gradually throughout the culture period. In the presence of dbcAMP or CT, cortisol secretion rates were higher on the first day in culture (4.4 ± 0.7 and 3.2 ± 0.3 /xg mg"1 protein 24 h"1, respectively) compared to those in HFA tissue maintained in the presence of ACTH (0.9 ±0.1 /xg mg"1 protein 24 h"1). However, by the third day in culture, cortisol secretion rates in HFA tissue maintained in the presence of dbcAMP and CT were comparable to those observed in the presence of ACTH.

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T7E1 100

yyyyyyyy LPPS

LPPS + ACTH

LPPS + dbcAMP

FIG. 3. Effect of ACTH, dbcAMP, and CT on specific binding of [125I]iodo-LDL by HFA membrane fractions. Membrane fractions were prepared as described in the text from tissue fragments that had been maintained for 72 h in medium containing LPPS (o), LPPS plus ACTH (a), LPPS plus dbcAMP (ta), and LPPS plus CT (•§). See Materials and Methods for details. Each data bar represents the mean of triplicate assays of specific binding of [125I]iodo-LDL. Adrenal tissue obtained from a single abortus was utilized for this experiment.

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ROLE OF cAMP IN HFA TISSUE p