Steroid concentrations in the outer and inner zones of the adrenal cortex of the guinea pig

J. steroid Biochem. Vol. 20, No. 5, pp. 1123-1127, 1984 Printed in Great Britain. All rights reserved

0022-473 l/84 $3.00 + 0.00 Pergaman Press Ltd

STEROID CONCENTRATIONS IN THE OUTER AND INNER ZONES OF THE ADRENAL CORTEX OF THE GUINEA PIG TETSUO NISHIKAWA* and CHARLES A. SmoTTt National Institute of Child Health and Human Development, Endocrinology and Reproduction Research Branch, National Institutes of Health, Bethesda, Maryland 20205, U.S.A.

(Received 2 August 1983) Summary-The outer (glomerulosa and fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig were separated and their steroid content determined. It was found that the con~ntration of 21-hydroxypregnenolone, deoxycorticosterone. corticosterone, aldosterone, 1I-deoxycortiso1, and cortisol was si~i~cantly higher in the outer cortical region, while the concent~dtion of pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone was significantly higher in the inner zone. The concentration of progesterone. 17-hydroxyprogesterone, and androstenedione was not different in the two zones. Examination of specific steroid ratios suggested the following: (I) 3,5’-01dehydrogenase/isomerase and 21-hydroxylase activities are reduced in the inner zone, (2) 17-hydroxylase and C,,.,, lyase activities appear to be equally active in the two zones (3) 1I/?-hydroxylase activity appears to be more active in the inner zone (4) 21-hydroxypregnenolone, deoxycorticosterone, corticosterone, 11-deoxycortisol. and cortisol along with aldosterone are produced principally in the outer zone.

On the basis of microanatomy the mammalian adrenal cortex has been subdivide into three principal regions or concentric zones [i]. The deveIopmenta1 and physiological basis for this interesting zonation is not well understood [2]. However, there have been several attempts to examine steroidogenesis in the different zones [3]. It is now generally accepted that aldosterone is formed and secreted by glomerulosa cells, but functional differences in steroidogenesis between the fasciculata and reticularis are less well understood. It has been considered that fascicuiata cells produce and secrete primarily the active glucocorticoid, while reticularis cells produce and secrete primarily C,, steroids, but this notion has not been firmly supported by experimental data. In an effort to pursue this problem, we have been establishing the guinea pig adrenal as an experimental model. The guinea pig, a cortisol producer, has an adrenal cortex which is grossly composed of an outer, yellow zone (zona glomerulosa and zona fasciculata) and an inner, brown zone (predominantly zona reticularis). We have previously reported that the soluble fraction *Present address: The Second Department of Internal Medicine, Chiba University School of Medicine, Inohana l-8-1, Chiba 280, Japan. tAddress for correspondence: Building IO, Room 8C-407, Bethesda, MD 20205, U.S.A. $The trivial steroid names used are: Pregnenolone, 3/S-hydroxy-5-pregnen-20-one; IFhydroxypregnenolone, 2 1-hydroxypreg3&17-dihydroxy-5-pregnen-20-one; nenolone, 3b,21-dihydroxy-.5-pregnen-20-one; 1717-hydroxy-4-pregnene-3, 20hydroxy-progesterone, 1I-dexycortisol, 17,2 1-dihydroxy-4-pregnenedione; 3,20-dione: androstenedione, 4-androstene-3,17-dione.

of the outer zone contained significantly more cortisol than the same fraction prepared from the inner was found for while the converse zone report essentially pregnenolone [4]. A recent confirmed this finding[5]. It has also been reported that specific binding proteins for pregnenolone and pregnenolone sulfate were concentrated in the inner zone [4]. More recently, we have confirmed that 3/j’-steroid sulfotransferase activity is highest in the inner cortical region [6], and that 3fl-sulfoconjugated steroids were present in significantly higher concentrations in the inner zone[7]. To further examine possible functional differences in steroidogenesis between the inner and outer zones, the content of a large number of steroids was assayed in homogenates of the two zones. Our aim was to simply examine the steroid profile at a single point in time. There are, of course, drawbacks to this approach, most notably contamination with extracellular fluid and blood. Nevertheless, it was felt that data derived from such an approach might yield interesting background information on distinctions in zonal steroidogenesis and metabolism. Such an approach has been previously used as indicated in a recent report [7]. MATERIALSAND METHODS

[7-3H]Pregnenolone~ (19.3 Ci/mmol), [7-‘H], 21-OH pregnenolone (44 Ci/mmol), [ 1.2-3H(N)]dehy(58.6 Ci/mmoI), droepiandrosterone @HA) [ 1,2(90 Ci/mmol), [ 1,2,6,7-‘H(N)]progesterone -‘H(N)1 7-OH progesterone (40.4 Ci/mmol), [1,23H(N)] androstenedione (46.1 Ci/mmol), [I ,23H(N)deoxycorticosterone (DOC) (41.8 Ci/mmol),

1123

1124

TETSUO NISHIKAWA and CHARLES A. STROTT

[I ,ZPH(N)] 1I-deoxycortisol (58.5 Ci/mmol), [I ,2-‘H(N)]corticosterone (40 Ci/mmol), [1,2-3H(N)]cortisol (56 Ci/mmol), and [ 1,2,6,7-3H(N)aldosterone (81.9 Ci/mmol) were purchased from New England Nuclear Corp. [7{~)-3HJ17-OH pregnenolone (10 Ci/ mmol) was purchased from Amersham. Purity of each labelled steroid was evaluated by thin-layer chromatography. Non-radioactive steroids were obtained from Steraloids, Research Plus, and Sigma Chemical Co. Stock solutions of labelled and unlabelled steroids were stored in absolute ethanol at -20°C. Celite 535 was purchased from Accurate Chemical Corp. Organic solvants and other chemical reagents were used as obtained from suppliers. Animals and tissue preparation Male guinea pigs (NIH inbred Strain 2) weighing between 700-900 g were killed by decapitation within 10 s of handling between 0900 and 1000 h. The adrenal glands were quickly removed and placed in an iced solution of 0.25 M sucrose, 10 mM Tris (pH 7.4), and 1.5 mM Na, EDTA (TES). Extraneous fat and fibrous material were removed. The glands were weighed, sliced in half lengthwise with a razor blade, and the inner zone separated from the outer zone as previously reported [4]. The separate zones were homogenized at 0°C in TES (1 g/IO ml) using a Teflon/glass homogenizer manually operated. The homogenates were spun at 100 g for 5 min in order to remove any unhomogenized tissue. The supernatants were quickly frozen and stored at -20°C until they were assayed. Protein concentrations were determined by the method of Lowry et a/.[&], using BSA as the standard. Extraction of steroids Aliquots of the homogenates (5~1000~1) were transferred to tubes to which approx. 3000 cpm of the appropriate steroid had been added in order to evaluate recovery. Water was added to bring the final volume to i ml. Samples were mixed with IOml of diethyl ether for extraction of steroids except corticosterone, 1I-deoxycortisot, cortisol, and aldosterone which were extracted with 10 ml of dichloromethane. Extracts were evaporated to dryness under reduced Table 1. Steroid concentrations Steroid .~.

..__.

Pregnenolone 17-OH Pregnenolone 21-OH Pregnenolone Progesterone DOC Corticosterone Aldosterone 1f.OH progesterone 1I-Deoxycortisol Cortisol DHA Androstenedione

pressure and then subjected to either celite or Sephadex LH-20 column chromatography. Chromatography Celite was mixed with 66% propylene glycoi:33% ethylene glycol (2: 1, w/v) and the mixture (1.4 g) was packed into disposable 5 ml glass pipettes according to the method of Abraham et aL[9]. The dried extract residues were rinsed with lO$ of ethanol, resuspended with 1 ml of isooctane, and transferred to the celite microcolumns. Steroids were eluted by stepwise increases of ethyl acetate in isooctane as follows: progesterone (6 ml, isooctane), pregnenolone (7m1, 5% ethyl acetate in isooctane), DOC and 17-OH progesterone (7 ml, 15% ethyl acetate in isooctane), 17-OH pregnenolone and 21-OH pregnenolone (7 ml, 25% ethyl acetate in isooctane). For the separation of DHA and androstenedione celite was mixed with ethylene glycol (1: 1 w/v) and the mixture (1.4 g) was packed into disposable 5 ml pipettes. The resuspended extracts as noted above were applied to the celite columns; androstenedione was eluted with 5 ml of isooctane and DHA with S’:/,ethyl acetate in isooctane. Cortisol was isolated using a celite column which consisted of celite and 2OO.i H,O-807; ethylene glycoi (3: 1, w/v). The dried extracts were resuspended with 20”/, ethyl acetate in isooctane and transferred to the columns. Cortisol was eluted with 7ml of 40% ethyl acetate in isooctane. Corticosterone, I I-deoxycortisol, and aidosterone were separated on LH-20 columns. The dried samples were dissolved in 0.25 ml of 98”! dichloromethane-2~~ methanol, and transferred to the top of the gel bed. Elution was performed with 98% dichloromethane--2% methanol. Corticosterone was eluted at 16-22 ml, 11-deoxycortisol at 22-30 ml, and aldosterone at 50-63 ml. All eiuates were dried under a gentle stream of air in a 37°C bath. Samples were resuspended with 1 ml of ethanol: 0.2 ml was used for estimation of recovery and the remainder subjected to radioimmunoassav. Recoveries ranged from 50 to 90%. Radioimmunoassa_ys Levels of 21-OH pregnenolone

in the outer and inner zones of the guinea-pig adrenal cortex WI* (4) (4) (8) (4) (8) (4) 14) (4) (4) (4) (4) 14)

Outer 83 * lot 18.4 + 7.1 712 f 173 2735 i 475 363 i 105 I030 f 375 7.2 k 1.4 489 + 74 2857 f 732 5070 2 940 4.1 kO.2 615&79

P value

Inner 1897 f 123 + 200 * 4064 + 17.3 * 297 i 0.9 + 322 f 71 * 1250 * 9.2 k 417 *

197 14.7 32.8 942 3.4

II

0.3 53 18 200 1.7 70

< 0.001 < cl.001 < 0.02 n.s.$
*Number of groups (one group consisted of three guinea pigs); trig/g tissue, Mean +_SE: $not significant.

were estimated

Adrenocortical

zonal steroid concentrations

1125

Table 2. Enzyme activities based on steroid ratios Outer

Inner

P value

A. 3~-Hydrox~teroid dehydrogena~lisomer~e

Pregnenolone Progesterone 17-OHPrognenolone 17-08 Progesterone 21-OH Pregnenolone DOC DHA Androstendione B. 21-hydroxylase Pregnenoione 2f-OH Pregnenolone Progesterone DOC 17-OH Progesterone 1I-Deoxycortisol C. 11-g Hydroxylase 1 1-Deoxycortisol

cortisol DOC Corticosterone D. 17-a hydroxylase Pregnenolone I?-OH Pregnenoione Progesterone 17-OHProgesterone E. CIFMLyase 17-OH Progesterone Androstenedione 17-OH Pregnenolone

0.03 + 0.01*

0.51 + 0.06


0.04* 0.02

0.41 + 0.09

to.01

2.29+ 0.21

10.16+ 1.44


0.007* 0.001

0.022+ 0.002


0.13 20.04

9.21i 2.30


10.3* 3.2

180.8& 44.4

to.01

0.19 +0.03

4.99+ 0.85


0.57 * 0.12

0.055+ 0.006


0.35+ 0.10

0.080f 0.005

<0.05

If.36 5 7.69

15.54+ 0.88

n.s.t

6.21f 1.78

12.91+ 2.51

n.s.

0.82 +0.13

0.82_e0.13

n.s.

15.27+ 3.41

< 0.05

4.30* 1.45

DHA

*Calculations based on data used to derive Table I, Mean rt SE. tNot significant

using a DOC antiserum which was kindly supplied by Dr Myron Weinberger (Indiana University). At the DOC antiserum dilution used (1: IOOOO),approx 20% of [3H121-OH pregnenoione was bound, and the linear portion of the standard curve covered the range of 50-2000 pg 171. The percentage cross reactivity to the DOC antiserum using labelled 21-OH pregnenolone was as follows: l%OH pregnenolone 17,21-OH pregnenolone (
Steroid concentrations in the outer and inner zones The concentrations of twelve steroids in the outer and inner zones of the adrenal cortex are shown in

Table 1. The concentrations of pregnenolone, 17-OH pregnenolone and DHA were significantly higher in the inner zone than in the outer zone, while the concentrations of 21-OH pregnenolone, DOC, corticosterone, aldosterone, 1I-deoxycortisol, and cortisol were significantly greater in the outer zone. There was no significant difference in the concentration of progesterone, 17”OH progesterone, and androstenedione between the two zones. Ratios of steroid substrate to steroid product were examined in an effort to evaluate the activity of various synthetic enzymes in the two zones. These data are recorded in Table 2. When the 5-ene/4-ene ratio was examined for four steroid combinations, a significant difference in each instance was noted between the two zones indicating that 3fl-01 dehydrogenase/isomerase activity is reduced in the inner zone. Three steroid combinations were examined for tl-hydroxylase activity; the results suggested that this enzyme activity is also reduced in the inner zone. When steroid combinations were examined for 1I/?-hydroxylase activity, it was found that this enzyme function is apparently greater in the inner zone in contradistinction to 21-hydroxylase activity.- On the other hand, no significant difference was noted

TETSUONLSHIKAWA and CHARLES A. STR~TT

1126

between the two zones for 17-hydroxylase activity. Steroid ratios for C,,_20 lyase activity indicated a reduced activity for 5-ene steroids in the inner zone, while there appeared to be no difference for the 4-ene steroids. Assuming that aldosterone is produced only by the zona glomerulosa, it can be used as a specific outer zone marker. Based on this concept, the ratio of various steroids to aldosterone was determined as a means of judging whether a particular steroid might be produced by both zones or primarily by the outer zone. A ratio that does not change significantly between the two zones or falls significantly in the inner zone suggests that the steroid is produced primarily in the outer zone. On the other hand a significant increase in the ratio in the inner zone suggests that the steroid is produced in the inner zone as well as possibly in the outer zone. The results suggest that 21-OH pregnenolone, DOC, I l-deoxycortisol, and cortisol are produced primarily by the outer zone. The ratio of corticosterone to aldosterone did increase significantly in the inner zone suggesting that corticosterone is produced in that zone. Such an interpretation in this case, however, is less clear than with the other steroids examined, in part, because corticosterone is an intermediate substrate in aldosterone formation. Thus, the corticosterone to aldosterone ratio also implies, as expected, that 18-hydroxylase activity is greater in the outer zone. DISCUSSION

The results presented in this report support the notion that differences in steroidogenic and/or metabolic activity exist between the outer and inner zones of the guinea pig adrenal cortex. The findings expand on previous reports which indicated additional functional differences between the two zones in this species [4,7, 10-121. The physiological basis for functional differences is not clear. The apparent primary function of the mammalian adrenal cortex is to produce both a potent mineral~orticoid and a glucocorticoid (the former is represented by aldosterone while the latter may be either cortisol or corticosterone); no other essential function aside from this has been established. It seem reasonably clear that aldosterone is produced by the glomerulosa cell [ 131. and is normally regulated by the reninangiotensin [14]. It seems equally clear that cortisol (depending upon the species) is produced by cells other than glomerulosa cells [2]. Confusion arises because the non-glomerulosa cells appear to be of two general types, i.e. fasciculata and reticuiaris cells. The distinction between the fasciculata and reticularis zones has been based principally on morphological grounds. Why there are two cell types in addition to the glomerulosa cells is not understood. Whether both the fasciculata and reticularis cells are normally involved in cortisol production is also poorly under-

stood; interpretation of data vary depending upon the species studied. To simplify matters, it is generally accepted that the fasciculata cell is probably the main glucocorticoid-producing cell, and that this cell is normally regulated by pituitary ACTH. The mystery cell seems to be the reticularis cell. Certain fundamental questions can be raised: (1) what is the developmental basis for the reticularis cell? (2) what is the function of the reticularis cell? and (3) how is the reticularis cell maintained and regulated? The morphological characteristics of the zona reticularis vary considerably from species to species. For example, in the human being the zona reticularis is composed of small, “compact” cells in comparison with the much larger cells of the zona fasciculata, while in the guinea pig the zona reticularis and zona fasciculata are composed of cells which are more nearly the same size. In some species the zona reticularis is small, such as in the rat where it comprises perhaps only S!,, of the total cortical volume [ 151. While at the other extreme, as in the guinea pig, the zona reticularis may comprise as much as two-thirds of the total cortical volume [4]. In some species, such as the human being, development of the zona reticularis demonstrates a striking relationship to sexual maturation [16]; this kind of phenomenon is apparently not the case in non-primates or primates other than gorillas [ 171. The relationship between the zona reticularis and DHA and DHA sulfate production is an interesting one. In the human being, development of the zona reticuiaris is correlated with an increasing production of these steroids, while in the guinea pig there is no such correlation. In the latter species, as noted above, the zona reticularis may constitute as much as two-thirds of the total cortical volume. but DHA and DHA sulfate production are minuscule [7]. It has been suggested that the zona reticularis might be the primary site of adrenal “androgen” production [IX]. In a recent report androstenedione was found to be more abundant in the zona reticularis of the guinea pig adrenal [S]. This, however, appeared not to be the case in our studies with the guinea pig. The principal C,, steroid produced by the guinea pig adrenal was androstenedione (as it was in the other report [5], but the concentration of this steroid was similar in the outer and inner zones. The fact that androstenedione was not more concentrated in the inner zone than in the outer zona fails to support the notion that this steroid might be responsible for the reduced ?I-hydroxylase activity in the inner zone as previously suggested [19]. In addition, the content of progesterone and 17-OH progesterone was similar in the two zones, and, thus, it would appear that neither of these steroids is responsible for the reduction in 21-hydroxylase activity in the inner zone 1191. The data presented in this report suggest that cortisol is produced primarily in the outer cortical region and confirm previous reports [4, 51. The data further suggest that part of the reason for this is

Adrenocortical

zonal steroid concentrations

reduced because of activities of 3j-01 dehydrogenase/isomerase and 21-hydroxylase enzymes in the inner zone (Table 2). We have measured the activity of both these enzyme systems using microsomes isolated from each zone. The activity of 3/I-01 dehydrogenase/isomerase was significantly reduced in the inner zone using pregnenolone and 21-OH pregnenolone as substrates (unpublished observations). The activity of 21-hydroxyiase, however, was significantly increased in the inner zone using 17-OH progesterone and 17-OH pregnenolone as substrates (unpublished observations). This latter finding confirms a recent report [l I]. Thus, it would appear that 21-hydroxylase activity in the inner zone, although more active in citro than in the outer zone, may be normally suppressed in uiuo. An alternate explanation for the low cortisol content in the inner zone could be an increased metabolic rate for cortisol in that zone. Along this line, it has been reported that ring A reduction of cortisol is highest in the inner zone of the guinea pig adrenal cortex (IO]. A third possibility for the low cortisol content in the inner zone is that cortisol is synthesized and then rapidly released from the reticuiaris cell in contrast to the fasciculata where cortisol is perhaps more slowly released. We favor the low production rate explanation, and this hypothesis is supported by studies which demonstrated that isolated inner zone cells, in contrast to outer zone cells, failed to increase cortisol production in response to ACTH and cAMP[20].

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