Steroid profiles formed by rat adrenocortical whole tissue and cell suspensions under different conditions of stimulation

STEROID PROFILES FORMED BY RAT ADRENOCORTICAL WHOLE TISSUE AND CELL SUSPENSIONS UNDER DIFFERENT CONDITIONS OF STIMULATION G. P. VINSON, B. J. WHITEHOUSE,* C. GODDARD and C. P. SIBLEY* Department of Biochemistry, St. Bartholomew’s Medical College, Charterhouse Square, London EClM 6BQ, and *Department of Physiology, Queen Elizabeth College, Campden Hill Road, London W8 7AH, England SUMMARY The production of five steroids, corticosterone, deoxycorticosterone (DOC), 18-hydroxydeoxycorticosterone (18-OH-DOC), 18-hydroxycorticosterone (18-OH-B) and aldosterone, has been measured following incubations of rat adrenal tissue. The following tissue preparations were used: whole gland minces, whole glands quartered, capsules and inner zones intact, or as cell suspensions. Stimulation by ACTH, LH and potassium was also studied. The steroid profile elaborated by the tissue varies in a striking manner depending both on the method of tissue preparation and on the mode of stimulation. In inner zones the variation is found in the relative amounts of corticosterone and 18-OH-DOC produced. In unstimulated cell suspensions, or in whole tissue subjected to preincubation, the corticosterone/l8-OH-DOC ratio is less than 1, possibly as little as 0.2. After stimulation, or in other preparations the ratio can rise to as much as 1.6. In capsules, the most important effect is on the capacity to produce the ll-oxygenated steroids as a whole. Even under conditions of maximal stimulation by ACTH or potassium, this capacity is greatly impaired in cell suspensions compared with whole tissue, while corticosterone is unaffected. The findings are not compatible with the theory that ACTH (or other stimulants) act at a single site in the biosynthetic pathway. They are compatible with the view put forward from these laboratories over the years, that control of steroidogenesis is also effected through maintenance of separated pools of steroid within the tissue which have different metabolic fates. In particular it appears that the 18-oxygenated steroids (in contrast to corticosterone) may be sequestered within tissue stores, and that in part their secretion is controlled by release from such stores.

INTRODUCTION

tissue in vitro has over the last 25 years yielded considerable insight into mechanisms of steroid hormone secretion. The methodology employed has developed from relatively simple incubation of whole tissue [l-5] to, in more recent years, superfusion of whole tissue, or separated glomerulosa and inner zone components [6,7], incubations of intact tissue with dialysis [8-111, utilisation of protease enzymes to produce cell suspensions [12,13], superfusion of such cell suspensions [14,15], and purification of individual cell types [16, IS]. “It has generally been found that the more sophisticated methodology, such as superfusion or preparation of cell suspensions, results in a more sensitive response of the tissue to stimulation, for example by ACTH, as judged by the ratio of stimulated to unstimulated production of corticosterone. This in turn has been interpreted as indicating a closer approximation to in vivo conditions in which the .response to ACTH is very sensitive indeed, especially in hypophysectomised animals. Suggested reasons for the apparent increased sensitivity under “improved” in vitro conditions are: increased accessibility of ACTH to the cells [19], removal of steroid products, or other inThe incubation

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of adrenocortical

hibitors from the system [20,21], or the decreased metabolism of ACTH by the adrenocortical tissue [22,23]. The rat adrenal cortex produces a range of other steroid products however which may not invariably all behave similarly to corticosterone. This paper examines the possibility that the steroid profile formed by rat adrenal tissue under different in vitro incubation conditions varies qualitatively as well as quantitatively, and that such variation gives important information on the mechanisms of steroidogenesis, the control of the adrenocortical secretion, and the nature of steroid secretion as a process distinct from biosynthesis.

MATERIALS AND

METHODS

Animals Rats of the Wistar strain were obtained from R. H. Tuck & Son., Chigwell, Essex, UK, and maintained

in the animal house at Queen Elizabeth College, or taken from the colony maintained at St. Barth+ lomew’s Medical College. They were fed on Spratt’s laboratory diet No. 1. 175

G. P. VINSONet at.

176 incubations

ticosterone) and the remainder oxidised with periodic acid to produce the y-lactones of the 18-oxygenated products. The dried DOC extract is also acetylated. All three fractions are then treated with heptafluorobutyric anhydride (0.1 ml, 20% in acetone) at 60°C for 30min to yield the 3-enolheptafluorobutyrat~ of all of the derivatives. Suitable aliquots of the extracts are then chromatographed directly on 60 cm columns (4mm i.d.) containing approximately 0.3% XE-60 on Gas Chrom Q support. Specificity, sensitivity, accuracy and precision of the methods have been described elsewhere [ZS].

1. Conventional. Rats were killed by cervical dislocation and the adrenals were quickly removed, cleaned and stored in beakers at 0°C until required for incubation. Glands were either quartered or minced, or decapsulated to yield largely glomerulosa (capsule) and inner zone fractions. Tissue from two glands was incubated in each flask containing 5 ml Krebs bicarbonate Ringer (potassium content 3.6 mM) with glucose. In some incubations, a preincubation period of 30min (from which the medium was discarded) preceded the main incubation of 2 h, to which stimulatory factors were added. In general 6 control and 6 experimental flasks were used in each experiment. 2. Cell suspensions. Adrenocortical tissue was incubated in Krebs bicarbonate Ringer containing 3.6 mM K+, 2OOmg/1OOml glucose and 1% w/v bovine serum albumin (Sigma, fraction V) (KRBGA) together with 2mg per ml collagenase (Worthington Biochemical Corporation) for one hour, following established methods [21,24]. Tissue was then gently disrupted by repeated pipetting, and cells centrifuged down at IOOg. The cells were then resuspended in fresh KRBGA and recentrifuged. Yields Of cells were approximately 90,000 cells/gland for capsules and 600,000 cells/gland for inner zones. Cells were then dispersed to flasks in small volumes of KRBGA, incubations were in 5 ml KRBGA for 2 h at 37°C under 95% 02, 5% coz. Extraction

and quantitation

Experiments

Conditions for the incubation experiments formed are summarised in Table 1.

per-

RESULTS

Stimulation changed the steroid profile in varying ways depending on the tissue preparation. This is particularly true with respect to the relative amounts of corticosterone and 18-OH-DOC in whole tissue or inner zones, and in the yields of 18-OH-B and aldosterone relative to other products in capsule incubations. Non-dispersed

tissue

In minced whole glands, after preincubation (Expt. 1, see Table I), the corticosterone/l8-OH-DOC ratio is frequently less than 1 (Fig. 1). The addition of ACTH under these conditions stimulates corticosterone significantly but not 18-OH-DOC in male glands. Neither compound is affected in female glands. The relatively refractory response of 18-OHDOC to ACTH stimulation is also seen when quartered male whole glands were used without a preincubation period (Expt. 2, Fig. 2). Under the same conditions, addition of 1 /~g per ml LH stimulated both compounds significantly. 18-OH-DOC was stimu-

of steroids

Five steroids, deoxycorticosterone (DOC), 18”hydroxy-DOC(l8-OH*DOC),corticosterone,l8*hydroxycorticosterone (18-OH-B) and aldosterone were measured using a glc method. In brief, in this method 5ml incubation media are first extracted with 2ml hexane to give a “DOC” fraction, and then with 2 x 2.5 ml ethyl acetate. The ethyl acetate fraction is divided, and part (usually l/3) is acetylated (for cor-

Table 1. Incubation experiments Expt. No.

Animals

Tissue preparation Whole glands, minced Whole glands, quartered Whole glands, quartered Capsules only Capsules only Inner zones, cell suspensions Inner zones, cell suspensions Capsule, cell suspensions Capsule. cell suspensions Capsule and inner zones, celi suspensions

l

Preincubation

yes no yes yes no (with collagenase) (with collagenase) (with collagenase) (with collagenase) (with collagenase, and sampling of medium at various time intervals, and after cell centrifugation)

Synacthen (Ciba-Geigy). t N.1.H. Ovine LH {Batch Si7).

Stimulation in experimental flasks lACTH 20 mu/ml (5.5 x 10-s M ACTH 20mU/~I 0; tLH 1&ml ACTH 20 mu/ml ACTH tOmU>ml 5.9 mM K+ (3.6mM Kf in controls) ACTH various concentrations LH various concentrations Potassium, various concentrations ACTH various concentrations Control only

Steroid profiles under different conditions of stimulation

3r

177

alone (after preincubation) (Expt. 4, Fig. 2). Here ACTH significantly increased corticosterone, DOC, 18-OH-B and aldosterone, but not 18-OH-DOC. Similar stimulatory effects were seen when the potassium content of the incubation medium was raised from 3.6mM to 5.9mM (Expt. 5, Fig. 3). CA

CA

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CA

CA

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ls-oHDOC

FEMALES

Fig. 1. Stimulation of steroidogenesis (following preincubation) in incubated minced whole adrenal glands of Wistar rats by ACTH (Expt. 1, see Table 1). C = control, A = ACTH stimulated. Corticost = corticosterone, II-OH-DOC = 18-hydroxydeoxycorticosterone. Values are pg per pair of incubated glands, means + S.E. (n = 6 throughout). Comparison of male corticosterone control and ACTH stimulated values, P < 0.005. Other values not significantly different from controls. lated by ACTH in quartered glands after preincubation (Expt. 3, Fig. 2), but even here the effect on 18-OH-DOC is less than on corticosterone; thus the ratio of corticosterone to l&OH-DOC in control flasks was 0.8 + 0.1 whereas after ACTH stimulation it was doubled to 1.6 + 0.09. When whole tissue was used there was no stimulation by ACTH of 18-OH-B or aldosterone. Stimulation of 18-OH-B and aldosterone occurred however when capsules were incubated

Cell suspensions

In these and similar experiments, the sensitivity of the response to stimulation was measured in terms of corticosterone production, for comparison with results reported from other laboratories. In our experiments, the ED-50 for ACTH stimulation of fasciculata/reticularis cells was 3.5 x lo- l1 M, and the effective ACTH concentration was maximal 1.8 x 10e9 M. The ED-50 for glomerulosa cells was similar, while in this case the maximal effective concentration of ACTH was 5 x lo- lo M. However, changes in steroid profile with stimulation were also seen in cell suspension incubations. In inner zone cell suspensions (Expt. 6, Fig. 4) basal levels of corticosterone production are quite variable, but always substantially less (often by a factor of 5) than 18OH-DOC. Stimulation with ACTH readily reverses their relative amounts, and maximally the corticosterone/l8-OH-DOC ratio is 1.4 (Expt. 6, Fig. 4).

Fig. 2. Production of steroids by rat adrenal glands under different conditions of incubation (Expts. 2-4, Table 1). C = control, A = ACTH stimulated. L = LH stimulated, DOC = deoxycorticosterone, l&OH-B = 18-hydroxycorticosterone. other abbreviations as in Fig. 1. Values are means + S.E. for pair of glands. (A) Whole adrenals quartered, without preincubation (Expt. 2). Corticosterone (comparisons with control): A, P -z 0.01; L, P < 0.001; 18-OH-DOC: L, P < 0.01. Other values not significantly different from controls. (B) Whole adrenals quartered, with preincubation (Expt. 3). Comparisons of ACTH stimulated with controls: DOC, P < 0.02; corticosterone, P < 0.001; 18-OH-DOC, P < 0.001; aldosterone, P < 0.05. (18-OH-B, not significant) (C) Capsules, with preincubation (Expt. 4). Comparisons of ACTH stimulated with controls: DOC, P < 0.001; corticosterone, P < 0.001; 18.OH-DOC, N/S; 18-OH-B, P < 0.01; aldosterone, P < 0.01.

G. P. VINWN et al.

178

1.0 0.8

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cl

- CONTROL

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- 5.4 mMK+

It is noteworthy that 18-OH-B production by inner zone cells is never stimulable by ACTH. On the other hand, LH produces a characteristic change (Expt. 7, Fig. 5). At low levels (0.1 and 0.2pg/ml LH), 18-OHDOC is actually depressed, while 18-OH-B shows a stimulation, which though slight is nevertheless quite reproducible. At increasing concentrations of LH, 18-OH-B is inhibited while 18-OH-DOC and corticosterone are stimulated. At the concentrations of LH shown here the basal corticosterone: IS-OH-DOC ratio is less than one, but at higher levels (not shown) it is greater than one. In contrast to inner zone results, cell suspension preparations materially alter the steroid profile formed by glomerulosa tissue. This is particularly well exemplified by experiment 8 (Fig. 6). Under varying conditions of stimulation by potassium, corticosterone is far the greatest product, by a factor of ten

(3.6 mMK+)

w 0.4

0.2 0 i 1MKDOC

QmoDara

lIoNa

Al-

Fig. 3. Production of steroids by capsules (not preincubated) with different potassium concentrations in incubation medium (Expt. 5). Abbreviations as for Figs 1 and 2. Comparisons of 54mM K+ with 3.6mM K+; corticosterone, P < 0.001; l&C?H-DOC, P i 0.001: 18-OH-B, P < 0.01; aldosterone. P < 0.01.

I

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60

60

100

120

140

160

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1 160

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Fig. 4. Production of steroids by inner adrenocortical cell suspensions, with varying stimulation by ACTH (Expt. 6, Table 1). Abbreviations as for Figs. 1 and 2. Values are ng produced in incubations of cells from 2 glands.

2040100

200

400

600

600

loo0

LH Inglml)

Fig. 5. Production of steroids by inner adrenocortical zone cell suspensions with varying stimulation by LH (Expt. 7. Table 1). Abbreviations as for Figs. 1 and 2. Values are yields in incubations of cells from two glands.

Steroid profiles under different conditions of stimulation

2

4

6

a

10

12

179

14

16

mMK’

Fig. 6. Production of steroids by capsule cell suspensions with varying stimulation by potassium (Expt. 8, Table 1). Abbreviations as for Figs. 1 and 2. Values are yields in incubations of cells from two glands.

in many cases. The other steroid products are all stimulated by increasing potassium (with maxima at slightly different concentrations), but the actual increment involved is considerably less than for corticosterone. Greater increases of products other than corticosterone are seen in ACTH stimulation (Expt. 9, Fig. 7), but even so the steroid profile is very different from that seen in whole capsules. At all leveis of ACTH corticosterone is the major product, and IS-OH-DOC is next greatest: it too is markedly stimulated by ACTH and in further contrast to Fig. 2c, 18-OH-B is only a relatively minor product. All products are stimulated by ACTH however, despite the great differences in individual yields. Experiment 10 (Fig. 8) illustrates the changes in steroidogenic capacity seen in capsule and inner zone tissue throughout the procedure for preparation of cell suspensions. During the initial preincubation with collagenase all of the 18oxygenated products in the capsule and 18-OH-DOC in the inner zones are

0.5

1

major products compared with corticosterone. Between the sample at 60min and the first supernatant following centrifugation of the cells (essentially the same medium: the procedure followed is simple mechanical d~ruption of the tissue by repeated pipetting at room tem~rature) there is a massive output of 1%OH-DOC and l&OH-B in the capsules, and 18-OH-DOC and corticosterone in the inner zones. Subsequent resuspension and centrifugation, and incubation of cells reduces the level of steroid produced to values consistent with unstirnulated levels in other experiments. DISCUSSION

Steroid profiles

The steroid profiles formed by whole capsules and inner zones of Wistar rat adrenals have been published elsewhere [26]. In general each tissue type pro-* duces a highly characteristic secretory pattern. In the

1.5

2 ACTH

2.5

3

3.5

”

5

Ing/ml)

Fig. 7. Production of steroids from capsule cell suspensions with varying stimulation by ACTk (Expt. 9, Table 1). Abbreviations as for Figs. 1 and 2. Values are yields in incubations of cells from two glands.

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Fig. 8. Production of steroids from (a) capsule (b) inner zone tissue during the process of preparation of cell suspensions. SI = supernatant following first centrifugation of cells after disaggregation of the tissue. S2 = supernatant following centrifugation of cells after further washing in incubation medium. Other abbreviations as for Figs. I and 2. Values are ng produced per pair of glands treated.

capsules, corticosterone and IS-OH-B are the major products, with smaller amounts of DOC, 18-OHDOC and aldosterone. Conversely in inner zones, which have a considerably higher overall steroidogenie capacity, co~icosterone and l&OH-DOC are the major products, and yields of DOC and 18-OH-B are relatively minute, while aldosterone is not detectable. The present experiments show that different tissue preparations can materially affect the overall steroid pattern in both tissue types. In inner zone preparations this amounts to changes in the corticosterone/l8-OH-DOC ratios. In whole minced or quartered tissue without preincubation, corticosterone output is generally greater than IS-OH-DOC, while after preincubation the ratio is less than 1 (Figs. 1 and 2). Formation of cell suspensions, which involves a more prolonged period of preincubation and exposure to collagenase, reduces the level of corticosterone relative to 18-OH-DOC even further (Fig. 4). More pronounced variation in steroid profile is seen in different types of capsule (largely z. glomeruloss) preparations. While in intact capsules l&OH-B is invariably a major product, (and 18-OH-DOC relatively minor) the capacity of capsule cell suspensions to produce any of the 18-oxygenated steroids is seemingly greatly impaired (Figs. 2 and 6).

Eflects of stimulation ACTH. In most studies thus far, the capacity of

rat adrenocortical tissue to respond to ACTH has largely been measured as the response of secreted corticosterone. In these terms the results reported here are largely consistent with the literature as a whole. It is well documented for example that preincubation of quartered whole tissue enhances its capacity to respond to ACTH (the literature on this topic has recently been summarised [21]). The degree of stimulation recorded here is well within the range reported by other authors. With regard to the cell suspensions. there is in the literature some inconsistency in the maximal degree of stimulation of fasciculata/reticularis cells by ACTH as judged by the stimulated/basal corticosterone output ratio. In general, many authors find that values of SO-100 for this ratio are maximal [19,24,27), but much higher values have been recorded by others [28]. More consistent indices of sensitivity are the ED-50 and the maximal effective concentration of ACTH. On these parameters there is more agreement in the literature, and the values obtained in our experiments accord well with those reported elsewhere [24,28]. Similarly the response of glomerulosa cells to increasing potassium concentration is in reasonably good agreement with the

Steroid profiles under different conditions of stimulation literature, with a maximum occurring at 8.4 mM K+ [19,27]. Stimulation, as well as tissue handling, produces characteristic effects on the secreted steroid profile. Thus ACTH stimulation of inner zone tissue has more marked effects on corticosterone than on 18-OH-DOC. This is most pronounced in minced tissue preparations, or in quartered glands without preincubation, when ACTH gave no effect on 18-OHDOC at all under conditions in which corticosterone was well stimulated (Figs. 1 and 2). (The apparent sex difference in the response of minced glands to ACTH in Fig. 1 has been discussed elsewhere [21]). Even where 18-OH-DOC is stimulated, (e.g. when quartered whole glands are incubated or in inner zone cell suspensions (Figs. 2 and 4)) it is seemingly much less affected than corticosterone, and there is a marked increase in the corticosterone/l8-OH-DOC ratio. Effects of ACTH on capsule preparations vary greatly with the nature of the preparation. In intact capsules (as in whole tissue) corticosterone is stimulated whereas 18-OH-DOC is less affected, 18-OH-B and aldosterone are also significantly stimulated, and the ratio of corticosterone to 18-OH-B is unaffected by stimulation (Fig. 2). These results are consistent with previous reports [S, 93. In cell suspensions on the other hand the capacity to form 18-OH-B and aldosterone is (relative to corticosterone) greatly reduced even under conditions of maximal ACTH or K+ stimulation: in these circumstances alone, 18-OHDOC is stimulated and becomes the second most prominent product (Figs. 6 and 7). LH. The possibility that LH has an intrinsic capacity to stimulate rat adrenocortical secretion was raised in experiments when it stimulated corticosteroid output in female glands under conditions in which they are barely responsive to ACTH [29]. Subsequent work by other authors showed that in uivo too, there were discrepancies between LH and ACTH effects and overall stimulation of steroid secretion was more rapid with LH than with ACTH [30]. Clearly, one problem is whether to attribute the activity to LH itself or to possible ACTH contamination. Radioimmunoassay data (generously provided by Prof. Lesley Rees) indicates that the NIH ovine LH as used in these experiments has an overall content of 0.1% ACTH (w/w) as judged by an ACTH,_,a standard. (Presumably in terms of activity this would be ap proximately equivalent to about 0.06% of ACTHi_a* as used in this study). With the cell suspension data (Fig. 5) the amounts of LH required to stimulate corticosterone production are in fact consistent with the view that they may be caused by ACTH. However the overall effect on the steroid profile is su@iciently different to imply that LH has at least some capacity to modify the adrenal response to ACTH. Most notable is the decline in 18-OH-DOC and increase in 18-OH-B seen at low levels: inner zone production of 18-OH-B is not stimulated by ACTH in cell sus-

181

pensions In addition, the reversal of the corticosterone/l8-OH-DOC ratio characteristic of ACTH stimulation is not marked except at higher levels of stimulation with LH than those shown. In contrast, ACTH gives a corticosterone/l I-OH-DOC ratio greater than 1 at the very lowest levels that stimulation can be detected. The characteristic effect of LH on the steroid profile is also seen in whole quartered glands (Fig. 2a), in which LH produced a significant elevation of 18-OH-DOC whereas ACTH did not. Potassium. The effects of stimulation with potassium while in reasonable agreement with results obtained by other authors [19,27] show the very marked effects of the method of tissue preparation. With intact capsules, (Fig. 3) the steroid profile is not greatly affected, and all of the compounds (except 18-OH-DOC) remain in fairly constant proportions to one another following stimulation. This is in great contrast with the cell suspension results (Fig. 6), when by comparison once again the capacity to produce 18-OH-B and aldosterone appears to be greatly impaired, even under the conditions of maximal stimulation. The capacity to produce corticosterone conversely was unaffected by the tissue preparation. Mechanisms of steroidogenesis

While for many years the view that the major site of ACTH action in the steroid biosynthetic pathway is at the point of cholesterol side chain cleavage [e.g. 311, the evjdence has accummulated suggesting that this cannot be the whole story. In particular the relative amounts of corticosterone and 18-OH-DOC produced by rat adrenocortical tissue have been found to change with experimental conditions in a manner which is incompatible with a “single site” theory. In the course of conventional incubations for example it was found that relative amounts of the two compounds varied with time quite massively (although the same was not true of their yields from radioactive precursors [32]). Suppression of endogenous ACTH (by cortisol pretreatment) inhibited corticosterone more than 18-OH-DOC production in subsequently incubated glands, and ACTH addition stimulated corticosterone more than II-OH-DOC [33, 8811-j. Results reported in this paper support this view, and indicate that corticosterone production by inner zones is generally more labile than 18-OH-DOC both as a result of tissue handling as well as of stimulation. It is fair to point out that this view is by no means a consensus, and in a recent paper Tan and Mulrow [34] point to the 0.9 correlation coefficient between corticosterone and l&OH-DOC in circulating plasma of rats under different conditions of ACTH treatment. However even in their data individual values for the corticosterone/l8-OH-DOC ratio vary from 1 to as much as 14, a finding which still requires explanation. In our hands the most dramatic variation in corticosterone to 18-OH-DOC ratio was found in incubations of adrenals from Brattleboro rats which suffer from diabetes insipidus, in which the ratio in inner

182

G. P. VINSON et al.

zone incubations could reach as much as 47 [2.5]. Clearly a single site for control of steroidogenesis in the steroid biosynthetic pathway is quite implausible. The findings are however compatible with an alternative view which we have advanced over the years. Based originally on ex~riments employing a system of incubation with dialysis, this view is that while (as the literature holds) corti~sterone may not be stored to any extent within the rat adrenal gland, other steroids, notably IS-OH-DOC may well be. To account for this it was postulated that some of the elaborated steroid product may be retained in the tissue through binding to a macromolecule, presumably a protein [S-l 11. Indeed it was postulated that where such binding occurred, particularly of DOC, l%hydroxylation was more likely than 1l/I-hydroxylation. Further work has supported this view. It has been found for example that 18-OH-DOC is indeed specifically retained within the tissue in the cytosol, and furthermore, that chromatography on Sephadex G-25 indicates that it is at least partially bound to a macromolecule [353. The amount of 18-OH-DOC so retained within the tissue can be varied by inhibition of membrane transport processes with ouabain, which can give rise to final yields of 18-OH-DOC as great in the tissue as in the medium: a concentration difference of 20-50 fold [36]. Corticosterone was simply not affected. It may be too that the extent of the storage capacity of the rat adrenal for 1%OH-DOC can be greatly under~timated by conventional techniques, in which it is assumed that all steroid is freely extractable with a solvent such as ethyl acetate. More recently we have found that ethyl acetate extraction of IS-OH-DOC from rat adrenocortical cytosol can be doubled either by heating to 90°C or by treatment with trichloracetic acid [37). Experiment 10 also supports the concept of an “invisible” store of steroid within the tissue. While during incubation the amount of steroid released into the medium was consistent with other whole tissue incubations, it is implausible that the massive outpouring of steroid following disaggregation of the tissue (Fig. 6) foilowed further steroid formation. More likely it appears to be a release triggered by mechanical handling, a phenomenon long recognised [38]. This may have particular significance in interpretation of steroidogenesis within the glomerulosa. In whole capsule tissue, 18OH-DOC (a relatively minor product) is not as greatly stimulated by ACTH as l&OH-B and aldosterone. Following preparation of a cell suspension (with a massive leaching of both IS-OH-DOC and Is-OH-B from the tissue), the capacity to form 18-OH-B and aldosterone is reduced. This leads to the conclusion that efficient aldosterone formation relies on two conditions, (I) a “store” of endogenous l&oxygenated precursor, (2) integrity of the tissue to facilitate accessibility to such a store. Acknowl~~gemenfs-.We are most grateful to Prof. Lesley Rees for assaying the ACTH content of NIH-LH, and to

Dr. D. Burley for the supply of Synacthen. This work was supported by an MRC project grant to GPV.

REFERENCES 1. Tepperman J.: Effects of purified ACTH added in oitro on the oxygen consumption and ascorbic acid content of surviving dog adrenal slices. E~ocrino~ogy 47 (1950) 384385. 2. Saffran M., Grad B. and Bayhss M. J.: Production of corticoids by rat adrenals in ~Gtw. ~~nc~jno~og~ 50 (1952) 639643. 3. Saffran M. and Bayliss M. J.: In lrirro bioassay of corticotrophin. Endocrinoloyp 52 (1953) 140-148. 4. Schonbaum E.. Birmingham M. K. and Saffran M.: Metabolism of glucose and steroid formation by rat adrenals in vitro. Can. J. Biochem. 34 (1956) 527-533. 5. Saffran M. and Schally A. V.: In aitro bioassay of corticotrophin: modification and statistical treatment. E~doc~~~o~og~ 56 (1955) 523532. 6 SalTran M., Ford P., Matthews E. K., Kraml M. and Garbeczewska L.: An automated method for fotlowing the production of corticosteroids by adrenal tissue in vitro. Can. J. ffioe~em. 4J (1967) 190-1907. 7 Baniukiewic~ S., Brodie A., Flood C., Motta M., Okamoto M., Tait J. F., Tait S. A. S., Blair-West J. R., Coehlan J. P.. Denton D. A.. Godine J. R.. Scoeeins B. A., Wintour E. M. and Wright R. b.: AdrenalYGiosynthesis of steroids in vim and in ciao using continuous superfusion and infusion procedures. In Funcrims ofthe udrenul correx (Edited by K. W. McKerns). North Holland. Amsterdam. Vol. I (1968) pp. 153-232. 8. Whitehouse 9. J. and Vinson G. P.: Compartmental arrangement of steroid precursors and the control of steroid hormone secretion in rat adrenal tissue in rirro. Acfa endocr., Cope& 68 (1971) 461-476. 9. Whitehouse 9. J. and Vinson G. P.: The effect of corticotrophin on the compa~mental arrangement of steroids and the control of steroid hormone secretion by the rat adrenal cortex. Steroids Lipid Res. 3 (1973) 110-117. 10. Vinson G. P. and Whitehouse B. J.: Compartmental arrangement of steroids formed from Cl-i4C]-acetate by rat adrenal zona glomerulosa and the effect of corticotrophin. Acra endocr., Copenh. 72 (1973) 737-745. II. Vinson G. P. and Whitehouse B. J.: The biosynthesis and secretion of aldosterone by the rat adrenal zona glomerulosa and the significance of the compartmental arrangement of steroids. Acta endocr., Copenk. 72 (1973) 746-752.

P. W. C., Island D. P., Liddle G. W., 12. Klop~nborg Michelakis A. M. and Nicholson W. E.: A method of preparing adrenal cell suspensions and its apphcability to the in vitro study of adrenal metabolism. Endocr~no~og~82 (1968) 10~3-1058. 13. Savers G., Portanova R.. Beall R. J.. Seelia S. and Malamed S.: Techniques for the isolation of cells of the adrenal cortex, the anterior pituitary and the corpus luteum. Acfn endocr., Copenh. suppl. 153 (1971) 1l-26. 14. Schulster D.: Regulation of steroidogenesis by ACTH in a superfusion system for isolated adrenal cells. Endocrinology 93 (1973) 700-704. 15. Lowry P. J. and McMartin C.: Measurement of the dynamics of stimulation and inhibition of steroidogenesis in isolated rat adrenal cells by using column perfusion. Eiochem. J. 142 (1974) 287-294. 16. Tait J. F.. Tait S. A. S., Gould R. P. and Mee M. S. R.: The properties of adrenal zona glomerulosa cells after purificalion by gravitational sedimentation. Proi. R. Sot. Land. (St. 185 (1974) 275-407.

Steroid profiles under different conditions of stimulation 17. Tait S. A. S., Tait J. F.. Gould R. P., Brown B. L. and Albano J. D. M.: The preparation and use of purified and unpurified dispersed adrenal cells and a study of the relationship of their CAMP and steroid output. J. steroid Biochem. 5 (1974) 775-787. 18. Bell J. B. G, Goutd R. P., Hyatt P. J., Tait J. F. and Tait S. A. S.: Properties of rat adrenal zona reticularis cells: Preparation by gravitational sedimentation. J. Endocr. 77 (1978) 25-41. 19. Haning R., Tait S. A. S. and Tait J. F.: In vitro effects of angiotensins, serotonin and potassium on steroid output and conversion of corticosterone to aldosterone by isolated adrenal cells. ~~oer~~o~ogy 87 (1970) 1147-1167. 20. Tait S. A. S., Tait J. F., Okamoto M. and Flood C.: Production of steroids by in uitro superfusion of endocrine tissue. I. Apparatus and a suitable analytical technique for adrenal steroid output. Endocrinology 81 (1967) 12131225. 21. Vinson G. P., Whitehouse B. J. and Goddard C.: The effect of sex and strain of rats on the in oitro response of adrenocortical tissue to ACTH stimulation. J. steroid Biochem. 9 (1978) 553560. 22. Birmingham M. K. and Kurlents E.: Inactivation of ACTH by isolated rat adrenals and inhibition of corticoid formation by adrenocortical hormones. Endocrinology 62 (1958) 4760. 23. Bennett H. P. J., Bullack G., Lowry P., McMartin C. and Peters J.: Fate of corticotrophins in an isolated adrenal cell bioassay and decrease of peptide breakdown by cell purification. Biochem. J. 138 (1974) 185-194. 24. Richardson M. C. and Schulster D.: Corticosteroidogenesis in isolated adrenal cells: effect of adrenocorticotrophic hormone, adenosine-3’,5’-monophosphate and /I-” adrenocorticotrophic hormone diazotized to polya~ilamide. J. Endocr. 55 (1972) 127-139. 25. Vinson G. P., Goddard C. and Whitehouse B. J.: Corticosteroid production in vitro by adrenal tissue from rats with inherited hypothalamic diabetes insipidus (Brattleboro strain). J. steroid Biochem. 9 (1978) 657-665. 26. Vinson G. P., Whitehouse B. J. and Goddard C.: Steroid 17-hydroxylation and androgen production by incubated rat adrenal tissue. J. steroid Biochem. 9 (1978) 677-683.

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27. Braley L. M. and Williams G. H.: Rat adrenal cell sensitivity to angiotensin II, a’-24ACTH, and potassium: a comparative study. Am. J. Physiol. 233 (1977) E402-E406. 28. Douglas J., AguiIera G., Kondo T. and Catt K.: Angiotensin II receptors and aldosterone production in rat adrenal glomerulosa cells. E~~cr~~~ogy 102 (1978) 685-696. 29. Vinson G. P., Bell J. B. G. and Whitehouse B. J.: Production of testosterone and corticosteroids by the rat adrenal gland incubated in vitro and the effects of stimulation with ACTH, LH and FSH. J. steroid Bio&em. 7 (1976) 407-411. 30. Philtips J. G. and Poolsanguan W.: A method to study temporal changes in adrenal activity in relation to sexual status in the female laboratory rat. J. Endocr. 77 (1978) 283-291. 31. Garren L. D., Gill G. N., Masui H. and Walton G. M.: On the mechanism of action of ACTH. Recent Prog. Hormone Res. 27 (1971) 433-478. 32. Vinson G. P. and W~tehouse B. J.: Interpretation of product mass- and specific activity-time curves in rat adrenal tissue incubated in vitro. Acta endocr. Copenh. 61 (1969) 709-719. 33. Vinson G. P. and Whitehouse B. J.: The relationship between corticosteroid biosynthesis from endogenous precursors and from added radioactive precursors by rat adrenal tissue in vitro. The effect of corticotrophin. Acta endocr. Cop&. 61 (1969) 695-708. 34. Tan S. Y. and Mulrow P. J.: Regulation of 18-hydroxydeoxycorticosterone in the rat. Endocrinology 102 (1978) 1113-1117. 35. Goddard C.: Mechanisms for steroid hormone production in the rat adrenal cortex. Ph.D. Thesis, University of London (1979). 36. Sibley C. P., Whitehou~ B. J., Vinson G. P. and Goddard C.: Studies on the distribution of steroid between medium and tissue following incubation of rat adrenals in vitro. J. Endocr., in press. 37. Vinson G. P. and Whitehouse B. J.: Unpublished observations, 38. Koritz S. B. and P&on F. G.: The stimulation in vitro by Ca ‘+, freezing and proteolysis of corticoid production by rat adrenaf tissue. J. Biot. Chem. 234 (1959) 3122-3128.