Molecular and Cellular E~do~rino~~~y 5 (1916) 0 North-Holtand Publishing Company

CHARACTERISTICS

255 --X7

OF THE RESPONSE OF HUMAN ADRENOCORTICAL

CELLS TO ACTH

J. KOLAN~WSKI

and J, CRABBE

Received 25 March 1976

The effects of adrenocorticotrophic hormone (ACTH) on human adrenocortical steroidogenesis were studied in adrenocortical cells which had been isolated from normal and hyperplastic glands by a technique combining trypsin digestion and mechanical dispersion, and incubated in the presence of ACTH or dibutyrgl cyclic AMP fdbcAMP). The response was measured in terms of cyclic AMP, cortisol, corticosterone, 11 -deoxycortisol and cortisone production. A classical sigmoid curve, calculated by non-linear, least square method, related the increase in CAMP production or in steroidogenesis to the log dose of ACTH. For the normal adrenocortical cells, the estimated concentration of ACTH inducing a half-maximal response was approximated 20 pg ACTH,_,4/ml for steroidogenesis, against 437 pgfml for CAMP production. The estimated Pm,, (per 107 cells/ml, on average) was 27 pmol CAMP/~ h and for steroidogenesis (in ng/2 h): 188 for cortisol, 106 for corticosterone, 37 for 1 Ideoxycortisol, and 32 for cortisone. dbcAMP (1.0 mM) stimulated steroidogenesis to a comparable extent. The cells from a hyperplastic adrenal gland exhibited a steroidogenic response to ACTH and dbcAMP which was 2-3 times greater than the response of a similar number of normal adrenocortical cells. Calculated per pmol CAMP generated, the ACTH-stimulated cortisol production by cells from hyperplastic gland was also increased with respect to normal celt response. These data suggest a prolonged effect of ACTH on cortisot biosynthetic pathway beyond the membrane step of cAMP generation.

Keywords:

human adrenal ceils; ACTH; cyclic AMP; steroidogenesis.

The aim of the present study was to evaluate some features of the human adrenocortical cell response to adrenocorticotrophic hormone (ACTS), more specifically to assess the sensitivity of adrenal cells to ACTH and the relationship between cyclic adenosine monophosphate (CAMP) generation and steroidogenesis. To this end, fragments of normal human adrenal cortex were submitted to cell dispersion by Sayers’ technique (Sayers et al., 197 I a,b); cells so isolated were incubated in the presence of ‘ACTH or dibutyryi cyclic AMP (dbcAMP). The response was evaluated in terms of cAMP generation on the one hand, and of cortisol, corticosterone, 1 l255

256

J. Kokznowski,

.J. Crabbt

deoxycortisol and cortisone production. The data indicate that, while cortisol is obviously the major secretory product, a relatively important amount of corticosterone, 1 1-deoxycortisol and cortisone is synthesized during the incubation with ACTH or dbcAMP. Despite the well-known qualitative differences between rat and man when it comes to adrenocortical steroidogenesis, the sensitivity of human adrenal cells to ACTH or to dbcAMP, and the relationship between CAMP generation and steroidogenesis are similar to what has been observed in the rat (Sayers et al., 1971a, b; Beall and Sayers, 1972; Kolanowski et al., 1974). The results obtained with normal human adrenocortical cells were also compared to the response of adrenocortical cells dispersed from a hyperpiastic gland, obtained during surgery for Gushing’s disease. This made it possible to evaluate the mechanism of adrenocortical hyperresponsiveness to ACTH resulting from previous prolonged stimulation with corticotropin (Kolanowski et al., 1975). The results are in keeping with the hypothesis that ACTH influences, in a lasting way the steroidogenie pathway distal to the initial steps involving adenylate cyclase activation, since adrenocortical cells from the hyperplastic gland exhibited an enhanced steroid production for a given amount of CAMP generated, as well as hyperresponsiveness to dbcAMP.

MATERLAL AND METHODS Fragments of human adrenal cortex, considered to be and henceforth referred to as normal, were obtained at surgery for pheochrolnocytoma in 2 patients and in one case of aldosterone-secreting adenoma. In all 3 patients, a normal adrenocortical function in terms of cortisol secretion had been documented before surgery. The hyperplastic glands were obtained during total adrenalectomy for bilateral adrenal hyperplasia in a patient presenting all characteristics of Cushing’s disease. After the removal of adrenal glands, fragments of adrenal cortex (of l-3 g approximately) were immediately immersed in 50 ml ice-cold Krebs-Ringer bicarbonate solution containing 0.2 g/100 ml glucose (KRBG) saturated with 95% O2 and 5% COZ (pH 7.45). The dispersion of adrenocortical cells was performed within 2 h of the removal of gland. The fragments of adrena cortex were trimmed of fat, and 0.7-I g of tissue was cut in small fragments and transferred to a siliconized 50-ml Erlenmeyer flask conta~ing 20 ml of KRBG to which 0.25~100 ml trypsin (TRL, Worth~gton Biochemical Co.) had been added. The solution, kept at ambient temperature, was continuously gassed with a mixture of 95% O2 and 5% CO:!. The following steps, in&ding five consecutive 20.min mechanical dispersions (500 rev./min at 37”C), centrifugation (100 g for 20 min at 4°C) and resuspension in 70 ml KRBG containing bovine serum albumin (BSA, 0.5 g/100 ml), lima bean trypsin inhibitor (LBI, Worthington Biochemical Corporation, 0.8 g/100 ml) and calcium chloride (to bring final calcium concentration to 7.65 mM), were performed according to Sayers et al. (1971)a,b).

Human adrenal cell response to ACTH

251

The resuspended pellets were then kept overnight at 4°C (in 95% 02 and 5% CO2 saturated solution) and submitted to incubation on the following morning. In one experiment, the viability and functional capacity of cells treated this way was verified by assessing the response to ACTH, in terms of CAMP production and steroidogenesis, immediately after cell dispersion and then after an overnight storage at 4°C. Aliquots of cell suspension (0.9 ml) were incubated for 2 h in teflon beakers placed in a Dubnoff shaker (60 oscillations/min), in an atmosphere of 95% O2 and 5% CO* at 37°C. ACTH tetracosapeptide (ACTH1_Zd; Cortrosyn-Organon) or N62’0-dibutyryl-adenosine-3’,5’-monophosphate (dbcAMP; Boehringer Mannheim, GmbH) were added to cell suspension in 0.1 ml vehicle to obtain final concentrations of ACTH ranging from 1 to 1 X lo6 pg/ml (see Results) and for dbcAMP, from 0.01 to IQ.0 mM. The vehicle was an aqueous solution of 0.9 g sodium chloride and 0.1 g BSA/lOO ml, acidified to pH 2.5 with 0.1 N hydrochloric acid. Cell suspensions to which 0.1 ml vehicle only had been added, were incubated in parellel to provide blank values. All incubations were performed in duplicate. Incubation was terminated by adding 5 ml methylene chloride to each beaker; the mixture was then transferred to lo-ml glass tubes, shaken mechanically and centrifuged for 10 min at 1500 g. The aqueous phase (0.5 ml) was used for CAMP determination by a modified Gilman technique (Brown et al., 1974) while 4 ml of methylene chloride extract were evaporated and the residue submitted to thin-layer chromatographic separation of steroids. Cortisol, corticosterone and 11 -deoxycortisol were measured by protein-binding assay (Kolanowski, 1974), as was cortisone (Kolanowski and Ortega, 1976). All results reported further are expressed as net CAMP or steroid production, i.e. the result obtained with cell aliquots incubated with vehicle only was subtracted from that obtained in the presence of ACTH or dbcAMP. The maximal response to ACTH (V,,,) and the concentration of ACTH inducing half-maximal response (A 5~, or the apparent dissociation equilibrium constant at 37°C) as well as the log dose-response curves for CAMP production and steroidogenesis, were computed using a non-linear, least square method (Cleland, 1967).

RESULTS Reliability of experimental conditions Response of freshly dispersed and stored adrenocortical cells to ACTH The response of isolated adrenocortical cells from the same pool was determined immediately after dispersion and after 16 h in the cold. A complete log dose-response curve to ACTH was documented each time, and the results concerning CAMP and cortisol production are summarized in table 1. The data indicate that only an insignificant (about 5%) loss in cell responsiveness occurred after the storage period, quite proportional in fact to the reduction in the number of viable cells (from

J. Kolanowski, J. CrabbP

258

Table 1 Comparison between the response to ACTH of adrenocortical cells incubated immediately after dispersion (fresh cells; 70,000 cells/ml) and after a 16-h storage at 4°C (stored cells; 67,000 cells/ml). The values (+ S.E.M.) reported are estimated Vmax (maximal response) and Aso, calculated from mean net observed values in response to ACTH at concentrations ranging from 1 to 1 X lo4 pg/ml. All incubations were performed in duplicate.

Cortisol

Fresh cells Stored cells

production

CAMP production

Vmax (ng/2 h)

A su !pg ACTH/ml)

Vmax (pmol/2

130.3 f 9.1 123.3 + 8.0

23 f 8 953

29.3 + 1.3 27.8 f 2.1

Net

Net F production lngl-

A 5,) (pg ACTHhl) 367 f 47 423 k 118

CAMPproduction

I pMol

/

h)

es/o--~

,

I 60 Net

B,S lnal

and

120 min

Eproduction

=

250[

0

15

30

60

120

mln

Fig. 1. Time-course experiment with incubated adrenocortical cells dispersed from hyperplastic adrenal glands. The cells were incubated in the presence of 1 X 10” pg ACTHt _24/ml. Incubation time varied from 2.5 min to 2 h. Upper part: net CAMP (o-- --- -0) and cortisol (F, 0~) production; lower part: net production of corticosterone (B, o---e), I ldeoxyand cortisone (E, o -)). Note difference in steroid production scale cortisol (S, AA) between upper and lower parts of graph.

Human adrenal cell response to ACTH

259

70,000 to 67,000 cells/ml of resuspension solution). The small changes in A 5~, i.e., in the reciprocal of affinity to ACTH (Sayers et al., 197la), are not significant. Storage of cell suspension in the cold did not result in any decrease in affinity of the system to ACTH, neither was maximal stimulation modified. Steroid production by cells incubated without ACTH was also unaffected. Therefore all experiments reported further were performed on dispersed cells kept overnight at 4”C, for reasons of convenience. Response

to theu~h~~line Accumulation of CAMP in response to ACTH (table 1) was low, at least when compared to the response of rat adrenal cells (Beal and Sayers, 1972; Kolanowski and CrabbB, 1974). in order to rule out a high phosphodiesterase activity as the reason for this observation, samples of cells were incubated in the presence of 0.3 mM theophylline. In the absence of ACTH, theophylline induced a slight increase in CAMP production and in steroidogenesis, corresponding to the effect exerted by 1 pg/ml of ACTH. On the other hand, theophylline added to samples incubated in the presence of ACTH (100 p&/ml) did not modify the steroidogenic or CAMP response to the peptide. Time-course experiment An attempt was made to evaluate the onset of the response to ACTH, and the relationship between the increase in CAMP generation and steroidogenesis. To this end, aliquots of cell suspension were incubated in the presence of 1 X lo6 pg ACTH/ml for periods of time ranging from 2.5 min to 2 h. (For this experiment the cells from hyperplastic adrenal glands were selected and exposed to a large amount of ACTH, so as to improve the precision of CAMP and steroid measurement, since these cells exhibit hyperresponsiveness.) The results, illustrated in fig. 1, indicate that CAMP generation could be detected before a steroidogenie response to ACTH was demonstrable. While nucleotide generation was clearly stimulated within the first 2.5 min of incubation with ACTH, the increase in steroidogenesis was not detectable until after 5 min of exposure to ACTH. CAMP generation increased rapidly during the first 30 min to slow down thereafter, whereas cortisol, corticosterone, 11-deoxycortisol and cortisone were produced at a constant rate through the 2-h incubation period. Response

to

ACTH

Normal adrenocortical

cells The complete log dose-response curves for CAMP, cortisol, corticosterone, 1 1-deoxycortisol and cortisone when normal dispersed adrenocorticaf cells were incubated with ACTH, are shown in fig. 2. For all parameters studied, a net stimulation occurred in response to ACTH concentrations as low as 1 pg/ml, indicating high sensitivity of adrenal cells to ACTH, and , by inference, little contamination by endogenous ACTH. Despite concomitant increases in CAMP accumulation and in steroidogenesis observed at all ACTH concentrations, the profiles for these two parameters were not parallel. While at low peptide con-

.f. Kolonowski, J. Chbbi

260

‘)

25 20

15 10 5

25

cf

I _ii,;,.,k ,, 1

35 30 25

.r. l

10 lo2 to3 lo4 10s P9 ACTH1+24/mi

E IӦ /zhr, 35

c* l

30

l

25

20

20

15

15

10

10

5

5

f

l

Fig. 2. Cortisol (F), CAMP, corticosterone (B), 1 l-deoxycortisol (S) and cortisone (E) production by isolated human adrenocortical cells in response to ACTHr_s4 at concentrations ranging from 1 to 1 X lo5 pg/ml. The cells (107,00O/ml on average) were dispersed from 3 normal adrenal glands. The points represent mean net observed values (minus steroid or CAMP production by aliquots incubated in absence of ACTH). The log dose-response curves were calculated by the non-linear, least square method.

centrations a definite enhancement of steroidogenesis was associated with only barely increased CAMP production, large doses of ACTH induced an additional increase in the concentration of CAMP without causing further stimulation of steroidogenesis (fig, 2). This results in quite different values of As0 for CAMP production of 437 + 61 (S.E.M.) pg ACTHim while for steroidogenesis, they

Human adrenal cell response

261

to ACTH

ranged from 13 f 3 (S.E.M.) pg ACTH/ml for 11-deoxycortisol, to 23.6 f 5.0 (S.E.M.) for corticosterone. These values of A so are comparable for all steroids measured here, expressing the parellel increase in cortisol, corticosterone, ll-deoxycortisol and cortisone production in response to increasing ACTH concentration (fig. 2).

B

CAMP

F

o---o

lnQ/2

(pMolesf2hr)

hrl

,100

700 250 600

1

.

500

200 liT -

400

150

.

300 100 200 50

100

.

1 PQ ACTH

l-24

.

10 lo2 lo3 lo4 lo5 lo6 PQ ACTH,_24/ml

Im’

S

E (nQ/

2hr)

(nQ

/ 2hr)

250 200 150

100 50

PQ

ACTH

1-2 d

ml

PQ ACTH

l-24’“”

Fig. 3. Net cortisol (F), CAMP, corticosterone (B), lldeoxycortisol (S) and cortisone (E) production by isolated adrenal cells harvested from hyperplastic glands (Gushing’s disease). All points represent t.he mean of duplicate samples; there were 125,000 cells/ml and ACTHl _24 concentrations ranged from I to 1 X lo6 pg/ml. Symbols are as indicated in fig. 2.

262

J. Kolanowski,

J. CrabbP

~y~e~Zasric adrenal G&V The net observed values and calculated log dose-response curves for CAMP production and steroidogenesis obtained with cells dispersed from hyperplastic glands are displayed in fig. 3. Despite a comparable number of cells (125,000 cells/ml vs 107,000 cells/ml on average in the case of normal adrenocortical cells) the production of cortisol, 1 I-deoxycortisol and corticosterone was considerably enhanced. No such amplification was seen however, for cortisone production. Net CAMP production in response to low ACTH concentrations was lower (l-50 pg/ml) or comparable (100-500 pg/ml) to that obtained with normal cells, by contrast, in response to ACTH concentrations greater than 1 X lo3 pg/ml, CAMP production by adrenocortical cells from hyperplastic gland was clearly amplified. Nevertheless, at all ACTH concentrations, the net cortisol production calculated in terms of pmol produced per pmol CAMP generated (fig. 4) was amplified in the case of cells from hyperplastic glands. This suggests an enhanced activity of the steroidogenic pathway beyond the CAMP generation step. The profile of the curve relating cortisol production per pmol generated CAMP to ACTH concentration, was quite similar for both types of adrenocortical cells (fig. 4). It results obviously from the shift to the right of the log dose-response curve for CAMP, for normal cells as well as for those harvested from hyperplastic glands (figs. 2 and 3). The estimated values for maximal responses in terms of CAMP generated and of

1

I aMole>

F /pMole generated c AMP net aroduction I 211r)

300

200

tot

1

10

to* pc,

103

104

105

ACTH, .241n~ I

Fig. 4. Net cortisol (F) production

by isolated human adrenal cells harvested from normal (*A) and hyperplastic (o- - - - - -0) adrenal glands, in response to ACTH1_24 at concentrations ranging from 1 to 1 X lo5 pg/ml. The values are expressed in pmol cortisol produced/ pmol CAMP generated.

263

Human adrenal cell response to ACTH

Table 2 Response to ACTH of human adrenocortical cells dispersed from normal (N; 107,000 cells/ml) and hyperplastic (H; 125,000 cells/ml) adrenal glands. The results were calculated by the nonlinear, least square method (estimates f S.E.M.). They are expressed in terms of maximal net steroid (ng/2 h) and CAMP (pmol/2 h) production (V max) and as ACTH concentration (pg ACTHt_a4/ml) inducing half-maximal response (Aso). CAMP Vmax

11-Deoxycortisol

Corticosterone

Cortisol

N

26.9 * 1 .O

187.9 f

H

87.8 f 3.6

663.4

Cortisone

7.7

105.6 +

4.4

37.0 f

1.5

32.0 f

1.0

f 19.9

199.7 f

6.0

160.5 f

7.5

22.8 +

1.0 4

‘450

N

437

1-I 4466

+61 f 962

20 238

4

24

f

5

13

f

3

23

f 32

80

i 12

62

f 15

265

i:

+

+ 49

different steroids produced, and for Aso for the same parameters, are given in table 2 for normal cells and those harvested from hyperplastic glands. For all parameters studied the cells from hyperplastic gland exhibited an increase in Vmax, or capacity of this response, while the A 5o values were 2-l O-fold higher, suggesting a decrease in affinity or sensitivity to ACTH (Sayers et al., 1971a). It must be stressed, however, that this apparent decrease in affinity results mainly from an additional ste-

Table 3 Response to CAMP of human adrenocortical cells dispersed from normal (N; 107,000 cells/ml, mean values from 3 experiments) and hyperplastic (H; 125,000 cells/ml) adrenal glands. The results are compared to the maximal observed net steroid production in response to ACTH. - __ ..__ Net steroid

production

(ng/2h)

dbcAMP 0.01

concentration 0.1

(mM) 1.0

Maximal response to ACTH 10.0

Cortisol:

N H

64 164

137 431

205 443

171 401

212 705

Corticosterone:

N H

44 48

98 183

114 203

109 214

119 224

11-Deoxycortisol:

N H

6 32

27 117

38 135

45 191

39 173

Cortisone:

N

2 1

9 11

17 16

13 21

33 24

H

264

J. Kohowski,

J. CrabbP

roidogenic response to large doses of ACTH, but the response to the lowest ACTH concentrations was also clearly amplified when compared to control experiments. Response to dbcAhU’ The mean net steroid production by adrenocortical cells incubated in presence of 0.01-10.0 mM dbcAMP is summarized in table 3. For all steroids measured, net production by the cells incubated with 1.0 mM dbcAMP was comparable to the maximal response to ACTH, usually achieved with peptide concentration of 1 X lo3 pg ACTH/ml cell suspension. In response to dbcAMP, the production of cortisol, 1 1-deoxycortisol and corticosterone by cells from hyperplastic glands was 23 times higher than that obtained with a similar number of normal cells. This degree of amplification of the steroidogenic response to dbcAMP was of the same magnitude as the amplification of steroidogenic response to ACTH, calculated per pmol CAMP generated. In terms of cortisone production, the response of cells from hyperplastic glands to dbcAMP was not amplified, as was also the case when ACTH was the stimulus (compare figs. 2 and 3).

DISCUSSION Reliability of experimental conditions The results reported here clearly indicate that adrenocortical cells dispersed from human adrenal cortex by the technique utilized previously in rat and guinea pig (Sayers et al., 1971a, b; Kolanowski et al., 1974), exhibit a normal functional capacity, even after several hours in the cold before the incubation. The latter conclusion is based not only on uniform capacity in terms of maximal CAMP or steroid production, but also on the rapidity of the response to ACTH. The time-lag between addition of ACTH to human adrenal cell suspension and the discernible onset of CAMP accumulation and of steroidogenesis, was quite similar to that observed with incubated rat adrenal cells (Beall and Sayers, 1972; Richardson and Schulster, 1972) or with adrenal gland perfused in situ in dog (Urquhart and Li, 1968) or calf (Edwards et al., 1973). Response of normal human adrenocortical cells to ACTH Relationship behveen CAMP generation and steroidogenesis Since ACTH-induced stimulation of steroidogenesis is associated with a concomitant increase in CAMP generation by adrenocortical cells, and since this cyclic nucleotide is capable of mimicking the physiological effects of ACTH, the hypothesis that CAMP is an obligatory intracellular mediator of ACTH has gained general acceptance. Some observations cast however doubt on this interpretation. For instance: Beall and Sayers (1972) have reported that, at low doses, ACTH can stimulate steroidogenesis without appreciable effect on CAMP production; secondly, large doses of ACTH can en-

Human adrenal cell response to ACTH

265

hance considerably CAMP accumulation without additional stimulation of steroidogenesis (Beall and Sayers 1972; Kolanowski and Crabbe, 1974); theophylline amplifies steroidogenic effects of added CAMP but not those of ACTH (Sayers et al., 1971a); and lastly, CAMP added to ACTH does not modify the steroidogenic response to the peptide (Pearlmutter et al., 1973). Moreover, it has been reported that CAMP is unable to mimic all physiological effects of ACTH (Jaanus et al., 1972). It is evident that the results presented herein cannot settle this controversy, neither is it possible on the basis of our data to disprove or confirm definitely the concept of ‘second messenger’. It is noteworthy, however, that at all ACTH concentrations the steroidogenic response was associated with enhanced CAMP generation, and that the latter phenomenon preceded the steroidogenic response to ACTH. results obtained with theophylline, which is known to inhibit phosphodiesterase activity, allow only the conclusion that the observed CAMP accumulation in response to ACTH probably approximates nucleotide generation. CAMP generation by human adrenal cells upon ACTH stimulation was low when compared to the response of rat adrenocortical cells, and this despite a high calcium concentration known to increase the adenylate cyclase activity (Sayers et al., 1971b, 1972; Jaanus et al., 1972). This represents another feature differentiating human and rat adrenal cell response to ACTH, besides the qualitative differences in steroidogenesis. On the other hand, the sensitivity of human adrenal cells to dbcAMP is comparable to what has been observed in the rat (Sayers et al., 1971a; Kolanowski and Crabbe, 1974), and in both species this nucleotide can reproduce the maximal steroidogenic response achieved with ACTH. Injluence of ACTH on steroidogenesis The present study indicates that ACTH stimulates in similar manner cortisol, corticosterone and 1 1-deoxycortisol secretions by human adrenocortical cells, and evidence is provided for cortisone synthesis by adrenal cortex. The latter observation confirms previous reports (Jenkins, 1965; Dazord et al., 1972). The cortisone-to-cortisol ratio in peripheral venous blood is much lower than that observed with incubated adrenocortical cells, prob-ably because of a much higher metabolic clearance rate for cortisone than for cortisol (Dazord et al., 1972). The same explanation can be offered for a relatively important 1 1-deoxycortisol production by human adrenal cells contrasting with very low peripheral blood levels (Kolanowski, 1974). Corticosterone-to-cortisol production ratio was also much larger than could be expected from corticosterone: cortisol ratio in peripheral blood (Kolanowski, 1974), confirming in man the results obtained with dog and calf adrenals perfused in situ with ACTH (Edwards et al., 1973). Hyperresponsiveness of cells from hyperplastic adrenal gland As has been reported (Kolanowski et al. 1975), after stimulation with ACTH the responsiveness of the human adrenal cortex to this peptide is increased. Since bi-

266

J. Kolanowski, J. Oahbt

lateral adrenal hyperplasia is understood as resulting from prolonged corticotropin hypersecretion, the cells harvested from hyperplastic adrenals were used to study the mechanism of this prolonged effect of ACTH. It has been possible to reproduce in vitro the ACTH-induced hyperresponsiveness, since the steroid production by these cells in response to ACTH was clearly enhanced when compared to the response of normal glands. The conclusion was formulated on the basis of in vivo studies that ACTH-induced adrenocortical hyperresponsiveness cannot result only from increased adrenal weight (Kolanowski et al., 1975). The present results confirm this interpretation, since for a given number of cells, the steroidogenic response was 2-3 times greater in the case of cells from a hyperplastic gland as compared to normal adrenocortical cells. No such amplification was observed for cortisone production, however, which is in keeping with reports indicating normal blood cortisone levels in patients with hypercorticism (Dazord et al., 1972; Kolanowski and Ortega, 1976). The study with isolated adrenocortical cells harvested from control and ACTHtreated guinea pigs led us (Kolanowski and Crabbe, 1974) to conclude that the mechanism involved in this prolonged effect of ACTH on steroidogenesis operates probably at sites distal to CAMP generation. The latter conclusion is further supported by the present study, since for a given amount of CAMP generated the production of cortisol was amplified, and this at all ACTH concentrations tested (fig. 4). Moreover, the response of cells from hyperplastic glands to dbcAMP was also clearly enhanced. Even from a theoretical point of view it would be difficult to assume that modifications in CAMP production by adrenal cells can represent an essential mechanism involved in what has been called a potentiation phenomenon (Kolanowski et al., 1975). At first, the role of CAMP is probably limited to activation of pre-formed enzyme protein(s) (Koritz and Wiesner, 1975; Schulster and Jenner, 1975) responsible for free cholesterol accumulation within the mitochondria with a consequently increased pregnenolone formation (Mahafee et al., 1974). On the other hand, no direct relationship exists between the CAMP content within adrenal cells and the magnitude of ACTH-induced steroidogenesis (Sayers et al., 1971a; Pearlmutter et al., 1973). By contrast, it can be assumed that ACTH exerts a lasting effect somewhere along the post-pregnenolone biosynthetic pathway of adrenocortical steroidogenesis. In cultured human fetal adrenal cells (Kan et al., 1975), or mouse adrenocortical tumor cells (Kowal, 1969), exposed chronically to ACTH or CAMP, pregnenolone and progesterone conversion to final steroids was increased. In both studies, there was evidence for prolonged stimulation of 1 lo-hydroxylation efficiency, in keeping with our observation on isolated adrenal cells from guinea pigs treated in vivo with ACTH (Kolanowski and Crabbe, 1974). A prolonged effect of ACTH on 17a-hydroxylase has also been reported (Slaga and Krum, 1973; Kan et al., 1975). Further investigation of effects exerted by ACTH on enzymes involved in biosynthesis of cortisol past the pregnenolone stage is needed to elucidate the mechanism of this prolonged action of ACTH.

Human adrenal cell response to ACTH

261

ACKNOWLEDGEMENTS This work was supported by Grant no. 3.4512.75 from the Fonds de la Recherthe Scientifique Medicale (Belgium). The authors wish to thank Prof. G. Alexandre and Dr. Verbeken-Soyez for the access to clinical material, Dr. Demo1 from Organon-Belge who generously supplied (Cortrosyn), Mrs N. Ortega for excellent techthe synthetic /&_a4-tetracosapeptide nical assistance and Mrs C. Schoonjans for her secretarial help.

REFERENCES Beail, R.J. and Sayers, G. (1972) Arch. Biochem. Biophys. 148,70-76. Brown, B.L., Albano, J.D.M., Barnes, G.D. and Ekins, R.P. (1974) Biochem. Sot. Trans. 2, 12. Cieland, W.W. (1967) Adv. Enzymol. 29, I-32. Dazord, A., Saez, J. and Bertrand, J. (1972) 3. Clin. Endocrinol. Metab. 35, 24 --34. Edwards, A.V., Hardy, R.N. and Malinowska, K.M. (1974) 3. Physiol. 239,477-498. Jaanus, S.D., Carchman, R.A. and Rubin, R.P. (1972) Endocrinology 91, 887-895. Jenkins, J.S. (1965) J. Clin. Endocrinol. Metab. 25, 649-654. Kan, K., Caspin, S. and Solomon, S. (1975) Abstr. Vol. 57th Ann. Meet. Endocrine Sot. p. 57. Kolanowski, J. (1974) J. Steroid Biochem. 5,55-64. Kolanowski, J. and Crabbk, J. (1974) J. Steroid Biochem. 5, 310. Kolanowski, J., Ortega, N. and Crabbe, J. (1974) Ann. Endocrinol. (Paris) 35,501-507. Kolanowski, J., Pizarro, M.A. and Crabbe, J. (1975) J. Clin. Endocrinol. Metab. 41,453-465. Kolanowski, J. and Ortega, N. (1976) Ann. Endocrinol. (Paris) (in press). Koritz, S.B. and Wiesner, R. (1975) Proc. Sot. Exp. Biol. Med. 149, 779-781. Kowal, J. (1969) Biochemistry 8, 1821- 1831. Mahaffee, D., Reitz, R.C. and Ney, R.L. (1974) J. Biol. Chem. 249,227-233. Pearlmutter, AX., Rapino, E. and Saffran, M. (1973) Endocrinology 92,679~686. Richardson, MC. and Schulster, D. (1972) J. Endocrinol. 55, 127-139. Sayers, G., Beall, R.J. and Seelig, S. (1972) Science 175, 1131-l 133. Sayers, G., Ma, R.M. and Giordano, N.D. (1971a) Proc. Sot. Exp. Biol. Med. 136, 619-622. Sayers, G., Swallow, R.L. and Giordano, N.D. (1971b) Endocrinology 88, 1063-1068. Schulster, D. and Jenner, C. (1975) 3. Steroid Biochem. 6, 389-394. Slaga, T.J. and Krum, A.A. (1973) Endocrinology 93,517-526. Urquhart, J. and Li, CC. (1968) Am. J. Physiol 214, 73-85,