polyhormonal regulation of avian and mammalian corticosteroidogenesis in vitro

Camp. Biochem. PhJrriol. Vol. 88A, No. I, pp. 131-140, 1987 0300-9629187 $3.00 -t 0.00 Printed in Great Britain lc 1987 Pergamon Journals Ltd

POLYHORMONAL REGULATION OF AVIAN AND MAMMALIAN CORTICOSTEROIDOGEN~SIS

IN VITRO

Rocco V. CARSIA, CoLm G. WANES and SASHA MALAMED

department of Animat Sciences, Rutgers University (R.V.C., C.G.S.), New Brunswick, New Jersey 08903, USA. Tel.: 201-932-9095 and Department of Anatomy, UMDNJ-Robert Wood Johnson Medicat

School (S.M.), Piscataway, New Jersey 08854, USA. Tel.: 251-463-4527

(Received S January 1987)

Abstract-l. The combined actions of ACTH, corticosterone and prolactin (PRL) in the acute regulation of corticosteroidogenesis were investigated using isolated adrenocortical cells from intact and hypophysectomized (hypox) rats (Rattus norvegicus) and from intact male domestic fowl (GuNus g&s domesticus).

2. Exogenous corticosterone suppressed to about 50% ACTH-induced corticosterone production of cells from either species. This suppression, in part, was due to ~orticosteron~ degradation.

3. oPRL, in the presence or absence of ACTH, raised corticosterone production of hypox rat cells, but not intact rat and domestic fowl cells.

4. In addition, oPRL counteracted the corticosterone-induced suppression of net ACTH-stimulated corticosterone production of hypox rat and intact domestic fowl cells, but not intact rat cells.

5. The potency of oPRL with domestic fowl cells was 4 times that with hypox rat cells. 6. Furthermore, in domestic fowl cells, the effect of oPRL was Ca2+-dependent.

Prolactin (PRL) and end product glucoeorticoids as well as ACTH can regulate glucocorticoid production. Results from experiments in uiuo (Mann et nl., 1977; Ogle and Kitay, 1979; Colby, 1979) and irz vitro (Advis and Ojeda, 1978; Giickman et al., 1979) suggest that PRL can stimulate adrenal steroid secretion and facilitate ACTH-induced corticosterone secretion, In addition, other experiments in viuo (Black et al., 1961; Hill and Singer, 1968) and in vitro (Peron et al., 1960; Bakker and DeWied, 1961; Fekete and Gorog, 1963; Saito et al., 1979; Car&a and Malamed, 1979, 1983; Carsia et al., 1983, 1984) have demonstrated that end product glucocorticoids can suppress adrenocortical glucocorticoid secretion, 1~ t&a, all these and probably other factors operate simultaneously to regulate steroidogenesis. However, the acute net effect of the actions of these agents on the adrenocortical cell per se cannot be predicted from the evidence of their separate actions. Thus, previous studies have permitted few conclusions about possible synergistic or exclusionary actions of these agents in combination at the cellular level.

Accordingly, in the present study, we tested the effect of various combinations of ACTH, ovine prolactin (oPRL) and exogenous corticosterone on endogenous corticosterone production by adrenocortical cells isolated from intact and hypox rats and from intact domestic fowl. The data presented here confirm previous evidence that exogenous corticosterone is a suppressant of net corticosterone production, and that PRL counteracts the suppressive action of corti-

*This work was supported by New Jersey American Heart Association Ch‘apters, Lhrited States Department of Agriculture (85~CRCR-I-1846) and the New Jersey Agricultural Experiment Station.

costerone (Carsia and Malamed, 1979, 1983; Carsia et al., 1984). In addition, we present new evidence indicating that this acute effect of PRL on isolated adrenocortical cells is Ca*+-dependent. Furthermore, we present new evidence that suggests that ACTH, in addition to inducing ~orticosteroidogen~sis, can also act to destroy exogenous corticosterone. Some of the combined effects of ACTH, PRL and exogenous corticosterone on the acute regulation of adrenocortical cell function were different in domestic fowl and rat adrenocorticai cells.

MATERIALS AND METHODS

Animals

Intact or 4-day-hypox male SpragueDawley rats (255-355 g; Charles River Breeding Laboratories, Wilming ton, MA) were maintained in a temperature (22”C), light (14-hr light, IO-hr dark photocycle) and humidity-controlled room and were provided feed (Forrnulab 5558, Ralston Purina, St Louis, MO) and water ad iibitum. Sexually mature White Leghorn roosters were fed a standard com- mercial diet and water ad libitutn and were individualiy housed at 22°C on a 16-hr light, 8-hr dark photocycle.

Collection of adrenaf glands

Rats were anesthetized with ether and killed by sectioning the abdominal aorta. White Leghorn roosters were killed by decapitation. The adrenals were removed, freed of adhering fat and connective tissue, and diced into pieces (about 2 mm’).

Adrenocortical cell isolation and j~cubatio~

The standard medium for cell isolation and incubation was Krebs-Ringer-HEPES buffer 124.2 mM HEPES (N-2-hydroxyeth$piperazine-iV’-2-ethane sulfonic acid), 118.5 mM NaCI, 4.75 mM KCl, 2.54 mM CaCl,, 1.25 mM KH,PO,, 1.25 mM MgS0,.7H,O, 1 I. 1 mM glucose], pH 7.5. Adrenocortical cells were isolated as previously described (Sayers, 1977) except that 0.2% collagenase (Type

131

132 Rocco V. CARSIA et al.

I; Sigma Chemicai Co.) and 0.01% lima bean trypsin inhibitor Worthington Biochemical Corp.) were used in- stead of 0.25% trypsin {Carsia et al., 1984). As previously reported (Malamed et al., 1970), few medullary cells ap- peared in the rat cell suspensions and zona fasciculata cells predominated. However, when domestic fowl adrenal tissue was used, at least 40% of the cells were medullary cells, whereas 60% were adrenocortical cells. Only adrenocortical cells were counted; these cells were easily identified in the light microscope because they contained many lipid drop- lets. The light microscopic identification of domestic fowl adrenocortical cells has been verified by electron microscopy (Carsia et al., 1985).

For incubation, additions to the standard ceil medium were 5.1 mM CaCl, (7.64mM final concentration), 0.5% BSA (Fraction V; Sigma Chemical Co.), 0.01% lima bean trypsin inhibitor and various concentrations of the following agents: ACTH-(l-24) (Cortrosyn; Organon, Inc.), oPRL (NIH SIO), and corticosterone. Prior to addition to cell suspensions, these agents were prepared in the following manner: ACTH was dissolved in a solution of 0.9% NaCl, and the pH adjusted to 2.6 with 1 .O N HCl, containing 0.1% BSA. oPRL was dissolved in the standard medium. Exogenous corticosterone was made up in the standard medium, including 0.8% ethanol. The final ethanol concentration in the cell suspensions did not exceed 0.04%. The incubation volumes (90% of the incubation volume was cell suspension, IO%, a solution containing various concentrations of agents) were 250 ,~l to 1.0 ml. Cells plus agents were incubated at either 37°C (rat cell suspensions) or 40°C (fowl cell suspensions) for 2 hr in a Dubnoff metabolic shaking water bath at 66-80 oscillations~min. After incubation, cell suspensions were boiled for 3 min and then frozen ( - 20°C) until assayed for total corticosterone. In each experiment, at least 89% of the cells were viable after incubation, as indicated by trypan blue dye exclusion (Liotta and Krieger, 1975).

Radioimmunoassay for corticosterone

Corticosterone, the major glucocorticoid secreted by rat adrenocortical cells (Sayers, 1977) and domestic fowl adre-

1

300 No corticostsrone T

nal tissue, in vitro (DeRoos, 1969; Nakamura and Tanabe, 1973) was measured by a modification of the radioimmunoassay procedure of Roy el al. (1974) using specific antibody (Miles Research Products). Cross-reactivities with the major Sa-degradation metabolites of corticosterone, 114, 2I-dihydroxypregn-Sa-ane-3,20-dione (5x-dihydro- corticosterone or 5a-DHB) and 3a, 1 la, 21-trihydroxy- pregn-5a-ane-20-one (38, 5a-tetrahydrocorticosterone or 38, Sa-THB) were 8.4% and 0.02%, respectively. As little as 0.1 ng corticosterone/ml could be detected. Radio- immunoassay of aliquots of the same pooled cell suspensions (performed with each radioimmunoassay) showed intra-assay and interassay coefficients of variation of 6. I % and 9,3%, respectively.

Analysis qf data

In an incubation to which exogenous corticosterone was added, the net corticosterone con~ntration at the end of incubation was determined by subtracting the concentration of exogenous corticosterone from the total concentration of corticosterone after incubation.

Data shown are the means f SEs of nine incubation replicates (three incubation replicates from each of three experiments). Analysis of variance (subsamples within sam- ples) followed by the Studentized Range test (Snedecor and Cochran, 1967) were used to analyze the data; means were deemed significantly different when P I 0.05.

RESULTS

Effects of ACTH, oPRL and exogenous corf~~ostero~e on adrenocortical cells from mutant rats

Figure 1 shows the effects of the combined actions of ACTH, oPRL and exogenous corticosterone on corticosterone production by adrenocortical cells isolated from intact rats. ACTH induced corticosterone production in a concentration-dependent manner. Although exogenous corticosterone did not suppress corticosterone production in the absence of ACTH,

Corticosterone (O.U3,ug/ml!

o No ACTH A 6.3 pgfml ACTH CI 25.0 pglml ACTH v 100.0 pglml ACTH

0 lOOO.Opg/ml ACTH

Ovine prolactin ( ng/ml 1

Fig. 1. Evidence that exogenous corticosterone suppresses ACTH-induced corticosterone production and that PRL does not affect corticosterone production by adrenocortical cells isolated from intact rats. Adrenocortical cells (25,000 cells/ml) were incubated with various concentrations of ACTH and oPRL in the presence or absence of exogenous corticosterone for 2 hr. Each symbol represents the mean of net corticosterone values from nine cell suspensions (three cell suspensions from each of three experiments).

SEs are represented by bars.

Polyhormonal regulation of corticosteroidogenesis 133

it did suppress corticosterone production induced by various concentrations of ACTH by about 50% (compare left and right panels). In contrast to the stimulatory effect of ACTH and the suppressive effect of exogenous corticosterone, oPRL had no effect on corticosterone production.

Effects of ACTH, oPRL and exogenous corticosterone on adrenocortical cells from hypox rats

Figures 2 and 3 show the effects of the combined actions of ACTH, oPRL and exogenous corticosterone on corticosterone production by adrenocortical cells isolated from 4-day hypox rats. Hypophysectomy reduced basal corticosterone production from 32.7 ng/ml to 7.3 t&ml (compare Fig. 1, Ieft panel with Figs 2 and 3), and corticosterone production induced by ACTH in the absence of oPRL and exogenous corticosterone (a 75% reduction; compare the scales of the ordinates of Figs 1,2 and 3). These results confirm earlier work (Sayers and Beall, 1973; Holmes ec al., 1980). As with cells from intact rats, exogenous corticosterone did not affect basal corticosterone production but it did suppress ACTH-induced corticosterone production of cells from hypox rats (Fig. 3). In contrast, oPRL had different effects on cells from intact and hypox rats: oPRL had no effect on cells from intact rats (Fig. I), but it did affect cells from hypox rats (Figs 2 and 3). oPRL stimulated both basal and ACTH- induced ~orticosterone production in a concentration-dependent manner (Figs 2 and 3). Moreover, oPRL acted more than additiveiy with ACTH to stimulate corticosterone production: maximal net corticosterone production attained with ACTH plus

o No ACTH; no corticosterone 0 Corticosterone (0.52 pgfml I L) ACTH (1.O q/ml 1 A ACTH 11.0 nglm! 1 +

corticosterone (0.52~g/ml) T

Ovine pro&tin ( ng/ml 1

Fig. 3. Evidence that exogenous corticosterone induces suppression and PRL counteracts corticosterone-induced suppression of ACTH-induced corticosterone production by adrenocortical cells isolated from hypox rats. Adre- nocortical ceils (25,~ ceils/ml) were incubated with various concentrations of oPRL with or without ACTH and with or without exogenous corticosterone for 2 hr. Each symbol represents the mean of net corticosterone values from nine cell suspensions (three cell suspensions from each of three

experiments). SEs are represented by bars.

I, -

ONO ACTH 140 A63pg/ml ACTH

o 25.0 pg/ml ACTH

0 100.0 pg/ml ACTH 120 0 1000.0 pg/ml ACTH /’

z ;-,lOO - c

z 80- ,o "

i

Ovine prolactin ( nglmt 1

Fig. 2. Evidence that PRL, in the presence or absence of ACTH, stimulates corticosterone production by adrenocortical cells isolated from hypox rats. Adrenocortical cells (25,000 cells/ml) were incubated with various concentrations of ACTH and oPRL for 2 hr. Each symbol represents the mean of corticosterone values from nine cell suspensions (three cell suspensions from each of three

experiments). SEs are represented by bars.

oPRL (118.9 ng/ml) was 1.7 times the sum of the net maximal corticosterone production values (69.2 ng/ml) attained with ACTH (51.5 ng/ml) alone and oPRL (17.7 ngjml) alone (Table 1). In addition, oPRL counteracted the suppressive effect of exogenous corticosterone on ACTH-induced corticosterone production (Fig. 3). Estimates of the half- maximal effective dose (ED,) values of oPRL for counteraction of the suppressive effect of hypophysectomy (218.7 f 22.6 ng/ml; mean & SE) and for counteracting corticosterone-induced suppression (221.5 k 18.3 ng/ml; mean f SE) of cells from hypox rats were similar.

Effects of ACTH, oPRL and exogenous corticosterone on adrenocortical cells from intact domestic fowl

Our previous work showed that corticosterone- induced suppression of endogenous corticosterone production occurred in cells isolated from the domestic fowl as well as in cells from the rat (Carsia et aZ., 1983, 1984). Acordingly, in the present study, we evaluated the influence of combinations of ACTH, oPRL and exogenous corticosterone on corticosterone production by domestic fowl cells. Figure 4 shows that, as seen with rat cells, oPRL did not affect basal corticosterone production or corticosterone production induced by ACTH. However, in contrast to the effect seen with cells from intact rats, oPRL (0.5 pg/ml) completely counteracted the

134 Rocco V. CA~WA et al.

Gorticosterone 10.47~.q/ml)

Ovine prolactin (0.5O~g/ml

CorticosteronelO.47~g/mll

ovine prola~t~n(O5O~g/ml)

0 1OL 106

ACTH f pglmt )

Fig. 4. Evidence that exogenous corticosterone induces suppression and PRL counteracts corticosterone-indu~d suppression of ACTS-induced corticosterone pr~uct~o~ by adrenocorticat cells isolated from intact domestic fowl. Adrenocortical cells (ISO, cells/ml) were incubated with or without ACTH, with or without oPRL and with or without exogenous corticosterone for 2 hr. Each column represents the mean of net corticosterone values from nine cell suspensions (three cell suspensions from each of three experiments). SEs are represented by bars on the columns.

suppressive effect of exogenous corticosterone on ACTH-jndu~d ~orticosterone productjo~ by cells from intact roosters.

As demonstrated with hypox rat cells, the effect of oPRL with rooster cells was con~ntration-dependent (Fig. 5). However, the ED, value of oPRL with rooster cells (55.3 _t 7.7 ng/mi; mean k SE) was about

=. o No ACTH, no corticosterone * Corticosterone 10.50 ,q/ml)

A ACTH (1.0 p~ug/ml) * ACW + corticosterone

Ovine prolixtin ( ng/ml)

Fig. 5. Evidence that exogenous ~rticosterane induces suppression and PRL counteracts corticosterone-jnduced suppression of ACTH-induced corticosterone production by isolated domestic fowl adre~~ortical cells. Cells (250,000 cells/ml) were incubated with various concentrations of oPRL with or without a maximal steroidogenic concentration of ACTH and with or without exogenous corticosterone for 2 hr. Each symbol represents the mean of net corticosterone values from nine ceti suspensions (three cell suspensions from each of three experiments). SEs are

represented by bars.

l/4 those with rat celts. Thus, compared to rat ceils, domestic fowl cells did not require the effect of hypophysectomy hefore isoiation to show the acute action of oPRL on co~icosterone-induced suppression of ACTH-induced corticosterone produ~tjon and were more sensitive to oPRL.

Domestic fowl cells were used in subsequent ex-

102 IO3 loL lo5 lo6 ACTH i pgiml I

0

Fig. 6. Evidence that the suppressive effect of exogenous corticosterone on ACT&induced cellular corticosterone production is bimodal and dependent on the concentration of ACTW. Isolated domestic fowl adrenocortical cells (2SO,O~ cells/ml) were incubate with various concentrations of ACTH, with or without exogenous corticosterone and with (right pune() or without (leji panel) oPRL for 2 hr. Each symbol represents the mean of net cortieosterone values from nine cell suspensions (three ceil suspensions

from each of three expe~ments). SEs are represented by bars.


periments because hypophysectomy was not required to demonstrate the effects of oPRL and thus, inter- pretations of results were not encumbered with possible complications resulting from the operation. Figure 6 shows further evidence that exogenous corticosterone lowers corticosterone production, whereas PRL enhances corticosterone production (in the presence of corticosterone).

Exogenous corticosterone did not change net corticosterone production in the absence of ACTH. How- ever, with concentrations of ACTH that were non- steroidogenic in the absence of exogenous corticosterone (10-1000 pg/ml), exogenous corticosterone caused a decrease in net corticosterone production such that the values were negative. The magnitude of the decrease in net corticosterone production was dependent on the concentration of ACTH and on the concentration of exogenous corticosterone. Minimal values of net corticosterone were attained with 100 pg ACTH/ml. In addition, the greater the exogenous corticosterone concentration the more negative the values of net corticosterone production. The negative net corticosterone values indicate a disappearance of immunoassayable ~orticosterone and suggest that corticosterone was degraded to metabolites that were not detected in the radioimmunoassay. In the presence of steroidogenic concentrations of ACTH (greater than 1000 pg/ml), exogenous corticosterone suppressed net corticosterone production (Fig. 6, left panel). This effect of exogenous corticosterone was concentration dependent: with a low concentration of exogenous corticosterone (0.26 pg/ml) there was suppression of net corticosterone production but this suppression was counteracted by high concentrations of ACTH. In contrast, a high concentration of exogenous corticosterone (0.53 flgiml) suppressed net corticosterone production induced by all steroidogenic concentrations of ACTH; with maximal steroidogenic concentrations of ACTH, suppression was about 50%.

In contrast to the suppressive effect of corticosterone on steroidogenesis, the effect of oPRL was positive. oPRL did not alter ACTH-induced corticosterone production (Fig. 6, right panel), but it did reduce the suppressive effect of exogenous corti- casterone. oPRL (0.5 pgg/ml) abolished the effect of the low suppressive concentration of exogenous corticosterone (0.26 pg/ml) and almost completely counteracted the effect of the high suppressive concentration of exogenous corticosterone (0.53 pg/ml).

Figure 7 shows the results from experiments with rooster cells incubated with various concentrations of Ca”+. Some of the effects of ACTH, oPRL and exogenous corticosterone on corticosteroidogenesis were Ca”+-dependent. In the absence of ACTH (Fig. 7, lower panel), Ca*+ stimulated corticosterone production in a concentration-dependent manner. Maximal Ca’+-stimulated corticosterone production (21.3 ng/ml) occurred with 5 mM CaZ+ (Table 1). [This was 6.2% of the maximal Ca’+-dependent corticosterone production in the presence of ACTH (345.9 ng/ml).] In the absence of ACTH, no differences in Ca*+ -stimulated corticosterone production were detectable when exogenous corticosterone, oPRL or exogenous corticosterone plus oPRL were added to the incubation medium.

boo ACTH ( l.o.ug/ml) T

l Corticosterone (O.LBjhg/ml

A Corticosterone + ovine prolactir

p 30 t

No ACTH d T

0 0 0.10 0.25 0.50 1.00 2.50 5.00 lo.00

CaC12 trnM)

Fig. 7. Evidence that some aspects of the effects of ACTH, PRL and exogenous corticosterone on cellular corticosterone production are Ca 2+-dependent. Isolated domestic fowl adrenocortical cells (250,000 cells/ml) were incubated with various concentrations of Ca2+ with or without a maximal steroidogenic concentration of ACTH, with or without oPRL and with or without exogenous corticosterone for 2 hr. Each symbol represents the mean of net corticosterone values from nine cell suspensions (three cell suspensions from each of three experiments). SEs are repre-

sented by bars.

As in the absence of ACTH, Ca2+ also enhanced corticosterone production induced by a maximal steroidogenic concentration of ACTH (1 .O pg/ml) (Fig. 7 and Table 1). In the absence of Ca*+ there was modest corticosterone production (8 1.2 ng/ml) which was 35 times the production in the absence of ACTH (2.3 ng/ml). However, with 5.0 mM Ca’+, ACTH- induced corticosterone production (345.9 ng/ml) was 4.3 times greater than in the absence of Ca*+ (81.2 ngiml). Thus, Ca*+ enhanced the maxima1 level of corticosteroidogenesis that could be stimulated by ACTH alone. Conversely, ACTH enhanced the maximal level of corticosterone production that could be stimulated by Caz+ alone. Also, maximal corticosterone production in the presence of 5.0 mM Ca*+ and 1 .O pgg/ml ACTH in combination (345.9 ng/ml) was 3.4 times greater than the additive values of maximal corticosterone production (102.5 ng/ml) by either agent alone (Table 1). There was additional evidence of ACTH and Ca2+ synergism: The ED,, value of Ca2+ for stimulating corticosterone production in the absence of ACTH was 7-8 times the value in the presence of ACTH f2.14 + 0.77mM vs 0.28 + 0.04 mM (mean + SE)]. Thus, ACTH enhanced cellular sensitivity to Ca2+.

Ca2+ also affected steroidogenesis induced by


Table I. Synergistic effect of steroidogenic agents on isolated adrenocortical cells

Adrenocortical cells

Steroidogenic agents

Net Bmax* (n&ml)

Additive net Bmax for each agent acting

separately (ng/mB Synergistic effect (O/o)+

Hypox rat

Domestic fowl

No ACTH, no oPRL 6.1 & 3.6 ACTH (I .O og/ml) 51.5 t_ 8,? oPRL (I .O pg/ml) 17.7 _+ 3.2

ACTH (1 .O ng/mlf + oPRL (1.0 #g/ml) 118.94 13.1 69.2 _t 9.3 171.8 & 16.2

No ACTH, no Ca*+ 2.3f2.1 ACTH (1 .O flg:ml) 81.2 i: 23.9 Ca*” (5.0 mM) 21.3 i4.9

ACTH (1 .O &ml) + Car+ (5.0 mM) 345.9 f 25.8 102.5 f 22.4 337.5 f 32.3

*The net Bmax values are the maximal corticosterone production values for each agent minus the basal values produced in the absence of steroidogenic agents during a 2-hr incubation. These values are depicted in Fig. 2 (hypox rat cells) and Fig. 7 (domestic fowl cells). Each value is the mean & SE from the data of three experiments.

tThe synergistic effect is the ratio of net Bmax value induced by two steroidogenic agents acting together to the additive net Bmax value induced by two steroidogenic agents acting separately x 100% (mean f SE). The net Bmax value induced by two steroidogenic agents acting together vs the additive net Bmax value induced bv two steroidoeenic agents acting separately are significantly different (P < 0.05).

ACTH in combination with oPRL and exogenous corticosterone (Fig. 7, upper panel). On the one hand, oPRL alone did not affect ACTH-induced corticosterone production, regardless of Ca*+ concentration. However, oPRL did counteract corticosterone-induced suppression of ACTH-induced corticosterone production, and this effect was Ca’+- dependent. Significant effect of oPRL was apparent at OSOmM Ca*+ and corticosterone-induced suppression was totally abolished with 2.50mM Ca*+.

DISClJSSlON

The results of these studies have provided new info~ation on the combined actions of ACTH, PRL and exogenous glu~ocorti~oids in the acute regulation of endogenous glucocorticoid production:

(1) PRL raises corticosterone production of hypox rat cells and counteracts the corticosterone-induced inhibition of steroidogenesis in cells from hypox rats and intact fowl

(2) Cat+, in the presence or absence of ACTH stimulates corticosterone production in domestic fowl cells and is needed for the actions of PRL.

(3) ACTH and PRL act synergistically to stimulate corticosterone production of cells from hypox rats.

In con&-mation of our previous work (Carsia and Malamed, 1979, 1983; Carsia er al., 1983, 1984), exogenous corticosterone, the natural end-product glucocorti~oid produced by the rat and the domestic fowl adrenal gland (Sayers, 1977; DeRoos, 1969; Nakamura and Tanabe, 1973) suppressed endogenous, ACTH-induced corticosterone production of isolated rat and domestic fowl adrenocortical cells by about 50% after 2 hr of incubation. In addition, we present new evidence indicating that exogenous corticosterone acutely suppressed ACTH-induced corticosterone production of cells isolated from hypox

rats (Fig. 3). The data from hypox rat cells suggest that the suppressive effect of exogenous corticosterone was not dependent on the residual effects of pituita~ factors.

In contrast to the uniform suppressive effect of exogenous corticosterone, the action of oPRL was different with cells from intact rats, hypox rats and intact domestic fowl. For example, oPRL did not stimulate basal or ACTH-induced corticosterone synthesis of cells from intact rats and roosters (Figs 1,4, 5, 6 and 7). However, oPRL did stimulate both basal and ACTH-induced corticosterone production of cells from hypox rats in a concentration-dependent manner {Figs 2 and 3). Moreover, oPRL acted with ACTH in a more than additive manner to stimulate corticosterone production by hypox rat cells (see Fig. 2 and data in Results).

In addition to the differences in PRL stimulation of corticosterone production among the three animal groups, there were also differences in PRL counteraction of the suppressive effects of exogenous corticosterone on corticosteroidogenesis. Although oPRL did not affect the suppressive action of exogenous corticosterone on cells from intact rats, it did acutely counteract the effect of exogenous corticosterone on cells from hypox rats (Fig. 3). These data are the first to show an acute, concentration-dependent action of oPRL on isolated adrenocortica’l cells from hypox rats; they are the first cellular evidence indicating the requirement for hypophysectomy in order to demonstrate a net positive effect of PRL on rat corticosteroidogenesis (Ogle and Kitay, 1979; Colby, 1979).

In contrast to rat adrenocortical cells, domestic fowl cells did not require the effect of hypophysectomy before isolation to show the action of oPRL in counteracting the suppressive effect of exogenous corticosterone (Figs 4-7). There were also species differences for the ED, values of oPRL in vitro: the EDS0 value for cells from hypox rats (about


220 ngfml) was 4 times greater than the value for cells from domestic fowl (about 55 ngjml). Thus, domestic fowl cells were considerably more sensitive to oPRL than were rat cells. Taken together, the data showing species differences for the hypophysectomy requirement and for the ED, values of oPRL in oitro suggest that the acute regulation of corticosteroidogenesis of the domestic fowl is more dependent on PRL than that of the rat.

Evidence from studies in vitro indicates that Ca*+ can stimulate mammalian corticosteroidogenesis in the presence (Sayers et al., 1972; Haskar and P&on, 1973; Perchellet and Sharma, 1979) or absence (Neher and Milani, 1978; Yanagabashi, 1979; Po- desta et al., 1980) of ACTH. In the present study, we show that both basal and ACTH-induced corticosterone production by domestic fowl cells is also dependent on Ca2+ (Fig. 7). The maximal level of corticosterone production that Ca2+ can induce is about 16 times greater in the presence of ACTH than in the absence of ACTH (Table 1). These data are consistent with work with rat adrenocortical cells (Sayers et al., 1971, 1972; Carsia et al., 1981) and are the first for domestic fowl cells showing that ACTH enhances the maximal level of corticosteroidogenesis that can be stimulated by Ca2+ as well as vice aersa. Moreover, ACTH increased the cellular sensitivity to Ca2+. In the presence of ACTH, the ED, value of CaZC was about l/8 the EDso value of Ca” in the absence of ACTH.

In addition to the Ca2+ dependence of ACTH action there was also Ca*+ dependence of oPRL counteraction of corticosterone-induced suppression of ACTH-induced corticosterone production (Fig. 7). In the absence of Ca’+, oPRL had no counteractive effect. However, significant counteraction was apparent with OSOmM Ca2+ and suppression was abolished with 2SOmM Cal’. Although little is known about the role of Ca2+ in PRL action on target tissues, there are reports indicating that PRL induction of RNA and casein synthesis is dependent on the extracellular Ca2+ concentration (Rillema, 1980; Cameron and Rillema, 1983). However, the present study is the first to show that the acute counteractive effect of PRL on corticosterone-induced suppression of ACTH-induced corticosterone production is Ca2+- dependent.

Our results provide two examples of the interaction of hormones to synergistically stimulate corticosterone production, that is, in a more than additive manner. Table 1 shows that ACTH and oPRL acting together on cells from hypox rats are almost twice as effective as when they act separately. To our knowl- edge this is the first quantitative report of this synergism between ACTH and PRL. Even more striking is the synergistic result of the combined actions of ACTH and Ca2+ on domestic fowl cells which is over three times as great as the sum of their separate steroidogenic effects. This is in agreement with data first reported by Sayers et al. (197 1, 1972) and Carsia et al. (1981) for rat cells. These findings of the synergistic actions of ACTH and PRL and of ACTH and Car+ suggest that these three steroidogenic agents operate via pathways that are not compIetely identical.

Some indications of how the combined actions of

ACTH, exogenous glucocorticoid and PRL acutely regulate glucocorticoid production may be seen in Fig. 6 (left panel). They suggest fme adjustments operating in addition to the established mechanisms for the regulation of corticosteroidogenesis. TWO modes of ACTH action in the presence of exogenous corticosterone are apparent. Incubation with concentrations of ACTH greater than zero but less than 1000 pg/ml (non-steroidogenic concentrations in the absence of exogenous corticosterone), resulted in negative net corticosterone values, thus indicating that some portion of the total corticosterone present at the start of incubation was metabolized to a product or products, such as &x-reduced metabohtes (Carsia et al., 1984), which reacted weakly, if at all, in the radioimmunoassay. Incubation with concentrations of ACTH greater than 1000 pg/ml (steroido- genie concentrations in the absence of exogenous corticosterone) resulted in positive net corticosterone values. However ACTH was limited in its ability to counteract the suppressive effect of high concentrations of exogenous corticosterone. The effect of a low concentration of exogenous corticosterone (0.26 pg/ml) was completely counteracted by ACTH, but the effect of a high concentration of exogenous corticosterone (0.53 pg/ml) was only partly counteracted by ACTH. These results are consistent with results of our earlier work with rat cells (Carsia and Malamed, 1983) which suggests that ACTH is a limited antagonist against exogenous corticosterone. Similarly, exogenous corticosterone is limited in its ability to suppress ACTH-induced corticosterone production. Our earlier work with isolated rat, beef and domestic fowl cells showed that exogenous glucocorticoid-induced suppression could reduce endogenous glucocorticoid production only to about 50% of the production induced by maxima1 steroido- genie concentrations of ACTH (Carsia et al., 1983). Thus, exogenous glucocorticoids could dampen but not abolish A~TH-induced steroidogenesis,

In the organism, glu~ocorticoid-induced suppression may function to prevent overstimulation of the adrenocortical cell. In addition, the work reported here with domestic fowl cells is the first to suggest that non-steroidogenic concentrations of ACTH facilitate corticosterone metabolism induced by exogenous corticosterone. This may be a useful mechanism for the quick removal of high corticosterone concentrations within the adrenal gland, when classical negative feedback results in low circulating ACTH levels. The result would be to decrease residual corticosterone secretion that may prove to be deleterious to the organism and to reduce high corticosterone concentrations that are thought to inhibit adrenal protein synthesis (Morrow et al., 1967) and mitochondrial function (Burrow, 1969) and to damage adrenal steroidogenic enzymes through the induction of free radical formation (Hornsby and Crivello, 1983). Presumably the metabolic products of glucocorticoids have little intrinsic activity in the periphery and within the adrenal gland.

Thus, our data suggest that in addition to its well known effect of stimulating corticosteroidogenesis, ACTH can act to remove residual corticosterone when corticosterone production is lowered via the


classical negative feedback loop. In contrast, PRL may act to dampen the suppression of corticosterone secretion. Thus PRL under certain conditions might ensure continued glucocorticoid secretion even with high circulating and adrenal concentrations of glucocorticoids. Indeed, oPRL acutely curtailed the effect of exogenous corticosterone over a wide range of ACTH concentrations (Fig. 6, right panel). That this steroidogenic effect of PRL might play an important role in adrenocortical response to stress is supported by studies with rats (Mueller et nl., 1976; Advis et al., 1977) and domestic fowl (Harvey et al., 1979) in which stressful stimuli increased circulating levels of PRL.

The concentrations of exogenous corticosterone and PRL used in these experiments are important in evaluating our results. The highest concentrations of exogenous corticosterone used (about 0.5 pg/ml) were roughly 3 times and 500 times greater than the normal plasma concentrations of rats (Rapp, 1969; Engeland, 1984) and domestic fowl {Schmeling and Nockels, 1978; Freeman et al., 1984) respectively. Furthermore, the free concentrations of corticosterone available to the adrenocortical cells were higher than those in plasma because corticosterone- binding globulin was not present in the medium. Nevertheless, due to the cytoarchitecture and blood flow dynamics in the rat adrenal gland (Harrison and Hoey, 1960; Kikuta and Murakami, 1982), the concentrations of corticosterone within the gland varies from 35flg/ml (Vance et al., 1969) to 104 pgjml (Tsang and Johnstone, 1973). Thus, the concentrations used in this study are well within the range of concentrations found in the rat adrenocortical cell environment in situ. However, evaluation of the results for domestic fowl cells is less clear-cut because the concentrations of glucocorticoids in celiular water within the domestic fowl adrenal giand have not been reported.

Evidence presented thus far suggests that ACTH, PRL and exogenous glucocorticoids can act simultaneously to provide a finely adjusting, fast-acting mechanism to regulate glucocorticoid production. The cellular mechanisms underlying this regulation have only recently begun to be investigated. Our previous work supports the hypothesis that at least part of the exogenous corticosterone-induced suppression of endogenous corticosterone production is due to the rapid degradation of corticosterone to SE-reduced metabolites (Carsia et al., 1984). The present study, in which we used a wide range of ACTH concentrations incubated with domestic fowl cells, supports our previous work: with 0.53 pg/ml exogenous corticosterone in the presence of concentrations of ACTH that were non-steroidogenic in the absence of exogenous corticosterone, as much as 3 1% of the exogenous corticosterone disappeared from the incubation medium.

Further support for a role of Sa-reductase in glucocorticoid-induced suppression are reports sug- gesting that ACTH is a limited antagonist of adrenal Sa-reductase (Ogle and Kitay, 1979; Colby, 1979; Ramirez et al., 1985) and that PRL is a potent inhibitor of adrenal 5cr-reductase in vivo (Ogle and Kitay, 1979; Colby, 1979) and in vitro with isolated adrenocortical cells (Carsia et al., 1984). Accord- ingly, if Sa-reductase ativity was an important com- ponent in the present study, then one might have expected similar relative actions of ACTH and oPRL on cort~costerone-induced suppression. In fact, ACTH had a limited effect in counteracting corticosterone-induced suppression (Fig. 7), whereas oPRL consistently counteracted suppression (Figs 3-7). Thus, our previous work (Carsia et ul., 1984) and the work presented here are the first studies to show that ACTH, PRL and exogenous corticosterone can acutely adjust adrenal Sa-reductase activity and, in turn, net corticosterone secretion.

The fixed maximal concentration (OS ~g/ml) of Our studies and those of other laboratories on the oPRL used in several experiments comprising this polyhormonal control of corticosteroidogenesis have study (Figs 4, 6 and 7) was 3 times greater than the raised a number of questions. Although ACTH is a circulating concentrations of PRL found in the weak inhibitor of adrenal Sa-reductase (Ogle and domestic fowl. This fixed concentration of oPRL was Kitay, 1979; Colby, 1979; Ramirez et al., 1985) it is used to ensure a maximal effect. Nevertheless, the the classic stimulator of corticosteroidogenesis. In concentrations of PRL used in our work were within contrast, PRL is a potent inhibitor of adrenal the range of circulating concentrations measured in 5a-reductase (Ogle and Kitay, 1979; Colby, 1979) the domestic fowl during stress (Harvey ef al., 1979). and might be expected to be a strong stimulator of Moreover, the EDSo value of oPRL with domestic steroid production. However, PRL is a weak stimu- fowl cells was about 55 ng/ml, well within the values lator of corticosteroidogenesis (Mann et al., 1977; obtained for non-stressed animals (Harvey et al., Advis and Ojeda, 1978; Glickman et al., 1979). In 1979). Thus, our data suggest that PRL can be a this connection it is unclear why hypophysectomy is physiologic, acute regulator of domestic fowl cortico- a requirement in rats but not in domestic fowl for the steroidogenesis. Evaluation of the results with rat exhibition of the effect of PRL. Another puzzle is cells is difficult because hypophysectomy was re- how these actions of ACTH and PRL combine to quired to demonstrate an effect of oPRL. In addi- counteract the corticosterone-induced suppression. tion, the ED, value of oPRL with hypox rat cells Further complicating an understanding of the coor- was approximately 220ng/ml or about 67 times dinated actions of ACTH, PRL, Ca’+ and exogenous greater than the plasma concentrations of PRL corticosterone on corticosterone production is the found in rats (Samuels and Bridges. 1983; Demarest synergistic relationship of ACTH, PRL and Ca** et al., 1985). Thus, even though other work indicates demonstrated in our studies. Also obscure are the that maintenance of rat adrenocortical function is mechanisms underlying the limitations of cortico- dependent on PRL (Ogle and Kitay, 1979; Colby, sterone-induced suppression of corticosteroido- 1979), our in vitro work raises a question concerning genesis. We have never observed suppression of more the role of PRL in the acute regulation of rat than about S&60% of the glucocorticoid production corticosteroidogenesis. occurring in the presence of exogenous glucocorti-


coid (Carsia and Malmed, 1979, 1983; Carsia et al., 1983, 1984) and only about 57% of this suppression can be explained by an increase in adrenal 5cr- reductase activity (Carsia et al., 1984). Thus, many aspects of the regulation of corticosteroidogenesis by heterologous hormones are still poorly understood

and require further study.

Acknowledgements-We thank Jean Gibney and Helen Weber for expert technical assistance, and MS C. J. Uckele for the preparation of this manuscript.

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