Nonsteroidal adrenal feedback demarcates two types

of pathways to CRF-ACTH release

CHARLES W. WILKINSON, WILLIAM C. ENGELAND, JEANETTE SHINSAKO,

AND MARY F. DALLMAN
Department of Physiology and Metabolic Research Unit, University of California,
San Francisco, California 94143

WILKINSON, CHARLES W., WILLIAM C. ENGELAND,
JEANETTE SHINSAKO, AND MARY F. DALLMAN. Nonsteroidal
adrenal feedback demarcates two types ofpathways to CRF-
ACTH release. Am. J. Physiol. 240 (Endocrinol. Metab. 3):
E136-E145, 1981.-We have tested the relationship between
corticosterone (B) and the number of adrenal glands on stim-
ulated corticotropin (ACTH) levels in rats. Rats were supplied
throughout the experiment with various doses of B in the
drinking fluid beginning 15-20 h before adrenal surgery. Bilat-
eral, unilateral, or sham adrenalectomy was followed on the 3rd
day by the stimulus of ether vapor or ether and laparotomy
with intestinal traction. Plasma ACTH levels 3 .min after the
stimuli were decreased by both the dose of B and the number
of adrenals, although prestimulus ACTH and B levels were
similar across groups. The decrease in ACTH after ether re-
sulted from an interaction between the dose of B and the
adrenal number; however there was no interaction between the
inhibitors after laparotomy with intestinal traction. These re-
sults show that I) in addition to B, some aspect of adrenal gland
number or mass inhibits the ACTH response to stimuli; and 2)
the adrenal number input serves as a marker to distinguish
between stimuli to ACTH secretion that are inhibited by B at
sites proximal and those that are inhibited by B at sites distal
to this input.
adrenal number; corticosterone; plasma corticotropin; adrenal
feedback models; rat
WE HAVE RECENTLY REPORTED that after unilateral ad-
renalectomy rats have augmented corticoliberin (CRF)
and corticotropin (ACTH) responses to the stimulus of
ether or laparotomy compared to the responsesof sham-
adrenalectomized controls (9). The increased magnitude
of the ACTH responsesoccurs from similar initial ACTH
and corticosterone levels in the two groups of rats and
before there are differences in circulating corticosterone
levels that might provide a differential rate-sensitive
feedback signal (5, 8). In other studies, we have shown
that unilaterally adrenalectomized rats bearing no adre-
nal medullas also hyperrespond to ether vapor compared
to sham-adrenalectomized controls (14); therefore, a dif-
ferential adrenal medullary response to ether is not re-
sponsible for the hyperresponsiveness in ACTH. In the
same experiments, there were no differences in plasma
renin concentration between the two groups before or
after ether vapor; therefore, it is unlikely that a differ-
ential angiotensin II response between the two groups
accounts for the differential ACTH responsiveness. We
El36 0193-1849/81/0000-0000$01.25
concluded that because the hyperresponsiveness in the
unilaterally adrenalectomized rats is evident by 2 min,
before there is a detectable difference from control in
plasma corticosterone levels, the afferent signal might be
related to total adrenal mass and might be mediated
neurally (9).
Models of corticosteroid feedback. Within the period
2-7 h after treatment with maximally effective doses of
dexamethasone, adrenocortical responsesto some stimuli
are inhibited completely, whereas the responsesto other
more intense stimuli are only partially or not inhibited in
rats (6, 22) and dogs (10). These experimental results
have been interpreted in two ways. One model proposes
that steroid feedback is saturable and that stimuli of
sufficiently high intensity will override the maximal feed-
back signal (21, 22, 27). A second model proposes that
there are low threshold, steroid-sensitive, saturable affer-
ent neural pathways to CRF secretion, and parallel high
threshold, steroid-insensitive pathways (5, 10, 24). In
previous experiments, we showed that the pituitary-ad-
renal responses to ether are blocked by prior treatment
with dexamethasone, whereas the responsesto ether and
laparotomy with intestinal traction are not (20).
In these experiments, we have tested the relationship
between corticosterone and adrenal number on the mag-
nitude of the ACTH responses to ACTH-releasing stim-
uli. Whether or not there is an interactive effect between
corticosterone and adrenal number depends on the stim-
ulus. These results show that adrenal number may serve
as a marker to distinguish between corticosterone feed-
back exerted at brain sites proximal or distal to the
adrenal number input (relative to the final common
pathway for ACTH secretion) and favor the interpreta-
tion that some afferent neural pathways to ACTH secre-
tion are corticosteroid-sensitive. As a byproduct, these
experiments also show that the relative compensatory
adrenal growth response after unilateral adrenalectomy
is independent of the degree of inhibition of ACTH
secretion by corticosterone.
METHODS
Male Sprague-Dawley rats weighing 70-90 g when they
arrived from the supplier (Simonsen Laboratories, Gil-
roy, CA) were housed 2-3/banging wire basket cage. The
rats were kept in a room with an automatically controlled
light cycle (lights on 0900-2100 h) and controlled tem-
Copyright 0 1981 the American Physiological Society
ADRENAL NUMBER INPUT TO PATHWAYS TO ACTH SECRETION
perature (23 + 1“C). Purina rat chow was available ad
libitum, and water was provided until 15-20 h before
surgery when it was exchanged for a dilute ethanol-saline
drinking fluid with or without corticosterone (B) that
was continued throughout the experimental period. We
used a range of doses of B: concentrations of 10, 15, 20,
40, or 80 ,ug/ml were provided in a vehicle of 0.5% NaCl:
0.5% ethanol. The dose of 160 ,ug/ml was provided in a
vehicle containing 2% ethanol. The steroid was dissolved
in absolute ethanol and then diluted appropriately. Fluid
intake was assessed by weighing water bottles to the
nearest gram every 6 or 12 h during the final 24 h of
experiments. In one experiment, the volume of drinking
fluid consumed was monitored throughout the experi-
mental period (Fig. 1).
The experimental design is shown in Fig. 1. Between
0900 and 1200 h on the day after provision of B in the
drinking fluid, rats were divided into three groups for
adrenal surgery under ether anesthesia: bilateral adre-
nalectomy, left adrenalectomy plus right sham adrenal-
ectomy, and bilateral sham adrenalectomy. Surgery was
accomplished in an average of 45 s, and rats were away
from their home cages for less than 3 min. In some
experiments, groups of rats from each surgical group
were killed at 6-h intervals during the final 24 h to
determine resting plasma ACTH and B levels. Between
0900 and 1200 h on the 3rd day after adrenal surgery and
the 4th day after providing B or vehicle, rats were ex-
posed either to ether vapor for 40 or 60 s in a closed 2-
liter beaker containing gauze squares and paper towels
satured with ether or were subjected, under ether anes-
thesis, to a midline abdominal incision and extrusion and
gentle manipulation of the entire length of the small
intestine, which was then returned to the peritoneal
cavity. The incision was closed with wound clips. This
Adrenal surgery
30
Volume drunk 20
by 2 rats/ 6 h
(ml) 10
0 12 24 36 48
Hours after start of B (IO-20pg/ml)
in drinking f hid
FIG. 1. Experimental design and mean volume of fluid intake per 2
rats per 6 h (n = 12 bottles weighed/group) in groups of rats after
adrenal surgery. There was no effect of adrenal surgery on corticoster-
El37
“laparotomy with intestinal traction” (6) requires 20 s to
accomplish after rats are anesthetized. Animals were
killed 3 min after the onset of exposure to ether.
On the final day of these experiments there were at
least three, and usually four people involved in anesthe-
tizing, performing laparotomies, killing the rats, and col-
letting adrenals, so that a large number of animals (120-
180) could be subjected to ACTH-releasing stimuli and
killed at 3 min (+ 10 s) after onset of the stimulus within
a total period of 2 h. All of this work was performed in
the animal room so that the time between picking up a
rat and killing it was less than 15 s. All rats were killed
by decapitation and trunk blood was collected in chilled,
heparinized plastic tubes. Blood was centrifuged at 4”C,
and plasma was collected and frozen until subsequent
assay for ACTH by radioimmunoassay (4, 19) and corti-
costerone by competitive protein binding radioassay (16).
Right adrenal glands were collected from rats with one
and two adrenals and were subsequently cleaned and
weighed to the nearest 0.01 mg on a microbalance. In
some experiments, adrenals were homogenized l:5OO (wt/
vol) in 80% physiological saline (0.9%):20% ethanol (~01:
vol) for determination of corticosterone content.
In two experiments, anterior pituitary glands were
collected from the three groups of rats at 0900 h on the
4th day. In one experiment, pituitaries were hemisected
and each half was placed in a lo-ml Teflon beaker con-
taining 2 ml Krebs-Ringer bicarbonate buffer containing
200 mg/lOO ml glucose. One equivalent of rat hypotha-
lamic extract (HE-RP-1) (generously supplied by the
National Pituitary Agency) was added to one-half of each
gland, and one equal volume of buffer was added to the
other half. The glands were incubated at 37°C in a
shaking-water bath in an atmosphere of 95% 02: 5% CO2
for 30 min. In the other experiment, hemisected pituitar-
Ether
Of
Lap. + Tr.
Resting
m-l --
---
Bilateral adrenalectomy
Unilateral adrenalectomy
- Sham odrenalectomy
m Lights off
I] Lights on
60 72 84
one (B) intake. Dose of B was 10, 15, or 23 ,ug/ml. Lap, laparotomy; Tr,
intestinal traction.
El38 WILKINSON, ENGELAND, SHINSAKO, AND DALLMAN

ies were quartered and preincubated for 3 h with hourly periods of darkness. Adrenal surgery did not affect daily
changes of medium before a final 30-min incubation with fluid consumption in any group during the following 3
or without addition of 1 eq of HE-RP-1. At the end of days; fluid intake averaged 30-34 ml. rat-‘. day-’ (Fig. 1).
the 30-min test incubations, medium was collected and Plasma corticosterone levels during the last day of the
frozen for subsequent ACTH assay. experiment varied as a function of the time of day in all
In each experiment, equal numbers of samples from groups except the group of bilaterally adrenalectomized
each experimental group were run in each assay, so that rats not supplied with B; however, in the other two
interassay variance was equally incorporated within the groups, peak plasma B occurred at 2100 h (lights out)
between-group variances. Significance of the differences when the fluid contained 10 or 40 ,ug B/ml, but not 160
among groups was determined by two- or three-way ,ug/ml. Because the peaks occurred before the onset of
analysis of variance followed by Tukey’s multiple-range substantial drinking, these results suggest that in rats
test for a posteriori contrasts (17). Linear regression with adrenal glands some ACTH-stimulated secretion of
equations and homogeneity of slopes were determined by endogenous corticosterone occurred at all but the highest
multiple-regression analysis (17). All analyses were per- dose of B in the drinking fluid.
formed on an IBM 370 computer. Plasma corticosterone levels were similar in the three
groups at 1500, 0300, and 0900 h at each dose of B (Fig.
3). At all doses, the plasma B levels were lower at 0900
RESULTS
than at 0300 h despite the fact that approximately equal
Patterns of fluid intake, B intake, and plasma B amounts of fluid were consumed between 2100 and 0300
concentrations. Unoperated rats drinking tap water con- h and between 0300 and 0900 h. This finding surprised us
sumed 24 t 0.2 mlrat-’ day-’ (n = 12 pairs, not shown);
l considerably. However, it may be that most of the intake
sham operation and provision of the ethanol-saline ve- during the second 6 h occurred early within that period
hicle resulted in a daily intake of 42 t 1 ml rat-‘. day-’ or that liver enzymes degrading corticosterone increase
(n = 9 pairs). Addition of B to the vehicle resulted in their activity during the dark period, thus acting more
levels of intake that were intermediate between those of efficiently on B between the hours of 0300 and 0900 than
water and vehicle. The dose of B did not appear to affect during the earlier hours of darkness. From these results,
the volume of fluid consumed because this was approxi- it seems likely that the three groups of rats were exposed
mately the same across all doses (Fig. 2). As in Fig. 1, B to equivalent amounts of B for at least 6 h before ACTH-
intake was similar in the three surgical groups at each releasing stimuli were applied at 0900 h.
dose of B in the drinking fluid; the daily dose of B Exogenous B causes adrenal atrophy but does not
provided by consumption of the drinking fluid ranged inhibit compensatory adrenal growth. Adrenal weight
between 0.26 and 4.68 mg/rat. was measured in the two groups of rats that had right
As anticipated, all groups of rats drank primarily dur- adrenals, and the data from all experiments were pooled
ing the 12 h of darkness when they normally engage in (Fig. 4). Analysis of variance yielded highly significant
greatest activity and eating (Fig. 1). When fluid intake effects of dose (P < 0.001) and of adrenal number (P <
was measured at 6-h intervals (last day, Fig. l), the rats 0.001) on right adrenal weight. There was a linear de-
were found to drink equal amounts during the two 6-h crease in adrenal weight that was proportional to the log

r] Sham adrenalectomy
a’ Unilateral adrenalectomy
m Bilateral adrenalectomy
80

60
Volume drunk
by 2 ratsin 24 h 40
(ml)
20

0
Vehicle IO 20 40 160
yg B/ml vehicle

Average B El 0 0.26 0.69 1.24 4.64

intake /rat I:.:...‘iJ 0 0.27 0.63 I.38 4.68

(mg/24 h) m 0 0.26 0.64 I I7

l 4.64
ADRENAL NUMBER INPUT TO PATHWAYS TO ACTH SECRETION El39

of the dose of B in both groups of rats. Compensatory Effect of dose of B on levels of ACTH and corticoster-
adrenal growth after unilateral adrenalectomy occurred one in 3 groups of rats. There was a consistent dose-
to approximately the same extent at each dose of B (the related inhibitory effect of B intake on resting ACTH
mean percent increase in right adrenal weight from left- levels in the three groups of rats (Table 1; Figs. 5 and 6).
adrenalectomized rats ranged between 16 and 39%; Fig. Analysis of variance of the plasma ACTH levels obtained
4, bottom). in the same samples from which the corticosterone data
Analysis of data from a single experiment in which the shown in Fig. 3 were obtained showed significant time-
effects of B (doses of 10, 20, 40, and 80 ,ug/ml) were of-day, dose, and adrenal number effects (Table 1). There
compared to the effects of either saline or ethanol-saline was one significant interaction, the dose of B and the
showed that the first significant decrease in adrenal number of glands. Because of the marked adrenal atro-
weight was caused by a dose of 20 lug B/ml (ANOVA, phy in the rats drinking 160 lugB/ml, we did not attempt
followed by Tukey’s multiple-range test). At the 80 and to measure resting ACTH levels; we believed these levels
160 pg B/ml doses, adrenals were markedly atrophic, would be below the sensitivity of the assay. The signifi-
weighing approximately 5-6 mg in the sham-adrenalec- cant time-of-day effect on ACTH levels at the 10 and 40
tomized groups; this adrenal weight is similar to that ,ug B/ml doses confirmed the inference drawn from the
found in rats of similar body weight 3 days after hypo- plasma corticosterone data (Fig. 3) that during the first
physectomy (7), suggesting that ACTH secretion had not hours of darkness ACTH levels are not markedly sup-
occurred for 3 days in these groups. pressed by relatively low doses of exogenous B.
In a separate experiment, rats were killed at 0300 and
IOgg BJ’ml 0900 h on the 3rd day after adrenal surgery (Fig. 5) and
plasma ACTH and B levels were determined. Although
Plasma 2~~~ 1.1..
.,.,.,.,.,.,.,._.
.;;,.,.,.;,
; ., 1 /k.;,...,~.:.:.:.:.:.:
:,;:::::.,
, ,:::*:~~::::.,
..::.y:,:.. .
::.._:.:,:,:,:,:.:.::.::::::‘.
.I..,....:.:.:.._.:,
.:::..:::::<
.&.::,.
_.,.,.,
:
:.:.:
:.:.:.:
:
:.:.:.:_:.:.:._,:,:.:
.,.,._.
_..._....
:::::::::::::,.::::
.::i:i:i:i:i:i:i:i:i’i:i:i:i:I
plasma B levels were similar in the three groups of rats
corticosterone IO 0 :j:;::
_,__
‘+x&j:,,
:;;;:‘i:giig?p.:.::::::::::::::::::.::: at both times for each dose, Fig. 5 shows clearly that only
(yg /lOOmI) ---.~~~~~~~:
............:.:.,.;,..~gg:.
.j._:
:i:::i’y!& %
WE., .y ;::, c.-- at the highest dose was ACTH suppressed equally in the
s..:.:.:.:.:.:.:.:.:.:.:.:.:
......~.~.......,......., :::i:i:iji:~i:jjjjj~:~. three groups, particularly at 0300 h. With doses of B
40yg B/ml
ranging between 10 and 20 pg/ml, it is clear that even at
ma l6Oyg B/ml
0900 h approximately 35% of the bilaterally adrenalec-
tomized rats have elevated plasma ACTH levels com-
pared to the other two groups (Fig. 6). Because rats were
Plasma housed 2-3/cage, we could not tell whether the individual
cof t icosterone 20 - bilaterally adrenalectomized animals with high ACTH
(~g/lOOml) levels consumed less steroid than those with low ACTH
levels. These data show that resting ACTH levels are
nearly the same at each dose of steroid in all three groups
of rats at the end of the dark period (0900 h) when stimuli
were applied, although they are not equal 6 or more
(hour) (hour)
hours earlier.
A --A Unilateral odrenolectomy o-o Sham odrenolectomy Effect of dose of B on levels of ACTH and adrenal
a----d Bilateral adrenalectomy corticosterone 3 min after stimulation of three groups of
FIG. 3. Plasma corticosterone (B) levels in 3 adrenal surgery groups
rats at 0900 h. Our goal in these experiments was to
are plotted as a function of time of day and dose of B (n = 6 rats/point; obtain partial, not total, inhibition of ACTH responses
means are accompanied by lines indicating t, SE). to stimuli in all three groups of rats and then to determine

IZ
r

8 FIG. 4. Right adrenal weight is in-

hibited in proportion to log of dose of
Right adrenal corticosterone (B) in drinking fluid. At
wet weight 6 each dose, weight of adrenals from left
(mg) adrenalectomized rats is greater than
that from sham-operated rats (n = l4-

/ 40 48/paint; means are accompanied by

% increase
lines indicating t SE).
20 EADX
- woo -100
fi Sham
l 1
- - 1 I I I I
JO
0.5% IO 20 40 80 160
ETOH: pg B/ml 0.5 % ETOH Saline
Saline
El40 WILKINSON, ENGELAND, SHINSAKO, AND DALLMAN

whether there was an effect of adrenal number on the 0, 1, and 2 adrenals 3 min after exposure to ether are
magnitude of the response. shown in Fig. 8. In contrast to the results obtained after
Three minutes after the onset of exposure to ether ether and laparotomy with intestinal traction, the ACTH
with subsequent laparotomy and intestinal traction, response to ether vapor alone is blocked in all three
plasma ACTH levels are determined by both the number groups at a dose of B just slightly above 40 pg/ml.
of adrenal glands and the dose of B (Fig. 7; Table 2). Analysis of variance of these data shows pronounced
Analysis of variance revealed no interaction between effects of both adrenal number and dose of corticoster-
adrenal number and dose of B for doses of 10,20, and 40 one, and additionally, shows an interactive effect (expt I,
,ug/ml (Table 2). Th e d oses of 80 and 160 pg B/ml were Table 2).
not included in the analysis because of the previously A partial in vivo bioassay confirmation of the ACTH
noted severe adrenal atrophy that resulted (Fig. 4) and results is shown by adrenal corticosterone concentrations
because the ACTH response was essentially abolished measured in rats with one and two adrenals 3 min after
(Fig. 7). exposure to ether (Fig. 9). As anticipated from the ACTH
Plasma ACTH concentrations measured in rats with levels, adrenal corticosterone concentrations decrease
markedly in both groups between the dosesof 20 and 40
pg B/ml*
TABLE 1. Plasma ACTH levels during day in three A second experiment studied the response to ether in
the three groups of rats supplied with 10, 15, or 20 pg B/
groups of rats
_c__I--_--_ ml drinking fluid (Fig. 10). In this experiment too, there
Plasma ACTH, pg/ml was an effect of adrenal number and of the dose of B on
ACTH levels; however, there was no interaction between
1500 2100 0300 0900 dose of B and adrenal number in this second ether
NCJ adrenals experiment in which very low doses of B were used.
Vehicle 287 t 142 482 t 72 379 -+ 127 430 t_ 214 When the regression of plasma ACTH concentration on
10 peg B/ml 220 t 49 285 t 119 229 +- 80 214 t 112 the dose of B was computed for each adrenal condition
40 ,~g B/ml 133 t, 142 248 t 253 156 t 158 43 t 33
for each of the two ether experiments, there was no
One adrenal difference. between experiments in the slopes of the
Vehicle 35 t, 17 119 t_ 72 97 t 55 69 t 65 regression lines (Table 3) and no dose-experiment inter-
10 pg B/ml 53 t 10 93 t, 31 54 t 18 39 + 17 action. However, for a given dose of B, the levels of
40 pug B/ml 30 t 15 42 -F 33 22 -+ 13 16 Z!I 6 ACTH at 3 min were different in the two experiments, a
Two adrenals difference reflected in the y-intercepts of the regression
Vehicle 22 t 21 85 t 47 53 t 25 37 t_ 36 lines (Table 3).
10 r_LgB/ml 24 t 13 54 t 37 33 t 14 14 t 3 When the data from the two ether experiments were
40 ,ug B/ml 21 Ifr 8 45 -i 27 41 t 46 13 t 5 pooled, there were significant effects of dose and adrenal
Analysis of
df I; P number (both P < O.OOl), and there was a significant
Variance
--- interaction between the two factors (P = 0.026). This
Dose of B 2 29.29 <O.OOl interaction is illustrated by the differences in slopes of
Time of day 3 7.50 <O.OOl the regression lines among the three surgical groups (Fig.
No. of adrenals 2 148.49 <O.OOl 11, top). The slope was different for each adrenal condi-
Interaction tion, and the extrapolated lines converged to a zero-
Dose x no. 4 13.13 <O.OOl ACTH intercept at a dose of B of approximately 46 ,ug/
Values are means t. SD; n = 5-6 rats/group. B, corticosterone. ml. In contrast, the slopes of the regression lines for the
Plasma ACTH values are given at times of day indicated in the three adrenal conditions after ether and laparotomy with
boxhead. Lights were off between 2100 and 0900 h. intestinal traction were not significantly different from

o-o
Sham adrenalectomy
‘\
‘\. \ A- --A
(pg/ml) ---a (+=& a--pa FIG. 5. Resting plasma ACTH levels
Unilateral adrenalectomy in 3 groups are progressively inhibited at
0300 and 0900 h by dose of corticosterone
(B). (n = . G/point;
. . means are accompa-
Plasma
4 n -se.-. n

B fromby left
.
6~~~ \- Bilateral adrenalectomy fi::d ‘lnesto indicating
right are 10+, 20J
“‘1’ k-dDoses
40 pg/Of
corticosterone 10
( NW)

Time of day (hours)

ADRENAL NUMBER INPUT TO PATHWAYS TO ACTH SECRETION El41

one another, and the lines do not converge (Table 3; Fig. HE-RP-1 in these experiments was similar to that of
11, bottom). normal rat pituitaries (9). These results show that neither
Effect of adrenal number is not atpituitary. To deter- the steroid regimen nor the surgical manipulation af-
mine whether the highest dose of corticosterone used in fected pituitary ACTH responsiveness to rat hypotha-
these studies (160 pg/ml) inhibited the pituitary’s capac- lamic extract. We also tested the responsiveness to HE-
ity to secrete ACTH and to determine whether the effect RP-1 of pituitaries from rats in the three surgical groups
of adrenal number on ACTH responses was at the pitui- after 10 pg B/ml. In this experiment, pituitaries were
tary, pituitary ACTH responses to a secretagogue were only hemisected and the ACTH release between 0- and
determined in vitro (Fig. 12). Neither the control nor the 30-min incubation without added secretagogue was com-
stimulated ACTH secretion differed among the three pared to that between 0 and 30 min with HE-RP-1. In
groups. The magnitude of the ACTH response to 1 eq of this experiment too, there was no effect of prior B treat-
ment or adrenal number in the pituitary donor on basal
iii ACTH (pg/ml) ACTH output or on stimulated ACTH release (not
P9 400 shown).
corticosterone
per ml 1A A 87 f 40 DISCUSSION
A 25*5 The results of these experiments demonstrate that in
IO 200- A l 16*4 addition to the feedback signal provided by corticoster-
one, there is a second inhibitory signal associated with
adrenal number that modulates stimulus-induced ACTH

TABLE 2. Analyses of ACTH values 3 min after ether or

ether and laparotomy with intestinal traction

Analysis of Variance

df F P

Ether I
Dose of B, 10,20,40 ,ug/ml 2 25.45 <O.OOl
No. of adrenals 2 31.89 <O.OOl
Interaction 4 6.01 <O.OOl

Ether II
200 A 56 f 23
Dose of B, 10,15,20 pg/ml 2 5.22 to.001
% A IIf No. of adrenals 2 30.34 <O.OOl
20 100 I A Interaction 4 1.57 0.191
0 II f 3
Lap III
0 v +A 1 Dose of B, 10,20,40 lug/ml 2 4.28 0.017
0 I 2 No. of adrenals 2 52.26 <O.OOl
Number of odrenol glands per rat Interaction 4 1.59 0.184
FIG. 6. Scattergram of resting ACTH levels at 0900 h in 3 adrenal Lap, laparotomy with traction; B, corticosterone. The experiments
surgery groups with increasing doses of corticosterone (B). Mean ACTH were performed in rats with 0, 1, or 2 adrenal glands drinking doses of
levels (right) are accompanied by & SE (n = 7-g/group). B ranging between 10 and 40 lug/ml.

500 \
f:
r \

PlasmaACTH FIG. 7. Adrenal number and dose of

3 minutes after corticosterone (B) determine magnitude
laparotomy + traction of ACTH response to laparotomy with
intestinal traction (n = 7-g/point; means
(Pg~mb 200 are accompanied by lines indicating t
i3E4).
fin\

100 yg corticosterone
40 per ml drinking fluid
0 m-ma.- -..

0 1 2
Number of
adrenal glands per rat
El42 WILKINSON, ENGELAND, SHINSAKO, AND DALLMAN

0\
800 - \
\
Plasm0 300 -
600 ACTH
Plasma 3 minutes 200 -
ACTH after Et20
3 minutes
after Et20400 (pg/ml) loo - IO yg corticostefone
I5
20 per ml drinking fluid
(pg/ml)
0 I 2
Number of
200
adrenal glands per rat
corticosterone FIG. 10. Adrenal number and dose of corticosterone (B) determine
per ml drinking fluid magnitude of ACTH response to ether (n = 8/group; means are
0[-f-y 40 accompanied by t SE).
0
0 1 2

Number of TABLE 3. Linear regression ofplasma ACTH

adrenal glands per rat concentration on dose of B for three experiments
FIG. 8. Dose of corticosterone (B) and adrenal number determine
magnitude of ACTH response to ether vapor (n = 8/paint; means are Group Slope y-Intercept
accompanied by lines indicating t SE). BilateraZ adrenalectomy
Ether I -17.50 1148.45
Ether II -14.73 721.67

10 - dk Lap III

Unilateral adrenalectomy
-3.66 516.94

Ether I -7.47 340.92

Ether II -9.06 261.59
Lap III -2.39 217.81

Adrenal corticosterone Sham adrenalectomy

Ether I -5.09 234.26
3 minutes after Et20 ’
Ether II -4.84 130.2 1
(m/lOOw) Lap III -3.33 185.00

Ether I and II, surgery with ether; Lap III, surgery with ether and
laparotomy with intestinal traction.

I I 1 I
0 tion products reaching the blood, an increase in the
10 20 40 80
activity of this enzyme during the last 6 h of darkness
yg cofticosterone
could decrease the amount of corticosterone reaching the
per ml drinking fluid
systemic circulation. Because we cannot express the re-
FIG. 9. Dose of corticosterone (B) determines right adrenal B con-
centrations 3 min after ether (n = 8/group; means are accompanied by sults as a function of plasma B concentration, we must
lines indicating t SE). use the dose of B per milliliter or daily B intake instead.
However, the dose expressed in this manner is nominal
secretion. Because basal ACTH secretion was not equally and is clearly greater than the dose of B actually reaching
suppressed in the three groups of rats by B supplied in the systemic circulation.
the drinking fluid (Table 1; Figs. 3, 5, and 6), the results Exogenous B inhibits resting and stimulated ACTH
from both stimuli must be considered to draw this con- secretion in each group of rats in a dose-dependent
clusion. The results also suggest that ether vapor excites fashion (Figs. 5-8,10, and by inference, 4). The threshold
steroid-sensitive afferent pathways to CRF and ACTH dose for inhibition of adrenal weight is 20 ,ug B/ml (Fig.
secretion, whereas laparotomy with intestinal traction 4) or a daily oral dose of approximately 0.6 mg B for rats
excites, in addition, corticosteroid-insensitive afferent weighing approximately 125 g. The decrease in adrenal
pathways. Because these conclusions involve different weight that occurred in both sham and unilaterally ad-
bodies of literature, they will be discussed separately renalectomized rats probably reflected an overall de-
below. crease in 24-h ACTH secretion and is similar to the
Effect of B ingestion on ACTH secretion. Plasma B effects observed after dexamethasone treatment (7). This
levels at 0900 h were not different after dosesof B ranging result suggeststhat 20 pg B/ml or 0.6 mg B/day orally is
between 10 and 80 pg/ml. It seems likely to us that the an appropriate replacement dose after bilateral adrenal-
uniformly low B levels found at 0900 h are a result of the ectomy. Resting ACTH levels in bilaterally adrenalecto-
normal increase in liver A4-steroid dehydrogenase levels mized rats drinking 20 lug B/ml support this conclusion
that were found to correlate directly with food intake in (Figs. 5 and 6). l

the rat (13). Because the liver filters all intestinal absorp- The threshold for suppression of stimulated ACTH
ADRENAL NUMBER INPUT TO PATHWAYS TO ACTH SECRETION El43

Ether time course of inhibition provided by injection of single

(2 experiments) doses of dexamethasone (25) or corticosterone (23) in
rats. The feedback efficacy of a given amount of B
supplied in the drinking water would be expected to
Regression lines calculated by least squares
depend on the time that stimuli are applied to the rats
(Data from experiments using 1 O-40 )-rg B/ml)
relative to the time of steroid ingestion.
Effect of adrenal number on stimulated ACTH secre-
Adrenal tion. There were significant effects of adrenal number on
number: Slope:
0 - -16.2
the levels of ACTH 3 min after stimuli between the doses
1 - -5.2 of 10 and 40 lugB/ml. Because the bilaterally adrenalec-
2 - -2.7 tomized rats were exposed to less B per 24 h than rats
with adrenal glands, it would be difficult to include
adrenal number in the anaysis if we had not used two
different stimuli to ACTH secretion to test this effect.
The different regression lines that emerged from each
f stimulus allow us to include all three adrenal conditions
Y Laparotomy with (0, 1, and 2) in our anaysis (Fig. 11). After ether, the slope
2 600 intestinal traction of the regression lines depends on adrenal number and
3 (3 experiments)
n 500\ c the lines for the three adrenal conditions converge at a
number: Slope:
dose of B a little greater than 40 r_lg/ml (Fig. 11, top). In
400 0 contrast, after laparotomy with intestinal traction, the
I-' '. 0 - -3.7
-'. \ -2.4 slopes of the B regression lines depend only on the dose
300 \\ 1 -
of B and not on the number of adrenal glands per rat
t \ \ 2 - -3.3
(Fig. 11, bottom). Taken together, these results show a
corticosterone-independent effect of adrenal gland num-
ber on the magnitude of the ACTH responsesto stimuli.
Either result (Fig. 11, top or bottom) in the absence of
"0 20 40 60 80 100 120 140 the other could be interpreted as a consequence of une-
$9 corticosterone/ml qual daily B exposure in the three groups of rats (0 < 1
FIG. 11. Interaction between exogenous corticosterone (B) and ad-
< 2). However, with both an interactive and a noninter-
renal number for two stimuli. Results from all experiments with ether active effect of adrenal number on the B-induced inhi-
(top) or laparotomy with intestinal traction (bottom) were pooled and bition of stimulus-induced ACTH secretion, it is clear
regression of ACTH concentration on dose of B was computed for doses that there is a B-independent adrenal feedback signal.
of B between 10 and 40 lug/ml. There is an interactive effect on ACTH We have previously speculated that the corticosteroid-
of adrenal number and dose of B after ether, but not after laparotomy
with intestinal traction (Table 2).
independent adrenal signal is mediated neurally (9). By
comparing stimulus-induced ACTH responses in rats
I Control with bilaterally transplanted adrenals to those with bi-
HE-RP- 1
laterally enucleated glands, we have since obtained fur-
ther functional evidence that there is a nonsteroidal
feedback signal from the adrenals. At 1 and 3 wk after
adrenal surgery, both groups of rats show greater ACTH
responsesto ether vapor than do sham-operated controls.
By 5 wk after the operations, both surgical groups are
again normoresponsive to ACTH-releasing stimuli (26).
Unless the assumption is made that transplanted adre-
nals become functionally reinnervated, these results are
0
not compatible with the interpretation that there is a
Number of adrenals/donor
neural feedback from the adrenal on ACTH secretion, as
FIG. 12. In vitro ACTH secretion per 30 min by 95 pituitaries from proposed by Hal&z and Szentagothai (12). These results
the 3 surgical groups without and with added rat hypothalamic extract. do suggest again that there is a nonsteroidal signal of
Horizontal lines above bars indicate t, SE. Donor rats received 160 pug adrenal mass because plasma and adrenal corticosterone
corticosterone (B)/ml in drinking fluid. levels do not increase between 3 and 5 wk.
When they proposed an adrenal neural feedback on
secretion also occurs at daily doses of oral B that range hypothalamic CRF secretion, Hal&z and Szentagothai
between 0.6 and 1.2 mg (Figs. 7-10). This dose is less suggested that some aspect of adrenal mass was moni-
than that used by others to study the effect of B on basal tored by the hypothalamus (12). Such a mechanism
and stress-induced ACTH secretion after adrenalectomy would explain not only the differential ACTH respon-
(1) or bilateral adrenal enucleation (2). siveness found in this study to depend on adrenal num-
All rats were exposed to ACTH-releasing stimuli at ber, but also the inhibitory effects of ACTH treatment
least 6 h, but less than 12 h, after they drank the exoge- on the responses of the adrenocortical system to various
nous B feedback signal. This would be expected to be a stimuli (5, 24). In addition to acutely promoting steroid
time of maximally effective feedback, judging from the secretion, chronic treatment with ACTH causesenlarged
El44
adrenal glands (11, 15). We do not know what variable of
adrenal mass is monitored by adrenal afferent nerves or
whether the adrenal number effects are neurally me-
diated. However, changes in both blood pressure and
capsular distention provoke changes in adrenocortical
afferent neural activity (18).
Another scheme can be proposed as a speculative
alternative to a neural hypothesis. If adrenocortical cells
secrete a factor at a relatively constant rate independ-
ently of ACTH concentration, this factor could be mon-
itored by the central nervous system to provide infor-
mation on the number of functional adrenocortical cells.
This information could then be used to alter the respon-
siveness of the CRF-ACTH pathway.
The results of these experiments show that when bi-
laterally adrenalectomized rats are given quantities of
corticosterone that return ACTH responsiveness to nor-
mal, they are actually over-treated with B but not treated
with an appropriate adrenal number signal.
Adrenal number input distinguishes between two
models of corticosteroid feedback inhibition. Some stim-
uli to the adrenocortical system excite corticosteroid
responsesin animals that are pretreated with maximally
effective dosesof steroid, whereas the responsesto other
stimuli are inhibited (6, 10,ZZ). These results have led to
two interpretations of the interaction between stimuli to
adrenocortical system activity and corticosteroid feed-
back. One interpretation currently accepted by many is
that stimuli that activate the adrenocortical system are
of variable intensity. Those that provoke adrenocortical
activity during the period 2-7 h after treatment with
maximally effective doses of corticosteroids simply ex-
ceed the inhibitory capacity of the feedback signal (21,
22, 27).
A second interpretation of the same data proposes that
there are two classesof afferent neural pathways to CRF
and ACTH secretion that are activated by stimuli to the
system. One class of afferent pathways is activated by
low-intensity stimuli, and its activity is inhibited by the
action of corticosteroids. A second class of afferent path-
ways is activated by high-intensity stimuli, and its activ-
ity is not affected by the action of corticosteroids (5, 10,
24
The results of the present experiments favor the latter
interpretation. The number of adrenal glands serves as
a marker that distinguishes the site of corticosteroid
feedback for the two stimuli applied in these studies. The
adrenocortical response to ether vapor is inhibited nearly
entirely by pretreatment with dexamethasone (522) and
is therefore a low-intensity stimulus in both models. The
magnitude of the ACTH response to ether was deter-
mined primarily by the dose of corticosterone. There was
a strong interaction between dose of corticosterone and
adrenal number (Table 3; Fig. 11). Therefore, there are
sites of B feedback both distal and proximal (relative to
the final common pathway for ACTH secretion) to the
adrenal number input (Fig. 13).
The adrenocortical response to laparotomy with intes-
tinal traction under pentobarbital (5) or ether (20) anes-
thesia is only slightly decreased by pretreatment with
maximally effective doses of dexamethasone. One model
of corticosteroid feedback on stimulus-induced ACTH
WILKINSON, ENGELAND, SHINSAKO, AND DALLMAN
secretion interprets laparotomy as a more intense stim-
ulus than ether; the other model postulates that laparot-
omy stimulates high threshold, steroid-insensitive-affer-
ent pathways to CRF and ACTH secretion. In the pres-
ent studies, there was no interaction between adrenal
number and dose of B (Fig. 11; Table 2). Therefore, the
adrenal number input is distal to the site of corticosteroid
feedback after ether and laparotomy plus intestinal trac-
tion (Fig. 13).
The absolute magnitude of the ACTH response to
ether and to laparotomy with intestinal traction is similar
in rats with two adrenal glands, as are the slopes of the
B regression lines (Tables 2 and 3; Fig. 11). Although
ACTH responsesto both stimuli at zero B are similar for
each adrenal condition, by decreasing the number of
adrenal glands, the differences in the slopes of the B-
dependent regression curves become increasingly appar-
ent [(slope ether)/(slope lap)]: 2 adr N 0.9; 1 adr N 2; 0
adr = 4. The schematic diagram shown in Fig. 13 postu-
lates that certain afferent pathways to CRF and ACTH
secretion are steroid-sensitive and others are not. The
components of the final common pathway (CRF- and
ACTH-secreting cells) are also inhibited by corticoste-
roids. If the model presented in Fig. 13 is correct, then
there should be a difference in slope between the regres-
sion curves for ether and for ether and laparotomywith
intestinal traction in rats with two adrenals. Although
both ether experiments resulted in calculated slopes of
about -5 and laparotomy with traction resulted in a
lower slope (-3), the experimental variation was such
that the differences in slopes between the two stimuli
were not significant in rats with two adrenals. Progressive
removal of adrenals reveals the differences between the
stimuli in terms of steroid feedback efficacy. The results
of the present experiments suggest strongly that steroid-
sensitive afferent neural pathways are more sensitive to
corticosteroid feedback inhibition than is the final com-
mon pathway because the ACTH response to ether alone
is inhibited by about 40 pg B/ml in all groups, whereas
complete inhibition of the ACTH response to laparotomy
with intestinal traction requires more than 40 pg B/ml.
We omitted from our analyses the ACTH results from
experiments using 80 and 160 rug B/ml in the drinking
fluid because these doses effectively inhibited the re-
Hypothalamo-
pituitary unit
Afferent neural
pathways (Final common
input pathway)
+ ACTH
(Neural?)
Adrenal number
(Mass?)
FIG. 13. A model that accommodates data. Corticosterone inhibits
afferent neural pathways stimulated by exposure to ether vapor, but
not additional pathways stimulated by laparotomy with intestinal trac-
tion. There are components proximal to adrenal number input (possibly
CRF neuron and corticotrope cells of pituitary) that are also sensitive
to inhibitory effects of corticosterone.
ADRENAL NUMBER INPUT TO PATHWAYS TO ACTH SECRETION El45

sponsesto both stimuli in all surgical groups. We inter- agrees with deductions from the literature on cortico-
pret this inhibition as an action of corticosterone on the steroid feedback (3).
final common elements of the adrenocortical system The help of Dr. Matilde A. Holzwarth is gratefully acknowledged.
(CRF- and ACTH-secreting cells). Sufficient doses of We thank Shirley Chi for the corticosterone assays and Annette Lowe
corticosterone supplied in an adequate schedule can act for illustrations.
This work was supported by Public Health Service Grant AM-06704
to cause atrophy of these elements. The finding of very C. W. Wilkinson is the recipient of National Institutes of Health
low adrenal weight in rats drinking these high doses of B Postdoctoral Fellowship AM-05681. M. F. Dallman is the recipient of
(Fig. 4) supports this speculation. Because the pituitary Public Health Service- Research Career Development Award AM-
responsesto a standard dose of HE-RP-1 were of normal 00072.
Present address of C. W. Wilkinson: Dept. of Medicine, Div. of
magnitude after the highest dose of B (160 pg/ml, Fig. Endocrinology and Metabolism, Mt. Sinai School of Medicine, New
II), the data also suggest that feedback efficacy of cor- York, NY 10029; of W. C. Engeland: Dept. of Physiology, Brown Univ.,
ticosteroids is exerted hierarchically, with steroid-sensi- Providence, RI 02912.
tive afferent neural pathways being more sensitive than Address reprint requests to: Dr. Mary F. Dallman, Dept. of Physi-
CRF neurons, which in turn are more sensitive than ology, Univ. of California, San Francisco, CA 94143.
corticotropes in the anterior pituitary. This conclusion Received 26 March 1980; accepted in final form 10 September 1980.

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