0013-7227/80/1065-1410$02.
00/0
Endocrinology
Copyright © 1980 by The Endocrine Society
Stimulus-Induced Corticotropin-Releasing Factor
Content and Adrenocorticotropin Release Are
Augmented after Unilateral Adrenalectomy,
Independently of Circulating Corticosteroid Levels*
WILLIAM C. ENGELAND,f FREDERICK SIEDENBURG, CHARLES W. WILKINSON,:}:
JEANETTE SHINSAKO, AND MARY F. DALLMAN§
Department of Physiology and Metabolic Research Unit, School of Medicine, University of California,
San Francisco, California 94143
ABSTRACT. There is evidence for a neural link between the
adrenal and hypothalamus that may mediate increased cortico-
tropin-releasing factor and ACTH secretion within seconds after
bilateral adrenalectomy. These studies on young male rats tested
the possibility that functional evidence for adrenal afferent
nerves might be revealed by the application of ACTH-releasing
stimuli after the acute ACTH and corticosteroid responses to
unilateral or sham adrenalectomy had subsided. Resting ACTH
and corticosterone levels were not different in the two groups 1,
3, or 7-10 days after adrenal surgery. Despite similar initial
conditions, the ACTH response to ether or to laparotomy with
intestinal traction (but not to ip saline injections) was greater
D URING the last 20 yr, anatomical and functional
evidence has slowly accumulated that supports the
hypothesis that there is an inhibitory,, neural feedback
mediated by capsular stretch receptors from the adrenal
glands to the hypothalamus that modulates ACTH re-
lease (1). Neurophysiological studies have shown that
spontaneously firing afferent adrenal nerve fibers change
their rates of discharge in response to mechanical defor-
mation of the capsule (2). The original anatomical evi-
dence (1) showed that neuronal cell nuclei in the hypo-
thalamic ventromedial nuclei (VMN) increased in vol-
ume after manipulations that 1) decreased adrenal size
(corticosteroid treatment), 2) eliminated the adrenals
(bilateral adrenalectomy), or 3) severed neural commu-
nication between adrenals and the central nervous sys-
tem (celiac ganglionectomy). Nuclear size decreased in
neurons in the VMN after treatments that increased
Received July 30,1979.
* This work was supported in part by USPHS Grant AM-06704 and
NASA-Ames University Interchange Agreement NCA2-OR665-602.
f Present address: Section of Physiology and Biophysics, Brown
University, Providence, Rhode Island 02912.
$ Recipient of USPHS Postdoctoral Fellowship AM-05681.
§ Recipient of USPHS Research Career Development Award AM-
00072. To whom requests for reprints should be addressed.
1410
Vol. 106, No. 5
Printed in U.S.A.
after unilateral adrenalectomy (P < 0.01) at times when plasma
corticosterone levels were not different. Hypothalamic cortico-
tropin-releasing factor-like activity was higher in unilaterally
adrenalectomized than in sham-operated rats 2 min after expo-
sure to ether (P < 0.05). We conclude that these results may
represent a functional demonstration of decreased inhibitory
neural feedback from the adrenal to the hypothalamus after
unilateral adrenalectomy, and discuss the possibility that the
absence of neural feedback may contribute to the well known
hyperresponsiveness of bilaterally adrenalectomized rats to
ACTH-releasing stimuli. (Endocrinology 106: 1410, 1980)
adrenal size, i.e. chronic stress or ACTH treatment. Ex-
tremely strong evidence for a neural link between the
adrenals and the hypothalamus was provided by the
demonstration that after unilateral adrenalectomy, nu-
clei in VMN neurons on the side ipsilateral to adrenal-
ectomy become smaller than those in sham-operated
rats, whereas those on the contralateral side increased in
volume (1). The uptake of [;)H]leucine into the VMN is
reported to be greater on the side contralateral to unilat-
eral adrenalectomy than on the ipsilateral side (3).
We have provided functional evidence for adrenal af-
ferent nerves by showing that the increase in corticotro-
pin-releasing factor (CRF) content is less and the in-
crease in plasma ACTH is greater seconds after comple-
tion of bilateral adrenalectomy or bilateral adrenal ma-
nipulation than after sham bilateral adrenalectomy (4).
Additionally, we have shown that unilateral lesions in
the hypothalamus (5, 6) or spinal cord (7) interfere with
compensatory adrenal growth provided that they are
placed on the appropriate side to the subsequent unilat-
eral adrenalectomy. We have also presented evidence
that the compensatory adrenal growth after unilateral
adrenalectomy is mediated neurally, since there is an
increase in the wet and dry weights and nucleic acid
 STRESS RESPONSE AFTER UNILATERAL ADRENALECTOMY 1411

content of the other gland 12 h after manipulation of the
neural and vascular supplies of one adrenal (8).
In the present experiments, we have examined the
effects of unilateral adrenalectomy compared to sham
operation on the CRF, ACTH, and corticosterone re-
sponses to subsequent stimuli. Some of these data have
been presented in an abstract (9).
Materials and Methods
Male Sprague-Dawley rats (Simonsen Laboratories, Gilroy,
CA) were received 2-10 days before an experiment and were
housed two or three per wire hanging basket cage in rooms with
controlled temperature (21-24 C) and lighting (12 h of light, 12
h of darkness). The rats were allowed food (Berkeley Diet-
Feedstuffs Processing; Purina rat chow) and tap water ad
libitum. Body weights at the time of experiments ranged be-
tween 90-250 g. In any single experiment, the weights varied by
10% or less.
Surgery
Left adrenalectomy was performed under ether anesthesia
via a dorsal skin incision. Surgical sham adrenalectomy was
performed similarly, except that the adrenal was only observed.
Skin incisions were closed with wound clips.
Stimuli
Three stimuli to ACTH secretion were used: exposure to
ether vapors for at least 40 sec; laparotomy with intestinal
traction under ether anesthesia; and 0.5 ml 0.9% saline injected
ip. After the stimuli, rats were returned to their home cages
until they were decapitated at the times indicated in Results.
tered. The quartered half-glands were put in 10-ml Teflon
buckets containing 2.0 ml Krebs-Ringer bicarbonate buffer (pH
7.2-7.3) and preincubated for 3 h. Figure 1 shows the time
course of ACTH release into the medium during this time and
the response to 1 equivalent of NIAMDD Rat HE-RP-1 (gen-
erously provided by the National Pituitary Agency). ACTH
released into the medium decayed exponentially during the
first 2 h of incubation and remained relatively stable for the
next 90 min. Figure 2 {left) shows that the ACTH released in
response to Rat HE-RP-1 was linear with time when the
sectioned hemipituitaries were exposed to the stimulus at 2.5 h.
Additionally, Fig. 2 {left) shows that the dose of HE-RP-1 used
did not contain enough ACTH to contaminate the response. A
30-min incubation with 0.5, 1.0, and 2.0 equivalents of Rat HE-
RP-1 produced the results shown in Fig. 2 {right). ACTH in
the medium was proportional to the log of the dose of Rat HE-
RP-1 added [F = 4.31 (2, 9); P < 0.05].
ACTH assay
Media from pituitary incubations were diluted 1:1 with horse
serum (Grand Island Biological Co., Grand Island, NY), and 0.5
ml incubation medium or 1.0 ml plasma was extracted by the
method of Rees et al. (10), with modifications. Corning glass
(960 mesh; 35 mg; Corning Glass Works, Corning, NY) was
2000r
+ 1 eq.
ACTH
in medium 1000 HE-RP-1
(pg/30 min)

Blood collection and assays

Trunk blood was collected in chilled, heparinized, plastic
centrifuge tubes and placed in ice water until centrifuged at 4
C. Plasma was separated and frozen at —20 or —70 C until
assay. For all assays, samples from a given experiment were
either assayed in a single assay or, when the assay capacity was
limiting, equal numbers of samples from each of the experimen-
tal groups were run within each assay so that interassay varia-
tion was incorporated equally in the results.
0 30 60 90 120 150 180 210
Time (minutes)
FIG. 1. ACTH released into the medium with time after starting in-
cubations of half-anterior pituitary glance cut into quarters. Each bar
represents the mean (±SEM) value of ACTH in the medium, except for
the two values at 180-210 min. At 180 min, 1 equivalent HE-RP-1 was
added to four hemipituitaries, and nothing was added to the other four
hemipituitaries.

Hypothalamic CRF activity assay

2000r 1000
Brains were removed from donor rats, and the medial basal
hypothalamus (~10 mg) was removed by cutting between optic
chiasm and mammillary bodies approximately 2 mm on either ACTH ACTH 800
side of the midline with scissors. The pieces of brain were then in medium 1000 in medium
(pg/30 min)
bisected into left and right halves, and pieces of tissue from five (P9)

to eight rats were pooled and homogenized in 0.1 N cold HC1, 600

and, after centrifugation, the supernatant was frozen for sub-

0 30 60 minutes 0.5 1.0 2.0
sequent assay. The hypothalamic extract was assayed in vitro Time after addition of Equivalents of HE-RP-I
using anterior pituitaries collected from male rats weighing 90- 1 equivalent of HE-RP-1
100 g. Assay rats were decapitated, pituitaries were removed FIG. 2. Left, ACTH released into the medium is linear with time after
from the sella turcica, the posterior lobes were separated and the addition of 1 equivalent HE-RP-1. Right, ACTH is released into
discarded, and the anterior lobes were bisected and then quar- the medium in proportion to the log of the dose of HE-RP-1.
 Endo 1980
1412 ENGELAND ET AL. Vol 106 No 5

added to 1 ml sample, agitated for 30 sec on a vortex mixer, and
centrifuged. The supernatant was removed, and the glass was
washed with 2 ml distilled H2O and centrifuged. The H2O was
discarded, and the ACTH was eluted from the glass with 50%
0.25 N HCl-50% acetone (vol/vol). The supernatant was trans-
ferred to new tubes, dried under N2, and then diluted serially
with assay buffer (10). aACTH-(l-24) was used as standard and
iodinated trace. Characteristics of the antiserum have been
described previously (11). Intraassay variation was 8%, and
interassay variation was 15%.
Corticosterone assay
Plasma and adrenal corticosterone values were determined
by a competitive protein-binding assay (12) using human
plasma as the source of transcortin.
Statistical analyses
Data were analyzed by one- or two-way analysis of variance
or by Student's two-tailed unpaired t test where appropriate.
Results
Resting plasma ACTH and corticosterone levels after
adrenal surgery
Mean resting plasma ACTH and corticosterone levels
collected at various times after surgery in 11 experiments
that contained groups of sham- and unilaterally adrenal-
ectomized rats are shown in Fig. 3. The overall means
from the results of separate experiments at each time are
indicated by the solid and dashed horizontal lines. In
no experiment was resting plasma ACTH significantly
elevated or plasma corticosterone significantly reduced
in unilaterally adrenalectomized rats compared to sham-
operated animals. The overall means show that both
plasma ACTH and corticosterone levels are slightly ele-
150
100
Plasma ACTH
(pg/ml)
50
vated in rats after unilateral adrenalectomy compared to
levels in sham-operated controls.
Augmented plasma ACTH response to some ACTH-
releasing stimuli in rats with one adrenal
Ether. After exposure to ether vapors, unilaterally adre-
nalectomized rats responded at 5 min with higher plasma
ACTH levels than sham-operated rats 1, 3, and 7 days
after adrenal surgery [F = 18.27 (1, 35); P < 0.001; Fig.
4]; however, plasma corticosterone levels were not differ-
ent at this time after ether in the two groups [F = 0.48
(1, 35); P = 0.49; Fig. 4]. There was a significant day
effect for both ACTH and corticosterone (P < 0.01 in
each case). In this experiment ACTH was not signifi-
cantly increased in unilaterally adrenalectomized rats 24
h after the operation; however, in the next experiment
and in others (not shown), the effect was present at 24 h.
Plasma ACTH and corticosterone levels and adrenal
corticosterone responses with time after ether, adminis-
tered 24 h after adrenal surgery, show that 2.5, 5, and 15
min after ether, plasma ACTH levels are elevated in rats
with one adrenal gland compared to levels in those with
two glands [F = 29.18 (1,40); P < 0.001; Fig. 5]. Moreover,
plasma corticosterone levels were similar in the two
groups at 2.5 and 5 min. Plasma, but not right adrenal,
corticosterone levels in the two groups differed at 15 min
[F = 4.96 (1, 40); P = 0.032]. In this experiment, 8 U
Sham adrenalectomy
Unilateral adrenalectomy
300
Plasma rf-
rh
ACTH
200 ftl
100
0

Mean of 5-8 rats

_ L
f t
n
Sham ADX °
20
30r Unilateral adrenalectomy *
Plasma
20
Overall mean corticosterone 10 -
Plasma Sham ADX
corticosterone
Unilateral adrenalectomy
(jug/100 ml)
(>ig/l00ml) 10 0
1
Time after adrenal surgery
1/2 I 3 7-10
Days after operations (days)
FIG. 3. There is no difference in resting plasma ACTH and corticos- FIG. 4. Plasma ACTH and corticosterone levels 5 min after ether
terone levels after unilateral adrenalectomy. Mean resting levels are vapors were administered to rats 1, 3, or 7 days after sham adrenalec-
from groups of five to eight rats killed at various times after unilateral tomy (cz=i) or unilateral adrenalectomy (ES-B). The vertical lines on
adrenalectomy (A) or sham unilateral adrenalectomy (A). or the bars indicate ±SEM. Each mean represents the results from five to
, Overall means at the various times. eight rats.
 STRESS RESPONSE AFTER UNILATERAL ADRENALECTOMY 1413

800 >4000 Laparotomy with intestinal traction. The two groups of

rats were tested 3 days after adrenal surgery with the
600 ADX stimulus of laparotomy with intestinal traction. Samples
were collected before, and 3 and 10 min after the stimulus
Plasma
400 (Fig. 6). Resting ACTH and corticosterone levels were
ACTH
similar, but the unilaterally adrenalectomized rats re-
(pg/ml)
sponded to the stimulus with higher plasma ACTH levels
200
than the sham-operated rats at 3 min (P < 0.05). At 10
min, ACTH levels were higher and corticosterone levels
0 were lower in rats with one adrenal than in those with
two (P < 0.01 in each case). Analysis of variance (adrenal
20 number and time) revealed a significant difference in the
Adrenal ACTH [F = 8.84 (1, 35); P = 0.005] but not in the
corticosterone 10 corticosterone [F = 3.30 (1, 35); P = 0.078] responses, due
(jug/100 mg) to adrenal number. There was a significant time effect in
0 both cases (P < 0.01).
Intraperitoneal saline injection. The two groups of rats
50 were subjected to saline injections 24 h after adrenal
Plasma surgery. In response to this slight stimulus to ACTH
corticosterone 25 secretion, there was no difference between the ACTH
(jug/100 ml) responses in the two groups (Table 1), although there
was a decrease in the magnitude of the plasma corticos-
0 terone response to the stimulus in rats with one adrenal
0 5 15 (5-min values), which may explain (via the diminished
Time after ether corticosterone rate-sensitive feedback signal) the higher
(min) ACTH levels at 10 min in the unilaterally adrenalecto-
15 min mized group.
after
8 U ACTH Hypothalamic content of CRF-like activity 2 min after
ip- ether vapors. Three experiments were performed on the
FIG. 5. Plasma ACTH and adrenal and plasma corticosterone concen-
trations with time after exposure to ether vapors or 15 min after ACTH 400
in rats 24 h after sham adrenalectomy or unilateral adrenalectomy
(eight per group). Mean values are accompanied by lines representing
±SEM. 300
Plasma
ACTH 200 4
ACTH were injected ip in other rats to test the maximum (pg/ml)
response at 15 min of right adrenal and plasma corticos- 100
terone levels in rats with one and two adrenals (Fig. 5).
Circulating ACTH levels were more than 4000 pg/ml in 0
both groups; however, adrenal corticosterone levels were
30
lower in both groups than the levels achieved 15 min
after exposure to ether vapor. Mean plasma corticoster-
one levels were similar 15 min after supramaximal doses Plasma 20
of ACTH to those observed in the two groups after corticosterone
exposure to ether vapors (Fig. 5). These results suggest (;jg/100 ml) 10
that the magnitude of the ACTH response to ether in
the sham-adrenalectomized rat stimulates the synthesis 0L
and secretion of corticosterone maximally. Therefore, the 0 10
increased ACTH levels found in unilaterally adrenalec- Time after laparotomy + traction
tomized rats after ether do not appear to serve any (min)
immediate purpose of increased adrenocortical stimula- FIG. 6. Plasma ACTH and corticosterone levels with time after lapa-
tion relative to that achieved in the sham-operated con- rotomy and intestinal traction in rats 72 h after sham adrenalectomy or
trols. unilateral adrenalectomy (eight per group).
 Endo • 1980
1414 ENGELAND ET AL. Vol 106 • No 5

TABLE 1. Plasma ACTH and corticosterone (B) responses of rats to ip I | Sham adrenalectomy
saline 24 h after unilateral or sham adrenalectomy (n = 6/group)

Unilateral adrenalec- \Z} Unilateral adrenalectomy

Sham adrenalectomy
Plasma tomy * p<0.05
time
(min) ACTH B (/ig/100 ACTH B (/ig/100
(pg/ml) ml) (pg/ml) ml)
500 rh
0 38 ± 15" 4.2 ± 0.5 38 ± 6 7.1 ± 1.1 Hypothalamic
2.5 105 20 9.0 ± 4.3 97 ± 18 6.5 ± 3.1 "CRF"
5.0 140 ± 41 44.1 ± 6.1 121 ± 47 26.3 ± 3.8
activity 250
10.0 40 ± 7 36.3 ± 2.4 102 23 35.5 + 4.2
(pg ACTH/30 min)
" Means ± SEM.
0
two groups of rats 7 days after adrenal surgery. In two of 500
the three experiments, performed at subjective lights-on
for the rats, hypothalamic CRF activity was significantly Plasma
higher in rats with one adrenal than in sham-adrenalec- ACTH 250
tomized animals (Fig. 7). In the third experiment, which (pg/ml)
was performed on rats at the time of their subjective 0
lights-off, the difference in CRF-like activity was not
significant. Plasma ACTH levels 2 min after ether were Experiment I II III
significantly greater in unilaterally adrenalectomized rats FIG. 7. Hypothalamic CRF-like activity and plasma ACTH levels 2
than in sham-operated animals in all three experiments, min after exposure to ether vapors in rats 7 days after sham adrenal-
although ACTH levels were lower in both groups in the ectomy or unilateral adrenalectomy (5-15/group). CRF-like activity
was assayed by determining ACTH released into the medium from
third experiment, which was performed at the time of quartered hemipituitaries, as described in Materials and Methods.
day when rats have the smallest CRF and ACTH re-
sponses to stimuli (13).
effect revealed by unilateral adrenalectomy occurs before
a significant elevation in circulating plasma levels of the
Discussion
major secretory product, corticosterone, we suggest that
The results of these experiments show clearly that the additional feedback signal is neural.
unilaterally adrenalectomized rats have an elevated hy- Evidence for a neural connection between the adrenal
pothalamic content of CRF-like activity and elevated and the hypothalamus, and for the negative influence of
plasma ACTH levels compared to those in sham-adre- adrenal volume sensors on the hypothalamic VMN was
nalectomized rats after some stimuli. The increased re- reviewed above. We believe that the data presented here,
sponsiveness to ether vapors or laparotomy with intes- showing increased responsiveness of the rat adrenocor-
tinal traction is not a result of a decreased corticosterone tical system to the stimuli of ether and laparotomy with
feedback signal in the unilaterally adrenalectomized rats. intestinal traction, are easily accommodated by postulat-
Resting corticosterone levels did not differ between the ing a decrease in the level of neural feedback that results
two groups in these experiments (Fig. 1), in our previous from decreased adrenal mass in unilaterally adrenalec-
studies (5-7, 14), or in studies by Mialhe et al. (15). tomized rats.
Increased plasma ACTH levels occur in unilaterally It seems likely from these results that the adrenal
adrenalectomized rats at a time after stress before cir- neural input may affect the responses to some stimuli
culating corticosterone levels differ from those of sham- but not others {i.e. ip saline injection). Resolution of this
adrenalectomized rats. These results strongly suggest possibility must await further experimentation using sev-
that there is a negative feedback signal from the adrenal eral other stimuli to the adrenocortical system.
which is not corticosterone that inhibits CRF and ACTH After bilateral adrenalectomy, both resting and stim-
secretion. Moreover, it seems likely that the adrenal ulated ACTH levels increase markedly (10, 13, 16). Al-
factor may not be a humoral signal at all, since its effects though it seems clear that the elevated resting levels can
are apparent at 2 min, a time before increased adrenal be reduced to normal by treating rats with replacement
secretion of corticosterone has appreciably altered doses of corticosterone (16), it was not possible to restore
plasma corticosterone from control levels. It is reasonable the response to laparotomy after adrenalectomy to that
to assume that all secretions from the adrenal cortex of sham-operated controls (16). The possibility was raised
occurring in response to ACTH will be elaborated more that another signal from the adrenal in addition to cor-
or less simultaneously. Because the adrenal feedback ticosterone was required to obtain stress-induced ACTH
 STRESS RESPONSE AFTER UNILATERAL ADRENALECTOMY
secretion of normal pattern and amplitude after bilateral
adrenalectomy (16). The data and discussion presented 9.
here for unilaterally adrenalectomized rats suggest that
the loss of neural feedback from the adrenal may also
10.
contribute to the hyperresponsiveness to ACTH-releas-
ing stimuli observed after bilateral adrenalectomy. Stud-
ies are currently in progress to examine this possibility. 11.
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