0013-7227/81/1091-0162$02.
00/0
Endocrinology
Copyright © 1981 by The Endocrine Society
Return of Pituitary-Adrenal Function after Adrenal
Enucleation or Transplantation: Diurnal Rhythms and
Responses to Ether*
CHARLES W. WILKINSON,! JEANETTE SHINSAKO, AND MARY F. DALLMAN|
Department of Physiology and Metabolic Research Unit, University of California,
San Francisco, California 94143
ABSTRACT. Adrenocortical diurnal rhythms and responses to
ether vapor were studied in rats 1, 3, and 5 weeks after bilateral
adrenal enucleation, autotransplantation, or sham transplanta-
tion in order to 1) determine whether diurnal rhythms in the
plasma corticosterone concentration and adrenal responsiveness
to ACTH are dependent on innervation of the adrenals, 2)
compare regeneration of function of transplanted and enucleated
adrenals, and 3) investigate adrenal mass-related nonsteroidal
inhibition of ether-stimulated ACTH secretion. Rats in both
enucleate and transplant groups exhibited significant morning-
evening differences in adrenal and plasma corticosterone con-
centrations and significant adrenocortical responses to ether 3
and 5 weeks, but not 1 week, after surgery. The morning-evening
differences in corticosterone concentration occurred in the ab-
sence of significant morning-evening variation in the plasma
f T l H E R E are circadian rhythms in circulating ACTH
A and corticosteroid concentrations in rats and other
mammals. The two rhythms are normally in phase, but
the plasma corticosterone rhythm of rats is amplified
relative to the ACTH rhythm. We have demonstrated
that this amplification is largely the result of a synchro-
nous rhythm in adrenal responsiveness to ACTH (1-3).
In light of our previous findings that compensatory ad-
renal growth after unilateral adrenalectomy is mediated
by adrenocortical nerves (4-6), the very rapid decline in
adrenal responsiveness to ACTH that occurs under some
experimental conditions (7) suggested that the rhythm in
adrenal responsiveness might also be mediated by adre-
nal nerves. Ottenweller et al. (8) have also proposed
neural mediation of rhythms in plasma corticosterone.
They found no diurnal rhythm in plasma corticosterone
Received August 19,1980.
Address requests for reprints to: Dr. Charles W. Wilkinson, Depart-
ment of Medicine, Division of Endocrinology and Metabolism, Mount
Sinai School of Medicine, New York, NY 10029.
* This work was supported by NIH Research Grant AM-06704. It
was presented in part at the 62nd Annual Meeting of The Endocrine
Society, Washington, D.C., June 18-20,1980.
f Recipient of NIH Postdoctoral Fellowship AM-05681.
$ Recipient of Research Career Development Award AM-OOO72.
162
Vol. 109, No. 1
Printed in U.S.A.
ACTH concentration, supporting our previous finding of a diur-
nal rhythm in adrenal responsiveness to ACTH. The responsive-
ness rhythm cannot be dependent on adrenal nerves unless
transplanted adrenals receive functionally specific reinnervation
within 3 weeks. The processes of regeneration of function after
enucleation and after transplantation are similar; there were no
differences in plasma or adrenal corticosterone values between
rats in enucleate and transplant groups at any time or under any
condition tested. As regeneration progressed, plasma ACTH
responses to ether declined in both enucleate and transplant
groups in the absence of changes in plasma corticosterone feed-
back. These results support our previous finding of adrenal mass-
related nonsteroidal suppression of ACTH responses to ether.
(Endocrinology 109: 162, 1981)
in rats 3 weeks after adrenal autotransplantation, al-
though the same rats exhibited a plasma corticosterone
response to ether 1 week later. Their interpretation was
that the transplanted adrenals were capable of respond-
ing to ACTH, but did not generate circulating cortico-
sterone rhythms because they lacked innervation.
After adrenal transplantation, regeneration occurs that
is histologically similar to regeneration after adrenal
enucleation (9). After transplantation, necrosis of the
medulla and most of the cortex occurs. After enucleation,
the empty capsule fills with blood, which is gradually
resorbed. In both cases, regeneration of the three cortical
zones occurs from the capsule and a thin rim of cortical
cells. Regeneration of the cortex is complete in approxi-
mately 5 weeks; the medulla does not regenerate. The
primary difference between transplantation and enuclea-
tion is that after enucleation, the vascular and neural
connections of the adrenal remain in contact with the
capsule. Enucleated adrenals show functional evidence
of reinnervation within 6 weeks, but not 3 weeks, as
indicated by the occurrence of neurally mediated com-
pensatory adrenal growth after unilateral adrenalectomy
(10). There is no evidence that functionally specific rein-
nervation of transplanted adrenals can occur.
 RESPONSIVENESS OF REGENERATING ADRENALS 163

TABLE 1. Mean data ± SEM (n = 6-9/group) from rats after sham operation (Sham), adrenal enucleation (Enuc), or adrenal transplantation
(Trans) under resting conditions and 5 min after exposure to ether vapor

Resting Ether
Weeks after surgery
Sham Enuc Trans Sham Enuc Trans
Plasma ACTH (pg/ml)
AM
1 33 ± 5 207 ± 40 218 ± 32 293 ± 16 740 ± 94 641 ± 67
3 25 ± 5 40 ± 9 48 ± 11 315 ± 49 350 ± 46 423 ± 42
5 35 ± 7 23 ± 3 30 ± 4 241 ± 39 302 ± 58 352 ± 55
PM
1 69 ± 17 522 ± 49 636 ± 42 278 ± 20 824 ± 72 786 ± 48
3 53 + 11 110 ± 28 139 ± 31 234 ± 31 435 ± 78 609 ± 68
5 82 ± 11 78 ± 13 98 ± 12 202 ± 30 241 ± 29 313 ± 46
Adrenal corticosterone (ng/100 g BW)
AM
1 138 ± 21 137 ± 24 105 ± 14 807 ± 135 154 ± 22 144 ± 17
3 160 ± 22 108 ± 23 136 ± 34 684 ± 48 451 ± 67 369 ± 25
5 174 ± 35 118 ± 24 121 ± 32 704 ± 75 527 ± 64 495 ± 89
PM
1 749 ± 125 186 ± 27 122 ± 13 921 ± 79 158 ± 7 112 ± 7
3 630 ± 136 523 ± 59 487 ± 31 893 ± 107 570 ± 38 522 ± 72
5 708 ± 73 569 ± 59 483 ± 42 721 ± 88 556 ± 63 602 ± 63
Plasma corticosterone (/xg/dl)
AM
1 0.8 ± 0.1 1.7 ± 0.2 2.0 ± 0.2 18.0 ± 1.0 4.6 ± 0.7 3.6 ± 0.4
3 0.7 ± 0.2 0.6 ± 0.1 1.1 ±0.2 12.3 ± 1.4 6.5 ± 0.6 7.2 ± 0.6
5 0.6 ± 0.1 0.8 ± 0.3 0.7 ± 0.2 12.8 ± 1.4 11.3 ± 1.6 7.5 ± 1.0
PM
1 18.0 ± 2.5 8.0 ± 0.9 4.6 ± 0.7 29.2 ± 2.0 8.1 ± 0.6 5.0 ± 0.6
3 14.0 ± 3.0 10.1 ± 1.2 12.9 ± 1.8 24.1 ± 2.2 16.2 ± 1.2 15.6 ± 1.5
5 19.2 ± 3.8 11.7 ± 1.3 11.0 ± 0.9 21.9 ± 1.3 14.6 ± 1.4 14.2 ± 1.5

To determine the role of adrenal nerves in the diurnal
rhythms of the plasma corticosterone concentration and
adrenal responsiveness to ACTH in rats, we investigated
morning-evening (AM-PM) differences in resting and
ether-stimulated pituitary-adrenocortical function at
three time intervals after bilateral adrenal enucleation,
autotransplantation, and sham transplantation.
Materials and Methods
Three hundred male Sprague-Dawley rats, weighing 100-200
g, were housed two per cage in a room with a standard 12-h
light, 12-h dark (LD 12:12) lighting schedule and regulated
temperature (23 ± 2 C). Rats were subjected to one of three
surgical procedures performed under ether anesthesia and com-
pleted within 20 min. For all procedures, a dorsal midline skin
incision was made, and the lateral abdominal wall was parted
to expose the adrenal. Bilateral adrenal enucleation was per-
formed by lifting each adrenal carefully without compromising
its vascular supply, slitting the capsule with microscissors, and
extruding the body of the gland with small blunt forceps. For
adrenal autotransplantation, each adrenal was removed, placed
on filter paper moistened with 0.9% saline, trimmed of fat, and
cut into quarters. The kidney ipsilateral to the removed adrenal
was then gently exteriorized, a slit was made in the renal
capsule, and the adrenal quarters were inserted into the result-
ing pocket. The kidney was then carefully replaced, the muscle
was sutured, and the operation was repeated on the contralat-
eral side. Two parts of the procedure are critical for successful
and complete transplantation: 1) removal of the adrenal must
include all of the capsule or the adrenal will regenerate in situ,
and 2) care must be taken in replacing the kidney to insure that
all of the transplanted tissue remains in the capsular pocket.
The sham operation consisted of locating but not touching each
adrenal, exteriorizing each kidney, slitting its capsule, replacing
the kidney, and suturing the muscle. The skin was closed with
wound clips in all cases. All rats were given a 0.5% saline
solution to drink in place of water for the first 7 days after
surgery.
Seven, 21, and 35 days after surgery, groups of rats from each
surgical condition were decapitated within 30 min of lights on
(0900 h) or lights off (2100 h). Each of these rats was removed
from a separate cage and decapitated within 10 sec of the time
the cage was initially touched in order to obtain valid resting
plasma ACTH concentrations. The remaining rat in each cage
was removed, placed for 1 min in a tank saturated with ether
vapor, returned to its home cage, and decapitated 5 min ± 5 sec
after the onset of exposure to ether. Trunk blood was collected
in heparinized plastic tubes, chilled immediately, and centri-
fuged at 4 C. Plasma was removed and frozen for subsequent
RIA of ACTH (11,12) and corticosterone. Corticosterone assays
were performed, using corticosterone antisera from Radioassay
Systems Laboratories (Carson, CA), on unextracted plasma in
which binding proteins had been heat denatured (13). After
164 WILKINSON, SHINSAKO, AND DALLMAN Endo • 1981
Vol 109 • No 1

TABLE 2. Separate three-way analyses of variance for each type of surgery

Plasma ACTH Adrenal B Plasma B

df
F P F P F P
Shams
Main effects
Weeks 2,78 1.2 NS 0.8 NS 3.7 <0.05
AMPM 1,78 0.1 NS 44.5 <0.001 143.2 <0.001
State 1,78 228.9 <0.001 50.1 <0.001 88.8 <0.001
Interactions
AMPM-state 1,78 8.5 <0.005 17.7 <0.001 6.0 <0.05
Enucleates
Main effects
Weeks 2,77 72.2 <0.001 46.2 <0.001 16.0 <0.001
AMPM 1,77 9.3 <0.005 53.7 <0.001 170.9 O.001
State 1,77 113.0 <0.001 26.6 <0.001 73.9 <0.001
Interactions
Weeks-AMPM 2,77 3.9 <0.05 9.0 <0.001 5.5 <0.01
Weeks-state 2,77 3.6 <0.05 6.9 <0.005 8.1 <0.001
AMPM-state 1,77 3.2 NS 24.5 <0.001 8.2 <0.005
Transplants
Main effects
Weeks 2,80 70.4 <0.001 51.0 <0.001 34.5 <0.001
AMPM 1,80 31.1 <0.001 35.8 <0.001 157.1 <0.001
State 1,80 159.5 <0.001 26.7 <0.001 43.5 <0.001
Interactions
Weeks-AMPM 2,80 8.5 <0.001 10.0 <0.001 20.1 <0.001
Weeks-state 2,80 3.2 NS 6.9 <0.005 5.3 <0.01
AMPM-state 1,80 3.3 NS 10.2 <0.005 6.3 <0.05
Independent variables are time after surgery (weeks), time of day (AMPM), and activation state (state). Specific interactions are included only
if there is a significant effect on at least one dependent variable. B, Corticosterone.

blood collection, all adrenal tissue was removed. All animals
that had received autotransplants were carefully inspected for
possible adrenal regeneration in situ and possible implantation
of the adrenal in the abdominal wall. Adrenal tissue was cleaned
and homogenized in 20% ethanol-80% saline for determination
of the corticosterone content by RIA.
The adrenal corticosterone concentration could not be ob-
tained directly because of the difficulty, particularly at 1 week,
of separating blood clots or necrotic tissue from enucleated or
transplanted cortical tissue for weighing. Therefore, the total
corticosterone content of adrenal tissue was determined. Adre-
nal weight increases linearly with body weight in male rats
between 120-300 g (14), but the adrenal corticosterone concen-
tration does not change with body weight in this range. There-
fore, measures of tissue corticosterone content were converted
to indirect indices of concentration by expressing them as
nanograms of corticosterone per 100 g BW (normalized adrenal
corticosterone content).
Because of the difficulty of drawing specific conclusions from
a four-way analysis of variance, the analysis was divided into
two sets of three three-way analyses to answer two primary
questions (15). The first question concerns changes over time
within each surgical condition, in diurnal rhythms and re-
sponses to ether (see Table 2). The second question concerns
differences among the three surgical groups at each time after
surgery (see Table 3). Pairwise comparisons among group
means were made using the Tukey multiple range test (a =
0.05) (15, 16).
Results
In sham-operated rats, there were no significant AM-
PM differences in the resting plasma ACTH concentra-
tion at any time after surgery, although there was a
consistent tendency for PM levels to be higher (Tables 1
and 2 and Fig. 1). There was a significant ACTH response
to ether in the sham groups in both the AM and PM at
all three times after surgery, but the increment in ACTH
concentration was consistently greater in the AM. In
sham-operated rats, neither resting nor ether-stimulated
plasma ACTH concentrations, either in the AM or PM,
changed with time after surgery.
In rats with enucleated or transplanted adrenals, AM-
PM differences in resting ACTH levels were significant
at 1 week, but not at 3 or 5 weeks (Tables 1 and 2 and
Fig. 1). Ether-elicited increases in plasma ACTH concen-
tration occurred in all enucleate and transplant groups,
except the PM transplant group, 1 week after surgery. In
this group, increases above the mean resting ACTH
concentration of 636 pg/ml may have been limited by
pituitary ACTH content. In rats with enucleated or
transplanted adrenals, AM and PM resting ACTH levels
and AM responses to ether declined significantly between
1-3 weeks after surgery but did not decline further be-
tween 3-5 weeks. Ether-elicited ACTH concentrations in
 RESPONSIVENESS OF REGENERATING ADRENALS 165

transplanted adrenals than in sham-operated rats. The

CO JL PM response of rats with enucleated adrenals was inter-
co mediate. At 5 weeks, there were no significant differences
.c
CO in ACTH concentrations among the three surgical groups
n rn in resting or ether-elicited ACTH concentrations at
either time of day.
5 min after ether In the sham-operated rats, resting AM-PM differences
in the plasma corticosterone concentration were highly
(0 Resting significant at all three times after surgery (Tables 1 and
•4—1

CO 2 and Fig. 2). There were significant plasma corticoster-

o one responses to ether at all times tested, except the PM,
3 5 weeks after surgery. The smaller increments in plasma
C
LJJ corticosterone responses in the PM than in the AM are
consistent with other reports (17,18) and with the ACTH
response increments in these experiments. Neither the
AM nor PM plasma corticosterone concentration 5 min
after ether exposure reflects the peak of the response.
The sampling time was selected to determine the peak of
the plasma ACTH response and to measure initial in-
600
creases in adrenal and plasma corticosterone concentra-
c tions. In sham-operated rats, neither resting nor ether-
IS stimulated plasma corticosterone concentrations in
Q. 400
(0 either the AM or PM changed significantly with time
c after surgery.
200
At 1 week, there were significant resting AM-PM dif-

0900 2100
n
0900 2100
•
0900 2100
Time of day
Weeks after surgery
FIG. 1. Plasma ACTH concentrations of rats 1, 3, and 5 weeks after
bilateral enucleation, autotransplantation, or sham transplantation of
the adrenals. Each bar represents the mean ± SEM of six to nine rats.
• , Samples from rats decapitated within 10 sec of cage opening (resting
levels). EH, Values from rats decapitated 5 min after the onset of a 1-min
exposure to ether vapor. In each group of four bars, the two on the left
represent samples taken within 30 min of lights on (0900 h) in the
morning; the two bars on the right in each group represent samples
taken within 30 min of lights off (2100 h) in the evening.
the evening declined between 3-5 weeks after surgery in
rats with transplanted adrenals and declined between 1-
3 weeks and again between 3-5 weeks in rats with enu-
cleated adrenals.
Comparisons among surgical groups (Table 3 and Fig.
1) show that at 1 week, resting and ether-stimulated
plasma ACTH concentrations at both times of day were
significantly higher in the enucleate and transplant
groups than in the comparable sham groups. At 1 week,
plasma ACTH values for each of the enucleate groups
were not different from the values for the respective
transplant groups. At 3 weeks, the only difference in
ACTH concentration among the surgical groups was a
significantly larger PM response to ether in rats with
ferences in plasma corticosterone concentrations in rats
with enucleated adrenals but not in rats with trans-
planted adrenals, although neither AM nor PM resting
levels differed significantly between the two surgical
groups (Tables 1 and 2 and Fig. 2). PM plasma cortico-
sterone concentrations increased with time after surgery
in both enucleate and transplant groups, and in both
cases, AM-PM differences in resting plasma corticoster-
one levels were significant at 3 and 5 weeks. One week
after surgery, there were neither AM nor PM plasma
corticosterone responses to ether in either enucleate or
transplant groups, despite the increases in plasma ACTH
concentration. Significant AM plasma corticosterone re-
sponses to ether occurred in both enucleate and trans-
plant groups 3 and 5 weeks after surgery, and both AM
and PM corticosterone concentrations after ether expo-
sure increased with time after surgery.
Within each surgical group, the response patterns of
normalized adrenal corticosterone content were the same
as those of plasma corticosterone concentration, with
two exceptions (Tables 1 and 2 and Fig. 3). There was no
PM increase in the adrenal corticosterone content 5 min
after ether exposure in any surgical group at any time,
and there was no resting AM-PM difference in adrenal
corticosterone levels in rats with enucleated adrenals 1
week after surgery. Resting AM-PM differences and AM
responses to ether were found in sham groups 1, 3, and
5 weeks after surgery and in enucleate and transplant
groups at 3 and 5 weeks.
166 WILKINSON, SHINSAKO, AND DALLMAN Endo • 1981
Vol 109 • No 1

TABLE 3. Separate three-way analyses of variance for each time after surgery

Plasma ACTH Adrenal B Plasma B

df
F P F P F P
lWeek
Main effects
Surgery 2,79 90.6 <0.001 95.6 <0.001 145.8 <0.001
AMPM 1,79 31.1 <0.001 16.9 <0.001 132.0 <0.001
State 1,79 119.9 <0.001 19.4 <0.001 82.6 <0.001
Interactions
Surgery-AMPM 2,79 8.1 <0.001 12.1 <0.001 32.5 <0.001
Surgery-state 2,79 3.5 <0.05 16.1 <0.001 44.6 <0.001
AMPM-state 1,79 10.3 <0.005 8.4 <0.005 6.7 <0.05
3 Weeks
Main effects
Surgery 2,80 13.1 <0.001 13.8 <0.001 11.7 <0.001
AMPM 1,80 7.1 <0.01 58.8 <0.001 171.2 <0.001
State 1,80 182.6 <O.OO1 41.9 <O.OO1 75.7 <O.OO1
Interactions
Surgery-AMPM 2,80 4.2 <0.05 0.5 NS 1.3 NS
Surgery-state 2,80 4.9 <0.01 4.5 <0.05 5.5 <0.01
AMPM-state 1,80 0.0 NS 11.7 <0.005 1.0 NS
5 Weeks
Main effects
Surgery 2,76 3.4 NS 6.2 <0.005 11.6 <0.001
AMPM 1,76 0.1 NS 51.2 <0.001 116.3 <0.001
State 1,76 136.3 <0.001 41.4 <0.001 46.7 <0.001
Interactions
Surgery-AMPM 2,76 0.1 NS 0.2 NS 5.1 <0.01
AMPM-state 1,76 7.6 <0.01 29.8 <0.001 13.4 <0.001

Independent variables are type of surgery (surgery), time of day (AMPM), and activation state (state). Specific interactions are included only if
there is a significant effect on at least one dependent variable. B, Corticosterone.
Comparisons across surgical groups showed that 1
week after surgery, AM and PM ether-stimulated and
PM resting levels of both plasma and adrenal cortico-
sterone were significantly lower in the enucleate and
transplant groups than in the sham groups (Table 3 and
Figs. 2 and 3). The failure of the extremely high ether-
elicited plasma ACTH concentration to increase adrenal
or plasma corticosterone levels suggests that the enucle-
ated and transplanted adrenals at 1 week were working
at maximal capacity and/or were functioning autono-
mously. The differences in normalized adrenal cortico-
sterone contents among surgical groups were not the
result of differential increases in body weight. In the
week after surgery, the mean weight gain for the sham
groups was 39 ± 2 g compared to 44 ± 2 and 41 ± 2 g for
enucleate and transplant groups, respectively. There
were no significant differences in body weight among
surgical groups at any time. At 3 weeks, there were no
differences in resting adrenal or plasma corticosterone
levels among surgical groups at either time of day, but
both AM and PM adrenal and plasma corticosterone
responses in sham-operated rats were greater at 5 min
after ether exposure than those in the other two surgical
groups. In the PM at 5 weeks, both resting and ether-
stimulated plasma corticosterone concentrations in the
sham-operated animals were higher than those in the
other two surgical groups, but no other differences in
adrenal or plasma corticosterone values were found
among the three surgical groups.
Discussion
The presence of AM-PM differences in plasma and
adrenal corticosterone levels within 3 weeks after bilat-
eral adrenal autotransplantation or enucleation argues
strongly against the hypothesis that adrenal innervation
or the adrenal medulla is necessary for the maintenance
of adrenocortical rhythms. Neurally mediated compen-
satory adrenal growth does not occur in unilaterally
adrenalectomized rats 3 weeks after bilateral adrenal
enucleation, even though such animals are capable of an
accelerated adrenal growth rate (10). If enucleated ad-
renals are not functionally reinnervated within 3 weeks,
it is extremely improbable that completely denervated
transplanted adrenals would receive functionally specific
neural input within this time.
Our findings of plasma corticosterone rhythms in male
rats 3 weeks after autotransplantation are at odds with
those of Ottenweller et al. (8) in female rats, but are
supported by Gibson's recent report (19) of diurnal cor-
 RESPONSIVENESS OF REGENERATING ADRENALS 167

30 1200.-

ih g 800 h-
pfc
g 20
CO T
T rr~i
11 co
JC —
Tf
i
CO 10 CO 400
•

o
n in ri 1
[:::vi:::::: N 5 min after ethei 5 min after ether
Resting
Resting to 800
co 20
CD
•«->
CO _r_ T T
JL "S
CO
400
O
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C l5 0 M \ \ m 1 1
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Q. 10
CO
c
ca
CO

0900 2100 0900 2100 0900 2100 0900 2100

0900 2100 0900 2100
Time of day
Time of day

1 3 5
Weeks after surgery
FIG. 2. Plasma corticosterone concentrations of rats 1, 3, and 5 weeks
after bilateral enucleation, autotransplantation, or sham transplanta-
tion of the adrenals. Each bar represents the mean ± SEM of six to
nine rats. Groups are organized and keyed as described in Fig. 1.
ticosterone rhythms in male rats 3.5 weeks after auto-
transplantation. It is possible that the regeneration proc-
ess occurs more slowly in female rats, and that by 4
weeks, when plasma corticosterone responses to ether
were observed in females, diurnal rhythms may also have
been present. Our data suggest that in regenerating ad-
renals, the capacity for steroidogenic responses to acute
increases in ACTH secretion returns at approximately
the same time as AM-PM differences in responsiveness
to ACTH.
The consistent finding of pronounced AM-PM differ-
ences in adrenal and plasma corticosterone levels in
sham-operated rats in the absence of significant AM-PM
differences in plasma ACTH concentrations confirms our
earlier findings of diurnal rhythms in adrenal responsive-
ness to ACTH (1-3). This interpretation of the data is
strengthened by consideration of the adrenal responsive-
ness measures plotted in Fig. 4. In bioassay systems,
adrenal corticosterone concentration and secretion rate
increase linearly with logarithmic increases in the dose
of ACTH (20, 21). Therefore, if there are no diurnal
changes in adrenal responsiveness to ACTH, the ratio of
Weeks after surgery
FIG. 3. Corticosterone content of adrenal tissue per 100 g BW of rats
1, 3, and 5 weeks after bilateral enucleation, autotransplantation, or
sham transplantation of the adrenals. Each bar represents the mean
± SEM of six to nine rats. Groups are organized and keyed as described
in Fig. 1.
the resting adrenal corticosterone concentration to the
logarithm of the resting plasma ACTH concentration
should remain constant throughout the day. Diurnal
fluctuations in this ratio indicate diurnal rhythms in
adrenal responsiveness to ACTH (2). Since there are no
AM-PM differences in the MCR of corticosterone in
female rats (3), the ratio of the resting plasma cortico-
sterone concentration to the logarithm of the resting
plasma ACTH concentration should also reflect adrenal
responsiveness to ACTH. Figure 4 shows that there is a
large difference between AM and PM values of both
ratios in sham-operated rats at all three times after
surgery.
There were no AM-PM differences in adrenal respon-
siveness to ACTH 1 week after surgery in either the
transplant or enucleate groups. Evening adrenal respon-
siveness to ACTH increased with time after surgery in
both groups. However, even at 5 weeks, the PM ratios of
plasma corticosterone to log ACTH concentration of the
enucleate and transplant groups did not return to control
values (Fig. 4, top). When the adrenal corticosterone
168 WILKINSON, SHINSAKO, AND DALLMAN Endo • 1981
Vol 109 • No 1

10,- adrenalectomized rats responded to ether exposure with

elevated hypothalamic corticosterone-releasing factor-
C 8 like activity and plasma ACTH concentrations relative
T3
to those in sham-adrenalectomized rats (23). These dif-
I 6
\
CO
ferences occurred in the absence of differences in resting
O)
o plasma ACTH or corticosterone levels and were evident
4
Q. within 2 min, before steroid-mediated feedback inhibition
O5
O 2
could have occurred. In another set of experiments in
which the resting plasma corticosterone concentrations
• o Shams of bilaterally, unilaterally, or sham-adrenalectomized rats
• ° Enucleates were equalized by administering corticosterone in the
• * Transplants drinking fluid, the ether-elicited ACTH secretion was
b.w.

400 — PM inversely related to the number of adrenals remaining in

E — AM the rats (24).
o \
X
300 In the present experiments, the mean resting and
o
ether-stimulated plasma ACTH concentrations of the
B/1

o 200
a were much greater than those of the sham groups, despite
c O) 100
0)
•o
(0
0
O)
c in plasma ACTH values could have been the result of
Weeks after surgery
FIG. 4. Adrenal responsiveness to ACTH of rats in each of the three
surgical groups 1, 3, and 5 weeks after surgery. Adrenal responsiveness
is expressed as the resting plasma corticosterone concentration (top) or
the normalized adrenal corticosterone content (bottom) divided by the
logarithm of the resting plasma ACTH concentration.
content was used to calculate the index of responsiveness
(Fig. 4, bottom), transplanted but not enucleated adrenals
were significantly less responsive than control adrenals
at 5 weeks. This indication of less than total return of
function 5 weeks after surgery is supported by the finding
that PM plasma corticosterone concentration 5 min after
ether exposure in the 5-week-enucleated and -trans-
planted groups is lower than that in sham-operated ani-
mals and by evidence that regenerated adrenals do not
regain normal size in relation to body weight within 3
months after enucleation even though they continue to
grow as the rats grow (10, 22).
Regardless of which index of responsiveness was used,
regenerating enucleated and transplanted adrenals did
not differ from one another with respect to either AM or
PM responsiveness to ACTH at any time after surgery.
These data reinforce the finding that plasma and adrenal
corticosterone resting levels and 5-min responses to ether
did not differ between the two conditions at any time.
Together, these results support the hypothesis that re-
generating enucleated and regenerating transplanted ad-
renals are functionally, as well as histologically (9), very
similar.
These experiments also provide support for our pre-
vious finding of an adrenal mass-related nonsteroidal
inhibition of ether-elicited ACTH secretion. Unilaterally
enucleate and transplant groups in the AM at 1 week
the fact that resting plasma corticosterone concentra-
tions did not differ among the groups. These differences
differences in the magnitude of the plasma corticosterone
feedback signal during the preceding 12 h and/or differ-
ential nonsteroidal feedback related to the mass of the
adrenals. However, 3 weeks after surgery, there were no
differences in AM or PM resting plasma corticosterone
concentrations among the surgical groups, but the PM
ACTH response to ether was elevated in the transplant
group relative to that in the sham-operated group. When
regeneration was near completion at 5 weeks, there were
no differences among surgical groups in the ACTH re-
sponses to ether. The finding of a gradual reduction of
ACTH responses as adrenal regeneration progressed is
consistent with the hypothesis of a nonsteroidal adrenal
mass-related input to the control of ACTH secretion.
The possibility that this input is mediated by neural
afferents from the adrenal is not consistent with the data
from these experiments, unless functional reinnervation
of transplanted adrenals can occur within 5 weeks.
Acknowledgments
We thank Angela DeCarlo for corticosterone assays, Annette Lowe
for illustrations, and Jeanne Connolly and Bonnie Iwamasa for typing
this manuscript.
References
1. Dallman MF, Engeland WC, Rose JC, Wilkinson CW, Shinsako J,
Siedenburg F 1978 Nycthemeral rhythm in adrenal responsiveness
to ACTH. Am J Physiol 235:R210
2. Wilkinson CW, Shinsako J, Dallman MF 1979 Daily rhythms in
adrenal responsiveness to adrenocorticotropin are determined pri-
marily by the time of feeding in the rat. Endocrinology 104:350
3. Kaneko M, Hiroshige T, Shinsako J, Dallman MF 1980 Diurnal
changes in amplification of hormone rhythms in the adrenocortical
system. Am J Physiol 239:R309
4. Engeland WC, Dallman MF 1976 Neural mediation of compensa-
 RESPONSIVENESS OF REGENERATING ADRENALS
tory adrenal growth. Endocrinology 99:1659
5. Dallman MF, Engeland WC, Shinsako J 1976 Compensatory ad-
renal growth: a neurally mediated reflex. Am J Physiol 231:408
6. Holzwarth MA, Dallman MF 1979 The effect of hypothalamic
hemi-islands on compensatory adrenal growth. Brain Res 162:33
7. Wilkinson CW, Shinsako J, Dallman MF 1978 Rapid reduction in
adrenocortical responsiveness to ACTH following restricted access
to water. Physiologist 21:128 (Abstract)
8. Ottenweller JE, Meier AH, Ferrell BR, Horseman ND, Proctor A
1978 Extrapituitary regulation of the circadian rhythm of plasma
corticosteroid concentration in rats. Endocrinology 103:1875
9. Skelton FR 1959 Adrenal regeneration and adrenal-regeneration
hypertension. Physiol Rev 39:162
10. Holzwarth MA, Shinsako J, Dallman MF 1980 Adrenal regenera-
tion: time course, effect of hypothalamic hemi-islands and response
to unilateral adrenalectomy. Neuroendocrinology 31:168
11. Rees LH, Cook DM, Kendall JW, Allen CF, Kramer RM, Ratcliffe
JG, Knight RA 1971 A radioimmunoassay for rat plasma ACTH.
Endocrinology 89:254
12. Dallman MF, DeManincor D, Shinsako J 1974 Diminishing corti-
cotrope capacity to release ACTH during sustained stimulation:
the twenty-four hours after bilateral adrenalectomy in the rat.
Endocrinology 95:65
13. Abraham GE, Manlimos FS, Garza R 1977 Radioimmunoassay of
steroids. In: Abraham GE (ed) Handbook of Radioimmunoassay.
Marcel Dekker, New York, p 591
14. Holzwarth MA, Wilkinson CW, Dallman MF 1980 Compensatory
adrenal growth in immature and mature male rats. Neuroendocri-
nology 31:34
169
15. Nie NH, Hull CH, Jenkins JG, Steinbrenner K, Bent DH 1975
Statistical Package for the Social Sciences, ed 2. McGraw-Hill, New
York
16. Winer BJ 1971 Statistical Principles in Experimental Design, ed 2.
McGraw-Hill, New York
17. Zimmermann E, Critchlow V 1967 Effects of diurnal variation in
plasma corticosterone levels on adrenocortical response to stress.
Proc Soc Exp Biol Med 125:658
18. Dunn J, Scheving L, Millet P 1972 Circadian variation in stress-
evoked increases in plasma corticosterone. Am J Physiol 223:402
19. Gibson MJ Circadian plasma corticosterone (B) rhythm and stress
responsiveness persist in rats with adrenal autotransplants. Ab-
stracts of the 62nd Annual Meeting of The Endocrine Society,
Washington DC, 1980, p 104 (Abstract)
20. Vernikos-Danellis J, Anderson E, Trigg L 1966 Changes in adrenal
corticosterone concentration in rats: method of bio-assay for
ACTH. Endocrinology 79:624
21. Saffran M, Schally AV 1955 In vitro bioassay of corticotropin:
modification and statistical treatment. Endocrinology 56:523
22. Pellegrino C, Trocigliani 1957 A cytochemical study of the adrenal
cortex regenerating after enucleation. J Physiol (Lond) 135:536
23. Engeland WC, Siedenburg F, Wilkinson CW, Shinsako J, Dallman
MF 1980 Stimulus-induced corticotropin-releasing factor content
and adrenocorticotropin release are augmented after unilateral
adrenalectomy, independently of circulating corticosteroid levels.
Endocrinology 106:1410
24. Wilkinson CW, Engeland WC, Shinsako J, Dallman MF 1981
Nonsteroidal adrenal feedback demarcates two types of pathways
to CRF-ACTH release. Am J Physiol 240:E136