Reprint Series

4 May 1984, Volume 224, pp. 452-459

SCIENCE

Stress Hormones: Their Interaction and Regulation

Julius Axelrod and Terry D. Reisine

Copyright 0 1984 by the American Association for the Advancement of Science


        Stress Hormones:
Their Interaction and Regulation

Julius Axelrod and Terry D. Reisine

The constancy of the "milieu inter-
ieur" is the condition of a free and
independent existence.

-CLAUDE BERNARD (I)

The body responds to increased physi-
cal or psychological demands by releas-
ing adrenocorticotropin (ACTH) from
the anterior pituitary, glucocorticoids
from the adrenal cortex, epinephrine
from the adrenal medulla, and norepi-
nephrine from sympathetic nerves.
These hormones serve to adapt the body
to stressors ranging from the mildly psy-
chological to the intensely physical by
affecting cardiovascular, energy-produc-
ing, and immune systems. It was the
19th-century physiologist Claude Ber-
nard who recognized the importance of
adaptive mechanisms with one of the
most cogent statements (cited above)
framed by a biological scientist (1). Wal-
ter Cannon referred to the complex bio-
logical responses necessary to maintain a
steady state in the body as homeostasis
(2). In a series of landmark experiments
during the early part of the 20th century,
Cannon recognized the importance of
the sympathomedullary system in react-
ing to stressful events evoked by acute

452

physical or psychobiological stressors
(3). He observed that the tissues liberate
a humoral agent which he termed "sym-
pathin." This was later identified as epi-
nephrine (adrenaline) and norepine@-
rine (noradrenaline) (4).

In 1936 Selye reported that diverse
noxious agents cause an enlargement of
the adrenal cortex as a consequence of
the "stress syndrome" (5). During the
following three decades many investiga-
tors observed that a variety of stressful
events cause a release of ACTH from the
anterior pituitary (6). The secreted
ACTH stimulates the synthesis of corti-
costeroids in the adrenal cortex. The
elevated corticosteroid levels in plasma
then inhibit the further release of ACTH
from the pituitary. In a series of elegant
experiments, Harris demonstrated that
the release of ACTH from the pituitary is
regulated by a corticotropin-releasing
factor (CRF) from the hypothalamus (7).
The CRF synthesized in the hypothala-
mus reaches the pituitary by a private
portal blood supply. It then stimulates
the secretion of ACTH from the pitu-
itary. After a long period of intensive
investigations CRF was isolated and pu-
rified, and its structure was character-

ized as a 41 amino acid peptide by Vale
and co-workers (8). CRF was thought to
be the major if not the sole means of
releasing ACTH from the pituitary. Re-
cent experiments indicate that ACTH
can also be released and regulated by
catecholamines and other hormones.

Catecholamines, Glucocorticoids, and

Sympathoadreno Activity

A variety of stressors cause an in-
creased activity of the sympathetic ner-
vous system and adrenal medulla (2).
This activity results in a discharge of
epinephrine and norepinephrine into the
blood stream and changes in the activity
of enzymes that synthesize catechol-
amines and in the concentrations of nor-
epinephrine and epinephrine in the brain.
With prolonged stress, marked compen-
satory changes in the activity of the
catecholamine biosynthetic enzymes ty-
rosine hydroxylase, dopamine P-hydrox-
ylase, and phenylethanolamine N-meth-
yltransferase (PNMT) occur. These
changes in enzyme activity are regulated
to varying degrees by glucocorticoids,
ACTH, and neuronal activity.

When mice are subjected to psychoso-
cial stressors through competition for
food and living space, they show in-
creases in blood pressure, adrenal
weight, and catecholamine concentra-
tions in the adrenal medulla (9). The
biosynthetic enzymes tyrosine hydroxy-
lase and PNMT are both increased in
mice experiencing excessive social stim-
ulation. Forced immobilization of rats

The authors are members of the Section on Phar-
macology, Laboratory of Clinical Science, National
Institute of Mental Health, Bethesda, Maryland
20205.

SCIENCE, VOL. 224


with the use of a model that combines
emotional stress (escape reaction) and
physical stress (muscle work) activates
both the sympathetic adrenal medullary
and adrenocortical systems and the pitu-
itary gland (IO). The activities of tyrosine
hydroxylase, dopamine P-hydroxylase,
and PNMT are increased in this type of
stress (II). Stress such as that associated
with swimming, electroshock, and insu-
lin also elevate these biosynthetic en-
zymes (II). Forced immobilization of
rats can cause an increase in the plasma
epinephrine and norepinephrine concen-
trations (12). Mild stressors such as
opening a cage door or handling a rat
produces an eightfold increase in plasma
epinpehrine concentrations.

stress the activity of tyrosine hydroxy-  these enzymes. As in the case of PNMT,
lase is rapidly elevated in the adrenal  removal of the pituitary gland of the rat
medulla without any change in the num-  results in a gradual reduction of tyrosine
ber of enzyme molecules (16). In chronic  hydroxylase and dopamine P-hydroxy-
stress, however, the activities of tyro-  lase activity in the adrenal medulla (22).
sine hydroxylase (17) and dopamine p-  *In hypophysectomized rats subjected to
hydroxylase are increased (18) in sympa-  immobilization stress the reduced dopa-
thetic nerves and the adrenal medulla as  mine P-hydroxylase activity in the adre-
a result of an increase in the number of  nal medulla can be restored by ACTH
enzyme molecules (16). If the pregangli-  and dexamethasone. From these experi-

The adrenal gland, important in stress
reactions, consists of at least two ana-
tomically and chemically distinct struc-
tures: an inner medullary area containing
catecholamine-producing   chromaffin
cells and an outer cortical region in
which glucocorticoids are synthesized.
In most mammals, the adrenal cortex
and medulla are contiguous and the main
catecholamine produced is epinephrine.
In some species, however, the chromaf-
fin tissue is separated from the adrenal
cortex and the predominant catechol-
amine found is norepinephrine (IS). This
suggested that a substance produced in
adrenal cortical tissue might gain access
to the medulla and regulate the conver-
sion of norepinephrine to epinephrine.
Such substances were thought to be glu-
cocorticoids whose synthesis is stimulat-
ed by ACTH. Whether or not giucocorti-
coids and indirectly ACTH modify the
formation of epinephrine in the adrenal
medulla was determined by subjecting
animals to hypophysectomy and, several
days later, measuring the activity of
PNMT, the epinephrine-forming enzyme
in the medulla (14). The activity of
PNMT showed marked decrease. Fur-
thermore, the repeated administration of
ACTH or the potent glucocorticoid
dexamethasone to hypophysectomized
rats restored PNMT activity to almost
normal values. PNMT activity in the
adrenal medulla is much lower in hy-
pophysectomized rats subjected to im-
mobilization stress (15), but this de-
crease can be prevented or reversed by
treatment with ACTH or glucocorti-
coids. These experiments indicated the
interrelationship of ACTH in the pitu-
itary and glucocorticoids in the adrenal
cortex in effecting the synthesis of epi-
nephrine (Fig. 1).

Summary. Stress stimulates several adaptive hormonal responses. Prominent
among these responses are the secretion of catecholamines from the adrenal
medulla, corticosteroids from the adrenal cortex, and adrenocorticotropin from the
anterior pituitary. A number of complex interactions are involved in the regulation of
these hormones. Glucocorticoids regulate catecholamine biosynthesis in the adrenal
medulla and catecholamines stimulate adrenocorticotropin release from the anterior
pituitary. In addition, other hormones, including corticotropin-releasing factor, vasoac-
tive intestinal peptide, and arginine vasopressin stimulate while the corticosteroids
and somatostatin inhibit adrenocorticotropin secretion. Together these agents appear
to determine the complex physiologic responses to a variety of stressors.

-

onic innervation to sympathetic nerves
of the superior cervical ganglia of the rat
are cut, the enzyme activity in cell bod-
ies and nerve terminals is not increased,
indicating that this mode of compensa-
tory regulation of the enzyme is a trans-
synaptic event (19). The transsynaptic
induction of tyrosine hydroxylase ap-
pears to be due to stimulation of acetyl-
choline nicotinic receptors (20). In-
creased stimulation of sympathetic
nerves also elevates the activity of dopa-
mine P-hydroxylase (18) and to a smaller
extent PNMT (21). Cutting the presynap-
tic nerves will block the increase of
dopamine P-hydroxylase and PNMT, in-
dicating a transsynaptic induction of

ments it appears that the stress-induced
increase in tyrosine hydroxylase is due
mainly to neuronal activity, whereas do-
pamine P-hydroxylase is affected by
both nerve activity and the pituitary ad-
renal axis and PNMT is controlled main-
ly by ACTH and glucocorticoids (Fig. 1).
By means of immunocytochemistry and
double labeling with radioisotopes, the
splanchnic nerve innervating the adrenal
medulla has been shown to induce de
novo synthesis of dopamine P-hydroxy-
lase and PNMT while glucocorticoids
inhibit degradation of these enzymes
(23).

Rats subjected to a variety of stressors
show considerably reduced concentra-

Fig. 1. Regulation of
catecholamine   bio-
synthesis in the adre-
nal  medulla.   The
expression of cate-
cholamines in the ad-
renal medulla is regu-
lated at a number of
different steus in the
biosynthetic-  path-

Anterior
pituitary

Af

way. Tyrosine hy-
droxvlase (TH) activi-
ty, ihe rate limiting
step in dopamine syn-
thesis, is affected by
nerve activity and to a
minor extent by glu-
cocorticoids of the
adrenal cortex. Dopa-
mine P-hydroxylase
(DBH) activity is reg-
ulated by nerve activ-
itv and elucocorti-

Nerve

The activity of tyrosine hydroxylase, a
rate-limiting enzyme in catecholamine
biosynthesis, can increase considerably
in response to stressors (II). In acute

coids. Pheiylethanol-
amine N-methyltrans-

Adrenal gland

ferase (PNMT), the enzyme that converts norepinephrine to epinephrine, is predominantly
regulated by glucocorticoids and to a small degree by nerve activity. Glucocorticoid synthesis is
stimulated by ACTH which is released from the anterior pituitary.

4 MAY 1984                                                                           453

ni


tions of brain norepinephrine (24). By
means of a technique for the precise
dissection of small areas of the brain (25)
it was observed that the stress-induced
depletion of norepinephrine occurs in
specific brain nuclei such as the nucleus
tractus solitarius, and the arcuate, peri-
ventricular, and ventromedial hypotha-
lamic nuclei (26). Epinephrine-contain-
ing neurons are also present in the brain
and localized in the brainstem (27). Im-
mobilization stress causes a selective
depletion of epinephrine in nucleus trac-
tus solitarius, locus ceruleus, and para-
ventricular and arcuate nuclei (28). Neu-
rons containing CRF have been found in

hypothalamic regions such as the para-
ventricular nucleus (29). Recent studies
have shown that inhibiting brain PNMT
activity enhances CRF immunoreactiv-
ity in the paraventricular nucleus, sug-
gesting that epinephrine may regulate the
activity of CRF-containing neurons (30).
Such an interaction between adrenergic
and CRF-containing neurons may be im-
portant in mediating stress-related re-
sponses.

Although a variety of stressors in-
crease the catecholamine concentrations
in urine (31), which can serve as a mea-
sure of stress, catecholamine concentra-
tions in plasma are generally considered

Table 1. Multireceptor control of cyclic AMP formation and ACTH release in mouse pituitary
tumor cells. An EDSo value is the concentration of drug at which 50 percent of the maximum

sttmulation occurs; an ICsO value is the concentration of drug at which 50 percent of the
maximum inhibition occurs. N.D., not determined.

Additions
(references)

ACTH release         Cyclic AMP

Stimulants
CRF (90)
P-Adrenergic agonists (48)
  (-)-Isoproterenol
  (+)-Isoproterenol
  (- )-Epinephrine
  (-)-Norepinephrine

VIP (31) . .
Forskolin (51, 91)
Cholera toxin (61)
Potassium (92)
Calcium ionophores
A23 187 (92)
Ionomycin (92)

Inhibitors
Dexamethasone*
  CRF (90)
  lsoproterenol (48)
  VIP (58)
  Forskolin (51)
Somatostatint
  CRF (93)
  Isoproterenol (93)
  VIP (93)
  Forskolin (93)
  Potassium (64, 92)
  Melittin (61)
  A23187 (61)
  Ionomycin (92)
  X537A (52, 92)

Receptor binding
Somatostatin (64)
PZ-Adrenergic (48)

Maximum
response
(percent of
basal)

EDso
(nM)

Maximum
response
(percent of
basal)

EDso
(nM

300 to 400

4 to 5

500

2

200 to 400         I          600         50
  100        N.D.         100       N.D.
200 to 400       50         600        100
200 to 400      200         400      1,000
  250          1         250          1
  500       1,ooO       4,000     10,000
  500        N.D.       4,000       N.D.
  400        N.D.         100       N.D.

400        N.D.         100       N.D.
400      5mo        N.D.       N.D.

Percentage
inhibition of
stimulated
response

Go
(nM)

Percentage
inhibition of
stimulated
response

Go
(nM)

100          I           0       N.D.
100           I           0       N.D.
60         IO           0       N.D.
90          5           0       N.D.

100          1       80 to 90         0.1
100           1       80 to 90         0.1
100          1       80 to 90         0.1
100          1       80 to 90         0.1
100          0.4      80 to 90        0.1
0        N.D.        N.D.       N.D.
0        N.D.        N.D.       N.D.
50        N.D.        N.D.       N.D.
100        N.D.        N.D.       N.D.

Dissociation
constant
  (nM)

Maximum
binding
(fmole/mg
protein)

I.7
11

141
64

I

"Cells were treated with IO-`M dexamethasone. The cells were washed and then dexamethasone and various
stimulants were added. ACTH released into the medium and intracellular cyclic AMP content were measured
after such manipulations.   YSomatostatin and various stimulants were added simultaneously to the cells.

   454

to be a more precise measure of the
stress-induced activation of the sympa-
thetic medullary system. Until recently it
was difficult to obtain a reliable measure
of plasma norepinephrine and epineph-
rine because of their extremely low con-
centrations. However, with the introduc-
tion of specific and highly sensitive ra-
dioenzymatic assays for catecholamines
it has become possible to determine their
concentrations (32) during basal condi-
tions and stressful situations in humans.
In general, plasma norepinephrine levels
reflect the activity of the sympathetic
nerves while epinephrine is a measure of
secretion from the adrenal medulla. Pos-
tural changes can cause two- to threefold
elevations in plasma norepinephrine but
negligible changes in epinephrine (33).
Public speaking results in a twofold in-
crease in plasma epinephrine and a 50
percent increase in norepinephrine (34).
The reverse is true in performing mental
arithmetic (3.5). During harassment, type
A individuals (coronary prone) have a
greater elevation of plasma epinephrine
than type B subjects (noncoronary
prone) (36). Depressed subjects show
increased basal levels of plasma norepi-
nephrine and epinephrine which has
been related to their degree of anxiety
(33. Propranolol, a P-adrenoreceptor
blocking drug, relieves somatic symp-
toms (tachycardia, tremors) or acute
panic state in anxious persons but ap-
pears to be of little use in other forms of
anxiety (38). Physical, cold, and thermal
stress can cause moderate to marked
elevation of plasma catecholamine (39).
Exercise also results in a rise in plasma
catecholamines (40) that depends on the
duration and severity of the exercise.
Stress due to surgery, hemorrhage, myo-
cardial infarction, hypoglycemia, and
hypoxia causes considerable increases in
plasma catecholamines (41). Hypoglyce-
mia induced by insulin or 2-deoxyglu-
case evokes the greatest increase in plas-
ma catecholamines, especially epineph-
tine. The high levels of catecholamine
achieved in hypoglycemia and myocardi-
al infarction may be enough to stimulate
B-adrenoreceptors on the anterior pitu-
itary. Studies in our laboratory have
revealed that insulin stress in rats stimu-
lates ACTH release from the pituitary
(42). This effect is blocked by proprano-
101 and since insulin stress raises plasma
catecholamine, the rise in ACTH release
appears to be due to the direct action of
epinephrine on the pituitary. It would be
of clinical interest to examine the effect
of propranolol on the plasma concentra-
tions of ACTH and glucocorticoids in
patients with hypoglycemia.

SCIENCE, VOL. 224


Multireceptor Release of

ACTH from Anterior Pituitary

Adrenocorticotropin is synthesized
and released from the anterior pituitary.
The availability of synthetic CRF (8, 43)
and of a mouse anterior pituitary cell line
(AtT-20/D16-16) that secretes ACTH
made it possible to study the mechanism
of release of ACTH and the involvement
of catecholamines, glucocorticoids, and
other hormones in this secretory pro-'
cess. Recent investigators of ACTH se-
cretion from the pituitary have usually
used primary cultures of the rat adeno-
hypophysis (44). Although much useful
information has been obtained with such
preparations, the heterogeneity of the
cell types and the low density of the
ACTH secreting cells (2 to 3 percent of
the total cell population) in the anterior
pituitary has limited the characterization
of factors directly controlling ACTH re-
lease. The AtT-20 cell line was used
previously to examine the processing of
ACTH from its precursor protein pro-
opiomelanotropin (POMC) as well as the
storage and secretion of ACTH and p-
endorphin (45). This cell line appears to
be homogeneous with regard to cell type
and, in contrast to primary anterior pitu-
itary cultures, predominantly releases
hormones of the POMC family of pep-
tides.

Normal anterior pituitary cells re-
spond to synthetic CRF by releasing
ACTH and P-endorphin (43). AtT-20
cells also secrete immunoreactive ACTH
and P-endorphin in response to CRF
(Table 1). Analogs of CRF show the
same order of potency in releasing
ACTH from AtT-20 cells as observed in
normal corticotrophs and, as shown in
intact animals and primary cultures of
the pituitary, glucocorticoids block the
CRF-stimulated release of ACTH in the
tumor cells. These findings prompted the
use of AtT-20 cells as a model for investi-
gating the cellular and molecular mecha-
nisms that regulate ACTH secretion
from the anterior pituitary.

There were previous indications that
catecholamines can stimulate ACTH re-
lease in vivo and in vitro (46). The com-
plexities involved in using the intact ani-
mal and primary cultures made it difficult
to interpret the precise mechanism
whereby catecholamines induced this re-
lease. Norepinephrine stimulated ACTH
release from AtT-20 cells in a calcium-
dependent manner (47). Ligand binding
studies with tritiated dihydroalprenolol,
a P-adrenoreceptor antagonist, indicated
the presence of a P-adrenoreceptor on
AtT-20 cell membranes which binds cat-

4 MAY 1984

echolaminergic agents with high affinity
(48). Isoproterenol, a P-adrenoreceptor
agonist, as well as epinephrine induced a
potent and stereoselective increase of
ACTH release from mouse tumor cells
which was calcium-dependent  and
blocked by the P-adrenoreceptor antago-
nist propranolol(48). Two subtypes of p-
adrenoreceptors are known, PI and p2
(49). &-Adrenoreceptors are most sensi-
tive to epinephrine whereas p,-adrenore-
ceptors are equally responsive to epi-
nephrine and norepinephrine. Pharmaco-
logic characterization showed that p2-
receptors are present on AtT-20 cells and
could mediate the release of ACTH (48).
The presence of pZ- but not p,-adrenore-
ceptors has been reported in the anterior
pituitary, but whether they are located
on a specific cell type has not been

Outside

Glucocortlcoids
      Gi*+
1

Catecholamlnes
VIP
I I   CRF
    I

determined (50). As in the case of CRF,
dexamethasone (Table 1 and Fig. 2)
blocks catecholamine-evoked secretion
of ACTH from AtT-20 cells (51). The
longer these cells have been incubated
with glucocorticoids the greater the de-
gree of inhibition of ACTH release.

In anterior pituitary membranes CRF
also stimulates adenylate cyclase activi-
ty (44). CRF increases the formation of
adenosine 3',5'-monophosphate (cyclic
AMP) in primary cultures and activates
cyclic AMP-dependent protein kinase,
an intracellular effector of cyclic AMP
(52). CRF also stimulates adenylate cy-
clase and cyclic AMP-dependent protein
kinase activity in AtT-20 cells (53). Sup-
port for a critical role of cyclic AMP in
regulating ACTH release comes from
studies with forskolin. This diterpene,

SRIF    R
                  K+

      I
    ?I
      I

Inside    I       Cyclic AMP
      I        0

0
ri
   0
  Ca*+

Nongranular
ACTH
   /

  Outside         4            +

Fig. 2. Molecular mechanisms involved in ACTH release. The release of ACTH can be
stimulated (+) by various hormones such as CRF. catecholamines acting on P-adrenoreceptors
(PI?), or a,-adrenoreceptors (not shown), VIP, or vasopressin (not shown). Each agonist acts on
separate and specific receptors. The hormone-induced secretion of ACTH involves a multitude
of intracellular second messengers. Secretagogues can activate adenylate cyclase (AC) to form
cyclic AMP. A guanine nucleotide stimulatory protein (N,) is required for hormone activation of
AC. Cyclic AMP activates protein kinase (PK) which catalyses the phosphorylation (P,) of a
protein substrate (KS). The phosphorylated KS may induce ACTH synthesis (Syn ACTS) or
the release of granular ACTH (in the circle). The secretion of nongranular ACTH appears to be
regulated differently from granular ACTH. Hormones and membrane depolarization, induced
by extracellular potassium (K') may stimulate calcium (Ca'+) influx or mobilization from
intracellular compartments. Other intracellular events (1) induced by secretagogues may
involve changes in phospholipid methylation, protein carboxymethylation, phosphatidylinositol
turnover, C-protein kinase, and glycosyltransferase activity. ACTH release can be inhibited (-)
by at least two hormones. Glucocorticoids can inhibit ACTH synthesis or release. Somatostatin
(SRIF) blocks the activation of AC or Ca" mobilization. k guanine nucleotide inhibitory
protein (NJ mediates SRIF's inhibition of AC. Coupling of N, with SRIF receptors leads to
either a direct blockage of the catalytic subunit of AC or an inhibition of N, activity.

455


which may directly activate adenylate
cyclase (.54), stimulates with equal po-
tency ACTH release (Table 1 and Fig. 2)
and cyclic AMP-dependent protein ki-
nase as well as the phosphorylation of
several distinct proteins in AtT-20 cells
(53). It is possible that these endogenous
proteins mediate the effect of cyclic
AMP on ACTH release. Isoproterenol
also stimulates adenylate cyclase activi-
ty (Fig. 2) and cyclic AMP-dependent
protein kinase in AtT-20 cells, indicating
that cyclic AMP may mediate the effect
of P-adrenoreceptor agonists on ACTH
release (53). The increase in ACTH se-
cretion induced by isoproterenol is
blocked by calcium antagonists such as
verapamil or by the absence of calcium
in the extracellular medium (51). The
activation of adenylate cyclase by iso-
proterenol is not prevented by calcium
ionophore blocking agents nor is the
stimulation of cyclic AMP-dependent
protein kinase altered by the depletion of
extracellular calcium (53). This suggests
that the actions of Ca*+ in the release of
ACTH occur at a step distal to kinase
activation by cyclic AMP. The secretion
of ACTH is stimulated by increasing
extracellular calcium concentrations.
Furthermore, isoproterenol and cate-
cholamines change the membrane po-
tential of AtT-20 cells and increase the
frequency of action potentials on these
cells by a calcium-dependent mechanism
(55).   Since   catecholamine-evoked
ACTH release from AtT-20 cells is also
dependent on the presence of calcium,
there are probably at least two intracellu-
lar mechanisms involving calcium or cy-
clic AMP that participate in corticotro-
pin secretion.

pituitary (57), also evokes the secretion
of ACTH from AtT-20 cells (Table I) in a
dose-dependent manner (51). This pep-
tide increases cyclic AMP accumulation
in these cells as well as cyclic AMP-
dependent protein kinase activity (53).
VIP also stimulates ACTH release from
human anterior pituitary tumor cells
(58). The effect of VIP on ACTH secre-
tion, like that of other hormones, is
blocked by glucocorticoids (51). Argi-
nine-vasopressin is also found in hypo-
thalamic neurons and was one of the first
hormones proposed to have CRF-like
actions. Vasopressin increases the secre-
tion of ACTH from primary cultures of
the anterior pituitary (59). This releasing
action is not mediated by cyclic AMP but
may involve some other intracellular ef-
fector system (56).

Inhibition of ACTH Release

In addition to the P-adrenoreceptor
control of ACTH release, an at-ad-
renergic-like mechanism may also be
involved (56). In primary cultures of the
anterior pituitary, ACTH release stimu-
lated by epinephrine and norepinephrine
is blocked by the potent and selective aI-
adrenergic receptor antagonist, prazo-
tin. Dopamine and serotonin antagonists
also block epinephrine-induced ACTH
secretion. at-Receptor agonists such as
phenylephrine and methoxyamine are
poor stimulators of ACTH release, sug-
gesting the presence of a nonclassical a,-
receptor mediated release of ACTH
which does not involve activation of
adenylate cyclase.

Besides synthetic CRF and catechol-
amines,  other hormones have been
found to stimulate ACTH release. Vaso-
active intestinal peptide (VIP), present in
the hypothalamus and known to stimu-
late prolactin release from the anterior

Glucocorticoids consistently block
basal and stimulate ACTH release from
the anterior pituitary in the intact animal
as well as cell preparations in vitro (Ta-
ble 1). Glucocorticoids may act through
several mechanisms to inhibit ACTH se-
cretion (60). Long-term treatment of ani-
mals or AtT-20 cells with dexametha-
sone reduces ACTH messenger RNA
activity,  indicating an inhibition of
ACTH synthesis at some pretransla-
tional site. The reduction of ACTH mes-
senger RNA production induced by glu-
cocorticoids correlates with their intra-
cellular glucocorticoid receptor binding
activity. Short-term treatment (2 hours)
of AtT-20 cells with dexamethasont
seems predominantly to effect hormone-
and cyclic AMP-stimulated ACTH re-
lease rather than ACTH synthesis, since
the rise in ACTH release promoted by
the calcium ionophore A23187 is not
reduced by brief glucocorticoid treat-
ment (53). Treatment of AtT-20 cells
with dexamethasone for short intervals,
while inhibiting isoproterenol-, CRF-,
and forskolin-stimulated ACTH release
(Table 1 and Fig. 2), does not affect the
activation of cyclic AMP-dependent
protein kinase by these secretagogues
(53). The ability of these secretagogues
to stimulate cyclic AMP accumulation is
not affected by short- or long-term treat-
ment with dexamethasone. The inability
of glucocorticoids to block CRF-stimu-
lated cyclic AMP formation was also
reported in primary cultures of the ante-
rior pituitary (44). Preliminary studies
have shown that the enzyme phospholi-
pase Az may be involved in releasing
ACTH since melittin, an activator of this

456

enzyme,  stimulates ACTH secretion
(61). Glucocorticoids have been ob-
served in some cell systems to induce the
synthesis of lipomodulin, a protein that
inhibits phospholipase A2 activity (62). If
glucocorticoids can rapidly induce either
the synthesis or the mobilization of this
protein, then the rapid inhibitory effects
of dexamethasone on ACTH release may
be related to a blockade of phospholi-
pase A2 activity. Thus, inhibition of
phospholipase A2 activity may serve as
another mechanism by which this class
of steroids act in the regulation of ACTH
secretion.

Somatostatin (SRIF) also inhibits
ACTH release in AtT-20 cells (Table 1).
This 14 amino acid peptide is of hypotha-
lamic origin and is known to block the
secretion of growth hormone, prolactin,
and thyroid-stimulating hormone from
the anterior pituitary (63). AtT-20 cells
have SRIF receptors which, when stimu-
lated, cause a reduction in ACTH secre-
tion (61, 64) evoked by potassium, CRF,
isoproterenol, VIP, cholera toxin, or
forskolin. SRIF reduces the ability of
these secretagogues to increase cyclic
AMP accumulation (61) (Table 1). These
observations suggest that SRIF can
block ACTH release by inhibiting the
activation of adenylate cyclase. SRIF
also inhibits forskolin-stimulated cyclic
AMP formation in cyc- variants of S49
lymphoma cells that are deficient in the
guanine nucleotide stimulatory protein
(NJ required for most hormones to acti-
vate adenylate cyclase (65). From these
data it was proposed that SRIF acted
through a guanine nucleotide inhibitory
protein (Ni) to reduce adenylate cyclase
activity (61, 65) as well as by a stimula-
tion of guanosine triphosphatase activity
(65). A useful agent in studying the man-
ner by which hormones inhibit adenylate
cyclase is a toxin derived from the nacte-
rium Bordetella pertussis. This toxin in-
duces the adenosine diphosphate (ADP)-
ribosylation of a 41,000-dalton protein
believed to be Ni (66). The toxin also
blocks the inhibitory effects of hormones
and guanine nucleotides on adenylate
cyclase in many tissues (66). The inhibi-
tion of growth hormone release by SRIF
from primary cultures of the anterior
pituitary is also blocked by the toxin
(67). In membranes of AtT-20 cells, Bor-
detella pertussis toxin induces the ADP-
ribosylation of a 41,000-dalton protein
and also prevents the inhibitory effect of
SRIF on forskolin-, CRF-, or isoprotere-
nol-stimulated cyclic AMP formation
and ACTH release (68). These findings
suggest that SRIF can act through Ni to
inhibit hormone-induced ACTH release.

SCIENCE, VOL. 224


Interactions of ACTH-Releasing Factors

The ACTH secretagogues can act indi-
vidually or in concert to regulate the
release of ACTH. Hypothalamic ex-
tracts are more potent in releasing
ACTH than any secretatogue alone.
These extracts appear to contain several
different CRF-like factors. In primary
cultures of the anterior pituitary, vaso-
pressin added together with synthetic
CRF induced a greater release of ACTH
than CRF alone, indicating that vaso-
pressin can potentiate the action of CRF
(59, 69). Although vasopressin does not
alter cyclic AMP accumulation in the
anterior pituitary, it causes a fourfold
potentiation in the stimulation of cyclic
AMP synthesis by CRF, suggesting that
vasopressin improves the efficiency of
coupling between CRF receptors and
adenylate cyclase. Epinephrine, by acti-
vating at-adrenoreceptors in the anterior
pituitary,  also potentiates synthetic
CRF's stimulation of ACTH release and,
like vasopressin, enhances the cyclic
AMP response to CRF (70). These find-
ings indicate a synergism between vaso-
pressin, a,-adrenergic agonists, and syn-
thetic CRF in releasing ACTH. &-
Adrenoreceptor agonists and CRF also
interact to regulate ACTH release. When
CRF and isoproterenol are added togeth-
er, the increase in ACTH secretion is
less than additive, suggesting that these
secretagogues act through a common
mechanism (51). This intracellular mech-
anism is distal to cyclic AMP accumula-
tion since the combined application of
CRF and isoproterenol produce additive
effects on cyclic AMP formation. VIP,
another ACTH secretagogue, appears to
release ACTH through a process inde-
pendent of CRF or P-adrenergic agonists
(50). VIP, together, with isoproterenol or
CRF, causes an additive increase in both
ACTH secretion and cyclic AMP pro-
duction. These findings indicate that
CRF, isoproterenol, and VIP may act on
separate compartments of adenylate cy-
clase as well as other second messenger
systems (Fig. 2).

Desensitization

Although hormones can induce rapid
and pronounced responses from cells,
their persistent presence can induce de-
sensitization (71). Corticotrophs become
refractory to CRF after prolonged expo-
sure to this peptide (72). This desensiti-
zation is manifest as a reduced maximal
ability of CRF to stimulate both cyclic
AMP formation and ACTH release.

4 MAY 1984

ACTH content is not affected by CRF
treatment, indicating that cells are not
depleted of the peptide hormone. Fors-
kolin-stimulated cyclic AMP accumula-
tion or ACTH release is not reduced by
CRF treatment, suggesting that both
adenylate cyclase and the intracellular
mechanisms mediating stimulus-secre-
tion coupling are unaffected in the desen-
sitized cells. Thus, either CRF receptors
are lowered in density or their coupling
to adenylate cyclase is impaired. Studies
in which anterior pituitary membranes
are exposed to `251-labeled CRF indicate
that desensitization involves the loss of
CRF receptors (73). Adrenalectomy, a
procedure that abolishes the glucocorti-
coid feedback inhibition of CRF release
in the hypothalamus, markedly de-
creased CRF receptor binding in the
pituitary 4 to 6 days after surgery. The
density of these sites returns almost to
normal after treatment with dexametha-
sane. Thus, CRF receptors can be down-
regulated and this may explain the de-
sensitization observed in primary cul-
tures. Recent studies have also shown
that the effect of CRF on ACTH release
in vivo is reduced after prior treatment of
animals with CRF, suggesting that de-
sensitization can occur under physiolog-
ic conditions (74).

Vasopressin  not only potentiates
CRF-stimulated ACTH release but can
also increase the ability of CRF to desen-
sitize its own receptor (75). Treatment of
primary cultures of the anterior pituitary
with a fixed concentration of arginine-
vasopressin and varying amounts of
CRF reduces the amount of CRF needed
to desensitize its receptors. Thus, vaso-'
pressin and CRF act synergistically to
release ACTH and regulate CRF recep-
tors.

P-Adrenoreceptors in many cell types
are also readily desensitized (76). This
was also found to be the case for mouse
pituitary tumor cells. Treatment of AtT-
20 cells with isoproterenol results in a
marked reduction of cyclic AMP forma-
tion and release of ACTH after restimu-
lation with the catecholamines (77). A
decreased binding of [3H]dihydroalpren-
0101 becomes apparent after 20 hours of
treatment with isoproterenol. The re-
duced binding is associated with a de-
creased density of j3-adrenoreceptors but
no change in receptor ligand affinity. The
desensitization of the cyclic AMP accu-
mulation and ACTH secretion responses
were observed before there was a de-
crease in receptor density. These find-
ings suggest that the desensitization of
the P-adrenoreceptor is a two-step pro-
cess. The first is rapid in onset and

shows a reduced capacity of catechol-
amines to elevate cyclic AMP accumula-
tion and ACTH release. The second step
is slower and is associated with a loss of
p-adrenoreceptors from cell membranes
(down regulation).

The rapid desensitization of the p-
adrenoreceptors to stimulation of cyclic
AMP synthesis and ACTH release with-
out changes in receptor density could be
due to the uncoupling of the receptor
from the adenylate cyclase complex, de-
creased activity of adenylate cyclase, or
changes in the ACTH secretory process.
These possibilities were examined by
first treating AtT-20 cells with isoproter-
enol to reduce the responsiveness to
cyclic AMP elevation and ACTH secre-
tion by about 50 percent (77). The cells
were then treated with forskolin to di-
rectly stimulate adenylate cyclase. The
generation of cyclic AMP and release of
ACTH in forskolin-treated cells were the
same as that of the fully sensitized cells.
This experiment indicates that during the
early desensitization of the P-adreno-
receptors, the adenylate cyclase and
ACTH secretory mechanism is normal
and that the desensitization is due to an
uncoupling of the receptor from adenyl-
ate cyclase.

P-Adrenoreceptor desensitization is
homologous (only desensitized to its
own receptor) since CRF- and VIP-stim-
ulated cyclic AMP accumulation and
ACTH release were unaffected by cate-
cholamine treatment of AtT-20 cells.
CRF receptors on normal corticotrophs
are also regulated independently of cate-
cholamine receptors (72, 75). The inde-
pendent nature of the desensitization of
these receptors indicates that the mecha-
nisms involved are specific for each re-
ceptor. Such a property would allow
corticotrophs to respond to some stimuli
despite the loss of responsiveness to
other CRF-like substances.

Somatostatin can also regulate the
sensitivity of its own receptor (78). Prior
exposure of mouse anterior pituitary
cells to SRIF lessens SRIF's antagonism
of CRF-, VIP-, isoproterenol-, and fors-
kolin-stimulated cyclic AMP accumula-
tion and ACTH release. Treatment with
SRIF increases the formation of cyclic
AMP in response to forskolin in these
cells. This increase is delayed in onset,
slow to recover, and is blocked by the
protein synthesis inhibitor cyclohexa-
mide. This suggests that prolonged treat-
ment of AtT-20 cells with SRIF desensi-
tizes SRIF receptors and causes a com-
pensatory sensitization of adenylate cy-
clase through a process requiring protein
synthesis. In cultures of brain cells. pro-

457


Fig. 3. Multihormonal control of ACTH re-
lease. ACTH release from the anterior pitu-
itary is regulated by a number of different
hormones. CRF and vasopressin, which are
present in hypothalamic neurons, reach the
anterior pituitary by a portal system and stim-
ulate (+) ACTH release. The secretion of the
releasing factors may be inhibited (-) by an
epinephrine input to the hypothalamus. Hy-
pothalamic VIP stimulates ACTH release
from both human and mouse tumor cells
suggesting a possible role of this peptide as a                          Epinephrine
CRF-like substance. Neuronal norepineph-                          I+)
rine or epinephrine released from the adrenal
medulla can also evoke ACTH release.
ACTH stimulates the synthesis of glucocort-
coids in the adrenal cortex. Glucocorticoids
can stimulate the synthesis of epinephrine or
inhibit ACTH release. Glucocorticoids can either act directly on the anterior pituitary to inhibit
the formation of messenger RNA for ACTH or inhibit ACTH release. In addition, these steroids
can act in the hypothalamus to effect CRF release (not shown). Somatostatin (SRIF) can block
the evoked release of ACTH from tumor cells and reduces the plasma concentrations of ACTH
in Nelson's syndrome (94, suggesting a role for this peptide in ACTH secretion.

longed application of SRIF produced an
adaptive response that was manifest as a
reduced ability of SRIF to stimulate cell
firing activity (79). Continued treatment
of anterior pituitary cells with SRIF re-
duces the subsequent ability of the pep-
tide to inhibit the release of growth hor-
mone and thyroid-stimulating hormone
(TSH) (80). These findings indicate that
SRIF receptors on many cell types can
be self-regulated. A model detailing the
multireceptor regulation in ACTH re-
lease in AtT-20 cells is shown in Fig. 2.

The Multireceptor Release of

ACTH in vivo

The studies showing that ACTH re-
lease in vitro is stimulated by multiple
factors raises the question of whether the
release of ACTH in vivo is under multi-
hormonal control. The injection of syn-
thetic ovine CRF in rats causes an imme-
diate increase in plasma ACTH concen-
trations (81). This stimulation is dose-
dependent and is neutralized by anti-
bodies to CRF. The ACTH release
induced by acute ether stress is partially
blocked by CRF antibodies, indicating
that molecules with immunologic charac-
teristics similar to synthetic CRF are
stress mediators (81). The lack of total
blockage of stress-evoked ACTH release
in rats by the CRF antibodies suggests
that hormones other than CRF are in-
volved in promoting the release of
ACTH in vivo.

Vasopressin also causes the release of
ACTH in the intact rat. This stimulation
is dose-dependent and prevented by a
vasopressin antagonist (81, 82). The
physiologic condition of the animal has
an important role in the stimulation of
ACTH secretion by vasopressin. Ani-

458

mals anesthetized with neuroleptics, opi-
ates, and Nembutal (conditions that
block CRF release) respond to vasopres-
sin with a smaller elevation in ACTH
release than that found in awake, freely
moving animals. Immunoneutralization
of CRF in nonanesthetized rats also low-
ers stimulation of ACTH release by
vasopressin. suggesting a dependence on
CRF for the ACTH releasing action of
vasopressin.  Vasopressin potentiates
CRF-stimulated ACTH release in anes-
thetized rats, indicating that vasopressin
and CRF act in a synergistic manner to
regulate ACTH release in vivo as well as
in vitro.

Catecholamines also appear to stimu-
late ACTH release in vivo by a direct
action on the anterior pituitary. Periph-
eral injections of epinephrine increase
plasma concentrations of ACTH in in'-
tact rats (46). The increase in plasma
ACTH caused by either epinephrine or
(-)-isoproterenol is stereospecifically
blocked by propranolol, suggesting that
P-adrenoreceptors are linked to the re-
lease of ACTH in vivo (46). P-Adrenore-
ceptor agonists have been proposed to
stimulate ACTH release in vivo by act-
ing through  "central mechanisms."
since hypothalamic lesions in female rats
prevented the increase in plasma ACTH
induced by (-)-isoproterenol (82, 83).
The process by which catecholamines
gain access to the brain to initiate these
central effects is not known. Previous
work has demonstrated that catechol-
amines do not cross the blood-brain bar-
rier (84). In contrast to these studies,
however, Mezey et al. (85) found that
(-)-isoproterenol stimulated ACTH re-
lease from male rats in which the hypo-
thalamus was separated from the pitu-
itary by either stalk-transection or le-
sions of the median eminence. The effect

of isoproterenol was blocked by pro-
pranolol but not by the selective PI-
adrenoreceptor antagonist practolol. Sal-
mefamol, a PI-adrenoreceptor agonist,
also stimulated ACTH release in stalk-
sectioned animals, indicating that p2-
adrenoreceptors can mediate the stimu-
lation of ACTH release by catechol-
amines in vivo. Isoproterenol-stimulated
ACTH release in stalk-transected ani-
mals is blocked by dexamethasone, sug-
gesting that the ACTH release induced
by P-adrenoreceptor agonists originates
from the anterior pituitary. These find-
ings are consistent with the previous
studies of Fortier (86) and McDermott et
al. (87) who used pituitary transplants to
examine the direct action of epinephrine
on an ACTH-mediated response. In
these studies, the anterior pituitary was
placed into the anterior chamber of the
eye of hypophysectomized rats. Injec-
tion of small quantities of epinephrine
into the eye reduced the level of circulat-
ing white blood cells (eosinophilia)
which is believed to accompany an in-
crease of ACTH release. Similar injec-
tions of epinephrine into the other eye
did not produce this response. These
data as well as the findings in stalk-
transected animals indicate that epineph-
rine can act directly on the anterior pitu-
itary possibly by way of P-adrenorecep-
tors to stimulate ACTH release.

Conclusion

Studies conducted in vitro and in vivo
indicate that the release of the stress
hormone ACTH is controlled by com-
plex regulatory mechanisms. Multiple
factors such as CRF, vasopressin. cate-
cholamines, and conceivably other hor-
mones stimulate ACTH release by di-
rectly acting on the anterior pituitary
(Fig. 3). Glucocorticoids and possibly
SRIF may inhibit the secretion of ACTH
also by a direct action on anterior pitu-
itary. Various hormones can indirectly
control ACTH release by acting on cen-
tral locations to modify the secretion of
these releasing or inhibiting factors. In
addition, corticotrophs become refrac-
tory to hormones that stimulate or inhibit
ACTH release. In most cases, the phys-
iologic significance of the desensitization
of hormone receptors on corticotrophs is
not known. However, down regulation
of glucocorticoid receptors in cortico-
trophs (88) may be responsible for the
reduced ability of dexamethasone to
lower plasma ACTH concentrations in
some depressed patients (89).

The release of ACTH is the final out-
come of the interplay among the hypo-

SCIENCE.VOL. 224


thalamus, adrenal cortex, adrenal medul-
la, and possibly other organs. Depending
on the type of stress experienced, it is
likely that a number of different hor-
mones may singly or in combination af-
fect the amount and duration of ACTH
secreted. The interaction of the various
stress mediators may act to fine-tune the
responsiveness of ACTH-secreting cells.

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We thank S. Sabol for providing the AtT-20 cells
We thank S. Sabol for providing the AtT-20 cells
and V. Hook, S. Heisler. and E. Mezey for
and V. Hook, S. Heisler. and E. Mezey for
collaboration in these studies. We also thank M.
collaboration in these studies. We also thank M.
Brownstein, M. Dratman, and M. Zatr for help-
Brownstein, M. Dratman, and M. Zatr for help-
ful suggestions.
ful suggestions.

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