Our experiments began several years ago as a
by-product of studies on the mechanism of the
eosinopenic action of ACTH. The results of
previous experiments convinced us that the
eosinopenia caused by ACTH stemmed from its
action on hypothalamic structures which had been
shown to control the blood eosinophils. To test
this working hypothesis we injected dogs
intracisternally (i.c.) with a commercial
preparation of ACTH. We not only found that the
eosinopenic action of ACTH was more pronounced
after intracisternal than after intravenous
injection, but we were fortunate to observe a
complex behavioral syndrome, unfamiliar to us,
which warranted further investigation. Most of
the dogs given ACTH i.c. exhibit apparently
normal behavior for about half an hour.
Thereafter all animals become apathetic and
exhibit diffuse muscular tremors which, at this
time, are more marked in the limbs. The rhythm
of respiratory activity assumes a peculiar
characteristic which will be described in more
detail. The dogs, are drowsy, yawn
frequently and after about one hour they
start to stretch in the way they usually do when
they awake from physiological sleep (figure).
The intervals between successive
stretching acts become shorter and shorter
until a stretching act begins immediately after
the preceding one (stretching crisis). Despite
this peculiar behavior the dogs seem to remain
in contact with the environment, as they are
responsive to calls and perform normal
activities without fear or aggressiveness.
Occasionally some anirnals exhibit
sialorrhea.
The stretching activity persists
approximately 24 to 72 hours according to the
amount of ACTH injected and terminates with
complete reintegration of normal behavior. Even
when given chronically i.c., crude ACTH
preparations are devoid of toxicity and on
successive administrations they maintain the
same degree of activity.
This report deals with our efforts to
identify the optimal amino acid sequence that
evokes such a syndrome and with our preliminary
attempts to localize the site of action of these
polypeptides.
The Stretching Syndrome is Evoked only by
ACTH and MSH Preparations
To establish whether or not the syndrome was
elicited specifically by our preparation of ACTH
(Cibacthen Ciba) dogs were injected i.c. with:
(a) purified pituitary hormones; (b) proteins
extracted from animal tissues other than
pituitary; and (c) several drugs eliciting blood
eosinopenia when injected i.c. Among the
hormonal products tested, only those with ACTH
and MSH activity elicited the stretching
syndrome. However, since MSH stimulates the
adrenal cortex and also produces eosinopenia
when injected intracisternally, the stretching
syndrome could still be directly or indirectly
related to a stimulation of the hypothalamic
structures that cause the fall in the
circulating eosinophils by eliciting ACTH
secretion from adenohypophysis. Consequently, a
number of drugs which cause eosinopenia when
injected i.c., were studied for their ability to
evoke stretching syndrome. As reported in
table2, doses of ACTH practically devoid of
eosinopenic action can elicit the stretching
syndrome. On the other hand drugs that induce
eosinopenia when injected i.c., fail to cause
the stretching syndrome. Finally, in dogs,
previously adrenalectomized and maintained with
a sufficient amount of hydrocortisone, ACTH
given i.c. evoked the stretching syndrome
without inducing eosinopenia.
Importance of the Route of
Administration
Probably the stretching syndrome has never
been reported before because ACTH acts on the
CNS only when injected in the cerebrospinal
fluid. By this route, the threshold dose of
ACTH, MSH and pure beta-MSH causing the
stretching syndrome is less than 0.006 IU., 5
gamma, and 10 gamma/kg respectively. In
contrast, the same drugs were inactive in doses
up to 10 IU., 1 mgrn. and 266 gamma/kg.
respectively when injected in the carotid
artery.
Effect of A CTH and ACTH Injected
Intracisternally in Several Animal
Species
Rabbits, cats, and rats given ACTH and MSH
alsô exhibit stretching crises
qualitatively similar to those of dogs. In
rabbits and rats the stretching syndrome is
preceded by increased grooming and scratching.
Rabbits yawn even more frequently than
dogs. In cats intracisternally injection of
ACTH caused a marked drowsiness interrupted by
stretching movements. However, doses of ACTH up
to 0.02 IU./kg. injected intrarachidially in men
caused nausea and vomiting but not stretching.
Because of these untoward side effects further
experiments in men were not carried out.
Relationship between Specific Hormonal
Properties and Stretching Syndrome
In dissociating or associating the
stretching syndrome with the properties MSH
and/or ACTH, it must be kept in mind that ACTH
possesses MSH activity and alpha-MSH can
stimulate adrenocortical functions. To judge
which of the two hormonal activities was more
closely related to the stretching syndrome, a
number of active preparations of ACTH and MSH
were dissolved in 0,1N NaOH and heated at
100° C for 30 minutes. This procedure which
destroys the ACTH properties of both
preparations fails to alter the MSH activity or
the ability to evoke the stretching syndrome. In
contrast both products lose all biological
activities, including the ability to elicit the
stretching syndrome, when dissolved in 0,1N HCl
and heated for six hours at 100° C. These
experiments suggest that the polypeptide chain
causing the stretching syndrome is definitely
shorter than that required to elicit
adrenocortical stimulation. Since MSH can evoke
the stretching syndrome, the chernical
configuration of the polypeptide causing the
syndrome might be closely related to the amino
acid sequence responsible for MSH activity.
Attempts to Identify the Chemical
Structure of the Agent Causing the
Syndrome
Since dog is most susceptible to the central
effect of ACTH, this species was selected for
these studies. Several ACTH preparations of
known biological activity and purity were
compared for potency in eliciting the stretching
syndrome. The activity of each product was
estimated by calculating the per cent of animals
exhibiting stretching crises after different
doses of each product. As shown in table 4, the
delta fraction is one-tenth as active as the
gamma, + gamma2 and alpha, + alpha2
corticotropins in stimulating
adrenocorticotropic function but it is at least
3 times more active than the alpha, + alpha2
fraction in eliciting the stretching syndrome.
In other experiments we compared the neurotropic
effect of a number of MSH polypeptides of known
chemical structure and specific biological
activity. As shown in table 5, despite some
relationship between MSH activity and stretching
activity, it does not seem probable that both
biological responses can be due to one and the
same amino acid sequence. Note the difference in
neurotropic activity between the purified a-MSH
and the a-MSH synthesized by Hofmann and Yajima.
The occurrence of the heptapeptide,
methionyl-glutamyl-histidyl-phenylalanyl-arginyl-tryptophanyl-glycine
in both adrenocorticotropins and melanotropins
might be the link to explain the efficacy of
both preparations in eliciting the stretching
syndrome; however, additional structural
features seem to play an important role for the
optimal stretching activity.
Delay in the Appearance of the
Symptomatology
Since the stretching syndrome begins about
one hour after the i.c. injection, the following
experiments were carried out to investigate
whether this delay was due to a slow diffusion
of the peptide into the active site(s) or to a
formation of an active metabolite. Dogs were
injected i.c., intraventricularly and
intrarachidially, at lumbar level, with either
ACTH or MSH. After i.c. and intraventricular
injection, the latency for the onset of the
stretching syndrome was practically the same. In
contrast, after intrarachidial injection, the
duration of latency increased by a factor of
four. In addition dogs were injected ic. with a
crude ACTH preparation in doses about 10 times
greater than the threshold dose. Samples of
liquor were withdrawn from these dogs at various
times (2-4-9 hours) after the injection (when
the stretching syndrome was fully developed),
and administered to smaller dogs (1 ml per
animal i.c.). The liquor of the donor dogs
always induced the stretching syndrome in the
receptor dog after the usual delay. These
experiments suggest that the delayed onset of
the stretching syndrome is due to a slow
diffusion of the active material from sites of
injection into the sites of action. This view is
also supported by other experiments in which
ACTH previously incubated at 37° C. for 6
hours either with liquor or with brain
homogenates and then injected i.c. into dogs
still elicited the stretching syndrome after the
usual latency. Moreover, the experiments
described above seem to indicate that the rate
of disappearance of ACTH is particularly
slow.
Effects of General Anesthetics on the
Stretching Syndrome
A hypnotic dose of phenobarbital temporarily
suppresses the stretching syndrome caused by a
small dose of ACTH. This antagonistic action
however can be overcome by increasing the dose
of ACTH. As shown in table 6 similarly to
phenobarbital, chloralose-urethane also
antagonizes the stretching syndrome. However,
the antagonistic effect of both drugs is
reversed by a thirty-fold increase of the ACTH
dose. The mechanism of this antagonism is still
under investigation.
Pharmacological Antagonists of the
Stretching Syndrome
Several drugs were tested as potential
antagonists of the stretching synrome evoked by
ACTH in either unanesthetized or anesthetized
dogs.
(a) Studies with unanesthetized dogs.
Atropine, scopolamine, chlorpromazine,
phenobarbital, reserpine, serotonin, melatonin,
STH were used in these experiments. Atropine,
scopolamine, chlorpromazine, phenobarbital
suppress the stretching syndrome; all the other
compounds tested were inactive.
(b) Studies with anesthetized dogs. Since
large doses of ACTH can still elicit the
stretching syndrome in anesthetized. animals, we
selected this experimental condition in order to
evaluate the pharmacological antagonists out
interference from environmental stimuli. Dogs
anesthetized with chloralose-urethane (50 and
250 mgm/Kg i.v. respectively) were given i.c. or
2 IU. per Kg of ACTH (Cibacthen). One hour after
the ACTH injection a single stretching movement
can be repeatedly evoked by tactile or pressure
stimuli applied to the limbs. Three hours af ter
the ACTH injection the anesthetized dogs show
spontaneous, regularly spaced, stretching
movements. The front legs are affected first but
later also the hind legs become involved in the
stretching syndrome. Tremors, muscular
hypertonia and characteristic alterations in the
respiratory rhythm precede and accompany the
stretching syndrome.
The effect of the drugs tested as
antagonists of the stretching syndrome was
evaluated by recording the number of stretchings
during fixed, successive (at least 6) time units
of 10 minutes before and after administration of
succinylcholine, myanesine, atropine, morphine,
chlorpromazine, diethazine, LSD, BOL, serotonin,
reserpine, GABA. As shown in TABLE 8,
chlorpromazine, morphine, atropine and
diethazine antagonized the effect of ACTH.
Effect of Intracisternally Injected ACTH
on Breath, Arterial Pressure, Glycemia and Body
Temperature
Depth and frequency of respiration were
altered by ACTH according to a recurrent rhythm
illustrated in figure 6. Periods of increased
depth and frequency wer interrupted by short
periods of apnea. Atropine and chlorpromazine,
which antagonize the stretching syndrome caused
by ACTH, also inhibit its effects on the
respiration.
ACTH i.c. neither affects arterial pressure
in dogs nor changes glycernia and body
temperature in rabbits.
Comments
Many symptoms of the stretching syndrome can
be considered an exaggeration of physiological
functions. Definite conclusions cannot be drawn
from our studies. However, a few working
hypotheses may be suggested. Several authors
have described extracorticotropic actions of
ACTH including neurotropic actions. On the other
hand, many considerations suggest to us a
neurohormonal role for MSH peptides:
(a) melanophores are derived from the neural
crest.
(b) the hypothalamus of hypophysectomized
rats takes up, selectively, injected MSH.
(c) the CNS and particularly the
hypothalamus contain materials with MSH
activity.
(d) Krivoy and Guillemin show that a-MSH
potentiates submaximal evoked potentials in the
cat spinal cord.
Our studies demonstrate that polypeptides
with ACTH and/or MSH activity cause a syndrome
which seems to be due to their stimulation of
certain specific functions of the CNS. This
central activity cannot be due to the
polypeptide sequence essential for the
corticotropic activity. In fact, ACTH and MSH,
if dissolved in alkali and heated for 30
minutes, retain both MSH and neurotropic
activity while they lose the corticotropic
activity. Furthermore, beta-MSH does not have
corticotropic activity, but induces the
stretching syndrome. Therefore, we had to
conclude that the stretching activity was due to
an amino acid sequence similar to that
responsible for the MSH activity. However, in
spite of a close relationship between stretching
and MSH activity, we are forced to exclude the
possibility that these two activities are
conditioned by the same amino acid sequence.
Indeed in the series of MSH polypeptides
studied, the capacity to induce the stretching
syndrome did not coincide with their melanophore
stimulating potency. Furthermore, crude ACTH
preparations are more active than pure a- and
b-MSH. At this point it might be pertinent to
mention that LSD, chlorpromazine and reserpine,
which mimic MSH on the frog's melanocytes,
injected i.c. fail to cause the stretching
syndrome. On the other hand, melatonin,
epinephrine and serotonin which block the MSH
effect on frog melanocytes do not antagonize the
neurotropic effect of MSH.
The delta1 fraction isolated by Bell et al.
from oxycellulose-ACTH was found to be the most
active compound in eliciting the stretching
syndrome. It represents 20% of the
oxycellulose-ACTH. It has a low ACTH activity
and 90% of the MSH activity of the starting
material. However, the last activity, unlike
that of a- and b-MSH, is not potentiated by
alkali treatment. Finally, the delta1 fraction
is not ketogenic and it is the most active
corticotropin in stimulating the aldosterone
synthesis. Since this fraction is not
homogeneous but a mixture of at least four
components we are continuing our efforts to
determine the most active neurotropic agent
among these fractions.
The antagonism against the stretching
syndrome displayed by different drugs known to
block the reticular formation, ie.
phenobarbital, atrpine, scopolamine,
chlorpromazine, diethazine and morphin indicates
that the reticular formation might be
concerned with the syndrome described. The
antagonistic effect of atropine and scopolamine
suggests that a cholinergic mechanism might be
involved. Some of the symptoms accompanying the
stretching syndrome may also be indicative of an
involvement of the reticular formation, namely
muscular tremors, muscular hypertonia and
respiratory dysrhythmia. Some others, such as
vomiting, scratching, sialorrhea and the
respiratory alterations too seem to indicate a
site of action in lower levels of the CNS.
Considering the overall observations it may
be suggested that a polyeptide, similar in
structure to MSH and ACTH and probably present
in the delta, fraction can antagonize the
sleeping state. Stretching and yawning are
two physiological acts that might be considered
as an effort of the body to delay the onset of
sleep and a mechanism to reinforce wakefulness
after sleep.
Discussion of the
Paper
I. H. PAGE: Do you think that your
experiments have a physiological meaning?
G. L. GESSA: We are now gathering
information to allow us to make a definite
statement as to the physiological meaning of the
findings presented. We would like to point out
the following: a specific effect is induced by
polyptides found in the CNS; all the animal
species studied are responsive to the active
polypeptides basically in the same manner,
with stretching and yawning which do not
differ qualitatively from those occurring
physiologically in "tired" animals; this effect
is highly reproducible, i.e., no tolerance is
induced toward the active polypeptides; the
minimum active dose is extremely small.
H. 0. J. COLLIER: Do these animals actually
go to sleep more readily or o they just yawn
and stretch? That is to say, is it
associated with sleepiness?
G. L. GESSA: The animals look drowsy and
apathetic, however, they do not sleep.
Actually, each stretching and yawning is
accompanied by what appears to be a behavioral
arousal. A similar phenomenon also occurs in
anesthetized animals, ie., each time the animal
stretches, it seems that it is momentarily
wakening from the anesthesia.
R. F. TiSLOW (Philadelphia): Intravenous
injection of mescaline in the dog produces some
of the symptoms you have shown (e.g.: arched
back , retroposition, stretching, etc.) after a
latent period of half an hour. Since mescaline
also produces a blood pressure drop, we were
under the impression that mescaline acted
through some release phenomenon.
P. H. BELL (Pearl River): Since we were
responsible for some of the fractions mentioned
by the speaker which were very effective in this
test, I should like to describe briefly some of
their properties, in particular the delta1
corticotrophin which proved to be the most
active. It is perhaps unfortunate that we call
them corticotrophins, particularly this delta1
fraction. It is true that it was obtained from
the anterior pituitary and had corticotrophin
activity. All commercial preparations of ACTH
which are prepared by the oxycellulose
purification method of Astwood have
approximately 5% of this delta1 fraction.
Since oxidized cellulose is an acidic
adsorbant, which quantitatively adsorbs this
component, it is reasonable to assume that the
delta1 fraction is either made up of basic
components or peptides with long runs of
arginine and lysines which are responsible for
the adsorption. Structure work on
beta-corticotrophin disclosed that the latter
case was responsible for its adsorption on
oxidized cellulose.
The delta1 fraction contained corticotrophin
activity when measured by the Sayer Assay.
However, we do know that it is made up of a
multiplicity of peptides. At least four can be
separated by paper chromatography. It also has
MSH activity which is of a different type than
the corticotrophins. Dr. Astwood, when working
with a similar fraction from crude ACTH, was
able to isolate a polybase basic material which
would form a picrate derivative which had the
proper melting point for spermine picrate. I
cannot tell you what the percentage of spermine
might be in the beta-corticotrophin used by the
speaker, but it would certainly be worthwhile to
test spermine for this interesting effect. One
other interesting property of this delta1
corticotrophin fraction which may be completely
irrelevant here but should be mentioned, is due
to the work of Gordon Farrell of Western
Reserve. Dr Farrell found that this fraction was
very active in stimulating the release of
aldosterone from the adrenal gland of a
decerebrate dog. This is probably unrelated to
your tests, but since this fraction has several
different components, I suggest that a little
fractionation would be in order so that a more
active and better characterized preparation
could be obtained.
W. A. KRivoy: Professor Rocha e Silva asked
a question which may be of general interest,
namely, whether or not the results that I
presented bear a relation to the results
presented by Professor Ferrari. We were unable
to show any effect on the spinal cord with
a-MSH, with oxytocin, or with highly purified
ACTH, although we could see an action on cat
spinal cord with a clinical sample of ACTH. The
latter has a five per cent impurity of a-MSH in
it, enough to stimulate the cat spinal
cord.
Our results on the fish may be related to
Professor Ferrari's observations in that the
latent period for the action on the fish is
approximately the same as the latent period for
the action on the dog, that is to say, something
of the order of thirty minutes to an hour for
maximal effect. In the cat, of course, there was
a latent period of about six minutes. However,
a-MSH had no action on the fish in the doses we
used.
