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mise à jour du
6 mai 2004
Ann NY Acad Sci
1963;104:330-345
Behavioral effects induced by intracisternally injected ACTH and MSH
Ferrari W, Gessa, GL, Vargiu L
University of Cagliari, Italy
 
Tous les travaux de MR Melis & A Argiolas 
Tous les travaux de M Eguibar & G Holmgren

Chat-logomini

stretching
 
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
stretching
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.