Central administration of peptides alters thermoregulation in the rabbit

Peptides, Vol. l, pp. 15-18. Printed in the U.S.A.

Central Administration of Peptides Alters Thermoregulation in the Rabbit J. M. L I P T O N A N D J. R. G L Y N

Physiology and Neurology Departments, Southwestern Medical School, University of Texas Health Science Center at Dallas, Dallas, TX 75235 R e c e i v e d 25 F e b r u a r y 1980 L I P T O N , J. M. A N D J. R. G L Y N . Central administration of peptides alters thermoregulation in the rabbit. P E P T I D E S

1(1) 15-18, 1980.--Sixteen peptides were injected intracerebroventricularly to test their effects on rectal temperature of rabbits in a thermoneutral environment. In initial tests 5 tzg a-MSH, ACTH(I-24), oxytocin, vasopressin and glucagon altered body temperature while ACTH(I-10), cholecystokinin, contraceptive tetrapeptide, gastrin, insulin, interferon, leupeptin, LHRH, panhibin (somatostatin), and proctolin did not. Bombesin also altered body temperature but in no consistent direction. In further tests on the effective peptides 1.25-5.0 p.g a-MSH and ACTH(1-24) produced dose-related decreases in rectal temperature as great as 1.0°C. The same doses of oxytocin and glucagon produced small, prolonged hyperthermias which did not exceed 0.4°C. Vasopressin caused rapid development of small increases in rectal temperature; temperature returned to normal in 2-3 hr. The results suggest that five of the peptides tested may have roles in central mediation of normal body temperature, hypothermia, hyperthermia and fever. Central peptides

Body temperature

a-MSH

ACTH

A number of peptides found within the brain may participate in the control of physiological functions. Some peptides alter body temperature when given centrally and it is therefore possible that these substances normally act on neuronal pathways which control thermoregulatory processes. For example, VIP and TRH produce hyperthermia when given centrally [11,25], and neurotensin causes hypothermia [3]. The effects on central temperature controls of many peptides have not been systematically studied, and the effects of still other peptides which are known or supposed to exist in the brain have not yet been determined. The purpose of the present experiments was to learn whether certain peptides affect body temperature when given centrally, in order to obtain an indication of their possible roles in normal thermoregulation, hypothermia, hyperthermia or fever. METHOD

Animals Male New Zealand albino rabbits 2-4 kg in weight were used. The animals were individually caged in a 21-23 ° environment. Lights were on 12 hr per day, and all experiments were done during the light phase. Food and water were available ad lib.

Surgical Procedure Animals were pretreated with ketamine hydrochloride and promazine (Ketaset Plus, Bristol Labs, 1 ml/2.5 kg, IM), and anesthesia was induced and maintained by inhalation of a methoxyfluorane (Metofane, Pitman-Moore, Inc.) and N20-O2 mixture. Each animal was placed in a modified Kopf rabbit stereotaxic instrument [13,22] fitted with anesthetic gas inhalation apparatus. An injection cannula (No. 201, David Kopf Instruments) was implanted into a lateral yen-

Oxytocin

Vasopressin

Glucagon

tricle by inserting it at a point 1.0 mm anterior to bregma and 2.7 mm lateral to the midline. The cannula was lowered until cerebrospinal fluid flowed freely up the tube. Four stainless steel screws were driven into the calvarium, and dental acrylic was applied to anchor the cannula to the screws. Benzathine penicillin G (Bicillin, Wyeth Laboratories) was given post-operatively (150,000 U, IM).

Peptides and Injection Procedure Non-pyrogenic saline, or one of the following peptides dissolved in saline, was given: ACTH(1-24) (Organon, MW=3,500); ACTH(1-10) (Peninsula Laboratories, 1,300); bombesin (Peninsula, 1,565); cholecystokinin (Peninsula, 1,064); contraceptive tetrapeptide (Peninsula, 501); gastrin (Calbiochem, 12,514); glucagon (Calbiochem, 3,550); insulin (Calbiochem, 11,466); interferon (Calbiochem, 13,00025,000); leupeptin (Peninsula, 485); L H R H (Peninsula, 1,201); a-MSH (Peninsula, 485); oxytocin (Peninsula, 967); panhibin (somatostatin) (Peninsula, 1,674); proctolin (Peninsula, 649); arginine vasopressin (Peninsula, 984). All intracerebroventricular (ICV) injections were 50/xl in volume followed immediately by a 20 /zl saline flush. All glassware was heated to 200°C for a minimum of 2 hr to destroy exogenous pyrogen, and commercial non-pyrogenic syringes were used for injections.

Temperature Recording Procedures Rabbits were restrained in conventional holders, and a thermistor probe (Yellow Springs International, No. 701) was inserted about 100 mm into the rectum. Temperature recordings were made every 10 min via an online computer (MINC 11, Digital Equipment Company) connected to a digital temperature recorder (Datalogger, United Systems

C o p y r i g h t © 1980 A N K H O International Inc.--0196-9781/80/010015-04500.90/0

16

LIPTON A N D G L Y N

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HOURS AFTER INJECTION FIG. 1. Hypothermias produced by ICV injections ofc~-MSH (upper graph) and ACTH(1-24) (lower) in rabbits in a 23°C environment.

Corp.). No injections were made until a stable baseline temperature was attained. All experiments were separated by at least 48 hr. Experiments were run in an environmental chamber at 23 ° (_+0.5°C). The average thermal response (ATR), the mean change in temperature (°C) over the duration of the response, was calculated for each rabbit.

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TIME AFTER INJECTION (HRS) FIG. 2. Hyperthermias caused by ICV injections of oxytocin (upper graph), vasopressin (middle), glucagon (lower) in rabbits in a 23°C environment.

TABLE 1 EFFECTS OF ICV ADMINISTRATIONOF PEPTIDES ON BODY TEMPERATUREOF RABBITSIN A 23°C ENVIRONMENT Peptide

RESULTS Five of the 16 peptides tested caused consistent changes in body temperature when given in 5 #g doses: c~-MSH, ACTH(1-24), glucagon, oxytocin and vasopressin (Figs. 1, 2). The remaining peptides, except for bombesin, had no effect on body temperature that was directly associated with the injection (Table 1). Bombesin caused both increases and decreases in body temperature and some biphasic responses with the average maximum response being a 0.6 ° hyperthermia two hours after injection. Because of the variability in response to bombesin and because temperature changes after other peptides were small and/or occurred long after the injection, several peptides, including bombesin, were not tested further. In additional tests on a - M S H and ACTH(1-24), both peptides caused hypothermias that were greatest 1-2 hr after injection (Fig. 1). The ATRs after random administration of 1.25, 2.5 and 5.0 p.g doses of ACTH(1-24) were related to the dose (r=0.99) and rectal temperature returned to normal within 4.5-6.0 hr. Hypothermias after c~-MSH were also directly related to the dose (r=0.99) but the differences in the magnitude of the effects among the three doses was greater than after ACTH(I-24). The hypothermias produced by

I

I

~-MSH ACTH(1-24) Oxytocin Vasopressin Glucagon ACTH(I-10) Bombesin Cholecystokinin Contraceptive tetrapeptide Gastrin Insulin Interferon Leupeptin LHRH Panhibin (Somatostatin) Proctolin

Dose (pmoles)

Mean max 8 T (°C)

Time to max A T (hr)

N

3.08 1.42 5.51 5.08 1.41 3.85 3,20 4.70

- 1.0 - 0.9 0.4 0.3 0.3 -0.2 0.6 0.1

1.67 1.17 1.0 0.33 1.33 1.0 2.0 0.17

7 7 I0 12 15 7 7 7

9.99 0.40 0.44 0.25 10.30 4.16

0.3 0.3 - 0.2 0.3 0.1 0.1

2.33 0.17 3.0 2.33 0.5 1.5

7 7 7 7 7 7

2.99 7.71

0.3 - 0.1

0.67 0.83

7 7

CENTRAL PEPTIDES

17

~-MSH were of shorter duration, and rectal temperature returned to baseline levels 3.5-5.0 hr after injection. Oxytocin and vasopressin injections produced hyperthermias with different characteristics (Fig. 2). Oxytocin caused dose-related hyperthermias (r=0.99) characterized by low amplitude and long duration. The two lower doses of vasopressin produced dose-related hyperthermias but the response to the highest dose was less than that caused by the intermediate dose (2.5 /~g). In all cases the vasopressininduced hyperthermias developed rapidly and lasted only 2.5-3.0 hr. Glucagon caused small, prolonged hyperthermias that did not vary significantly with dose (Fig. 2). Increases in temperature began within l0 rain after injection and persisted for at least five hours. DISCUSSION These experiments show that a-MSH, ACTH(1-24), oxytocin, vasopressin and glucagon affect body temperature when given centrally to the normothermic rabbit. These substances are thus added to the list of peptides [ 10] that may have a role in central mediation of body temperature control in this species. ACTH has long been known to produce defervescence when given to patients with febrile illness. Kass and Finland [18] noted that the amplitude and duration of fevers produced by pyrogen injection in man and rabbits were reduced by prior intramuscular injection of ACTH. They concluded that ACTH affects hypothalamic temperature controls and that no alteration of the fundamental pathologic processes of the illness are produced by the peptide. There were no tests of the effects on body temperature of ACTH alone in their experiments but peripheral administration of this substance in another study produced decreases in body temperature of both normothermic and febrile rabbits [15]. This effect on fever may occur naturally since it is well-established that pyrogen induces ACTH secretion [2, 9, 17, 24]. ACTH-like immunoreactivity has been detected in cells and fine beaded axons throughout the brainstem of the rat [30]. The region of heaviest concentration was the medial basal hypothalamus with cell bodies positively stained in the arcuate nucleus. Dense fiber distributions were seen surrounding the third ventricle with lighter activity in other brain regions. The same anatomical distribution was observed in animals that had been hypophysectomized prior to examination which suggests that the ACTH-like activity derives from local synthesis within the brainstem tissue. These findings support the previous observations of ACTH in median eminence, basal hypothalamus and limbic regions [21] in normal and hypophysectomized rats [20, 21]. Radioimmunoassay of corticotropin showed immunoreactive ACTH in the hypothalamu s of rats, rabbits, dogs, monkeys and humans [26]. ACTH was found in the hypothalamus, midbrain, pons, medulla, thalamus, preoptic area, and limbic system of rats and rabbits; in the hypothalamus, preoptic area, midbrain, amygdala, and thalamus of dogs; but only in the hypothalamus of monkeys and human beings. Our findipg that central administration of ACTH(1-24) causes dose-related decreases in body temperature along with previous evidence that (1) peripheral administration of ACTH causes hypothermia and inhibits fever, (2) adrenal cortical activity is elevated in the presence of pyrogens, and (3) that ACTH occurs naturally in brain tissue associated with central control of body temperature in many species,

suggests that the substance may have a role in central thermoregulatory processes. The smaller ACTH molecule, ACTH(1-10), had no effect on body temperature when given centrally which suggests that amino acids 11-24 may be most important to production of hypothermia. It may be that a portion of the central ACTH molecule is an important inhibitor of neurotransmission in certain temperature control pathways. The combined evidence opens the possibility that central ACTH release may normally act to attenuate the amplitude and duration of fever. Hypothermia was also produced by ICV injection of c~-MSH. ACTH(1-24) contains within it the peptide backbone of a-MSH, and it is thought that normallyoccurring ACTH(1-39) serves as a precursor molecule that is enzymatically cleaved and processed to a-MSH [23]. Thus it may be that the shared amino acid sequence of the two molecules is responsible for the hypothermia. That a-MSH may have a role in normal central thermoregulatory processes is supported by the finding that this substance is synthesized within the brain and that it is concentrated in hypothalamic cells and fibers [14, 16, 28]. A diurnal rhythm of immunoreactive a-MSH has been found in discrete regions of the rat brain [27] and the concentration of a-MSH in the medial preoptic region increases at a time when preoptic temperature is lowest [1]. These relations support the idea that a-MSH has a role in circadian variations in thermoregulatory as well as behavioral and neuroendocrine processes [27]. Vasopressin, oxytocin and glucagon increased body temperature when injected ICV. After vasopressin relatively brief hyperthermias occurred which were maximum with the intermediate dose (2.5 /~g). It is not clear how the present results on vasopressin in the rabbit relate to the antipyretic effect observed by Kasting, Veale and Cooper [19] when Pitressin was infused into the septal region of the sheep. It is not likely that pyrogenic contaminants are responsible for the hyperthermias seen in our experiments because the temperature curves are not consistent with the long term fever characteristic of central pyrogen injections. To learn whether the difference in results depends upon differences in the substance injected (vasopressin vs. Pitressin), the site of infusion or differences in responses of the two species requires further experimentation. The finding that central administration of oxytocin produced dose-related hyperthermia suggests that this peptide may also be important to central thermoregulatory processes. Little is known about the physiological significance of oxytocin within the CNS although fibers containing this peptide can be traced from magnocellular nuclei of the hypothalamus to widespread neural structures including the substantia gelatinosa of the spinal cord [8]. In the periphery oxytocin causes milk ejection and uterine contraction but there is no known effect of oxytocin in male animals which were used in the present experiments. The finding that glucagon alters body temperature when given centrally is a new observation. In the periphery glucagon acts primarily on the liver to cause an increase in glucose output and a subsequent rise in blood glucose concentration. This could have a beneficial effect during exposure to cold, but there is as yet no reason to tie together peripheral and central effects of glucagon. The effects of bombesin on body temperature were not consistent. In previous research this peptide given centrally in the cold caused hypothermia in rats [4-7]. However, recent studies [29] indicate that bombesin simply disrupts cen-

18

LIPTON AND GLYN

tral temperature controls and causes dysthermia in both hot and cold environments. Most of the peptides tested failed to affect body temperature, a finding which increases the probability of specificity of those that did alter thermoregulation. It may be that the inactive peptides influence central temperature under certain conditions (e.g. in e x t r e m e ambient t e m p e r a t u r e s , in particular species) or at higher doses than used here. H o w e v e r , our negative results on L H R H are similar to those of others who failed to find consistent effects on body temperature after central injections in the cat [25], m o u s e [3] and rat [12]. Panhibin (somatostatin) has also b e e n shown to h a v e no significant effect on body temperature w h e n given centrally to

the m o u s e [3] and to the rat [6] in the cold, but it appears to inhibit the temperature effects of o t h e r neuropeptides, specifically neurotensin,/3-endorphin and b o m b e s i n [7]. It thus appears unlikely that L H R H and panhibin have a direct role in central temperature control but, in the case of panhibin, there may be a modulatory effect on o t h e r central mediators in regulation against cold.

ACKNOWLEDGMENT Supported by National Institute of Neurological and Communications Disorders and Stroke grant No. 10046.

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baum. Basel: Karger, 1980, pp. 186-194. 8. Buijs, R. M. Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat. Cell Tiss. Res. 192: 423-435, 1978. 9. Chowers, I., H. T. Hammel, J. Eisenman, R. M. Abrams and S. M. McCann. Comparison of effect of environmental and preoptic heating and pyrogen on plasma cortisol. Am. J. Physiol. 210: 606-610, 1966. 10. Clark, W. G. Changes in body temperature after administration of amino acids, peptides, dopamine, neuroleptics and related agents. Neurosci. Biobehav. Rev. 3: 179-231, 1979. II. Clark, W. G., J. M. Lipton and S. I. Said. Hyperthermic responses to vaso-active intestinal polypeptide (VIP) injected into the third cerebral ventricle of cats. Neuropharmacology 17: 883-885, 1978. 12. Cohn, M., S. J. Cohn and M. L. Cohn. A clue to the mechanism of action of aspirin: role of thyrotropin releasing hormone (TRH). Soc. Neurosci. Abstr. 4: 406, 1978. 13. Crawford, I. L., J. I. Kennedy and J. M. Lipton. A simple "planilabe" for rapid establishment of the stereotaxic horizontal zero plane in rabbits. Brain Res. Bull. 2: 297-298, 1977. 14. D~sy, L. and A. PeUetier. Immunohistochemical localization of alpha-melanocyte-stimulating hormone (t~-MSH) in the human hypothalamus. Brain Res. 154: 377-381, 1978. 15. Douglas, W. W. and W. D. M. Paton. The hypothermic and antipyretic effect of preparations of ACTH. Lancet, Feb. 16, 342-345, 1952.

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