Neuropeptides and temperature regulation

J. therm. Biol. Vol. I I, No. 2, pp. 79-83, 1986

0306-4565/86 $3.00 + 0.00 Pergamon Journals Ltd

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NEUROPEPTIDES AND TEMPERATURE REGULATION L. JANSK~',t S. VYBJRAL,l J. MORAVEC,I J. ]S~ACH,~ZEL,t W. RIEDEL,2 E. and C. S I M O N - O P I a ~ K M A N N 2

SIMON 2

tDepartment of Comparative Physiology, Faculty of Science, Charles University,Vini~n~ 7, 129 00 Prague 2, Czechoslovakia 2Max-Planck-lnstitut ffirPhysiologische und Klinische Forschung, W.G. Kerckhoff-lnstitut, D-6350 Bad Nauheim, F.R.G.

Aktract--l. A method is described, which makes it possible to analyse, both qualitatively and quantitatively, the effect of neuropeptides on individual thermoregulatory outputs. Use of the method also facilitates speculation on neuronal sites in the anterior hypothalamus, which might be specifically affected. 2. Data show that neurotensin shifts the threshold Tcomto higher temperatures whilst bombesin shifts the threshold T~., downwards and reduces the efficiency of individual thermoregulatory outputs. 3. Data suggest that peptidergic receptor sites in the brain are specifically involved in thermoregulation and confirm that the effect of neuropeptides on resting nerve terminals consists in inhibiting spontaneous transmitter release. Key Word Index--Neuropeptides; bombesin; neurotensin; thermoregulation; transmitter release.

manipulation of core body temperature (T~o~), while leaving the peripheral temperatures relatively unaffected. This simplifies operation of the system regulating thermal homeostasis and makes it possible to determine the threshold core temperatures and the efficiencies of individual thermoregulatory outputs as a function of core temperatures under normal conditions and after injections of neuropeptides. The experimental set-up used is shown in Fig. 1. Bombesin (BOM), or neurotensin (NT) in nanogram quantities were/njected into anterior or posterior hypothalamus of conscious rabbits. In other experiments the effect of arginine vasotocin (AVT), bombesin (BOM), neurotensin (NT) and methionine-enkephalin (met-ENK) on the nervus phrenicus-diaphragm preparations of golden hamsters was tested electrophysiologically. Using a standard microelectrode technique the quantal spontaneous release of acetylcholine (ACh) was measured as frequency of miniature end plate potentials (mepp) in vitro at a bath temperature of 20°C. All neuropeptides were added to the bath to give a final concentration of 10 -6 M. This experimental design enabled us to study the action of neuropeptides on single nerve terminals in the resting state.

INTRODUCTION

The discovery of biologically active peptides within the central nervous system has eficited considerable interest in their physiological role. Substantial evidence has accumulated that neuropeptides are involved in a variety of processes including those controlling foodintake, reproduction, sleep, hibernation and the perception of pain. In addition, several studies have indicated that various neuropeptides are involved in the control of body temperature. It has been found that intraventricular or central administrations of ACTH, a-MSH, angiotensin, bombesin, neurotensin, substance P and vasopressin induce hypothermia, while administration of endorphins, enkephalins, T R H and somatostatin induce hyperthermia (see Clark and Lipton, 1983). Some of these neuropeptides can elicit both hyperthermic and hypothermic effects, depending on experimental conditions and on species used. The data, however, are as yet insufficient to establish the precise role of neuropeptides in thermoregulation. Only few peptides have been studied thoroughly enough to allow general conclusions about their specific effects on individual thermoregulatory outputs, e.g. on cold thermogenesis (CT), shivering and nonshivering therrnogenesis (NST), peripheral vasomotor tone (PVMT: vasoconstriction or vasodilation), respiratory evaporative heat loss (REHL: panting) or sweating. Until this information is available it will not be possible to conclude that neuropeptides really participate in body temperature control under physiological conditions.

RESULTS AND DISCUSSION

From the data obtained (Jansk~, et al., in press; Vybiral et al., 1986) it became evident that in normal rabbits during intestinal cooling T~or~ decreases slightly and the cold thermogenesis due to shivering (CI') increases proportionally with the decreasing T ~ (Fig. 2). A decrease of about l°C in To,~ induced maximal cold thermogenesis. During warming with the T~o~ increasing, the activity of heat dissipating mechanisms (e.g. vasodilatation, expressed as release of the PVMT and panting, the later measured as frequency of respiration and expressed as REHL),

MATERIALS AND M E T H O D S

In order to clarify the mode of neuropeptide action on thermoregulation, a method of intestinal cooling was used (lnomoto et al., 1982), which enables the 79

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Fig. 1. Experimental set-up enabling to determine the threshold Tom and the efficiencyof individual thermoregulatory outputs in the rabbit after injections of neuropeptides into the hypothalamus each thccmorql~tory output remained unchang~, while the ~ com temperature for inducing CT, PVMT and REHL was shifted upwards. Thus, inj~ions of NT into the anterior hypoti~damus induced a dgniltcant hyperthcrmic effect. The hypm-thermic response to NV in the rabbit contrasts with hypothvmmia obsecvcd after NT in other spec~ (mic~ rats, guinea pigs, ~ ) (Bissctte e t al., 1982). It is known that stimulalton of central dopami~ (DA) receptors c a u ~ hypothcrmia in several spedes and it was suggested, therefore, that

i n ~ proportionally to the increasing T~o~. An inctea~ of T~, of about 2oC induced maximal ~ v ~ t a w h i l ¢ an increase of I oc induced maximal fion. The T~., at which the thermo- rcgu~tnry outputs are ~ t in action is defined as the ~ t o r y thrv~old. The slopes of rclatiom~ip6 b~w~m T , , and the intensities of individllal th~raor~.datory outputs indi.~te ¢~..'ency of regulatory medmnim~. From Fig. 2, tt is ewdent that after injection of 250lag of NT into the anterior hypothalamus both the efficiency and the capacity of

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the effect o f NT is mediated by dopaminergic pathways. Since it was found that in rabbits activation of dopaminergic receptors induces hyperthermia, the different response to NT may be attributed to the different role of DA in the control of body temperature of the rabbit. The effect of BOM differs markedly from that of NT (Fig. 3). When 250 ng of BOM were injected into the anterior hypothalamus, the metabolic heat production started to increase at lower T~,~ and the relationship between the metabolic rate and T ~ was significantly reduced in its slope. When the intensity of the PVMT was plotted against Tco,~, it became evident that in this ease BOM also significantly reduced the slope of this relationship without changing the T~.,, at which the maximum vasodilation was attained. Further analysis of the data confirmed that the threshold T ~ for inducing REHL and the efficiency of this thermoregulatory output were reduced. Thus, during cooling BOM exerted an hypothermic effect, because of the lowering of the threshold T,~, while during warming it provoked hyperthermia, because of the inadequate panting. On the basis of data obtained the mode of central bombesin or neurotensin action on thermoregulatory centres can be explained proceeding from the Bligh's neuronal model of body temperature control (Bligh, 1979). The model is based on the presumption that neurons in the preoptic area of the anterior hypothalamus (POAH) receive inputs from warm and cold sensors located both on the body periphery and in the body core. It is presumed that these interneurons transmit warm or cold signals by separate, but interlinked pathways. The specific feature of this system is that the warm and cold pathways can reciprocally inhibit each other. As deep body temperature increases, activity of warm pathways is elevated and activity of cold pathways is depressed, the final result being an activation of heat loss mechanisms. In

cold-exposed animals, in contrast, cold pathways are activated, while warm ones are inhibited so that the heat production mechanisms are set into action. Through a balanced activation o f warm and cold pathways, heat production and heat loss are adequately controlled to yield a regulated and stable internal body temperature. Peripheral vasomotor tone (PVMT), which is controlled by the sympathetic nervous system, is presumably regulated through a separate neuronal pathway, which also integrates the activity of both warm and cold pathways. Effect of BOM on neural circuits in the anterior hypothalamus observed in our experiments, which, as shown here, consists in a downward shift of the threshold T ~ of all thermoregulatory outputs and in the reduction of the efficiency o f CT and of the PVMT, can be interpreted as a predominant inhibition of the cold pathway. The lowered efficiency of the REHL indicates that BOM also exerts some inhibitory influence on the warm pathway, however (Fig. 4). The effect of BOM on the warm pathway seems to be different from that on the cold pathway because there is not an upward shift of the threshold T~o~ for induction of the R E H L after BOM. If the effect of BOM on the warm pathway were the same as on the cold one, then the interthreshold zone would be widened. This, however, was not observed in our experiments. The effect of NT can be more precisely localized. Since NT induces an upward shift of the threshold Tcorc only, it can be presumed that it influences mainly those sites in the warm pathway which are located behind interneurons interlinking the warm pathway with the cold pathway (Fig. 5). Our data provide the first evidence that peptidergic receptors in the anterior hypothalamus are specifically involved in the body temperature control. This is further supported by our findings that both neuropeptides are ineffective in other areas of the

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hypothalamus, namely in its posterior part. Another important feature of neuropeptide action is that although it persists for many hours, or even days, it is fully reversible. The que~.ion still remains how the elementary neuronal processes are affected by neuropeptides. There is general agreement that in the central nervous

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lease. So far, however, no data on neuropeptid¢ action were obtained at mammalian neuromuscular junction, which represents the simplest system at which the effect of neuropeptid~ can be studled, We performed our studies on gokl~a hamster neuromuscular junctions (Moravec and ]ansk~, 1954),

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because in hibernators peptides are considered to play an important role in inducing hibernation (Dawe, 1978). It was observed that all neuropeptides induced a marked decrease of the quantai spontaneous release of ACh. As it can be seen from Fig. 6, the effect of all neuropeptides was very similar, the depression being between 30-50% of the control values. The effect of all neuropeptides was not reversible even after 2 hours of washing out by the Liley physiological solution. In a Ca 2+ free medium, however, the inhibitory effect of BOM on the spontaneous quantal release of the transmitter is fully reversible (Moravec, 1986). Application of Ca 2+ to the inner side of the nerve terminal membrane indicates that the difference between the effect of BOM in the Ca 2+ free medium and in the Liley physiological solution is due to the changes in Ca 2÷ receptor sites at the inner side of the nerve terminal membrane (Moravec, 1984). This effect could be either due to a very strong binding of neuropeptides to target sites, or due to a very high concentration of peptides used. Therefore, in case of met-ENK the dose-response curve was also measured. As evident from Fig. 7 there was no difference in neuropeptide effect in doses ranging from 10 -5 to 10 -~2 M. Therefore, we must consider the possibility that the effect of neuropeptides in target sites is brought about by a particular mechanism, so far unknown. The results confirm the general view that neuropeptides inhibit transmitter release from nerve terminals acting at the presynaptic membrane. Why in a simple system, such as the neuromuscular junction, this effect is independent of the chemical structure of

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the substance used, while in the central nervous system the peptides induce a structure specific response, remains to be elucidated. REFERENCES

Bissette G., Luttinger D., Mason G. A., Hernandez D. E. and Loosen P. T. (1982) Neuroteusin and thermo~gulation. Ann. N.Y. Acad. Sci. 400, 268-282. Bligh J. (1979) The central neurology of mammalian thermorcgulation. Neuroscience 4, 1213-1236. Clark W. G. and Lipton L M. (1983) Brain and pituitary pcptides in thermoregulation. Pharmac. Ther. 22, 249-297. Dawe A. R. (1978) Hibernation trigger researchupdated. In Strategies in Cold (Edited by Wang L. C. H. and Hudson J. W.), pp. 541-566. Academic Press, New York. lnomoto T., Mercer J. B. and Simon E. (1982) Opposing effects of hypothalamic cooling on threshold and sensitivity of metabolic response to body cooling in rabbits. J. Physiol., Lond. 322, 139-150. Jansk~ L., Riedel W., Simon E., Simon-Oppermann C. and Vybiral S. (1986) Effect of bombesin on thermoregulation in the rabbit. Eur. J. Pharmac. To be published. Moravec J. (1984) In vitro bombesin binding at nerve terminals of golden hamsters in normal and Ca-free solution. Proc. II. Syrup. on Peripheral and CNS Synapses, Varna, Bulgaria. Moravec J. (1986) Neuropeptides reduce quantal spontaneous release of transmitter in skeletal muscle of golden hamster. (Different recoveries in Ca2+ and Ca'+-free solutions). Physiol. Bohemoslov. 35, In press. Moravec J. and .l'ansk~, L. 0984) Effect of neuropeptides on spontaneous quantal transmitter release. Physiol. Bohemoslov. 33, 547. Vybiral S., Nach~.zelJ. and Jansk~ L. (1986) Hyperthermic effect of neurotensin in the rabbit. Pfliigers Arch. ges. Physiol. 406, 312-314.