Effect of dermorphin on thermoregulation in rats at selected ambient temperatures

Peptides, Vol. 17, No. 2, pp. 241-245, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/96 $15.00 + .OO

SSDI 01%~9781(95)02132-9

ELSEVIER

Effect of Dermorphin on Thermoregulation in Rats at Selected Ambient Temperatures T . G . EMEL’YANOVA,

* ’ A. B. USENKO,*

V. I. DEIGIN,? E. P. YAROVAt

AND A. A. KAMENSKYt

*N. N. Semenov Institute of Chemical Physics RAS, Department of Matter Structure, Kosygin str. 4, 117977 Moscow, Russia fLomonosov Moscow State University, Department of Human and Animal Physiology, Moscow, Russia Received 16 May 1995 EMEL’YANOVA, T. Cl., A. B. USENKO, V. I. DEIGIN, E. P. YAROVA AND A. A. KAMENSKY. EfSecfof dermotphinon thermoregulation in rafs at selected ambient temperatures. PEPTIDES 17(2) 24 l-245, 1996.-1ntrapexitoneal administration of dermorphin caused dose-dependent changes in rats core temperature and tail skin temperature (indicative of compensatory thermoregulatory vasoreactions in rats). The character of these changes depended strongly on the environmental temperature at which the inversion of the dermorphin-induced thermoregulatory effect was observed. In the cold environment (4-7°C) dermorphin caused a significant, stable, dose-dependent hypothermia. In the thermoneutral environment (27-28°C) dermorphin also caused hypothermia, but this effect was less pronounced. In the hot environment (3 l -32°C) dermorphin caused hyperthermia. Dermorphin-induced changes in tail skin temperature indicate that dermorphin suppresses the thermoregulatory peripheral compensatory vasomotor reactions. Pretreatment with naloxone attenuated dermorphin-induced effects on core temperature and partially enhanced vasomotor effects of dermorphin. The data obtained indicate that dermorphin affects the core temperature regulation via p-opiate receptors, whereas vasomotor effects of the peptide are probably mediated via naloxone-insensitive receptors. Dermorphin Vasodilatation

Naloxone

Thermoregulation

Hypothermia

OPIOID-INDUCEDchanges in the body temperature of animals have been studied since the 1960s ( 19). Gpioid peptides cause both hypothermia and hyperthermia, as well as biphasic response in animals (8,9). The character of these thermoregulatory effects depends to a large extent on peculiar features of the experimental conditions used, namely, on animal species, the degree of their restraint, the dose level, and route of drug administration, and also on the environmental temperature (2,6,13,21) . Dermorphin (DM), an amphibian skin peptide (Tyr-D-AlaPhe-Gly-Tyr-Pro-Ser-NH,), is unique in having a D-amino acid in its sequence. The presence of DM binding sites has been shown in mammalian central nervous system and peripheral organs (22). It has been also found that DM is the first among the naturally occurring peptides to be a highly potent and highly specific agonist towards the morphine (cl) receptors ( 1,18). This opioid peptide exhibits long-term analgesic effects in laboratory animals and isolated preparations ( 3). Autonomic effects of DM depend to a large extend on methodological variables. Centrally injected DM produces bradycardia and respiratory depression in rabbits (20,27), evokes changes in renal function (17), stimulates respiratory and locomotor activity (24)) and raises heart rate and blood pressure in rats ( 10). Subcutaneously injected DM stimulates prolactin secretion and inhibits gastrointestinal motility in rats ( 10).

Hyperthermia

Vasoconstriction

There is some evidence that DM causes the changes in body temperature of animals. It has been demonstrated that centrally injected DM produces a hypothermic response in conscious rabbits ( 20,27 ) , and temperature- and dose-dependent responses in rats (4). The purpose of the present study was to clarify the influence of the dose level and environmental temperature on the core temperature and peripheral vasomotor response to IP-injected dermorphin in rats. METHOD Male outbread albino rats, weighing 180-250 g, were used in the experiments. All experiments were carried out in previously handled, awake, and fed animals. Each animal was used only once. In each experiment 10 animals were divided into two groups: five rats received the injection of a control solution and the other five rats received the injection of DM dose. Two or three experiments were carried out for each tested dose (lo-15 animals in each experimental group). The experiments were conducted at different environmental temperatures: 1) in the cold environment (4-7°C); 2) in the comfort environment ( thermoneutral environment, 27-28°C) (14); and 3) in the hot environment (31-32°C).

’ Requests for reprints should be addressed to T. G. Emel’yanova.

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temperature value the basal temperature value (the mean of the three values measured in the same rat 30, 20, and 10 min prior to IP injection). In the cold environment rat basal colon temperature was 36.6 & 0.2”C (means ? SEM; ANOVA) and basal tail skin temperature was 9.6 + 0.4”C; in the thermoneutral environment these values were 37.8 -C 0.2“C and 28.3 t O.S’C, respectively; in the hot environment these values were 38.6 +- 0.2”C and 34.5 ? 0.8”C. DM was synthesized in the National Engineering Center of Peptide Preparations “Peptos” (Moscow); naloxone (NL) was obtained from Sigma (USA). The drugs were administered IP (1 ml/kg body weight) as a distilled water solution. The doses used were 0.5, 5, 50, 500, and 5000 ,ug/kg body weight for DM and 1 mglkg body weight for NL. Control animals were injected by equivalent volumes of distilled water. In the experiments with NL each animal received two injections with a 15mitt interval: group l-distilled water + distilled water; Group 2-NL + distilled water; group 3-distilled water + DM; group 4-NL + DM. All values were compared to the control values. The statistical significance of differences in temperature changes was verified by means of a nonparametric Mann-Whitney U-test. RESULTS

In the cold environment the low dose of DM (0.5 pug/kg) caused a small decrease in core temperature [Fig. 1 (A)]. The 0

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1. The effect of different doses of [P-injected dermorPhin on core temperature (A) and tail skin temperature (B) changes in rats exposed to the cold environment (4-7’C). *p < 0.05 vs. control.

The air temperature in the chamber ( Tch), the colon temperature ( T,), and the temperature of the tail skin (T,,) were recorded during the experiment. The tail is known to be a specific organ of thermoexchange in the rat, and because of it the tail skin temperature changes reflect the activity of peripheral blood vessels (vasoconstriction or vasodilatation) . Copper-nickel thermocouples were used for continuous recording of the temperature. Each thermocouple was placed into the guide flexible plastic tube 2 mm in diameter. To exclude the damage to the animal each colon guide tube had a sealed oval tip. Each animal had two thermocouples taped to its tail; the first one was inserted for a depth of 60 mm into the colon and the second one was taped at the root of the tail. The signal from the thermocouple was transferred to A/D converter in the selected time intervals (2 mitt). Codes from A/D converter were transferred to a PC, in which they were recorded and processed. During the experiment each animal was housed in an individual partial-restraint plastic box (200 X 55 X 60 mm), where the rat could not turn about. Ten boxes with rats were placed into a temperature-regulated chamber. The animals were adapted to experimental conditions for 2 h. Intraperitoneal injections were carried out only when the core temperature of each rat was stabilized for at least 30 min. For injections, the rats were not removed from their boxes or from the temperature-regulated chamber. The temperature was recorded 30, 20, and 10 min prior to injection and then at lo-min intervals during 2 h after the injection. Changes in core ( AT,)/tail skin (AT,,) temperature of each injected rat were calculated by subtracting from postinjection

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2. The effect of different doses of IP-injected dermorphin on core temperature (A) and tail skin temperature (B) changes in rats exposed to the thermoneutral environment (27-28°C). *p < 0.05 vs. control.

DERMORPHIN

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EFFECT ON THERMOREGULATION

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(AT,, = 2.09 t 0.89”C 10 min after IP injection) was replaced by the long-term vasoconstriction [Fig. 2( B )I. The decrease in a dose from 5 mg/kg to 500 pg/kg resulted in attenuation of the second phase of the vasomotor reaction. DM at a dose of 50 pgl kg caused only the first phase, vasodilatation. DM at doses of 5 and 0.5 pg/kg had no significant effect on T,,. In the hot environment DM (5 mg/kg, 500 and 50 pg/kg) caused a delayed dose-dependent hyperthermic response, with the maximum T, changes (0.71 ? 0.33”C at a dose of 5 mg/kg) 80 min after IP injection [Fig. 3(A)]. DM at dose of 5 mg/kg caused a relatively small, significant, short-term vasodilatation (AT, = 0.33 2 0.23”C 10 min after IP injection) [Fig. 3(B)]. Low doses of DM had no significant effect on T,,. In the experiments with NL we have used a more effective DM dose level: 500 pg/kg in the cold and thermoneutral environments, and 5 mg/kg in the hot environment. In the cold environment NL alone produced a weak vasoconstrictive response and insignificant decrease in core temperature [Fig. 4( A, B)]. However, when injected 15 min prior to DM administration, NL attenuated the peptide-induced hypothermic response [Fig. 4 (A)]. On the other hand, NL and DM combined injection had a greater effect on T,, changes than either DM or NL alone [Fig. 4(B)]. In the thermoneutral environment NL alone evoked a weak decrease in T,, ( -0.29 2 0.08”C 40 min after IP injection), ac-

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FIG. 3. The effect of different doses of IP-injected dermorphin on core temperature (A) and tail skin temperature (B) changes in rats exposed to the hot environment (31-32°C). *p < 0.05 vs. control.

hypothermic response increased with doses from 5 to 500 pg/ kg. Core temperature changed significantly 10 min after administration of DM, and hypothermia was sustained during all the recording period. DM at a dose of 500 pg/kg was more effective, as hypothermic response attained its maximum ( - 1.80 ? 0.32”C) 40 min after IP injection. Further increases in the DM dose up to 5 mg/kg resulted in a diminished hypothermic response. Figure 1(B ) illustrates DM-induced changes in tail skin temperature in rats exposed to the cold environment. Animals in the cold develop peripheral vasoconstriction, as reflected in the slow continuous decrease in tail skin temperature. The responses to DM were similar to the control response except that the largest dose of DM produced biphasic vascular response [Fig. 1 (B)] The first short-term phase, vasodilatation (AZ’,, = 0.33 + 0.21”C 10 min after IP injection), was replaced by the long-term stable second phase, vasoconstricdon (T,, decreased to a maximum level of -0.90 It 0.23”C). In the thermoneutral environment DM at doses of 50 and 500 pg/kg produced a significant hypothermia that was sustained over the period of recording [Fig. 2(A)] _ The highest dose of DM (5 mg/kg) caused a biphasic response-namely, short-term, less pronounced hypothermia (AZ’, = -0.06 + 0.13”C 10 min after IP injection), replaced by slowly developed hyperthermia, which attained its maximum (0.33 + 0.21”C) 80 min after IP injection. Low doses of DM (0.5 and 5 pgg/kg) had no significant effect on T,. The highest dose of DM caused a clearly pronounced biphasic vascular response; short-term vasodilatation

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Time postinjection (min) FIG. 4. The effect of naloxone (1 mgkg), dermorphin (500 j&kg), and their combined injection on core temperature (A) and tail skin temperature (B) changes in rats exposed to the cold environment (4-7°C). Differences from control, *p < 0.05; differences from DM-treated group, op < 0.05; differences from NL-treated group, #p < 0.05.

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level and the environmental temperature. The inversion of DM thermoregulatory effect is similar to those of peptide and nonpeptide opioids (7,9 ). Moreover, IP-injected DM-induced core temperature responses followed a pattern similar to that obtained after central administration of DM (4). It has been demonstrated that peripherally injected DM is able to penetrate the blood-brain barrier ( 1,22). These data suggest that IP-injected DM is acting on the central mechanisms of the core temperature regulation. It seems likely that DM either directly modifies the activity of central thermosensors or alters the activity in the afferent sensor pathways, with consequent disturbance of the compensatory mechanisms. DM-induced changes in tail skin temperature are also of some interest. The large dose of DM produced biphasic vasomotor reaction, and in this case, the short-term vasodilatation was replaced by long-term vasoconstriction. Moreover, in the thermoneutral environment (when any thermal stress was absent) the vasomotor response was more pronounced and was observed even after DM was injected at doses of 50 and 500 ,ug/kg. Under these conditions the vasomotor reaction was accompanied by the corresponding core temperature changes. It has been shown that peripherally injected DM (8 mg/kg) did not influence either the basal heart rate or blood pressure in rats ( 11). Therefore, our data indicate that DMinduced changes in peripheral vasomotor reactions are of a A

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FIG. 5. The effect of naloxone (1 mg/kg), dermorphin (500 @kg), and their combined injection on core temperature (A) and tail skin temperature (B) changes in rats exposed to the thermoneutral environment (2728°C). Differences from control, *p < 0.05; differences from DMtreated group, op < 0.05; differences from NL-treated group, #p < 0.05. -0.5

companied by a slowly developed peripheral vasoconstriction [Fig. 5 (A, B )] . Under these conditions NL also weakened DMinduced hypothermic response [Fig. 5(A)]. At the same time, NL inhibited the first phase of the DM-induced vascular response (vasodilatation) [Fig. 5(B)]. The second phase of vasomotor response on NL and DM combined injection replicated that after NL alone. In the hot environment NL alone revealed no significant influence on core temperature and tail skin temperature [Fig. 6( A, B)]. However, NL partially weakened the DM-induced hyperthermia [Fig. 6(A)], blocked DM-induced short-term vasodilatory response, and enhanced vasoconstriction in DM-treated animals [Fig. 6(B)].

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DISCUSSION

Intraperitoneal injection of dermorphin produced a dosedependent hypothermia in rats exposed to the cold environment. In the thermoneutral environment the DM-induced hypothermic response was less pronounced; at the same time a large dose of DM evoked a slowly developing hyperthermia. In the hot environment DM produced a hyperthermic response. The data obtained show, in our opinion, that the magnitude and direction of IP-injected DM-induced core temperature changes may be considered as a function of both the dose

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FIG. 6. The effect of naloxone (1 mg/kg), dermorphin (5 mg/kg), and their combined injection on core temperature (A) and tail skin temperature (B) changes in rats exposed to the hot environment (31-32°C). Differences from control, *p < 0.05; differences from DM-treated group, OP < 0.05; differences from NL-treated group, #P < 0.05.

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local character and likely may be related with the thermoregulatory responses to DM injection. In view of the fact that peripherally injected DM demonstrated thermoregulatory activity at dose level that did not evoke changes in heart rate, blood pressure, and nociception ( 11,22), data obtained allow us to conclude that DM-induced thermoregulatory responses are not related with, and do not depend on, its cardiovascular or analgesic effects. Our data suggest, therefore, that peripherally injected DM, like other opioids ( 12,23), depresses the thermoregulatory system leading to a fall in body temperature in the cold environment and to a rise in body temperature in the hot one, and also reduces the corresponding compensatory vasomotor responses. According to our data, DM affects T,, at low doses, which do not cause any changes in tail skin temperature. Moreover, DMinduced core temperature changes do not correlate with those of the vasomotor reactions. At the same time, DM-induced vasomotor reactions partially facilitate the core temperature changes and maintain the core temperature at a new level. The data allow us to conclude that the effect of DM on the regulation of core temperature and on the vasomotor (compensatory) responses is probably realized via at least two independent and unrelated pathways.

This conclusion is also confirmed by some data obtained in the experiments with NL. On the one hand, the drug reduced both the hypothermic and the hyperthermic effects of DM. On the other hand, in some cases, NL enhanced DM-induced vasomotor responses. There exists some contradictory information about the influence of NL on the thermoregulatory effects of opioids. Some findings suggest that the effects of opioids are completely blocked by NL or naltrexone (5,13,25); other findings indicate that this is not the case (2,6). Our data are in good agreement with the results ( 15,16,28 ) demonstrating that NL weakens thermoregulatory effects of opioids. It has been shown that DM is highly potent and speciftc agonist towards the ~1receptors (1) . Inasmuch as thermoregulatory effects of DM are similar to morphine-induced effects, the functional similarity probably reflects actions on similar subset of NL-sensitive receptors designated p ( 15 ) . The resistance of DM-induced vasomotor responses to antagonism by NL indicates that DM stimulates a mixture of receptors including NL-insensitive subtypes (6) ( 18). Taking our data into account, one may conclude that DM influences the core temperature regulation through p-opiate receptors, whereas vasomotor effects of the peptide are probably mediated by another subtypes of opiate receptors.

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