Role of endogenous cyclo(His-Pro) in cold-induced hypothermia in the desert rat (Mastomys natalensis)

Peptides. Vol. 15. No. 8, pp. 1471-1474, 1994 Copyright Q 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0196-9781/94 $6.00 + .OO

Pergamon 0196-9781(94)00126-X

Role of Endogenous Cyclo(His-Pro) in ColdInduced Hypothermia in the Desert Rat

(Mastomys natalensis) RAKESH

SHUKLA,*

N. H. RAHMANI,* HARUO MIZUMA,? CHANDAN PRASADP’

RIKHAB

CHAND

SRIMAL*

AND

*Division of Pharmacology, Central Drug Research Institute, Lucknow, India and -f-Section Oj’Endocrinology, Department of Medicine, Obesity Research Program, Louisiana State University Medical Center, New Orleans, LA 70112 Received SHUKLA,

1994

R. C. SRIMAL AND C. PRASAD. Role of‘e~do~guw~rs eyc/o(lJis-Pro) in coldPEPTIDES 15(8) 147 l-1474. 1994.-Central administration of exogenous cyclo(His-Pro) (CHP) is known to produce hypothermia in rodents. In the present study. we examined the role of endogenous CHP in cold-induced hypothermia in the desert rat, ;2la.srom.1s nataknsis. The results of these studies show that a rise in hypothalamic CHP content accompanied a decrease in rectal temperature during cold exposure. Immunoneutralization ofendogenous CHP resulted in a significant decline in cold-induced hypothermia. In addition, central administration of cyclo(AlaGly). a structural analogue of CHP. also led to a decrease in cold-induced hypothermia. The results of these studies show that changes in endogenous CHP levels may affect body temperature regulation. induced

R., N. H. RAHMANI.

20 May

hypothermia

hlustom~:~

Endogenous

H. MIZUMA,

in /he desert rut (Ma.sto~ny.s nata/ensi.s).

nutalensih

cyclo(His-Pro)

Cyclo(His-Pro) Hypothermia Cyclo(Ala-Gly)

Thermoregulation

Musfo~n~s nutahsis is a species of rodent native to the desert region of Natal, South Africa, where days are very hot and nights are very cold. The ability of this rodent species to withstand extreme temperature changes makes it a very useful animal model in which to study thermoregulation. Although M. nalalensis has been bred in captivity since 1968 ( I7), it still possesses many unique thermoregulatory properties that are similar to those of its wild counterpart. For example, the time required to attain maximal hypothermia at an ambient temperature of 10°C is much greater for M. natalcnsis than for white albino rats (see Fig. I). Histidyl-proline diketopiperazine or cyclo(His-Pro) (CHP) is a cyclic dipeptide endogenous to the central nervous system of many animal species; the administration of exogenous CHP elicits a variety of biologic activities (15.16), including modulation of body temperature regulation (7,12.13). For example, both peripheral and central administration of CHP leads to a time- and dose-dependent hypothermia in rats (7.12,13). In contrast. CHP attenuates the hypothermic effects of a number of drugs, including ketamine (3). tetrahydrocannabinol (2), and alcohol ( 14).

’ Requests for reprints should be addressed I542 Tulane

Ave., New Orleans.

to Dr. Chandan

Prasad.

Cold-induced

hypothermia

This background information on the thermoregulatory pharmacology of exogenous CHP led us to examine whether this endogenous peptide may play a role in cold ambient temperature-induced hypothermia in ,W. nutulrnsis. The results of the studies summarized here show that indeed cold-induced hypothermia in nl. nufa/cn.c.i.v is secondary to a rise in brain CHP levels. METHOD

Adult male albino rats (Charles Foster, 150-200 g) and desert rats (M. nutaknsis, 60-80 g) were bred and maintained in a light- and temperature-controlled vivarium of the Central Drug Research Institute. Lucknow. India.

Indwelling cannulae were implanted in the lateral ventricles ofl2f. natukn.cis as described elsewhere (20). Briefly, the animals were anesthetized with pentobarbitone sodium (35 mg/kg, IP) and the heads fixed in a rat stereotaxic instrument (Narashige,

Section

LA 70 1 12.

1471

of Endocrinology.

Department

of Medicine,

LSU Medical

Center,

1472

SHUKLA

Japan). A burr hole of 1.O mm diameter was made 2 mm lateral and 3 mm anterior to the interaural plane after exposing the bone. The cannula (12 mm long. 20-ga stainless steel tube) containing a stylet was fixed (2 mm from lower end) onto a small stainless steel plate and lowered 2 mm vertically into the burr hole with an electrode holder. The steel plate was then fixed to the skull bone with dental cement. The animals were allowed to recover in individual cages for I week before experimentation began. To confirm cannula placement site after the experiment. rats were injected with IO ~1 of 1% solution of methylene blue and killed by a pentobarbitone overdose, and the brains were sectioned and examined for staining of the ventricular wall.

serum albumin

as the immunogen. The specificity of the CHP in this study has been reported elsewhere (I I ). More recently, we further characterized the specificity of this antibody to show that it exhibits < 0.005%) cross-reactivity with 24 additional cyclic dipeptides containing the following pairs of amino acids: His/Ala, His/Val. His/Leu, His/Glu. His/Gin, His/ Ser. His/Lys. His/Phe. D-His/Phe, His/Gly, His/Met, His/Trp, His/His, Ser/Pro.N-methyl-Tyr/Pro, l-methyl-His/Pro. l-methylHis/Sar, Lys/Phe, Tyr/Arg, Tyr/D-Arg. D-Tyr/D-Arg, acetyl-Tyr/ Arg, Phe/Arg, and Trp/Arg. Peptides were dissolved in pyrogen-free saline (0.9%>NaCl in water, w/v) and filtered through a Maxflow microfilter of 0.22 km pore size. Glasswares were made pyrogen-free by baking at 18°C for 5 h. antibody

used

Administration of Peptides and AntihodJ and Temperature Measurements Two hours before experimentation, the cannulated animals were placed at the desired ambient temperature (10 or 24°C). Rectal temperature was measured with a thermistor probe inserted 2 cm into the rectum and connected to an electronic telethermometer. Temperature measurements were made at 0.5-h intervals beginning 1 h prior to and continuing up to 5 h after drug administration. The drugs were administered to the lateral cerebral ventricle through a chronically implanted cannula in a total volume of IO ~1. using a Hamilton microsyringe fitted with a #26 hypodermic needle with a rubber disc fixed at the desired length. The stylet of the cannula was replaced immediately after drug administration. Bruin Dissection

and Cjv3fHi.vPro)

Rudioimmunoussa~~

M. natukensis were housed in a modified BOD (Biological Oxygen Demand) incubator maintained at an ambient temperature of IO or 24°C and an RH of 50% for a total of 4 h. At the end of this period, the animals were decapitated and their brains removed from the skull and dissected into seven regions by a procedure similar to that described by Glowinski et al. (5). CHP was extracted from the tissues with 0.4 M cold perchloric acid followed by neutralization with potassium bicarbonate; the neutralized samples were lyophilized to dryness and then reconstituted in buffer for radioimmunoassay (RIA). A detailed description of tissue extraction and RIA is published elsewhere (1 I). The pellet was used for protein determination by the method of Lowry et al. (lo), and CHP contents were expressed as pg CHP/mg protein. All samples were assayed in duplicate using two concentrations of the sample. In a typical assay. significant tracer displacement was affected by 40 pg CHP per tube, and 50% displacement was produced by about 400 pg CHP. The useful range of the standard curve extended to about 2.56 ng. The intra-assay coefficient of variation derived from IO replicate samples was 4.9 -t 0.3%, and the interassay coefficient of variation derived from six independent assays of the same sample was 9.9 +- 1.1%.

RESLJLTS

The Time Reynired to .-lttuin Musimul H,pothermia Longer rn M. nutulen.c.i.s Compared to .-llh;no Rut.v

The data are presented as mean * SEM and were analyzed statistically using one-way ANOVA followed by Tukey-Kramer multiple comparison tests. A nonpaired t-test was employed for comparisons between the two groups. Peptides and CHP AntihodJx Cyclo(His-Pro), cyclo(Gly-Gly), and cyclo(Ala-Gly) were purchased from Sigma Chemical Co. (St. Louis, MO). CHP antibody was raised in rabbits using CHP conjugated to bovine

Is

Adult albino rats and ,M. nutukensis were housed in a modified maintained at an ambient temperature of 10°C and an RH of 50%. At 0.5-h intervals, rectal temperature was recorded as described and the time to attain maximal hypothermia for each animal was determined. The results of this study show that M. nutulensis took significantly longer to attain maxlmal hypothermia compared with albino rats (albino rat = 210 + 7.3. ,Mastom~:r = 330 f 11.5 min, n = 6. p < 0.0001).

BOD incubator

Cold Ambient Tcmperatwe Raises Brain Qrlo(His-Pro) Levelc The data presented in Fig. I show the distribution of CHPlike immunoreactivity (CHP-LI) in seven brain regions (striatum. hippocampus. hypothalamus. cortex. cerebellum, pons-medulla. and midbrain) of M. nutulensir housed at an ambient temper-

Sir

Statistical AnalJwY

ET AL.

HIP

HYP

BRAIN

Cor

Cer

P&M

M-0

REGION

FIG. 1. Regional distribution of cyclo(His-Pro)-like immunoreactivity in the brains of Mastom~s nafalensis housed at two different ambient temperatures. Desert rats (n = 5 per group) were housed at 10 or 24°C for a total of 4 h. and then the regional brain distribution of CHP (pg/ mg protein) was measured as described under the Method section. The data are presented as mean + SEM. Desert rats housed at 24°C exhibited an even regional distribution of CHP-LI (ANOVA: F-ratio = 8.35, p = 0.0001). In animals kept at low ambient temperature (IO”C), however. there was a differential change in regional CHP levels with increases. decreases, and no significant change in some regions.

ENDOGENOUS

CYCLO(His-Pro)

1473

AND THERMOREGULATION

sulted in a significant reduction in cold-induced hypothermia when compared with control animals receiving normal rabbit serum (NRS = -2.07 f 0.39”C, CHP-Ab = -0.95 F 0.22”C, n = 6. p = 0.032). The amount of CHP antibody administered was sufficient to bind approximately 100 ng CHP in in vitro binding or S-IO times the total M. nata/en.tis brain content of the peptide. The results of this study, therefore. suggest that coldinduced hypothermia may be secondary to a rise in the hypothalamic CHP level. Antugonism of Cold-Induced HJ;oothermia in M. nataknsis hi, CJrk$Ala-Gly)

m E ‘X

-2.5

9 I

-3.0 p NRS

CHP-Ab

TREATMENT

FIG. 2. Immunoneutralization ofendogenous cyclo(His-Pro) attenuates cold-induced hypothermia in desert rats. Desert rats were divided into two groups (n = 6). housed at an ambient temperature of 10°C. administered either NRS or CHP-Ab. and changes in rectal temperature followed as described under the Method section. The data are presented as mean + SEM, and the rectal temperature differences in two groups of rodents were analyzed

statistically

using nonpaired

r-test.

ature of 10 or 24°C for 4 h (n = 5). In animals maintained at 24”C, there was an uneven regional distribution of CHP-LI (ANOVA: F-ratio = 8.35, p = 0.0001). The order ofdistribution of peptide in different regions, from the highest level in striatum to the lowest level in the midbrain, was the same as that presented on the x-axis of Fig. I. In animals housed at 10°C and exhibiting hypothermia. however, differential changes in the regional CHP concentrations occurred; the peptide level increased in the hypothalamus (24°C = 579 f 159. 10°C = 1449 * 63. p = 0.002). decreased in the striatum (24°C = 1017 ? 177, 10°C = 367 t 73, p = 0.01 I), hippocampus (24°C = 825 f 115. 10°C = 329 f 62, p = 0.004) and cerebellum (24°C = 342 + 67, 10°C = 50 ? 24. p = 0.003) but remained unchanged in the cortex (24°C = 409 +- 87. 10°C = 425 f 38. p = 0.870) pons-medulla (24°C = 204 _+46. 10°C = I 12 i 11.p = 0.084). and midbrain (24°C = 119 t 53, 10°C = 225 + 37. p = 0.087). An examination of the data (Fig. I) on mean regional CHP levels at two ambient temperatures (24 and 10°C) shows that housing rats at 10°C. a condition eliciting hypothermia, is associated with a remarkable rearrangement of brain CHP distribution. For example, at an ambient temperature of 24°C. the highest and the lowest levels of CHP-LI were found in the striaturn and the mid-brain, respectively. with other regions being in between. However, when animals were housed at 10°C. the distribution pattern changed remarkably with the hypothalamus. a brain region thought to play a major role in thermoregulation (9) containing the highest level of CHP-LI. Because exogenous CHP is known to elicit hypothermia (7,12,13), the question arises whether the increase in hypothalamic CHP is the cause or the effect of cold-induced hypothermia in M. natalensis. The following experiment was designed to answer that question. ImmlmonPl~trali=ution Attenuates Cold-Induced

of Endogenous CJrlo(His-Pro) Hl;aothermia

in M. natalensis

The data presented in Fig. 2 show that intraventricular administration of rabbit anti-cyclo(His-Pro) to M. natulensis re-

The above data suggest that it is possible that an analogue of CHP may block the action of endogenous CHP and, therefore, attenuate cold-induced hypothermia. To this end we are in the process of testing a series of analogues of CHP; the results of two such analogues are presented in Table I. lntraventricular administration of 20 pg cyclo(Ala-Gly), but not cyclo(Gly-Gly), resulted in a signilicant (p < 0.001) reduction in cold-induced hypothermia when compared with control animals receiving pyrogen-free saline. DISCUSSION

The maintenance of homeothermia in mammals is a complex process that requires close integration between heat production and the heat loss pathways that require the action of a variety of endogenous mediators such as biogenic amines ( I. 19). acetylcholine (4). and neuropeptides (2 I) at multiple recognition sites within the central nervous system, particularly the hypothalamus. Among the many known neuropeptides, a role for exogenous CHP in body temperature regulation in rodents is well established (2.3.7.12- 14.18). For example, central administration of exogenous CHP to rats produces a time- and dosedependent hypothermia (7.12). Within the brain. CHP not only acts at the anterior hypothalamic/preoptic area (AH/POA) of the hypothalamus to elicit hypothermia. but intraventricularly administered [‘H-Pro]CHP was predominantly concentrated in the AH/POA subarea of the hypothalamus (6). The results of the studies presented here show that i) there is a concomitant rise in hypothalamic CHP content and a de-

TABLE

I

ANTAGONISM OF COLD-INDUCED HYPOTHERMIA BY CYCLO(Ala-Gly) Maxmal Change ,n Rectal

Treatment

Temperature (“C)

Control (Vehicle) Cyclo(Gly-Gly) Cyclo(Ala-Gly)

~ 1.82 -t 0.49 (5) -1.80 + 0.46 (5) + I .36 ?Z 0.09 (5)

The data are presented as mean 2 SEM with the number of observations in parentheses. Animals were administered with peptides (20 fig in 10 ~1) or vehicle (pyrogenfree saline) intraventricularly as described in the Method section. One-way ANOVA: F = 21.61 I, p = 0.0001. p-Value derived from the Tukey-Kramer multiple comparison test: control vs. cyclo(Gly-Gly). >O.OS: control vs. cyclo(AlaGly).
1474

SHUKLA

crease in core temperature at an ambient temperature of lO”C, and ii) cold-induced hypothermia was attenuated by immunoneutralization of endogenous CHP or the administration of cyclo(Ala-Gly), an analogue of CHP. These data have led us to suggest for the first time a role for endogenous CHP in thermoregulation. However, we can only speculate on the mechanism by which CHP elicits hypothermia at low ambient temperature at this time. Central administration of CHP has been shown to lower body temperature in a thermoneutral environment (24-25°C). leading to even greater hypothermia at low ambient temperatures (4-5°C) with no significant effect on core temperature at high (3 1“C) ambient temperatures (7.12). These data are consistent with a mechanism whereby CHP may act to reduce body heat conservation and production rather than a modulation of the set point of body temperature. The failure of CHP to affect body temperature in a hot environment may be due to the fact that, under such conditions, both body heat conservation and production are minimally active. In contrast. immunoneutralization of endogenous CHP at an ambient temperature of 24°C did not result in a significant change in core temperature (NRS = 0.866 * 0.14. CHP-Ab = 0.383 i 0.202, n = 6. p = 0.078). These data suggest that the site at which CHP acts to reduce the heat production mechanism (peripheral and/ or central) may not be very sensitive to small changes in CHP

ET AL.

concentration. Such a mechanism is also consistent with CHP attenuation of an increase in the rectal temperature of hypothermic neonatal rats housed at 24°C (18). It is also possible, however, that CHP may facilitate the heat loss system. It is now generally agreed that dopamine within AH/POA mediates hypothermia by acting as a functional component of the heat loss system (9). In addition to the hypothalamus, the nigral dopaminergic system exerts an influence on thermoregulation through a yet poorly defined mechanism (9). Within the hypothalamus. CHP acts at AH/POA, a region in which dopamine interacts to promote the heat loss system to elicit hypothermia (7). This fact, combined with attenuation of CHP hypothermia by dopaminergic antagonists (13). suggests that CHP might act as a dopaminergic agonist in eliciting hypothermia. A series of studies of the relationship between CHP and dopaminergic mechanisms in the striatum and hypothalamus has led to the conclusion that the peptide may indirectly act like a dopaminergic agonist by inhibiting dopamine reuptake (6.8). In conclusion, these data suggest that endogenous hypothalamic CHP may play an important role in the thermoregulatory process. ACKNOWLEDGEMENTS We

thank Ms. Anne Compliment for her timely and excellent editorial help. This is publication #25 from the Obesity Research Program.

REFERENCES I. Beckman, A. L.; Eisenman. J. S. Microelectrophoresis of biogenic amines on hypothalamic thermosensitive cells. Science 170:334336; 1970. 2. Bhargava, H. N. The effect of thyrotropin-releasing hormone and

cyclo(His-Pro) on delta-9-tetrahydrocannabinol-induced hypothermia. Life Sci. 26:845-850: 1980. 3. Bhargava, H. N. Antagonism of ketamine-induced anesthesia and hypothermia by cyclo(His-Pro) and thyrotropin-releasing hormone. Neuropharmacology 20:699-702: 198 1. 4. Crawshaw. L. 1. Effect of intracranial acetylcholine injection on thermoregulatory responses in the rat. J. Comp. Physiol. Psycho]. 83:32-35;

1973. J.; Iversen, L. L. Regional studies of catecholamines in the rat brain. J. Neurochem. 13:655-664: 1966. Ikegami, H.; Prasad, C. Neuropeptide-dopamine interactions V. cyclo(His-Pro) regulation of striatal dopamine transporter complex. Peptides 11:145-148: 1990. Jacobs, J. J.: Prasad. C.: Wilber. J. F. Cycle (His-Pro): Mapping hypothalamic sites for its hypothermic actton. Brain Res. 250:205209; 1982. Jikihara, H.; Ikegami, H.; Koike, K.: et al. lntraventricular administration of histidyl-proline diketopiperarine [cyclo(His-Pro)] suppresses prolactin synthesis and secretion: A possible role of cyclo(HisPro) as dopamine uptake blocker in rat hypothalamus. Endocrinology 132:953-958; 1993. Lee, T. F.; Mora. F.; Myres, R. D. Dopamine and thermoregulation: An evaluation with special reference to dopaminergic pathways. Neurosci. Biobehav. Rev. 9:589-598; 1985. Lowry, 0. H.; Rosebrough, N. J.: Farr. A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275; 195 1. Mori, M.; Prasad, C.; Wilber. J. F. Specific radioimmunoassay of cycle (His-Pro), a biologically active metabolite of thyrotropin-releasing hormone. Endocrinology 108: 1995- 1997: 198 I.

5. Glowinski, 6. 7. 8.

9. 10. 1 I.

12. Prasad, C.; Matsui. T.; Williams, J.; Peterkofsky. A. Thermoregulation in rats: Opposing effects of thyrotropin-releasing hormone and its metabolite histidyl-proline-diketopiperazine. Biochem. Biophys. Res. Commun. 85: 1582- 1587: 1978. 13. Prasad. C. Neuropeptide-dopamine Interactions 1. Dopaminergic mechanisms in cyclo(His-Pro)-mediated hypothermia in rats. Brain Res. 437:345-348: 1987. 14. Prasad. C.; Balasubramanian. P. Cycle (His-Pro) and the development of tolerance to the hypothermic effect of ethanol. Neuropeptides 12:75-79: 1988. 15. Prasad. C. Neurobiology of cyclo(His-Pro). Ann. NY Acad. Sci. 553: 232-251: 1989. 16. Prasad, C. Bioactive cyclic dipeptides. Peptides (in press). 17. Pringle. G.: King, D. F. Some developments in techniques for the study of the rodent filarial parasite Lifomo.roid~~.scurinit I.A preliminary comparison of the host efficiency of the multimammate rat, Pruoru.ts j.~lasfom~~.s~ nara~cnsis, with that of the cotton rat, S&n&on hi.ypid~r.s. Ann. Trop. Med. Parasitol. 62: 462-468: 1968. 18. Sato. T.: Kato. T.: Miyamoti. C.: et al. Effect of thvrotropin-releasing hormone and cyclo(His-Pro) on the homeothermic development of neonatal rats. Acta Endocrinol. 1 13: 18 I- 188: 1986. 19. Shukla, R.; Srimal. R. C.: Dhawan, B. N. The effect of intracerebroventricular administration of catecholamines and their antagonists on rectal temperature of Mnstom]:s nata1msi.s. Naunyn Schmiedebergs Arch. Pharmacol. 318:38-42: 1981. 20. Shukla, R.: Srimal, R. C.: Dhawan. B. N. A technique for chronic cannulation ofthe lateral cerebral ventricle in Mustorn?:~ nuru/cwsi.c. Ind. J. Exp. Biol. 19:97-98: 198 I. 21. Yehuda. Y.: Kastin, A. J. Peptides and thermoregulation. Neurosci. Biobehav. Rev. 4:459-47 1: 198 I.