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Cyclo(His-Pro) Modulation of Body Temperature at Hot Ambient Temperature in the Desert Rat (Mastomys natalensis)
Peptides, Vol. 18, No. 5, pp. 689–693, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(97)00130-7
Cyclo(His-Pro) Modulation of Body Temperature at Hot Ambient Temperature in the Desert Rat (Mastomys natalensis) RAKESH SHUKLA,* RIKHAB CHAND SRIMAL* AND CHANDAN PRASAD†1 *Division of Pharmacology, Central Drug Research Institute, Lucknow, India †Section of Endocrinology, Department of Medicine, Louisiana State University Medical Center, New Orleans, LA 70112 Received 20 November 1996; Accepted 13 January 1997 SHUKLA, R., R. C. SRIMAL AND C. PRASAD. Cyclo(His-Pro) modulation of body temperature at hot ambient temperature in the desert rat (Mastomys natalensis). PEPTIDES 18(5) 689–693, 1997.—Cyclo(His-Pro) (CHP) has been shown to facilitate cold-induced hypothermia in the desert rat Mastomys natalensis. In the present study, we examined the role of endogenous CHP in hyperthermia induced by hot ambient temperature (407C) in the above rodent species. The results of these studies show that housing rodents at 407C resulted in a altered distribution of CHP in the brain, with a rise in hypothalamic content accompanied by an increase in rectal temperature. While administration of exogenous CHP decreased hyperthermia, immunoneutralization of endogenous CHP increased hyperthermia. The results of these studies show that changes in endogenous CHP levels may affect body temperature regulation. q 1997 Elsevier Science Inc. Mastomys natalensis
Cyclo(His-Pro)
Hyperthermia
Thermoregulation
Endogenous cyclo(His-Pro)
METHOD
HISTIDYL-PROLINE diketopiperazine or cyclo(His-Pro) (CHP) is a cyclic dipeptide endogenous to neuronal and nonneuronal tissues of many animal species (15, 16). Administration of exogenous peptide elicits a variety of biologic activities, including modulation of body temperature regulation (7, 12, 13). For example, both peripheral and central administration of CHP lead to 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). More recently, we examined whether endogenous CHP may play a role in cold ambient temperature-induced hypothermia in Mastomys natalensis . The results of the study show that since CHP immunoneutralization attenuated cold-induced hypothermia, the decrease in body temperature must be secondary to a rise in brain CHP levels (21). M. natalensis 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. natalensis has been bred in captivity since 1968 (17), it still possesses many unique thermoregulatory properties that are similar to those of its wild counterpart. Since CHP attenuated cold-induced hypothermia in our earlier study (21), it was of interest to examine what role the peptide may play in thermoregulation at high ambient temperatures.
Adult desert rats (M. natalensis, 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 of M. natalensis 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, Japan). A burr hole of 1.0 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-gauge 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 one week before experimentation began. To confirm the cannula placement site after the experiment, rats were injected with 10 ml of 1% solution of methylene blue and sacrificed with a pentobarbitone overdose and the brains sectioned and examined for staining of the ventricular wall. Two hours before experimentation, the cannulated animals were placed at the desired ambient temperature (407C or 247C). 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 half-hourly interval be-
1 Requests for reprints should be addressed to Dr. Chandan Prasad, Section of Endocrinology, Department of Medicine, LSU Medical Center, 1542 Tulane Ave, New Orleans, LA 70112 USA; Fax: (504) 568-4159; E-Mail: [email protected].
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FIG. 1. Regional distribution of cyclo(His-Pro)-like immunoreactivity in the brains of Mastomys natalensis housed at two different ambient temperatures. Desert rats (n Å 7 per group) were housed at 407C or 247C for a total of 4 h, and then the regional brain distribution of CHP (pg/mg protein) was measured as described under text. The data are presented as mean { sem. Desert rats housed at 247C did not exhibit any significant difference in regional distribution of CHP-LI (ANOVA: F-ratio Å 9.85, p Å 0.131). In animals kept at high ambient temperature (407C), however, there was a differential change in regional CHP levels with increases, decreases, and no significant change in some regions.
ginning 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 10 ml, using a Hamilton microsyringe fitted with #26 hypodermic needle with a rubber disc fixed at the desired length. The stylet of the cannula was replaced immediately after drug administration. M. natalensis were housed in a modified biological oxygen demand (BOD) incubator maintained at an ambient temperature of 407C or 247C and an relative humidity (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 (11). The pellet was used for protein determination by the method of Lowry et. al. (10) 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 10 replicate samples was 4.9 { 0.3%, and the inter-assay coefficient of variation derived from six independent assays of the same sample was 9.9 { 1.1%. 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. Cyclo(His-Pro) was purchased from Sigma Chemical Co., St. Louis, MO. CHP antibody was raised in rabbits using CHP conjugated to bovine serum albumin as the immunogen. The specificity of the CHP-antibody used in this study has been reported elsewhere (11). CHP was dissolved in pyrogen-free saline (0.9% NaCl in water, w/v) and filtered through a Maxflow microfilter of 0.22 mm pore size. Glassware was made pyrogen-free by baking at 1807C for 5 h. RESULTS
The data presented in Fig. 1 show the distribution of CHPlike immunoreactivity (CHP-LI) in seven brain regions (striatum, hippocampus, hypothalamus, cortex, cerebellum, pons-medulla, and midbrain) of uncannulated M. natalensis housed at an
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FIG. 2. Exogenous cyclo(His-Pro) attenuates hyperthermia produced by high ambient temperature in desert rats. Desert rats were divided into two groups (n Å 6), housed at an ambient temperature of 407C, administered either saline or CHP (10 mg/rat), and changes in rectal temperature followed as described under text. The data are presented as mean { sem, and the rectal temperature differences in the two groups of rodents were analyzed statistically using nonpaired t-test.
FIG. 3. Immunoneutralization of endogenous cyclo(His-Pro) augments hyperthermia produced by a high ambient temperature in desert rats. Desert rats were divided into two groups (n Å 6), housed at an ambient temperature of 407C, administered either normal rabbit serum (NRS) or CHP antibody, and changes in rectal temperature followed as described under text. The data are presented as mean { sem, and the rectal temperature differences in the two groups of rodents were analyzed statistically using nonpaired t-test.
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ambient temperature of 407C or 247C for 4 h ( n Å 5). In animals maintained at 247C, there was an uneven regional distribution of CHP-LI (ANOVA: F-ratio Å 9.85, p Å 0.13); however, the regional differences were not statistically significant. The distribution of peptide in different regions, from the highest level in striatum to the lowest level in the pons-medulla, is shown in Fig. 1. However, in uncannulated animals housed at 407C and exhibiting hyperthermia, differential changes in the regional CHP concentrations occurred. For example, the peptide level increased in the cortex (247C Å 573 { 152, 407C Å 2094 { 578, p Å 0.03), pons-medulla (247C Å 431 { 50, 407C Å 1283 { 468, p Å 0.046), and hypothalamus (247C Å 843 { 172, 407C Å 1401 { 197, p Å 0.047); decreased in the hippocampus (247C Å 675 { 158, 407C Å 186 { 63, p Å 0.025) and mid-brain (247C Å 1279 { 328, 407C Å 325 { 56, p Å 0.021); but remained unchanged in the striatum (247C Å 1099 { 282, 407C Å 1284 { 178, p Å 0.595) and cerebellum (247C Å 673 { 180, 407C Å 775 { 321, p Å 0.784). An examination of the data (see Fig. 1) on mean regional CHP levels at two ambient temperatures (247C and 407C) shows that housing uncannulated rats at 407C, a condition eliciting hyperthermia, is associated with a remarkable rearrangement of brain CHP distribution. For example, at an ambient temperature of 247C, the highest and the lowest levels of CHP-LI were found in the striatum and the pons-medulla, respectively, with levels in the other regions falling in between. However, when animals were housed at 407C, the distribution pattern changed remarkably, with the cortex containing the highest level of CHP-LI. While exogenous CHP is known to elicit hypothermia at ambient temperatures of 57C to 227C (7, 12, 13), the effect of the peptide on thermoregulation at high ambient temperature has not yet been explored. To this end, we examined the effect of intraventricular CHP on high ambient temperature-induced hyperthermia in M. natalensis.. The data presented in Fig. 2 show that housing animals at 407C led to a time-dependent increase in rectal temperature that reached a maximum of 3.57C within 120 min and remained high until the measurements were terminated at 240 min. It is interesting to note that during the above period, no animal died due to hyperthermia, suggesting that M. natalensis have adapted To survive in high ambient temperatures. In animals injected intraventricularly with CHP (10 mg/rat), the rise in rectal temperature was slower, and it took 180 min to achieve maximal hyperthermia. Since exogenous CHP attenuated emergence of hyperthermia at 407C (Fig. 2), we wondered whether immunoneutralization of endogenous CHP would augment hyperthermia. The data presented in Fig. 3 show that intraventricular administration of rabbit-anti-cyclo(His-Pro) to M. natalensis resulted in a significant reduction in hyperthermia for the first 120 min and a significant increase in hyperthermia for the rest of the experimental period. The amount of CHP antibody administered was sufficient to bind approximately 100 ng CHP in in vitro binding or 8 to 10 times the total M. natalensis brain content of the peptide. The mechanism underlying a delay in the emergence of hyperthermia after antibody administration is not clear from the available data. However, one could that it may be due to the kinetics of neutralization of endogenous CHP at high ambient temperatures. Alternatively, the anti-CHP antibody administered into the intraventricular space may diffuse slowly to different neuroanatomic loci yielding a time-dependent changing pattern of brain CHP distribution leading to a delay the appearance of hyperthermia.
DISCUSSION
The maintenance of homeothermia in mammals is a complex process that requires close integration between heat production and the heat-loss pathways, which depend on the action of a variety of endogenous mediators such as biogenic amines (1, 19), acetylcholine (4), and neuropeptides (22) 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 ( 3H-Pro]-CHP was predominantly concentrated in the AH/POA subarea of the hypothalamus (6). The results of the present and an earlier study (21) show that exposure of M. natalensis to high and low ambient temperatures results in a redistribution of CHP in the brain, with a concomitant rise in hypothalamic CHP content and a decrease/increase in core temperature, depending on the ambient temperature. Furthermore, exogenous CHP attenuates hyperthermia and augments hypothermia resulting from high and low temperatures, respectively. Immunoneutralization of endogenous CHP led to temperature changes at high and low ambient temperatures that were opposite those associated with exogenous CHP administration. These data suggest a role for endogenous CHP in thermoregulation and the possibility of pharmacological use of CHP and its analogs in the maintenance of normothermia. However, at this time, the mechanism by which CHP elicits its thermoregulatory effects remains speculative. Central administration of CHP has been shown to lower body temperature in a thermoneutral environment (24-257C) and to lead to even greater hypothermia at low ambient temperatures (4-57C), with no significant effect on core temperature at 317C (7, 12), and hyperthermia at 407C. 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. Such a mechanism is also consistent with CHP attenuation of an increase in the rectal temperature of hypothermic neonatal rats housed at 247C (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 influences thermoregulation through an as-yet poorly defined mechanism (9). Within the hypothalamus, CHP acts at AH/ POA, a region in which dopamine interacts to promote the heatloss 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 have 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.
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REFERENCES 1. Beckman, A.L.; Eisenman, J.S. Microelectrophoresis of biogenic amines on hypothalamic thermosensitive cells. Science 170:334– 336; 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; 1981. 4. Crawshaw, L.I. Effect of intracranial acetylcholine injection on thermoregulatory responses in the rat. J. Comp. Physiol. Psychol. 83:32– 35; 1973. 5. Glowinski, J.; Iversen, L.L. Regional studies of catecholamines in the rat brain. J. Neurochem. 13:655–664; 1966. 6. Ikegami, H.; Prasad, C. Neuropeptide-dopamine interactions V. cyclo(His-Pro) regulation of striatal dopamine transporter complex. Peptides 11:145–148; 1990. 7. Jacobs, J.J.; Prasad, C.; Wilber, J.F. Cyclo (His-Pro): Mapping hypothalamic sites for its hypothermic action. Brain Res 250:205–209; 1982. 8. Jikihara, H.; Ikegami, H.; Koike, K.; Wada, K.; Morishige, K.; Kurachi, H; Hirota, K.; Miyake, A.; Tanizawa, O. Intraventricular administration of histidyl-proline diketopiperazine [cyclo(His-Pro)] suppresses prolactin synthesis and secretion: A possible role of cyclo(His-Pro) as dopamine uptake blocker in rat hypothalamus. Endocrinology 132:953–958; 1993. 9. 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. 10. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. 11. Mori, M.; Prasad, C.; Wilber, J.F. Specific radioimmunoassay of cyclo (His-Pro), a biologically active metabolite of thyrotropin-releasing hormone. Endocrinology 108:1995–1997; 1981.
12. Prasad, C.; Matsui, T.; Williams, J.; Peterkofsky, A. Thermoregulation in rats: Opposing effects of thyrotropin-releasinghormone and its metabolite histidyl-proline-diketopiperazine. Biochem. Biophys. Res. Commun. 85:1582–1587; 1978. 13. Prasad, C. neuropeptide-dopamine interactions I. Dopaminergic mechanisms in cyclo(His-Pro)-mediated hypothermia in rats. Brain Res 437:345–348; 1987. 14. Prasad, C.; Balasubramanian, P. Cyclo (His-Pro) and the development of tolerance to the hypothermic effect of ethanol. Neuropeptides 12:75–79. 15. Prasad, C. Neurobiology of cyclo(His-Pro). Ann. NY Acad. Sci. 553:232–251; 1989. 16. Prasad, C. Bioactive cyclic dipeptides. Peptides 16:151–164; 1994. 17. Pringle, G.; King, D.F. Some developments in techniques for the study of the rodent filarial parasite Litomosoides carinii I. — A preliminary comparison of the host efficiency of the multimammate rat, Praomys (Mastomys) natalensis, with that of the cotton rat, Sigmodon hispidus. Ann. Trop. Med. Parasitol. 62:462–468; 1968. 18. Sato, T.; Kato, T.; Miyamori, C.; Kajiwara, S.; Murata, A.; Imura, E.; Sakura, N.; Hashimoto, T. Effect of thyrotropin-releasing hormone and cyclo(His-Pro) on the homeothermic development of neonatal rats. Acta Endocrinol. 113:181–188; 1986. 19. Shukla, R.; Srimal, R.C.; Dhawan, B.N. The effect of intracerebroventricular administration of catecholamines and their antagonists on rectal temperature of Mastomys natalensis . N-S Arch. Pharmacol. 318:38–42; 1981. 20. Shukla, R.; Srimal, R.C.; Dhawan, B.N. A technique for chronic cannulation of the lateral cerebral ventricle in Mastomys natalensis. Ind. J. Exp. Biol. 19:97–98; 1981. 21. Shukla, R.; Rahmani, N.H.; Mizuma, H.; Srimal, R.C.; Prasad, C. Role of endogenous cyclo(His-Pro) in cold-induced hypothermia in the desert rat (Mastomys natalensis ). Peptides 15:1471–1474; 1994. 22. Yehuda, Y.; Kastin, A.J.; Peptides and thermoregulation. Neurosci. Biobehav. Rev. 4:459–471; 1981.
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Cyclo(His-Pro) Modulation of Body Temperature at Hot Ambient Temperature in the Desert Rat (Mastomys natalensis) RAKESH SHUKLA,* RIKHAB CHAND SRIMAL* AND CHANDAN PRASAD†1 *Division of Pharmacology, Central Drug Research Institute, Lucknow, India †Section of Endocrinology, Department of Medicine, Louisiana State University Medical Center, New Orleans, LA 70112 Received 20 November 1996; Accepted 13 January 1997 SHUKLA, R., R. C. SRIMAL AND C. PRASAD. Cyclo(His-Pro) modulation of body temperature at hot ambient temperature in the desert rat (Mastomys natalensis). PEPTIDES 18(5) 689–693, 1997.—Cyclo(His-Pro) (CHP) has been shown to facilitate cold-induced hypothermia in the desert rat Mastomys natalensis. In the present study, we examined the role of endogenous CHP in hyperthermia induced by hot ambient temperature (407C) in the above rodent species. The results of these studies show that housing rodents at 407C resulted in a altered distribution of CHP in the brain, with a rise in hypothalamic content accompanied by an increase in rectal temperature. While administration of exogenous CHP decreased hyperthermia, immunoneutralization of endogenous CHP increased hyperthermia. The results of these studies show that changes in endogenous CHP levels may affect body temperature regulation. q 1997 Elsevier Science Inc. Mastomys natalensis
Cyclo(His-Pro)
Hyperthermia
Thermoregulation
Endogenous cyclo(His-Pro)
METHOD
HISTIDYL-PROLINE diketopiperazine or cyclo(His-Pro) (CHP) is a cyclic dipeptide endogenous to neuronal and nonneuronal tissues of many animal species (15, 16). Administration of exogenous peptide elicits a variety of biologic activities, including modulation of body temperature regulation (7, 12, 13). For example, both peripheral and central administration of CHP lead to 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). More recently, we examined whether endogenous CHP may play a role in cold ambient temperature-induced hypothermia in Mastomys natalensis . The results of the study show that since CHP immunoneutralization attenuated cold-induced hypothermia, the decrease in body temperature must be secondary to a rise in brain CHP levels (21). M. natalensis 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. natalensis has been bred in captivity since 1968 (17), it still possesses many unique thermoregulatory properties that are similar to those of its wild counterpart. Since CHP attenuated cold-induced hypothermia in our earlier study (21), it was of interest to examine what role the peptide may play in thermoregulation at high ambient temperatures.
Adult desert rats (M. natalensis, 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 of M. natalensis 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, Japan). A burr hole of 1.0 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-gauge 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 one week before experimentation began. To confirm the cannula placement site after the experiment, rats were injected with 10 ml of 1% solution of methylene blue and sacrificed with a pentobarbitone overdose and the brains sectioned and examined for staining of the ventricular wall. Two hours before experimentation, the cannulated animals were placed at the desired ambient temperature (407C or 247C). 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 half-hourly interval be-
1 Requests for reprints should be addressed to Dr. Chandan Prasad, Section of Endocrinology, Department of Medicine, LSU Medical Center, 1542 Tulane Ave, New Orleans, LA 70112 USA; Fax: (504) 568-4159; E-Mail: [email protected].
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FIG. 1. Regional distribution of cyclo(His-Pro)-like immunoreactivity in the brains of Mastomys natalensis housed at two different ambient temperatures. Desert rats (n Å 7 per group) were housed at 407C or 247C for a total of 4 h, and then the regional brain distribution of CHP (pg/mg protein) was measured as described under text. The data are presented as mean { sem. Desert rats housed at 247C did not exhibit any significant difference in regional distribution of CHP-LI (ANOVA: F-ratio Å 9.85, p Å 0.131). In animals kept at high ambient temperature (407C), however, there was a differential change in regional CHP levels with increases, decreases, and no significant change in some regions.
ginning 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 10 ml, using a Hamilton microsyringe fitted with #26 hypodermic needle with a rubber disc fixed at the desired length. The stylet of the cannula was replaced immediately after drug administration. M. natalensis were housed in a modified biological oxygen demand (BOD) incubator maintained at an ambient temperature of 407C or 247C and an relative humidity (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 (11). The pellet was used for protein determination by the method of Lowry et. al. (10) 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 10 replicate samples was 4.9 { 0.3%, and the inter-assay coefficient of variation derived from six independent assays of the same sample was 9.9 { 1.1%. 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. Cyclo(His-Pro) was purchased from Sigma Chemical Co., St. Louis, MO. CHP antibody was raised in rabbits using CHP conjugated to bovine serum albumin as the immunogen. The specificity of the CHP-antibody used in this study has been reported elsewhere (11). CHP was dissolved in pyrogen-free saline (0.9% NaCl in water, w/v) and filtered through a Maxflow microfilter of 0.22 mm pore size. Glassware was made pyrogen-free by baking at 1807C for 5 h. RESULTS
The data presented in Fig. 1 show the distribution of CHPlike immunoreactivity (CHP-LI) in seven brain regions (striatum, hippocampus, hypothalamus, cortex, cerebellum, pons-medulla, and midbrain) of uncannulated M. natalensis housed at an
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FIG. 2. Exogenous cyclo(His-Pro) attenuates hyperthermia produced by high ambient temperature in desert rats. Desert rats were divided into two groups (n Å 6), housed at an ambient temperature of 407C, administered either saline or CHP (10 mg/rat), and changes in rectal temperature followed as described under text. The data are presented as mean { sem, and the rectal temperature differences in the two groups of rodents were analyzed statistically using nonpaired t-test.
FIG. 3. Immunoneutralization of endogenous cyclo(His-Pro) augments hyperthermia produced by a high ambient temperature in desert rats. Desert rats were divided into two groups (n Å 6), housed at an ambient temperature of 407C, administered either normal rabbit serum (NRS) or CHP antibody, and changes in rectal temperature followed as described under text. The data are presented as mean { sem, and the rectal temperature differences in the two groups of rodents were analyzed statistically using nonpaired t-test.
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ambient temperature of 407C or 247C for 4 h ( n Å 5). In animals maintained at 247C, there was an uneven regional distribution of CHP-LI (ANOVA: F-ratio Å 9.85, p Å 0.13); however, the regional differences were not statistically significant. The distribution of peptide in different regions, from the highest level in striatum to the lowest level in the pons-medulla, is shown in Fig. 1. However, in uncannulated animals housed at 407C and exhibiting hyperthermia, differential changes in the regional CHP concentrations occurred. For example, the peptide level increased in the cortex (247C Å 573 { 152, 407C Å 2094 { 578, p Å 0.03), pons-medulla (247C Å 431 { 50, 407C Å 1283 { 468, p Å 0.046), and hypothalamus (247C Å 843 { 172, 407C Å 1401 { 197, p Å 0.047); decreased in the hippocampus (247C Å 675 { 158, 407C Å 186 { 63, p Å 0.025) and mid-brain (247C Å 1279 { 328, 407C Å 325 { 56, p Å 0.021); but remained unchanged in the striatum (247C Å 1099 { 282, 407C Å 1284 { 178, p Å 0.595) and cerebellum (247C Å 673 { 180, 407C Å 775 { 321, p Å 0.784). An examination of the data (see Fig. 1) on mean regional CHP levels at two ambient temperatures (247C and 407C) shows that housing uncannulated rats at 407C, a condition eliciting hyperthermia, is associated with a remarkable rearrangement of brain CHP distribution. For example, at an ambient temperature of 247C, the highest and the lowest levels of CHP-LI were found in the striatum and the pons-medulla, respectively, with levels in the other regions falling in between. However, when animals were housed at 407C, the distribution pattern changed remarkably, with the cortex containing the highest level of CHP-LI. While exogenous CHP is known to elicit hypothermia at ambient temperatures of 57C to 227C (7, 12, 13), the effect of the peptide on thermoregulation at high ambient temperature has not yet been explored. To this end, we examined the effect of intraventricular CHP on high ambient temperature-induced hyperthermia in M. natalensis.. The data presented in Fig. 2 show that housing animals at 407C led to a time-dependent increase in rectal temperature that reached a maximum of 3.57C within 120 min and remained high until the measurements were terminated at 240 min. It is interesting to note that during the above period, no animal died due to hyperthermia, suggesting that M. natalensis have adapted To survive in high ambient temperatures. In animals injected intraventricularly with CHP (10 mg/rat), the rise in rectal temperature was slower, and it took 180 min to achieve maximal hyperthermia. Since exogenous CHP attenuated emergence of hyperthermia at 407C (Fig. 2), we wondered whether immunoneutralization of endogenous CHP would augment hyperthermia. The data presented in Fig. 3 show that intraventricular administration of rabbit-anti-cyclo(His-Pro) to M. natalensis resulted in a significant reduction in hyperthermia for the first 120 min and a significant increase in hyperthermia for the rest of the experimental period. The amount of CHP antibody administered was sufficient to bind approximately 100 ng CHP in in vitro binding or 8 to 10 times the total M. natalensis brain content of the peptide. The mechanism underlying a delay in the emergence of hyperthermia after antibody administration is not clear from the available data. However, one could that it may be due to the kinetics of neutralization of endogenous CHP at high ambient temperatures. Alternatively, the anti-CHP antibody administered into the intraventricular space may diffuse slowly to different neuroanatomic loci yielding a time-dependent changing pattern of brain CHP distribution leading to a delay the appearance of hyperthermia.
DISCUSSION
The maintenance of homeothermia in mammals is a complex process that requires close integration between heat production and the heat-loss pathways, which depend on the action of a variety of endogenous mediators such as biogenic amines (1, 19), acetylcholine (4), and neuropeptides (22) 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 ( 3H-Pro]-CHP was predominantly concentrated in the AH/POA subarea of the hypothalamus (6). The results of the present and an earlier study (21) show that exposure of M. natalensis to high and low ambient temperatures results in a redistribution of CHP in the brain, with a concomitant rise in hypothalamic CHP content and a decrease/increase in core temperature, depending on the ambient temperature. Furthermore, exogenous CHP attenuates hyperthermia and augments hypothermia resulting from high and low temperatures, respectively. Immunoneutralization of endogenous CHP led to temperature changes at high and low ambient temperatures that were opposite those associated with exogenous CHP administration. These data suggest a role for endogenous CHP in thermoregulation and the possibility of pharmacological use of CHP and its analogs in the maintenance of normothermia. However, at this time, the mechanism by which CHP elicits its thermoregulatory effects remains speculative. Central administration of CHP has been shown to lower body temperature in a thermoneutral environment (24-257C) and to lead to even greater hypothermia at low ambient temperatures (4-57C), with no significant effect on core temperature at 317C (7, 12), and hyperthermia at 407C. 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. Such a mechanism is also consistent with CHP attenuation of an increase in the rectal temperature of hypothermic neonatal rats housed at 247C (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 influences thermoregulation through an as-yet poorly defined mechanism (9). Within the hypothalamus, CHP acts at AH/ POA, a region in which dopamine interacts to promote the heatloss 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 have 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.
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REFERENCES 1. Beckman, A.L.; Eisenman, J.S. Microelectrophoresis of biogenic amines on hypothalamic thermosensitive cells. Science 170:334– 336; 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; 1981. 4. Crawshaw, L.I. Effect of intracranial acetylcholine injection on thermoregulatory responses in the rat. J. Comp. Physiol. Psychol. 83:32– 35; 1973. 5. Glowinski, J.; Iversen, L.L. Regional studies of catecholamines in the rat brain. J. Neurochem. 13:655–664; 1966. 6. Ikegami, H.; Prasad, C. Neuropeptide-dopamine interactions V. cyclo(His-Pro) regulation of striatal dopamine transporter complex. Peptides 11:145–148; 1990. 7. Jacobs, J.J.; Prasad, C.; Wilber, J.F. Cyclo (His-Pro): Mapping hypothalamic sites for its hypothermic action. Brain Res 250:205–209; 1982. 8. Jikihara, H.; Ikegami, H.; Koike, K.; Wada, K.; Morishige, K.; Kurachi, H; Hirota, K.; Miyake, A.; Tanizawa, O. Intraventricular administration of histidyl-proline diketopiperazine [cyclo(His-Pro)] suppresses prolactin synthesis and secretion: A possible role of cyclo(His-Pro) as dopamine uptake blocker in rat hypothalamus. Endocrinology 132:953–958; 1993. 9. 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. 10. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. 11. Mori, M.; Prasad, C.; Wilber, J.F. Specific radioimmunoassay of cyclo (His-Pro), a biologically active metabolite of thyrotropin-releasing hormone. Endocrinology 108:1995–1997; 1981.
12. Prasad, C.; Matsui, T.; Williams, J.; Peterkofsky, A. Thermoregulation in rats: Opposing effects of thyrotropin-releasinghormone and its metabolite histidyl-proline-diketopiperazine. Biochem. Biophys. Res. Commun. 85:1582–1587; 1978. 13. Prasad, C. neuropeptide-dopamine interactions I. Dopaminergic mechanisms in cyclo(His-Pro)-mediated hypothermia in rats. Brain Res 437:345–348; 1987. 14. Prasad, C.; Balasubramanian, P. Cyclo (His-Pro) and the development of tolerance to the hypothermic effect of ethanol. Neuropeptides 12:75–79. 15. Prasad, C. Neurobiology of cyclo(His-Pro). Ann. NY Acad. Sci. 553:232–251; 1989. 16. Prasad, C. Bioactive cyclic dipeptides. Peptides 16:151–164; 1994. 17. Pringle, G.; King, D.F. Some developments in techniques for the study of the rodent filarial parasite Litomosoides carinii I. — A preliminary comparison of the host efficiency of the multimammate rat, Praomys (Mastomys) natalensis, with that of the cotton rat, Sigmodon hispidus. Ann. Trop. Med. Parasitol. 62:462–468; 1968. 18. Sato, T.; Kato, T.; Miyamori, C.; Kajiwara, S.; Murata, A.; Imura, E.; Sakura, N.; Hashimoto, T. Effect of thyrotropin-releasing hormone and cyclo(His-Pro) on the homeothermic development of neonatal rats. Acta Endocrinol. 113:181–188; 1986. 19. Shukla, R.; Srimal, R.C.; Dhawan, B.N. The effect of intracerebroventricular administration of catecholamines and their antagonists on rectal temperature of Mastomys natalensis . N-S Arch. Pharmacol. 318:38–42; 1981. 20. Shukla, R.; Srimal, R.C.; Dhawan, B.N. A technique for chronic cannulation of the lateral cerebral ventricle in Mastomys natalensis. Ind. J. Exp. Biol. 19:97–98; 1981. 21. Shukla, R.; Rahmani, N.H.; Mizuma, H.; Srimal, R.C.; Prasad, C. Role of endogenous cyclo(His-Pro) in cold-induced hypothermia in the desert rat (Mastomys natalensis ). Peptides 15:1471–1474; 1994. 22. Yehuda, Y.; Kastin, A.J.; Peptides and thermoregulation. Neurosci. Biobehav. Rev. 4:459–471; 1981.
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