Characterization of opioid peptides and opioid receptors in the brain of jerboa (Jaculus orientalis) a hibernating rodent

PII S0361-9230(97)00282-7

Characterization of Opioid Peptides and Opioid Receptors in the Brain of Jerboa (Jaculus orientalis),

a Hibernating Rodent

NOUREDDINE BOURHIM,*†

MOSTAFA KABINE* AND M’HAMED SAID ELKEBBAJ*

*Laboratoire de Biochimie, Biologie Cellulaire et Mole´culaire (Unite´ de Biochimie) Faculte´ des Sciences Ain chock, B.P. 5366, Casablanca, Morocco, †Laboratoire de Neuroendocrinologie Expe´rimentale Faculte´ de

Me´decine Nord, INSERM (U297), Boulevard Pierre Dramard, 13326 Marseille Cedex 15, France

ABSTRACT: The present study was undertaken to investigate the biochemical characteristics of the opioid receptors and opioid peptides in the jerboa (Jaculus orientalis) brain, a sub- desert rodent of Morocco. We have demonstrated the presence of d , m , and k sites in the jerboa brain. The endogenous opioid peptides methionine-enkephalin, b -endorphin, and dynorphin were evaluated in different physiological states of the animal (active and hibernating). The circulating methionine-enkephalin in different states of the animal (active, hibernating, exposure to cold conditions, and fasting) was evaluated in the plasma. Our results indicate that in the hibernating state the opioid recep- tors level decreased, whereas the concentration of opioid pep- tides increased. These findings suggest that both opioid recep- tors and opioid peptides could be involved in the adaptation of the jerboa to survive under thermal stress. © 1997 Elsevier Science Inc.

KEY WORDS: Hibernation, Brain, Methionine-enkephalin, b -En- dorphin, Dynorphin, Fast, Cold acclimatation.

INTRODUCTION

The action of opiates and opioid peptides in the nervous system are mediated by interactions with at least three classes of opioid receptors, the d , m and k receptors [12,20,27]. Opioid receptors in the membranes of nerve cells are key elements in a regulatory system that allows opioids outside the cell to modify intracellular events and alter cell function. These receptors mediate the diverse effects of endogenous opioid peptides and opiate drugs. Activation of opioid receptors characteristically depresses neuronal firing and inhibits central nervous system pathways ascending from the spi- nal cord to the cerebral cortex, and blocks hypothalamic and neurohypophyseal hormone release; opioids also cause hypoten- sion, respiratory depression, and decrease of food intake (see [29]

for review). All these phenomena have also been observed during mammalian hibernation [31].

Hibernation or winter torpor is a successful adaptation ex- ploited by some mammals, regulated by a yet unknown endoge- nous mechanism. Numerous neuroactive substances, including neurotransmitters and opioids, have been hypothesized to be im- portant in controlling the entry and maintenance of hibernation [32]. Hibernators have also the ability to survive long periods of food shortage by a marked reduction in the rate of their energy

expenditures and to substantially reduce the heart rate, body tem- perature, and food intake, which are the same phenomena observed after administration of opioids [7,8]. The literature data on the functional activity of opioid receptors in hibernators are contra- dictory. Using the autoradiographic method, a decrease in the level of m -binding sites has been observed in ground squirrels (Citellus lateralis) [6]. In other studies, no difference was detected in the level of binding of the antagonist [

H]naloxone in the brain of hibernating and active ground squirrels (Spermophilus lateralis) [2,33]. However, the biochemical and behavioral characteristics of these receptors in the active and hibernating state are unknown.

The present study was undertaken to investigate the biochem- ical characteristics of the opioid binding sites in the awake-active state and induced hibernation of jerboa (Jaculus orientalis) (Fig.

1), a subdesert rodent of Morocco, and to characterize opioid receptors. This animal is known as a true hibernator [3,5,14] in comparison with the other desert species of jerboa (Jaculus jacu- lus) (Fig. 1) [4], which is considered as a nonhibernating animal [14]. Indeed, jerboa (Jaculus orientalis) presents the same charac- teristics of all other mammalian hibernators, such as cardiac bra- dycardia, respiratory depression, and hypothermia. The same phe- nomena were observed when a nonhibernating animal was exposed to administration of opioids [8]. Our aim in this work was to characterize opioid receptors and opioid peptides in the brain of the jerboa and to investigate the effect of cold and fast on the level of free methionine-enkephalin (Met-enk). Using tritiated ligands, we have characterized the three types of opioid binding sites ( d , m , and k ) in the brain of active and hibernating animals. By radio- immunoassay (RIA), we have evaluated the Met-enk, b -endorphin, and dynorphin immunoreactivity in different regions of the jerboa brain in the two states (active and hibernating) and we have estimated the circulating Met-enk in jerboa plasma in four states (active, hibernating, exposed to cold conditions, and fasting).

MATERIALS AND METHODS

Animals

Jerboas (Jaculus orientalis) were captured in the subdesert land of Eastern Morocco (see Fig. 1) and were transferred to the laboratory in a preacclimated room (22 6 2°C), with food (sun- flower seeds, barley, maize, and carrots) and free access to water.

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The light cycle during the entire experiment was set to 14-h light and 10-h dark (but it cannot be excluded that the dark period may have been occasionally interrupted).

Induced Hibernation

To induce hibernation, young adult jerboas of both sexes, 4 – 6 months old and 100 –150 g of body weight, were transferred to the cold room and kept at 4 6 1°C for 3 weeks of adaptation with food and free access to water. At the end of the three weeks, food was removed and hibernation was soon induced; animals of this group of jerboas are defined as ‘‘hibernating jerboas.’’

Exposure to Cold Conditions

To study the effect of cold, young adult jerboas of both sexes, 4 – 6 months old and 100 –150 g of body weight, were transferred to the cold room and kept at 4 6 1°C for 2 weeks with food and free access to water. This group of jerboas represent the experi- mental animals ‘‘exposed to cold conditions.’’

Fasting

Young adult jerboas of both sexes, 4 – 6 months old and 100 – 150 g of body weight, were kept in the laboratory at 22 6 2°C without food for a period of 24 h to 96 h; this group includes the

‘‘fasting jerboas.’’

Control jerboas were kept at room temperature with food and free access to water.

Membrane Preparation for Binding Assays

Brains were potterized in 50 mM Tris-HCl buffer, pH 7.4 and were centrifuged twice at 200 3 g for 5 min. Pellets were resus- pended and disrupted by homogeneization with a potter. The resulting homogenates were centrifuged at 1000 3 g for 10 min at 4°C to remove nuclei and cellular debris. The resulting supernatant was centrifuged at 30,000 3 g for 15 min at 4°C. The supernatant was discarded and the pellets were resuspended in buffer to a final protein concentration of 0.8 –1 mg/ml. Protein was measured by the method of Bradford [9] with bovine serum albumin (BSA) as a standard.

Opioid Receptor Binding Assays

Binding was conducted in assay buffer, in a total volume of 1.0 ml containing 0.5 ml of crude membranes (0.8 to 1 mg protein), 0.1 ml radioligand, and 0.4 ml buffer containing specific effectors to mask one or more sites. Briefly, d -sites have been identified by [

H] [D-Ala

, DLeu

] enkephalin (DADLE) in the presence of 0.1 m M morphiceptin (to prevent DADLE binding to m -sites), m -sites with [

H] [D-Ala

, Me-Phe

, Gly-ol] enkephalin DAGO in the presence of 0.1 m M [D-Ser

, Leu

] enkephalin, Thr

(DSLET) (to prevent DAGO binding to d -sites), and k -sites by [

H] Ethylketo- cyclazocine (EKC) in the presence of 5 m M DADLE (which at this concentration masks d and m sites) [10,11]. Nonspecific binding was performed by 10 m M levorphanol. For equilibrium binding, incubation conditions were 30 min at 37°C. Separation of free and bound radioligand was obtained by rapid filtration in vacuo through Whatman GF/B glass-filter circles (2.5 cm in diameter), presoaked in a 0.3% solution of polyethylenimine in water and 0.01% polylysine [15]. Filters were washed with 15 ml assay buffer, dried, provided with 5 ml scintillation cocktail (Instagel Packard), incubated at 4°C overnight. Radioactivity was deter- mined in a scintillation counter (Intertechnique SL 3000) with 52%

efficacy for [

H]. All assays were performed in triplicate.

Radioimmunoassays (RIA)

Methionine-enkephalin. Extraction of peptides from the jerboa brain structures was performed as reported elsewhere with slight modifications [16]; after decapitation, the brain was dissected according to Glowinski and Iversen [17]. Tissues were frozen on dry ice in 2 ml of acetic acid (0.5 N), boiled for 10 min, chilled for 10 min in ice bath, and homogenized with polytron (Brinkman).

The homogenate was centrifuged at 12,000 3 g for 10 min at 4°C, the supernatant was collected, lyophilyzed, and reconstituted in 2 ml of phosphate-buffered saline (PBS)/BSA 0.1%, pH 7.4. Anti- Met-enk antisera previously characterized [13] were obtained from Dr. A. Cupo; the final dilution of anti-Met-enk antiserum was 1:120,000. The Met-enk was iodinated in the laboratory using the chloramine T method [18], and was purified by HPLC using an Octadesyl silane column (Lichrosorb C18, 250 3 5 mm, Merck) and eluted with a linear gradient 10 – 60% of acetonitrile in trieth- ylamine formate (TEAF) 0.1 M pH 3.0, with a flow rate of 1 ml/min. The incubation was carried out in a final volume of 0.3 ml in PBS/0.1% BSA, pH 7.4, containg 0.1 ml antibodies (1:120,000), 0.1 ml of

I Met-enk and 0.1 ml of extract. After incubation of 48 h at 4°C, the complex was precipitated by 0.1 ml of g -globuline in PBS (12 g/ml) and 0.1 ml of polyethylene glycol (PEG) 6000, 30% in H

O. The supernatant was eliminated and the radioactivity of the pellet determined in a g counter (Cristal Packard).

b -Endorphin. Extraction was performed as indicated for Met- enk. The anti- b -endorphin antiserum was obtained in the labora- tory after injection of b -endorphin to rabbit. Antiserum 334 was obtained in the laboratory after injection of human- b -endorphin FIG. 1. Geographical distribution of jerboa in Morocco, derived from

Aulagnier and Thevenot, 1986 [4].

(Bachem Marura del Rey, CA) coupled to BSA by glutaraldehyde (Sigma). The characteristics of the antiserum obtained with a similar procedure were described in a previous study [21]. The antiserum was used at the dilution of 1:10,000 in the RIA. Sen- sivity of the essay was 20 pg/tube. Iodination of b -endorphin was performed in the laboratory by the technique that utilizes chlora- mine-T [18]. The separation of free and bound b -endorphin was performed by immunoprecipitation.

Dynorphin 1–13. The extraction was performed as indicated for Met-enk. The dynorphin 1–13 antiserum was a generous gift from Dr. A. Goldstein (Alto, CA), The characterization of this antiserum was previously described [16]. Iodinated dynorphin was prepared by chloramine-T method [18]. The final dilution of the antiserum in the RIA was 1:20,000. Sensitivity of the assay was 2.2 fmol/tubes. The separation was performed as indicated for Met-enk.

Plasma Extraction Techniques

Plasma (0.2– 0.4 ml) was acidified with 3 vol perchloric acid (0.2 N) and centrifuged at 2000 3 g for 10 min. The supernatant was filtered by reverse-phase octadecylsilyl silica (C-18) cartridge (Sep-Pak, Waters Co.), preequilibrated in 0.25 M triethylamine formate (TEAF) buffer pH 3.2, and washed with 15 ml TEAF.

Data Analysis

Analysis of binding isotherms was performed by linear regres- sion methods from the Scatchard plots of the data [28] or by mathematical modeling using the ligand program [23,24] as de- scribed in previous studies from our laboratory [10,11].

Statistics

Statistical difference between control (active jerboas) and hi- bernating jerboas sample means was determined by the paired Student t-test. A p-value lower than 0.05 was considered signifi- cant.

Materials

[

H] DADLE (specific activity 45 ci/mmol) and [

H] EKC (specific activity 15 ci/mmol) were purchased from New England Nuclear Corporation. [

H] Etorphine and [

H] Bremazocine were purchased from Amersham. Levorphanol was a gift from Hoff- mann La Roche. Morphiceptin, DSLET, DADLE and Met-enk, dynorphin were purchased from Sigma and b -endorphin was pur- chased from Peninsula. All the other chemicals were purchased from Merck (Darmstadt, Germany) or Sigma.

RESULTS

Characterization of d , m , and k Opioid Receptors in the Jerboa Brain

The binding of ligands specific for each of the examined opioid receptor type is summarized in Table 1. Specific binding of the ligand of d and m receptor [

H] DADLE, in the presence of morphiceptin, to prevent the binding of [

H] DADLE to m recep- tors, indicate a B

of 160 fmol/mg of protein and a dissociation constant (K

) of 0.4 nM (Table 1). Scatchard analysis of specific binding of the highly selective m ligand [

H] DAGO, in the presence of DSLET, to prevent binding to d receptors, shows a B

for m receptors of 53 fmol/mg of protein and a K

of 0.38 nM (Table 1). To label the k -receptors, [

H] EKC was used in the presence of 5 m M DADLE to prevent binding to d and m sites. The results indicate a B

of 90 fmol/mg of protein and a K

of 0.14 nM (Table 1).

Characterization of Total Opioid Binding Sites in Jerboa Brain Membranes

We have used [

H] bremazocine, a nonselective opioid ligand that binds to d -, m -, and k - receptors, to detect the total opioid binding sites densities in jerboa brain membranes. The results (Table 1) pointed out a high density of opioid binding sites (260 fmol/mg protein) and a K

of 0.6 nM.

Hibernation Effect on the Opioid Receptors

The effect of induced hibernation on the binding capacities and affinities is shown in Fig. 2A. The B

of d receptor types was reduced from 160 fmol/mg to 53 fmol/mg of proteins. For m -sites, the results indicate a decrease from 52 fmol/mg to 32 fmol/mg and k -sites were reduced from 90 fmol/mg of protein to 50 fmol/mg of protein. The affinities were moderately affected by hibernation in all cases (Fig. 2B). To evaluate the effect of hibernation on total receptors, we have used the nonselective ligand; [

H] bremazo- cine. The results indicate that the density of opioid binding sites was reduced from 260 fmol/mg to 115 fmol/mg and the K

was increased to 1.32 nM, but this variation was not statistically significant (Fig. 2A and B).

Distribution of Met-Enk, b -Endorphin, and Dynorphin in the Jerboa Brain

To make a correlation between opioid binding receptors and endogenous ligands, we have quantified by means of RIA Met- enk, b -endorphin, and dynorphin in different regions of the jerboa brain.

Met-enk. The highest level of met-enk immunoreactivity was found in the cerebral cortex, hypothalamus, and striatum, with the lowest level in the hippocampus and neural lobe of the pituitary.

During the induced hibernation, we observed a significant increase in the immunoreactivity in the cortex, striatum, and hippocampus.

In contrast, no significant modification was detected in the hypo- thalamus and pituitary (Fig. 3A).

b -Endorphin. As shown in Fig. 3B, the concentration of b - endorphin varied greatly in different regions of the jerboa brain.

The highest level of this peptide was found in the hypothalamus.

In all cases the concentration of b -endorphin increased in the induced hibernation.

Dynorphin. The highest concentration of dynorphin 1–13 was TABLE 1

CALCULATION OF EQUILIBRIUM SATURATION BINDING PARAMETERS OF [³H] DADLE, [³H] DAGO, [³H] EKC, AND [³H] BREMAZOCINE WITH JERBOA NEURAL MEMBRANES

Radioligand

Receptor

Type K_d B_max

[

H] DADLE (0.1 m M

Morphiceptin) d 0.40 6 0.20 160 6 40

[

H] DAGO (0.1

m M DSLET) m 0.23 6 0.12 52 6 10

[

H] EKC (5 m M

DADLE) k 0.14 6 0.10 90 6 30

[

H] Bremazocine d , m and k 0.60 6 0.10 260 6 30

Comparison of the binding in the active and hibernating state derived

from the Scatchard plot. Results are means 6 SEM of three separate

experiments.

found in the hypothalamus and striatum (Fig. 3C). In induced hibernation the amount of immunoreactivity was increased in all brain samples from different regions.

Estimate of the circulating peptide (Met-enk) in jerboa plasma.

Fig. 4 presents the effect of cold conditions, induced hibernation, and fasting on the circulating Met-enk level. In cold conditions, the concentration of met-enk was increased by 2.14-fold in compari- son with controls. In induced hibernation this ratio decreased to 1.54-fold. In contrast, the fasting conditions caused a decrease of the circulating peptide in the jerboa plasma.

DISCUSSION

Our results indicate that the three types of opioid receptors ( d , m , and k ) are present in the jerboa brain. The Scatchard analysis of the saturation binding indicated the presence of one class of high binding affinity. In hibernating jerboa brain membranes, the dis- tribution of d , m , and k was reduced. To correlate the opioid binding site diminution and natural endogenous ligand behavior, the quantification of Met-enk (endogenous ligand of d -receptors), b -endorphin (endogenous ligand of m -receptors), and dynorphin (endogenous ligand of k -receptors) in different regions of the jerboa brain indicated an enhancement of the level of all endoge- nous ligand during hibernation. This increase could be explained by several ways and the following hypotheses can be put forth: (a)

during hibernation of the jerboa, the body temperature was de- creased to 5 6 2°C, and at this temperature the major peptidase activity could have been reduced. The increase of peptides in the nervous tissue could also be ascribed to a reduced release. (b) Opioid peptide synthesis was enhanced to play an analgesic role and/or to reduce pain of the animal and favor the entrance in hibernation. This hypothesis is supported by the finding that ad- ministration (ip) of low doses of naloxone to jeboas in the hiber- nation state causes a premature arousal of the animal, at variance with the control animals that received saline solution (0.9% NaCl) (unpublished observation). In contrast, the decrease of the opioid

FIG. 3. Regional distribution of Met-enkephalin (A), b-endorphin (B), and dynorphin (C) immunoreactive material, in the jerboa brain in awake- active and hibernating jerboas. All values are means 6 SEM for three animals. Extracts were prepared as discussed in Materials and Methods.

p , 0.05 in all cases, control (active animal) vs. hibernation.

FIG. 2. Comparison of the binding capacities (B

) (A) and affinities (K

) (B) in the active and hibernating state derived from the Scatchard plot.

Results are means 6 SEM of three separate experiments. p , 0.05 in all

cases, control (active animal) vs. hibernation.

binding sites observed in the present study could be explained by a downregulation of the receptors, due to the accumulation of opioid peptides in brain structures during hibernation as shown above, and/or to a saturation of opioid receptors by endogenous opioids. The decrease of receptor expression during hibernation could also be explained by a reduction of the receptor internaliza- tion (which is a temperature-dependent phenomenon).

All these hypotheses are supported by the fact that DADLE (an analogue of leucine-enkephalin, which binds to d -receptors) has been shown to induce many physiological changes that may favor hibernation [25]. It is, therefore, possible that the maintenance of hibernation may be associated with an increased activity of brain opioids. This increase may be manifested in the peripheral circu- lation and could represent the ‘‘hibernation induction trigger’’ as defined by others [26]. To verify this hypothesis we have quanti- fied the Met-enk peptide in the plasma in two physiological states (active and hibernating). Our results indicate that in all cases the level of opioid peptides was enhanced in hibernation in compari- son with the active state. These findings support the hypothesis that the ‘‘hibernation induction trigger’’ could be represented by an opioid peptide. This increase of Met-enk in plasma could also derive from a reduction of the peripheral catabolism. In other words, the observed effects could represent an epiphenomenon of hibernation rather than an adaptive or causative phenomenon.

The results obtained in the study of b -endorphin and dynor- phin, which are the natural ligands for m and k receptor, respec- tively, are similar to those of Met-enk, i.e., an increased expression in induced hibernation. This drastic change in endogenous b - endorphin and dynorphin concentration during hibernation may suggest a role of these peptides in inducing hibernation. Indeed, it has been demonstrated that stimulation of k -opioid receptors (re- ceptors for dynorphin) by specific agonist induced respiratory depression, bradycardia, and hypothermia [1,19,30]. All of these are physiological manifestations of hibernation; it may, thus, be speculated that the observed increase of dynorphin expression in the jerboa brain may be involved in regulating the transition from the active to hibernating states.

To investigate the effect of cold and fast on the level of free Met-enk, we have quantified the circulating peptide in the plasma of the jerboa. In the present study, to induce hibernation we have used animals preacclimated in the cold room without food. Our results suggest that cold alone induces an increase in the peptides expression; in contrast, fasting alone causes a decrease of the level

of Met-enk concentration, and the association of cold and fasting causes an increase of Met-enkephalin level and induces hiberna- tion. All these results suggest that the opioidergic system could be implicated in the induction of hibernation. Indeed, we have ob- served that the level of the peptide (Met-enk) was increased in the induced hibernation state (154% in comparison with the active jerboas). Furthermore, the administration of naloxone (antagonist of opioids) to the jerboa during hibernation induces an untimely awake period of the animal (unpublished observation). The same results were observed by others [22]. It was also reported that the injection of an opioid agonist into the brain of the ground squirrels (Spermophilus tridecemlineatus) induces premature hiberna- tion [25].

In summary, our results indicate that opioid receptors ( d , m , and k types) and endogenous peptides (Met-enk, b -endorphin, and dynorphin) from the jerboa brain may play an important role during hibernation. The present evidence is still not sufficient to prove that only the opioidergic system could be implicated in this adaptative phenomenon, but the present results support our work- ing hypothesis. Future studies, aimed at the investigation of mRNA level of peptides and opioid receptors in the active and hibernating animal, could clarify this problem and could provide also the answer to the question as to whether peptide expression increase during hibernation in the jerboa is controlled at the transcriptional level. These studies are at present in progress in our laboratory.

ACKNOWLEDGEMENTS

This work was supported by INSERM-FRANCE (U 297) and CNCPRST-MAROC. The authors thank Ms. W. Berrada for her help in the preparation of Fig. 1.

REFERENCES

1. Adler, M. W.; Geller, E. B. Physiological functions of opioids: Tem- perature regulation. In: Herz, A. H.; Simon, E. J., eds. Handbook of experimental pharmacology, vol. 104/II, Opioids II. New York: Per- gamon Press; 1992:205–238.

2. Aloia, R. C.; Angle, M. L.; Orr, G. R.; Raison, J. K. A critical role for membranes in hibernation. In: Heller, H. C.; Musacchia, K. J.; Wang, L. C. H., eds. Living in the cold. New York: Elsevier; 1986:19 –26.

3. Andjus, R. K.; Elhilali, M.; Veillat, J. P.; Baddouri, K. H. Tolerance of one species of jerboa (Jaculus orientalis) to prolonged exposure to deep hypothermia. J. Physiol. (Paris) 68:531–542; 1974.

4. Aulagnier, S.; Thevenot, M. Les mammife`res sauvages du Maroc.

Trav. Inst. Sci. Se´rie Zool. 41:61–91; 1986.

5. Baddouri, K. H.; Butlen, D.; Imbert–Teboul, M.; Lebouffant, F.;

Marchetti, J.; Chabardes, D.; Morel, F. Plasma antidiuretic hormone levels and kidney responsiveness to vassopressin in the jerboa, Jaculus orientalis. Gen. Comp. Endocrinol. 54:203–215; 1984.

6. Beckman, A. L. Functional aspects of brain opioid peptides system in hibernation. In: Heller, A. C.; Musacchia, X. J.; Wang, L. C. H., eds.

Living in the cold. New York: Elsevier; 1986:225–234.

7. Beckman, A. L.; Llados–Eckman, C.; Stanton, T. L. Physical depen- dence on morphine fails to develop during the hibernation state.

Science 212:1527–1529; 1981.

8. Belluzi, J. D.; Grant, N.; Garsy, V.; Sarontakes, D.; Wise, C. D.; Stein, L. Analgesia induced in vitro by central administration of enkephalin in rat. Nature 260:625– 626; 1976.

9. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248 –254; 1976.

10. Castanas, E.; Bourhim, N.; Giraud, P.; Boudouresque, F.; Cantau, P.;

Oliver, C. Interaction of opiates with opioid binding sites in the bovine adrenal medulla. I. Interaction with d and m sites. J. Neurochem.

47:677– 687; 1985.

11. Castanas, E.; Bourhim, N.; Giraud, P.; Boudouresque, F.; Cantau, P.;

FIG. 4. Effect of cold, hibernation, and fasting on the plasma level of

Methionine-enkephalin. All values are means 6 SEM.

Oliver, C. Interaction of opiates with opioid binding sites in the bovine adrenal medulla. II. Interaction with k sites. J. Neurochem. 47:688 – 699; 1985.

12. Chang, K.-J. Opioid receptors: Multiplicity and sequelae of ligand receptor interactions. In: Conn, M., ed. The receptors, vol. 1. New York: Academic; 1984:1– 81.

13. Cupo, A.; Jarry, T. Detection of methionine enkephalin at 10

mole level. J. Neuroimmunol. 8:57– 67; 1985.

14. Elhilali, M.; Veillat, J. P. Jaculus orientalis: A true hibernator. Mam- malia 39:401– 404; 1975.

15. Gioannani, T.; Howard, A. D.; Hiller, J. M.; Simon, E. J. Purification of an active opioid-binding protein from bovine striatum. J. Biol.

Chem. 260:15117–15121; 1985.

16. Giraud, P.; Castanas, E.; Patey, G.; Oliver, C.; Rossier, J. Regional distribution of methionine enkephalin-Arg

-Phe

in the rat brain: Com- parative study with the distribution of other opioid peptides. J. Neu- rochem. 41:154 –160; 1983.

17. Glowinski, J.; Iversen, L. Regional studies of catecholamines in the rat brain. I. Disposition of [

H] norepinephrine, [

H] dopamine and [

H]

DOPA in various regions of the brain. J. Neurochem. 13:655– 669;

1966.

18. Hunter, W. M.; Greenwood, F. C. Preparation of

I labelled growth hormone of high specific activity. Nature 194:495– 496; 1962.

19. Illes, P. Modulation of transmitter and hormone release by multiple neuronal opioid receptors. Rev. Physiol. Biochem. Pharmacol. 112:

141–233; 1989.

20. Iwamoto, E. T.; Martin, W. R. Multiple opioid receptors. Med. Res.

Rev. 1:411– 440; 1981.

21. Lissitzky, J. C.; Giraud, P.; Conte de Volx, B.; Gillioz, P.; Boudour- esque, F.; Eskay, R. L.; Oliver, C. b -Endorphin is present in high concentration in the hypophyseal portal vessels of rat. Neurosci. Lett.

19:191–195; 1980.

22. Margules, D. L.; Goldman, B.; Finck, A. Hibernation: An opioid- dependent state? Brain Res. Bull. 4:721–724; 1979.

23. Munson, P. J.; Rodbard, D. Ligand: A versatile computerized approach

to characterization of ligand binding system. Anal. Biochem. 107:

220 –239; 1980.

24. Munson, P. J.; Rodbard, D.; Klotz, I. M. Number of receptor sites from Scatchard and Klotz graph: A constructive critic. Science 220:279 – 281; 1983.

25. Oeltgen, P. R.; Nilekani, S. P.; Nuchols, P. A.; Spurrier, W. A.; Su, T. P. Opioid and hibernation: Further studies on d opioid receptors ligands selectivity induced hibernation in summer-active ground squir- rels. Life Sci. 43:1565–1574; 1988.

26. Oeltgen, P. R.; Walsh, J. N.; Hamann, S. R.; Randal, D. C.; Spurrier, W. A.; Myers, R. D. Hibernation ‘‘trigger’’ opioid-like inhibitory action on brain of the monkey. Pharmacol. Biochem. Behav. 17:1271–

1274; 1982.

27. Pert, C. B.; Snyder, S. H. Opiate receptor: Its demonstration in nervous system tissue. Science 179:1011–1014; 1973.

28. Scatchard, G. The attraction of proteins for small molecules and ions.

Ann. NY Acad. Sci. 51:660 – 672; 1949.

29. Simonds, W. F. The molecular basis of opioid receptor function.

Endocr. Rev. 9:200 –212; 1988.

30. Van Giersbergen, P. L. M.; De Lang, H.; De Jong, W. Effects of dynorphin A(1–13) and fragments of b-endorphin in blood pressure and heart rate of characterized rats. Can. J. Physiol. Pharmacol. 69:

327–333; 1991.

31. Wang, L. C. H. Is endogenous opioid involved in hibernation? In:

Cynthia, C.; Florant, G. L.; Wunder, B. A.; Horrwitz, B., eds. Life in the cold: Ecological, physiological and molecular mechanisms? Boul- der, CO: Westview Press; 1993:297–304.

32. Wang, L. C. H.; Lee, T. F.; Jourdan, M. L. Seasonal differences in thermoregulatory responces to opiates in a mammalian hibernator.

Pharmacol. Biochem. Behav. 26:565–571; 1987.

33. Wilkinson, M.; Buchanan, G.; Jacobson, W.; Youglais, E. V. Brain opioid receptors in the hibernating bats, Myotis lucifugus: Modifica- tion by low temperature and comparison with rat, mouse, hamster.

Pharmacol. Biochem. Behav. 25:527–532; 1986.