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The dynamic relationship between mu and kappa opioid receptors

in body temperature regulation

Xiaohong Chen *, Daniel B. McClatchy, Ellen B. Geller, Ronald J. Tallarida, Martin W. Adler

Center for Substance Abuse Research, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, PA 19140, USA

Received 20 December 2004; accepted 8 April 2005

Abstract

Previous studies demonstrated that intracerebroventricular (icv) injection of a kappa opioid receptor agonist decreased, and a mu agonist

increased, body temperature (Tb) in rats. A doseresponse study with the selective kappa antagonist nor-binaltorphimine (nor-BNI) showed that a

low dose (1.25 nmol, icv) alone had no effect, although a high dose (25 nmol, icv) increased Tb. It was hypothesized that the hyperthermia induced

by nor-BNI was the result of the antagonist blocking the kappa opioid receptor and releasing its inhibition of mu opioid receptor activity. To

determine whether the Tb increase caused by nor-BNI was a mu receptor-mediated effect, we administered the selective mu antagonist CTAP (1.25

nmol, icv) 15 min after nor-BNI (25 nmol, icv) and measured rectal Tb in unrestrained rats. CTAP significantly antagonized the Tb increase

induced by icv injection of nor-BNI. Injection of 5 or 10 nmol of CTAP alone significantly decreased the Tb, and 1.25 nmol of nor-BNI blocked

that effect, indicating that the CTAP-induced hypothermia was kappa-mediated. The findings strongly suggest that mu antagonists, in blocking the

basal hyperthermia mediated by mu receptors, can unmask the endogenous kappa receptor-mediated hypothermia, and that there is a tonic balance

between mu and kappa opioid receptors that serves as a homeostatic mechanism for maintaining Tb.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Mu and kappa opioid receptors; Body temperature; CTAP; nor-BNI; Rat

Introduction

Among its many functions, the opioid system plays an

important role in regulating Tb (Adler et al., 1983; Baker and

Meert, 2002; Geller et al., 1986; Wilson and Howard, 1996).

Mu opioid agonists, such as morphine, DAMGO or PL017,

given icv or directly into the preoptic anterior hypothalamus in

rats, produce hyperthermia which is blocked by icv injection of

the mu-selective antagonist CTAP orh-FNA (Appelbaum and

Holtzman, 1986; Bradley et al., 1991; Handler et al., 1992,1994; Spencer et al., 1988). Icv injection of the kappa opioid

receptor agonists dynorphin A117, U50,488H, U69,593 or

spiradoline induces hypothermia in rats (Adler and Geller,

1993; Adler et al., 1983; Cavicchini et al., 1988, 1989; Spencer

et al., 1988) and the kappa-selective antagonist nor-BNI can

block the hypothermic effect (Adler and Geller, 1993; Adler et

al., 1983; Cavicchini et al., 1988; Cavicchini et al., 1989;

Handler et al., 1992, 1994; Spencer et al., 1988). The

successful cloning of the mu (Chen et al., 1993), delta (Evans

et al., 1992; Kieffer et al., 1992) and kappa (Li et al., 1993;

Yakimova et al., 1998; Yasuda et al., 1993) opioid receptors

have allowed the use of antisense oligodeoxynucleotide to

explore functions of these opioid receptor systems. Antisense

studies further confirmed that the kappa opioid receptor

mediates hypothermia and the mu opioid receptor mediates

hyperthermia in rats (Chen et al., 1995, 1996b). The role of the

delta opioid receptor in thermoregulation is less clear and the

effect seems to depend on the delta1/delta2 selectivity of theligand used (Benamar et al., 2004; Broccardo and Improta,

1992; Handler et al., 1992; Salmi et al., 2003; Spencer et al.,

1988; Tepperman and Hirst, 1983).

In conducting a doseresponse study with the selective kappa

opioid receptor antagonist nor-BNI (Portoghese et al., 1987;Tor-

tella et al., 1989), we found that a low dose (1.25 nmol, icv) of

nor-BNI alone has no effect on Tb and that a high dose (25 nmol,

icv) can increase Tb. Yakimova et al. (1998) investigated the

effect of mu and kappa agonists on spontaneous activity and

temperature response characteristics of POAH neurons of rats in

a brain slice preparation. The results showed that most of the

0024-3205/$ - see front matterD

2005 Elsevier Inc. All rights reserved.doi:10.1016/j.lfs.2005.04.084

* Corresponding author. Tel.: +1 215 707 5305; fax: +1 215 707 1904.

E-mail address: chenyang@temple.edu (X. Chen).

Life Sciences 78 (2005) 329 333

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neurons responding to the kappa opioid agonists are sensitive

to mu opioid receptor activation and that no co-localization

was observed between kappa and delta opioid receptors. These

results from morphological data lend support to our hypothesis

of a functional balance between mu and kappa opioid re-

ceptors. We propose that the reason for the Tb increase

induced by icv injection of the high dose of nor-BNI is that itantagonizes the endogenous kappa opioid receptor and releases

its inhibition of mu opioid receptor activity. To determine

whether the Tb increase caused by nor-BNI was a mu-opioid-

receptor-mediated effect, the present studies examined whether

the selective mu opioid receptor antagonist CTAP could block

the nor-BNI-induced Tb changes. We also investigated whether

CTAP, itself, can have effects on Tb, and whether any Tbchange caused by CTAP was kappa-opioid-receptor-mediated

by determining if the selective kappa opioid receptor anta-

gonist nor-BNI could block the CTAP-induced Tb changes.

Materials and methods

Animals

Male Sprague Dawley rats, weighing 150 175 g, were

housed in groups of 67 for at least 1 week in an animal room

maintained at 22T2 -C and approximately 50% relative

humidity. Lighting was on a 12/12 h light/dark cycle (lights

on at 7:00 and off at 19:00). Cannulae were implanted into the

lateral ventricle according to standard procedures in our

laboratory (Adams et al., 1993). Rats were anesthetized with a

mixture of ketamine hydrochloride (100 150 mg/kg) and

acepromazine maleate (0.2 mg/kg). A cannula made of PE-10

tubing (outer diameter 0.61 mm) was implanted into the right

lateral ventricle using the following stereotaxic coordinates: A

5.4, LR 1.5, H 3.5, according to Pellegrino and Cushman (1967),

system A. The animals were housed individually after surgery.

Experiments began 1 week postoperatively. Each rat was used

only once. At the end of the experiment, sites of injection were

verified using microinjection of bromobenzene blue.

Tb measurement

The rats were placed into individual plastic cages in an

environmental room kept at 21 T0.3 -C and 52T2% relative

humidity. After a 1-h acclimation period, a thermistor probe(YSI series 400, Yellow Springs Instrument Co., Inc.,

Yellow Springs, OH) was lubricated and inserted approxi-

mately 7 cm into the rectum; Tb measurements were read

from a digital thermometer (Model 49 TA, YSI). During the

readings, the tail of the rat was held gently between 2

fingers and the animal was otherwise free to move about.

The first three readings were taken at 30-min intervals. To

allow for adaptation to the procedure, the first reading was

discarded and the subsequent two averaged to establish a

baseline. In this way, each animal served as its own control.

Experimental values were then compared to the pre-drug

baseline values obtained for each animal at 15, 30, 45, 60,

90, 120 and 180 min.

Drugs

Drugs were dissolved in 0.9% saline. The mu opioid

antagonist CTAP [H-D-Phe-Cyc-Tyr-D-Try-Arg-Thr-Pen-Thr-

NH2(cyclic)] and the kappa opioid agonist dynorphin (1

17) were produced by Multiple Peptide Systems, San Diego,

CA, for NIDA. The kappa opioid antagonist norbinaltorphi-mine dihydrochloride (nor-BNI) was obtained from Research

Biochemicals Inc., Natick, MA.

Injections

Unrestrained rats received an icv injection of 5 Al followed

by a 3-Al saline flush which was completed in 30 s. The control

group received 8 Al of saline.

Statistical analysis

The data are expressed as the mean and standard error.Statistical analysis of difference between groups was assessed

with a two-way analysis of variance (ANOVA) followed by

Duncans test. p 0.05, ANOVA followed by

Duncans test), but higher doses (>6.25 nmol) caused a

significant increase in the basal Tb over a period of 60 min as

compared to the saline group (p

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temperature increase at 30 min for nor-BNI. Nonlinear curve-

fitting (Tallarida, 2000) produced the smooth curve shown.

CTAP at doses 0.1 nmol and 1 nmol, produced no

significant change (p >0.05) in the basal Tb over a period of

180 min as compared with the corresponding saline group (Fig.

2). At doses of 5 nmol and 10 nmol, CTAP produced a

significant decrease (p < 0.05) in the basal Tb in the first 60 min

as compared with the corresponding saline group (Fig. 2). Fig.

2 inset shows the CTAP dose-response curve for temperature

decrease at 30 min.

Antagonistic effect of nor-BNI on Tb increase induced by

dynorphin

Rats were divided into 3 groups of 59 each and given an

icv injection of saline+saline, saline+dynorphin (4.65 nmol)

or nor-BNI (1.25 nmol)+ dynorphin (4.65 nmol). Nor-BNI was

given 30 min prior to the injection of dynorphin. The results

shown in Fig. 3 indicate that icv injection of dynorphin (4.65

nmol) induced hypothermia, and the kappa antagonist (1.25

nmol) blocked this hypothermia (p < 0.01, compared to thecorresponding control group).

Antagonistic effect of CTAP on Tb increase induced by nor-BNI

Rats were divided into 4 groups of 67 each and given an

icv injection of saline + saline, saline+ CTAP (1 nmol), nor-BNI

(25 nmol)+saline or nor-BNI (25 nmol)+CTAP (1.0 nmol),

respectively. CTAP was administered 15 min after the injection

of nor-BNI. The results shown in Fig. 4 indicate that icv

injection of a high dose (25 nmol) of nor-BNI can induce an

increase in Tb (p < 0.01, compared to the saline group) and

CTAP (1.0 nmol) can antagonize the effect.

The same schedule of administration was performed withnor-BNI 1.25 nmol. The results in Fig. 5 showed that the low

dose (1.25 nmol) of nor-BNI did not produce an increase in Tb.

Antagonistic effect of nor-BNI on Tb increase induced by CTAP

Rats were divided into 4 groups of 410 each and given

an icv injection of saline+ saline, nor-BNI (1.25 nmol)+ sa-

-1.5

-1

-0.5

0

0.5

1

1.5

-30 0 30 60 90 120 150 180

Saline + Saline

Saline + Dynorphin (4.65 nmol)

nor-BNI (1.25 nmol) + Dynorphin (4.65 nmol)

2Tb(C)

Time (min)

Fig. 3. Effect of icv injection of nor-BNI on Tb change induced by icv injection

of dynorphin A117. p

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line, saline+nor-BNI (1.25 nmol) or nor-BNI (1.25 nmol)+C-

TAP (10 nmol). Nor-BNI was given 30 min before CTAP.

The results shown in Fig. 6 indicate that icv injection of a

high dose (10 nmol) of CTAP can decrease Tb (p

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released, resulting in the effects demonstrated with high doses

of these selective antagonists. These results strongly support

our hypothesis that there is a tonic balance between mu and

kappa opioid receptors that serves as the homeostatic mecha-

nism for maintaining Tb.

Acknowledgments

This work was supported by grant DA 13429, DA 00376

(MWA) and DA 09793 (RJT) from NIDA.

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