Chen Etal 05 Mu Vs Kappa Receptors Body Temp

Life Sciences 78 (2005) 329 – 333 www.elsevier.com/locate/lifescie

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 (T b) in rats. A dose – response 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 T b. 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 T b 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 T b in unrestrained rats. CTAP significantly antagonized the T b increase induced by icv injection of nor-BNI. Injection of 5 or 10 nmol of CTAP alone significantly decreased the T b, 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 T b. 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 T b (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 or h-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 A1 – 17, 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 * Corresponding author. Tel.: +1 215 707 5305; fax: +1 215 707 1904. E-mail address: [email protected] (X. Chen). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.04.084

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 the ligand 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 dose–response study with the selective kappa opioid receptor antagonist nor-BNI (Portoghese et al., 1987;Tortella et al., 1989), we found that a low dose (1.25 nmol, icv) of nor-BNI alone has no effect on T b and that a high dose (25 nmol, icv) can increase T b. 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

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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 receptors. We propose that the reason for the T b increase induced by icv injection of the high dose of nor-BNI is that it antagonizes the endogenous kappa opioid receptor and releases its inhibition of mu opioid receptor activity. To determine whether the T b increase caused by nor-BNI was a mu-opioidreceptor-mediated effect, the present studies examined whether the selective mu opioid receptor antagonist CTAP could block the nor-BNI-induced T b changes. We also investigated whether CTAP, itself, can have effects on T b, and whether any T b change caused by CTAP was kappa-opioid-receptor-mediated by determining if the selective kappa opioid receptor antagonist nor-BNI could block the CTAP-induced T b changes.

Drugs

Materials and methods

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 Duncan’s test. p < 0.05 was taken as a significant level of difference.

Male Sprague–Dawley rats, weighing 150–175 g, were housed in groups of 6–7 for at least 1 week in an animal room maintained at 22 T 2 -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.

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

Results Effect of icv injection of nor-BNI and CTAP on the basal T b Rats were divided into 6 groups, each receiving icv injections of saline or nor-BNI (1.25, 3.125, 6.25, 12.5, or 25 nmol) and saline. Lower doses (1.25 and 3.125 nmol) produced no significant effect on the basal T b over a period of 180 min as compared to the saline group ( p > 0.05, ANOVA followed by Duncan’s test), but higher doses (> 6.25 nmol) caused a significant increase in the basal T b over a period of 60 min as compared to the saline group ( p < 0.01). The results are shown in Fig. 1. Fig. 1 inset shows the dose–response curve for Temperature increase

Animals

Drugs were dissolved in 0.9% saline. The mu opioid antagonist CTAP [H-D-Phe-Cyc-Tyr-D-Try-Arg-Thr-Pen-ThrNH2(cyclic)] and the kappa opioid agonist dynorphin (1– 17) were produced by Multiple Peptide Systems, San Diego, CA, for NIDA. The kappa opioid antagonist norbinaltorphimine dihydrochloride (nor-BNI) was obtained from Research Biochemicals Inc., Natick, MA.

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The rats were placed into individual plastic cages in an environmental room kept at 21 T 0.3 -C and 52 T 2% 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 approximately 7 cm into the rectum; T b 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.

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Time (min) Fig. 1. Effects of icv injection of nor-BNI on T b. p <0.01 for nor-BNI (6.25 – 25 nmol) group vs saline group. N = 6 – 7 per group. Each point represents the mean + SE. Inset: dose – response curve for temperature increase (-C) at 30 min for nor-BNI.

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Fig. 2. Effects of icv injection of CTAP on T b. p < 0.01 for saline group vs CTAP 5 and 10 nmol group. N = 3 – 5 per group. Each point represents the mean + SE. Inset: Dose – response line for temperature decrease (-C) at 30 min for CTAP.

temperature increase at 30 min for nor-BNI. Nonlinear curvefitting (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 T b increase induced by dynorphin Rats were divided into 3 groups of 5– 9 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 1.5

Fig. 4. Effect of icv injection of CTAP on T b change induced by icv injection of nor-BNI 25 nmol. p < 0.01 for nor-BNI + saline group vs. nor-BNI + CTAP group. N = 6 – 7 per group. Each point represents the mean + SE.

nmol) induced hypothermia, and the kappa antagonist (1.25 nmol) blocked this hypothermia ( p < 0.01, compared to the corresponding control group). Antagonistic effect of CTAP on T b increase induced by nor-BNI Rats were divided into 4 groups of 6 – 7 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 T b ( p < 0.01, compared to the saline group) and CTAP (1.0 nmol) can antagonize the effect. The same schedule of administration was performed with nor-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 T b. Antagonistic effect of nor-BNI on T b increase induced by CTAP Rats were divided into 4 groups of 4 –10 each and given an icv injection of saline + saline, nor-BNI (1.25 nmol) + sa-

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Time (min) Fig. 3. Effect of icv injection of nor-BNI on T b change induced by icv injection of dynorphin A1 – 17. p < 0.01 for saline + saline group vs. saline + dynorphin group. N = 5 – 9 per group. Each point represents the mean + SE.

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Time (min) Fig. 5. Effect of icv injection of CTAP on T b change induced by icv injection of nor-BNI 1.25 nmol. No significant differences. N = 5 – 6 per group. Each point represents the mean + SE.

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line, saline + nor-BNI (1.25 nmol) or nor-BNI (1.25 nmol) + CTAP (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 T b ( p < 0.01, compared to the saline group) and that nor-BNI (1.25 nmol) can block this effect. Discussion The findings presented in this study indicate that (1) high doses of the kappa opioid receptor antagonist nor-BNI can induce hyperthermia that can be blocked by the mu opioid receptor antagonist CTAP; and (2) high doses of CTAP can induce hypothermia that can be blocked by the kappa opioid receptor antagonist nor-BNI. A plausible mechanism for these findings is that nor-BNI blockade of kappa opioid receptors allows endogenous mu opioid receptor activity to be unopposed, resulting in hyperthermia, and that CTAP blockade of mu opioid receptors allows endogenous kappa opioid receptor activity to be unopposed, resulting in hypothermia. The data shown in Fig. 1 indicate that blockade of kappa opioid receptors with nor-BNI results in a significant temperature increase (at 30 min). Similarly, mu receptor blockade with CTAP produced a reduction in body temperature (Fig. 2). Previous studies showed that icv injection of mu opioid receptor agonists (i.e. PL017, morphine, DAMGO) in rats produces hyperthermia which is blocked by icv injection of the mu opioid receptor antagonists CTAP or beta-FNA (Adler and Geller, 1993; Appelbaum and Holtzman, 1986; Bradley et al., 1991; Handler et al., 1992, 1994; Spencer et al., 1988), and that icv injection of the kappa opioid receptor agonists, dynorphin, U50,488H, U69,593, dynorphin or spiradoline, induces hypothermia in rats which can be blocked by the kappa opioid receptor antagonist norBNI (Adler and Geller, 1993; Adler et al., 1983; Cavicchini et al., 1988, 1989; Handler et al., 1992, 1994; Spencer et al., 1988). These results demonstrated that hyperthermia induced by opioid agonists is mediated by the mu opioid receptor and

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Time (min) Fig. 6. Effect of icv injection of nor-BNI on T b change induced by icv injection of CTAP. p < 0.01 for saline + saline group vs. nor-BNI + CTAP group. N = 4 – 10 per group. Each point represents the mean + SE.

hypothermia induced by opioid agonists is mediated by kappa opioid receptors in the brain. This hypothesis was also confirmed by the results of studies in which selective agonists were administered into the preoptic anterior hypothalamus of rat brain (Xin et al., 1997). Lower doses (1.25 and 3.125 nmol) of nor-BNI alone and lower doses (0.1 and 1.0 nmol) of CTAP alone each had no effect on T b, while higher doses (6.25 –25 nmol, icv) of nor-BNI or higher doses (5.0 and 10.0 nmol, icv) of CTAP produced hyperthermia or hypothermia respectively. We can offer two possible explanations. One is that although nor-BNI is very selective for kappa receptors, high doses of nor-BNI can act non-selectively as an agonist on mu opioid receptors, and that although CTAP is very selective for mu receptors, high doses of CTAP can act non-selectively as an agonist on kappa opioid receptors as well. However, if nor-BNI or CTAP can produce hyperthermia or hypothermia, respectively, directly by activating mu or kappa opioid receptors, respectively, then nor-BNI or CTAP at the same dose should produce analgesia, but they do not (Chen et al., 1996a). A more likely explanation is that nor-BNI remains selective for kappa opioid receptors and, by blocking them, allows endogenous mu opioid receptor activity to be seen, indicating the existence of a tonic balance between mu and kappa opioid receptors. CTAP has high affinity and selectivity for the mu opioid receptor (Kramer et al., 1989). It can block the hyperthermia induced by icv injection of the mu opioid receptor agonist PL017 (Handler et al., 1994). Dynorphin has been shown to be a selective kappa opioid agonist (Goldstein et al., 1979) and nor-BNI is a selective kappa antagonist (Portoghese et al., 1987). In a previous study, we found that a high dose (25 nmol) of dynorphin produced hypothermia and that nor-BNI blocked this effect (Handler et al., 1992, 1994). For the present work, we injected a low dose (4.65 nmol) of dynorphin that decreased T b about 1 -C (shown in Fig. 3). Icv injection of 1.25 nmol norBNI blocked this hypothermia, confirming that the effect induced by low doses of dynorphin is mediated by kappa opioid receptors. As shown in Fig. 5, CTAP (1.0 nmol, icv) itself had no effect on T b and neither did it affect T b after a low dose of nor-BNI (1.25 nmol, icv), also suggesting that there is no interaction on T b between CTAP (1.0 nmol) and nor-BNI (1.25 nmol). However, the results shown in Fig. 4 indicate that CTAP (1.0 nmol, icv) can significantly antagonize the T b increase induced by icv injection of 25 nmol of nor-BNI during the first 45 min of measurement (p < 0.05), which suggests that the mu opioid receptor is involved in the nor-BNI-induced increase of T b. Additional evidence supporting the existence of a tonic balance is that nor-BNI (1.25 nmol, icv) can significantly block the T b increase induced by icv injection of 10 nmol of CTAP (p < 0.05, Fig. 6), which indicates that the kappa opioid receptor is involved in the CTAP-induced decrease of T b. We thus propose that the nor-BNI blockade of kappa opioid receptors allows endogenous mu opioid receptor activity to be released and that the CTAP blockade of mu opioid receptors allows endogenous kappa opioid receptor activity to be

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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 mechanism for maintaining T b. Acknowledgments This work was supported by grant DA 13429, DA 00376 (MWA) and DA 09793 (RJT) from NIDA. References Adams, J.U., Tallarida, R.J., Geller, E.B., Adler, M.W., 1993. Isobolographic superadditivity between delta and mu opioid agonists in the rat depends on the ratio of compounds, the mu agonist and the analgesic assay used. The Journal of Pharmacology and Experimental Therapeutics 266, 1261 – 1267. Adler, M.W., Geller, E.B., 1993. Physiological functions of opioids: temperature regulation. In: Herz, A., Akil, H., Simon, E.J., (Eds.), Handbook of Experimental Pharmacology, vol. 104/II. Springer-Verlag, Berlin, pp. 205 – 238R Opioids II. Adler, M.W., Hawk, C., Geller, E.B., 1983. Comparison of intraventricular morphine and opioid peptides on body temperature of rats. In: Lomax, P., Scho¨nbaum, E. (Eds.), Environment, Drugs and Thermoregulation. Karger, Basel, pp. 90 – 93. Appelbaum, B.D., Holtzman, S.G., 1986. Stress-induced changes in the analgesic and thermic effects of opioid peptides in the rat. Brain Research 377, 330 – 336. Baker, A.K., Meert, T.F., 2002. Functional effects of systemically administered agonists and antagonists of mu, delta, and kappa opioid receptor subtypes on body temperature in mice. The Journal of Pharmacology and Experimental Therapeutics 302, 1253 – 1264. Benamar, K., Rawls, S.M., Geller, E.B., Adler, M.W., 2004. Intrahypothalamic injection of deltorphin-II alters body temperature in rats. Brain Research 1019, 22 – 27. Bradley, E.A., Geller, E.B., Piliero, T., Adler, M.W., 1991. Actions of Aselective opioid agonists and antagonists on body temperature in the rat. The FASEB Journal 5, A861. Broccardo, M., Improta, G., 1992. Hypothermic effect of D-Aladeltorphin II, a selective fl opioid receptor agonist. Neuroscience Letter 139, 209 – 212. Cavicchini, E., Candeletti, S., Ferri, S., 1988. Effects of dynorphins on body temperature of rats. Pharmacological Research Communications 20, 603 – 604. Cavicchini, E., Candeletti, S., Spampinato, S., Ferris, S., 1989. Hypothermia elicited by some prodynorphin-derived peptides: opioid and non-opioid actions. Neuropeptides 14, 45 – 50. Chen, Y., Mestek, A., Liu, J., Hurley, J.A., Yu, L., 1993. Molecular cloning and functional expression of a A-opioid receptor from rat brain. Molecular Pharmacology 44, 8 – 12. Chen, X.H., Adams, J.U., Geller, E.B., DeRiel, J.K., Adler, M.W., Liu-Chen, L.-Y., 1995. An antisense oligodeoxynucleotide to A-opioid receptors inhibits A-agonist-induced analgesia in rats. European Journal of Pharmacology 275, 105 – 108. Chen, X.-H., Geller, E.B., Adler, M.W., 1996a. Electrical stimulation at traditional acupuncture sites in periphery produces brain opioid-receptormediated antinociception in rats. The Journal of Pharmacology and Experimental Therapeutics 277, 654 – 660. Chen, X.-H., Geller, E.B., DeRiel, J.K., Liu-Chen, L.-Y., Adler, M.W., 1996b. Antisense confirmation of A- and n-opioid receptor mediation of morphine’s effects on body temperature in rats. Drug and Alcohol Dependence 43, 119 – 124.

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