Intrathecally injected nicotine enhances the antinociception induced by morphine but not β-endorphin, d -Pen2,5-enkephalin and U50,488H administered intrathecally in the mouse

Neuropeptides(1996)30 (4), 373-378 © PearsonProfessionalLtd 1996

Intrathecally injected nicotine enhances the antinociception induced by morphine but not -endorphin, D-Pen2,5-enkephalin and U50,488H administered intrathecally in the mouse H.- W. Suh, 1 D.- K. Song, 1 K.- J. Lee, 2 S.- R. Choi, 1 Y.- H. Kim ~ ~Departmentof Pharmacology,Instituteof NaturalMedicine 2Departmentof Pediatrics,Collegeof Medicine,HallymUniversity,Chunchon,Kangwon-Do,S. Korea

Summary The effect of nicotine injected intrathecally (i.t.) on the inhibition of the tail-flick response induced by morphine, ~-endorphin, g-Pen2,S-enkephalin (DPDPE), or {(trans-3,4-dichloro-N-methyI-N- [2-(1-pyrrolidinyl)cyclohexyl] benzeocetamide)} (U50,488H) administered i.t. was studied in ICR mice. The i.t. injection of nicotine alone at doses from 1 to 12 ~g produced only a minimal inhibition of the tail-flick response. Morphine (0.2 ~g), ~-endorphin (0.1 ~g), DPDPE (0.5 ~g) or U50,488H (6 ~g) caused only slight inhibition of the tail-flick response. Nicotine injected i.t. dose dependently enhanced the inhibition of the tail-flick response induced by i.t. administered morphine (0.2 ~g). However, i.t. injected nicotine at the same doses was not effective in enhancing the inhibition of the tail-flick response induced by ~-endorphin, DPDPE, or U50,488H administered i.t. Our results suggest that stimulating nicotinic receptors located in the spinal cord may enhance the antinociception induced by morphine administered spinally. However, the activation of nicotinic receptors at the spinal level may not be involved in modulating the antinociception induced by I}-endorphin, DPDPE, and U50,488H administered spinally.

INTRODUCTION Recent studies have demonstrated that nicotine is involved in the regulation of nociception. Nicotine is known to have both hyperalgesic and antinociceptive effects when it is given acutely or chronically. 1-7 For example, systemic injection of nicotine produces antinociception.8,9 Iwamoto5 has reported that nicotine microinjected into the pedunculopontine tegmental Received 4 November 1995 Accepted 15April 1996 Correspondence to: Hong-Won Suh, Department of Pharmacology, College of Medicine, Hallym University, 10kchun-Dong, Chunchon, Kangwon-Do, 200-702, S. Korea. Tel: (0361) 58-1654; Fax: (0361) 58-1652,

nucleus or the nucleus raphe magnus produces antinociception. In addition, Hamann & Martin6 reported that nicotine microinjected into the region of central gray or caudally into the posterior medulla produces antinociception. However, hyperalgesic effects were observed when nicotine was administered into the region of dorsal posterior mesencephalic tegmentum.~ In contrast to the action of nicotine at supraspinal levels, nicotine injected i.t. does not appear to produce profound antinociception.l°-12 We and others have previously reported that the intrathecal (i.t.) injection of various types of opioids produces a profound antinociception. For example, the antinociception induced by morphine is mediated by

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stimulating specific bt-opioid receptors. 1>1z This hypothesis is based on the findings that [~-funaltrexamine, CTOP (D-Phe-Cys-Tyr-D-Tyr-Orn-Thr-Pen-Thr-NH2), or naloxone effectively antagonizes antinociception induced by morphine2 >~7 In addition, [~-endorphin administered i.t. produces the antinociception via the stimulation of g- or K-opioid receptors in the spinal cord. This contention is supported by the findings that the antinociception induced by [~-endorphin administered i.t. is effectively antagonized by CTOP or nor-binaltorphimine, which selectively antagonizes antinociception induced by g- and ~:-opioid receptors, respectively. However, [~-endorphin (1-27) and natrindole administered i.t., which selectively antagonize antinociception induced by e- and 8-opioid receptors, respectively, do not affect the antinociception induced by [3-endorphin28,19 I)-Pen2,5-enkephalin (DPDPE) produces the antinociception via stimulating 8-opioid receptors, a°-22 Furthermore, several lines of evidence have demonstrated that the antinociception induced by US0,488H is mediated by the stimulation of ~c-opioid receptors. This contention is based on the findings that the antinociception induced by U50,488H was blocked by WIN44,441-3 or nor-binaltorphimine, a ~-opioid receptor antagonist, injected systemically or i.t.23-25 Several lines of evidence indicate that nicotine exerts its pharmacological effects by interacting with endogenous opioid systems. For example, nicotine causes changes in endogenous enkephalin systems of the central nervous system and the release of enkephalin. 2¢27In addition, nicotine increases the opioid gene expression. 28,29 Furthermore, nicotine or cigarette smoking also elevates opioid levels in serum. 3° However, the interaction of nicotine with opioids in the regulation of antinociception at the spinal cord level has not been well characterized. Therefore, the effect of nicotine injected i.t. on the antinociception induced by various types of opioids, such as morphine, [3-endorphin, DPDPE and U50,488H, injected i.t. was examined in the present study.

response, mice were gently held with one hand with the taft positioned in the apparatus (EMDIE Instrument Co., Maidens, VA, Model TF6) for radiant heat stimulation. The tail-flick response was elicited by applying radiant heat to the dorsal surface of the tail. The intensity of heat stimulus in the tail-flick test was adjusted so that the animal flicked its tail within 3-5 s. The tail-flick latency was measured before (r0) and after (T 1) the injection of opioid agonists. The inhibition of the tail-flick or hotplate response was expressed as 'percent maximal possible effect (% MPE)' which was calculated as [(T 1- T o)/(T 2- T 0)] x 100, where the cut-off time (t 2) was set at 10 s for the tail-flick test. Intrathecal injection Intrathecal (i.t.) administration was performed following the method of Hylden & Wilcox32 using a Hamilton syringe with 30 gauge needle. The i.t. injection volume was 5 B1 and the injection site was verified by injecting a similar volume of 1% methylene blue solution and determining the distribution of the injected dye in the spinal cord. Dye injected i.t. was distributed both rostrally and caudally but over a short distance (about 1 cm) and no dye was found in the brain. The success rate for the injections was consistently found to be over 95% before the experiments were undertaken. Experimental protocol Mice were injected i.t. with various doses (from 1 to 12 btg) of nicotine alone, or nicotine in the presence or absence of morphine (0.2 gg), [~-endorphin (0.1 gg), DPDPE (0.5 gg) or U50,488H (6 btg). The tail-flick responses were tested 20, 20, 10 and 10 min after the i.t. injection of morphine, [~-endorphin, DPDPE or U50,488H, respectively. The times used were chosen based on preliminary time course studies; at the times used, there was a maximal inhibition of the tail-flick responses induced by each opioid agonist.

MATERIALS AND METHODS Experimental animals

Statistics

Male ICR mice weighing 23-25 g were used for all the experiments. The animals were housed 5 per cage in a room maintained at 22 + 0.5°C with an alternating 12 h light-dark cycle. Food and water were available ad libitum. Animals were used only once.

The data are presented as mean + SEM. One-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test when more than one dose was administered, was used for statistical evaluation. The median antinociceptive doses (EDso) and 950/0 confidence intervals were calculated according to the method described by Litchfield & Wilcoxon,33 with the aid of a computer program described by Tallarida & Murray. 34 P values of less than 0.05 were considered to indicate statistical significance.

Assessment of antinociception Antinociception was determined by the tail~flick test. 31 For the measurement of the latency of the tail-flick

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01 1 033 066 01212 i.t. Nicotine (lag) Fig. 1 Effect of nicotine injected intrathecally (i.t.) on inhibition of the tail-flick response induced by morphine administered i,t. After measuring the basal tail-flick latency, morphine (0.2 lag) was administered i.t. in the presence or absence of nicotine (from 1 to 12 lag). The tail-flick response was measured 20 rain after i.t. morphine injection. The vertical bars denote the standard error of the mean. The number of animals used for each group was 10. *P < 0.05 compared with group of mice injected with morphine i.t.

Drugs

RESULTS

Morphine hydrochloride was purchased from Sam-Sung Pharm. Co. (Seoul, Korea). ~-Endorphin and D-Pen 2,5enkephalin were purchased from Peninsula Laboratory Inc. (Belmont, CA). U50,488H was purchased from Research Biomedicals Inc. (Natick, MA). Nicotine was purchased from Sigma Chemical Co. (St Louis). Morphine, f3-endorphin, DPDPE, U50,488H and nicotine were dissolved in sterile saline (0.9% NaC1 solution).

Mice were injected i.t. with a fixed dose of morphine (0.2 p-g), ~-endorphin (0.1 p-g), DPDPE (0.5 p-g), or U50,488H (6 p-g) in the presence or absence of various doses (from 1 to 12 p-g) of nicotine. Nicotine administered i.t. alone slightly increased inhibition of the tail-flick response, as shown in Figure 1. The i.t. injection with morphine (0.2 p-g), [3-endorphin (0.1 p.g), DPDPE (0.5 ~tg) or U50,488H (6 p-g) alone caused a sight inhibition of the tail-flick response (Figs 1, 3, 4 and 5). The inhibition of the tailflick response induced by morphine was potentiated by nicotine injected i.t. in a dose-dependent manner (Fig. 1). As shown in Figure 2, morphine, at doses from 0.075 to 0.3 nmol, caused dose-dependent increases in the inhibition of the tail-flick response in mice pretreated with with saline. The i.t. injection of nicotine (6 p-g) enhanced the inhibition of the tail-flick response induced by morphine administered i.t.; the dose-response curve was shifted to the left and EDs0 value for morphine for inhibition of the tail-flick response in mice treated with nicotine was decreased about 5-fold below that of saline-treated controls (Fig. 2). On the other hand, the inhibition of the tail-flick response induced by [~-endorphin, DPDPE, or U50,488H administered i.t. was not changed by the same doses of nicotine administered i.t. (Figs 3-5).

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Fig. 2 Effect of nicotine injected intrathecally (i.t.) on inhibition of the tail-flick response induced by morphine administered i.t. After measuring the basal tail-flick latency, nicotine (6 lag) in the presence or absence of various doses of morphine was administered i.c.v. The tail-flick response was measured 20 min after i.c.v, morphine injection. The vertical bars denote the standard error of the mean. The number of animals used for each group was 10.

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Although several previous reports have demonstrated that i.t. nicotine does not cause a profound inhibition of the tail-flick response, the interaction between nicotine and opioids for producing antinociception at the spinal cord level has not been well characterized. We found, in Neuropeptides (1996) 30(4), 373-378

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Fig. 3 Effect of nicotine injected intrathecally (i.t.) on inhibition of the tail-flick response induced by J~-endorphin administered i.t. After measuring the basal tail-flick latency, 13-endorphin (0.1 pg) was administered i.t. in the presence or absence of nicotine (from 1 to 12 #g). The tail-flick response was measured 20 min after i.t. ~-endorphin injection. The vertical bars denote the standard error of the mean. The number of animals used for each group was 10.

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Fig. 4 Effect of nicotine injected intrathecally (i.t.) on inhibition of the tail-flick response induced by DPDPE administered i.t. After measuring the basal tail-flick latency, DPDPE was administered i.t. in the presence or absence of nicotine (from 1 to 12 ~g). The tail-flick response was measured 10 min after i.t. DPDPE injection. The vertical bars denote the standard error of the mean. The number of animals used for each group was 10.

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Fig. 5 Effect of nicotine injected intrathecally (i.t.) on inhibition of the tail-flick response induced by U50,488H administered i.t. After measuring the basal tail-flick latency, U50,488H was administered i.t. in the presence or the absence of nicotine (from 1 to 12 I~g). The tailflick response was measured 10 rain after i.t. U50,488H injection. The vertical bars denote the standard error of the mean. The number of animals used for each group was 10.

Neuropeptides (1996) 30(4), 373-378

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Spinal nicotine in opioid antinociception

the present study, that nicotine appears to selectively enhance the antinociception induced by morphine administered i.t. However, i.t. injected nicotine does not seem to modulate aminociception induced by [~-endorphin, DPDPE or U50,488H injected i.t. The results suggest that stimulation of nicitonic receptors by nicotine in the spinal cord is coupled with g-opioid receptors to show the potentiating effect for the production of antinociception. However, stimulation of spinal nicotine receptors appears not to be coupled with either 8- or K-opioid receptors at the spinal cord level for the enhancement of the production of antinociception. We found recently that the same doses of nicotine injected supraspinally also enhance the antinociception induced by morphine administered supraspinally (unpublished observation). Thus, it is likely that nicotine receptors may be functionally coupled with g-opioid receptors to produce synergistic antinociceptive effect at supraspinal sites. We have previously observed that nicotine injected supraspinally enhances the antinociception induced by ]3-endorphin administered supraspinally, suggesting that the activation of nicotine receptors by nicotine at supraspinal sites may be functionally coupled with c-opioid receptors to show the potentiation for the production of antinociception (unpublished observation). However, we found in the present study that nicotine injected i.t. did not affect the aminociception induced by [~-endorphin administered i.t. The exact reason for the differential effects of nicotine on [3-endorphin-induced antinociception administered supraspinally and spinally is not known curremly. However, several studies suggest that this differential action might be due to the activation of ctifferem receptor types by ]3-endorphin at supraspinal and spinal levels. [3-Endorphin administered supraspinally produces the antinociception by stimulating copioid receptors while the aminociception induced by [3-endorphin administered spinally appears to be mediated by g- and ~c-opioid receptors. This suggestion is supported by the findings that supraspinally administered [3-endorphin-induced antinociception is antagonized by an c-opioid receptor antagonist [[3-endorphin (1-27)], but not a g-opioid receptor antagonist (]3-funaltrexamine or CTOP), administered supraspinally. TM The aminociception induced by [3-endorphin injected i.t. is effectively antagonized by CTOP or nor-naltorphimine, but not by naltrindole or ICI174,864.16 Thus, it appears that only a-opioid receptors stimulated by [~-endorphin located at supraspinal sites are selectively coupled with nicotine receptors to show enhancing effect for the production of antinociception. In the present study, we found that nicotinic receptors in the spinal cord are not functionally coupled with 8or K-opioid receptors for enhancing the antinociceptive © Pearson Professional Ltd 1996

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effect. Our results are consistent with the finding that nicotine injected supraspinally does not affect the antinociception induced by DPDPE or U50,488H administered supraspinally (unpublished observation). Several lines of evidence indicate that the antinociceptive effect induced by DPDPE given i.t. is mediated by the stimulation of 8-opioid receptors while that induced by U50,488H given i.t. is mediated by the stimulation of ~opioid receptors? °-2s For example, the antinociceptive effect induced by DPDPE injected i.t. is effectively antagonized by ICI174,864 administered i.t. 22 In addition, the antinociception induced by US0,488H was blocked by WIN 44,441-3 or nor-binaltorphimine (a K-opioid receptor antagonist) injected systemically or i.t. 2s-2s Thus, it appears that at both supraspinal and spinal levels, nicotinic receptors may not be functionally coupled with 8or ~:-opioid receptors for enhancing the antinociceptive effects induced by DPDPE or U50,488H.

ACKNOWLEDGEMENTS

This work was supported by a Research Gram from the Korea Ginseng and Tobacco Research Institute (1995) and KOSEF (961-0704-046-2). REFERENCES

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