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Keywords:

  • dynorphin;
  • kappa-opioid receptor;
  • mice;
  • nicotine;
  • spinal cord;
  • tolerance

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The aim of the present study was to explore the possible role of kappa/dynorphin system in the development of tolerance to nicotine antinociception in mice. First, we observed that kappa-opioid receptor (KOP-r) participates in the acute spinal antinociception produced by nicotine (3 and 5 mg/kg, s.c.) since the pre-treatment with the selective kappa antagonist nor-binaltorphimine (3 mg/kg, i.p.) attenuated this response in the tail-immersion test but not in the hot-plate test nor in locomotor responses. Possible changes in the expression of KOP-r were investigated in tolerant mice to nicotine antinociception by using autoradiography of [3H]CI-977 binding. The density of KOP-r decreased in the spinal cord of tolerant mice. In addition, bi-directional cross-tolerance between nicotine (3 and 5 mg/kg, s.c.) and the selective kappa agonist U50,488H (10 mg/kg, s.c.) was found in the tail-immersion test. Recent evidences indicate that an up-regulation of dynorphin levels in the spinal cord and subsequent activation of NMDA receptors participate in the development of tolerance to opioid and cannabinoid antinociception. In this study, dynorphin content in the lumbar spinal cord was similar in control and nicotine tolerant mice. Furthermore, the administration of the NMDA antagonist MK-801 (0.03 and 0.01 mg/kg, i.p.) before each daily nicotine injection did not modify the development of nicotine tolerance. In summary, these data indicate that KOP-r is directly involved in the development of tolerance to nicotine antinociception by a mechanism independent from dynorphin and NMDA receptors.

Abbreviations used
DOP-r

δ-opioid receptor

KOP-r

κ-opioid receptor

MOP-r

μ-opioid receptor

nAchRs

nicotine acetylcholine receptors

nor-BNI

Nor-binaltorphimine

Nicotine produces a variety of behavioural responses including effects on locomotor activity, cognitive performance and pain perception. These responses are mediated by the activation of pentameric ligand gated-ion channels which are the nicotinic acetylcholine receptors (nAChRs) (Gotti et al. 2006). In the nervous system, these receptors are composed of the combination of different α (α2–α10) and β (β2–β4) protein subunits (Le Novère et al. 2002). Chronic nicotine treatment produces tolerance to most of its pharmacological responses, but the mechanisms underlying these effects are not well known. The development of tolerance is initially associated with nAChRs up-regulation and desensitization (Quick and Lester 2002; Robinson et al. 2007). Tolerance to nicotine seems to involve neuronal adaptation at both drug–receptor interaction level and intracellular processes. Thus, L-type calcium channels and calcium-dependent calmodulin protein kinase II antagonists prevent the development of tolerance to nicotine-induced antinociception (Damaj 2005).

Several studies have explored the participation of the endogenous opioid system in different nicotine pharmacological effects. Accordingly, mu-opioid receptor (MOP-r) and peptides derived from pre-proenkephalin are involved in nicotine rewarding responses and participate in its acute antinociceptive responses and the expression of nicotine physical dependence (Berrendero et al. 2002, 2005). In agreement with these results, bi-directional cross-tolerance between morphine and nicotine antinociceptive effects has been previously demonstrated (Zarrindast et al. 1999; Biala and Weglinska 2006). Recently, Galeote et al. (2006) have shown an increase in the functional activity of MOP-r in the spinal cord of mice tolerant to nicotine-induced antinociception. This effect could be an adaptive mechanism to counteract the establishment of this process since nicotine tolerance was developed faster in MOP-r knockout mice. In addition to MOP-r, KOP-r has been reported to modulate some behavioural effects of nicotine, such as locomotion and place aversion associated to nicotine withdrawal (Hahn et al. 2000; Ise et al. 2002). Nevertheless, the possible role of KOP-r in the development of nicotine antinociceptive tolerance remains unknown.

On the other hand, although different peptide products are derived from preprodynorphin (dynorphin A, dynorphin B, α- and β-neoendorphin, Leu-enkephalin), several studies have reported an involvement of spinal dynorphin A (1–17) fragment in cannabinoid and opioid antinociceptive tolerance (Vanderah et al. 2000; Gardell et al. 2002). An increased expression of spinal dynorphin is pronociceptive and promotes antinociceptive tolerance induced by the chronic administration of these drugs. Although dynorphin was identified as an endogenous KOP-r agonist with lower affinity for DOP-r and MOP-r (Goldstein et al. 1979), provided of antinociceptive effects (Ossipov et al. 1996), this peptide has significant non-opioid activity (Vanderah et al. 2001). Thus, activation of NMDA receptors by dynorphin produces excitatory and nociceptive effects (Laughlin et al. 2001) promoting cannabinoid and opioid tolerance.

The present study was designed to investigate the role of the kappa/dynorphin system in the development of tolerance to nicotine antinociceptive effects. First, the role of KOP-r in acute nicotine antinociception was investigated. Autoradiographic techniques were used to study the potential changes induced by chronic nicotine treatment in the density of these opioid receptors in the spinal cord and brain. Bi-directional cross-tolerance between nicotine and the KOP-r agonist U-50,488H was also assessed, as well as the possible role of dynorphin and NMDA receptors in nicotine tolerance.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Animals

Experiments were performed in C57BL/6 (Charles River, France) male mice weighing 24–28 g at the beginning of the experiments. Mice were housed five per cage in a temperature (21 ± 1°C) and humidity-controlled (55 ± 10%) room with a 12-h light/12-h dark cycle (light between 8:00 am and 8:00 pm). Food and water were available ad libitum. Mice were habituated to their new environment and handled for 1 week after arrival, and before starting the experimental procedure. The observer was blind to treatment in all the experiments. Animal procedures were conducted in accordance with the guidelines of the European Communities Directive 86/609/EEC regulating animal research and approved by the local ethical committee (CEEA-IMAS-UPF).

Drugs

(-)-Nicotine hydrogen tartrate salt [(-)-1-methyl-2(3-pyridyl)pyrrolidine] and (-)U-50,488H [trans-(1S,2S)-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide hydrochloride] (Sigma, Madrid, Spain) were dissolved in physiological saline (0.9%) and administered by subcutaneous route. Nicotine doses used were calculated as nicotine hydrogen tartrate salt. Nor-binaltorphimine dihydrochloride (nor-BNI), and MK-801 (Sigma) were dissolved in physiological saline (0.9%) and administered by intraperitoneal route. All drugs were injected in a volume of 10 mL/kg. The route of administration of the different drugs was chosen based on previous studies using several experimental designs (Berrino et al. 2003; Balerio et al. 2005; Berrendero et al. 2005).

Acute responses induced by nicotine

To evaluate the participation of the KOP-r in locomotor responses and antinociceptive effects induced by acute nicotine administration, mice were pre-treated with the KOP-r antagonist nor-BNI (3 mg/kg, i.p.), 24 h before nicotine (1, 3, and 5 mg/kg, s.c.) or saline injection. The dose of nor-BNI (3 mg/kg) used in this study has been previously reported to block the antinociceptive effects of the kappa agonist U-50,488H (Narita et al. 2003a). Locomotor measurements for each mouse were taken 5 min after nicotine or saline injection by using individual locomotor activity boxes (9 cm × 20 cm × 11 cm; Imetronic, Passac, France). The boxes were provided with two lines of six infrared beams to evaluate both horizontal and vertical activity during 10 min under a dim light (20–25 lux). The antinociceptive responses for each mouse were determined 15 and 16 min after nicotine or saline injection by using the tail-immersion and hot-plate tests respectively, as previously reported (Simonin et al. 1998). In the tail-immersion test, the water temperature was maintained at 48 ± 0.5°C using a thermo-regulated water circulating pump (Clifton, North Somerset, UK). The time to withdraw the tail was determined and a cut-off was set up at 15 s to prevent tissue damage. In the hot-plate test, the heated surface of the plate was kept at a temperature of 50 ± 0.1°C (Columbus Instruments, Columbus, OH, USA). The nociceptive threshold evaluated was the jumping response. In absence of jumps, a 240 s cut-off was used to prevent tissue damage. The data obtained were expressed as absolute values.

Assessment of tolerance

To study the possible role of KOP-r in the development of nicotine tolerance, bi-directional cross-tolerance between nicotine and the KOP-r agonist U-50,488H was investigated. Tolerance development to nicotine or U-50,488H-induced antinociception was performed as previously reported (Narita et al. 2003a; Galeote et al. 2006). Briefly, C57BL/6 mice were injected by subcutaneous route every day at 10.00 am, 14.00 pm and 18.00 pm with nicotine (5 mg/kg, s.c.) or saline during 12 days to achieve nicotine tolerance. To develop antinociceptive tolerance to U-50,488H, mice were treated subcutaneously with U-50,488H (10 mg/kg, s.c.) or saline once daily (10.00 am) for a period of 12 days. To assess tolerance, the antinociceptive responses to nicotine and U-50,488H were measured on days 1, 2, 4, 6, 8, and 10 by using the tail-immersion test, 15 min after the first daily injection (data not shown). Bi-directional cross-tolerance between nicotine and U-50,488H was determined in mice chronically treated with these compounds after an acute administration of nicotine (1.5, 3, and 5 mg/kg, s.c.), U-50,488H (5, 10, and 20 mg/kg, s.c.) or saline (2 h following the morning injection on the 12th day). U-50,488H doses used in the present study have been previously demonstrated to induce clear antinociceptive effects mediated by KOP-r activation (Narita et al. 2003a,b). Both, locomotor activity and nociception were measured in these mice. Locomotor measurements for each mouse were taken 5 min after acute injection, by using individual locomotor activity boxes (9 cm × 20 cm × 11 cm; Imetronic). The boxes were provided with two lines of six infrared beams to evaluate both horizontal and vertical activity during 10 min under a dim light (20–25 lux). The antinociceptive responses for each mouse were determined 15 and 16 min after acute injection by using the tail-immersion and hot-plate tests, respectively (as described above). The data obtained were expressed as percentage of maximum possible effect using the following equation: (MPE %) = (test latency − control latency)/(cut-off time − control latency) × 100.

In a second experiment, the role of NMDA receptors in the development of tolerance to nicotine antinociceptive effects was studied. For this purpose, the NMDA receptor antagonist MK-801 (0.01, 0.03, and 0.1 mg/kg, i.p.) was administered 30 min before each nicotine (5 mg/kg, s.c.) or saline injection. As described above, nicotine-induced antinociception was evaluated on days 1, 2, 4, 6, 8, 10, and 12 by using the tail-immersion test, 15 min after morning nicotine or saline injection. MK-801 (0.1 mg/kg) produced a blockade of the acute nicotine antinociceptive response probably because of an interaction with the nicotinic receptors. Consequently, we used lower doses of this NMDA antagonist (0.01 and 0.03 mg/kg) since MK-801 is about 100 times more potent at blocking NMDA receptors than the nicotinic receptors (Briggs and McKenna 1996; Zakharova et al. 2005). Similar low doses of MK-801 have been used to describe different behavioural effects in mice involving NMDA receptors (Juni et al. 2006; Tang et al. 2006).

Dynorphin immunoassay

To study the possible modification of dynorphin levels in the spinal cord of nicotine tolerant mice, C57BL/6 animals were decapitated 1 h after the last nicotine or saline injection. The spinal cords were quickly removed and placed on ice and the lumbar spinal cord sections dissected. Immediately, they were frozen by immersion in 2-methyl-butane and stored at −80°C until use. Tissue extraction and immunoassay of dynorphin A (1–17) were carried out as previously described (Malan et al. 2000). Briefly, thawed tissue was placed in 1 N acetic acid, disrupted using a Polytron homogenizer and incubated for 30 min at 95°C. After centrifugation at 14 000 g for 20 min (4°C) the supernatant was lyophilized and then stored at −80°C. Protein concentration of the supernatant was determined by DC-micro plate assay (Bio-Rad, Spain), following manufacturer’s instructions. Immunoassays were performed using a commercial enzyme immunoassay system using an antibody specific for dynorphin A (1–17) (Bachem/Peninsula Laboratories, Belmont, CA, USA). Dynorphin content in the tissue extracts was calculated from a standard curve using known concentrations of dynorphin A (1–17).

Autoradiographic procedures

Spinal cord and brain slicing

To study possible modifications in the density of KOP-r in the brain and spinal cord of nicotine tolerant mice, C57BL/6 animals were decapitated 1 h after the last nicotine or saline injection. Their brains and spinal cords were quickly removed and rapidly frozen by immersion in 2-methyl-butane surrounded by dry ice. All samples were stored at −80°C during a similar period of time until processed for analyses of KOP-r binding. Brain coronal and lumbar spinal cord sections were cut in a cryostat (20 μm-thick), thaw-mounted on gelatin/chrome-coated slides, dried briefly at 30°C and stored at −80°C until used. In a previous study, we observed an increase of the functional activity of MOP-r (Galeote et al. 2006) in the lumbar spinal cord of nicotine tolerant mice. The analysis of the same structure helps the comparison between the results obtained in both studies.

Autoradiography of KOP-r binding

The autoradiographic procedure was performed as detailed previously (Kitchen et al. 1997). To determine total binding, sections were pre-incubated for 30 min in 50 mM Tris–HCl pH 7.4 containing 0.9% NaCl, before incubation for 60 min in 50 mM Tris–HCl pH 7.4 with 2.5 nM [3H]CI-977. Non-specific binding was determined in the same buffer for 60 min in the presence of 1 μM naloxone. After incubation, slides were washed three times during 5 min with ice-cold 50 mM Tris–HCl buffer (pH 7.4) and rapidly dried in cold-air. Autoradiograms were generated by apposing the labeled tissues, together with autoradiographic standards ([3H] microscales, Amersham, Madrid, Spain), to tritium-sensitive film (Hyperfilm-[3H], Amersham) for a period of 6 weeks or 18 weeks for brain or spinal cord sections respectively, and developed for 4 min at 20°C. Developed films were analyzed and quantified in a computerized image analysis system (MCID, St. Catharines, ON, Canada) using the standard curve generated from [3H]-standards. Measurements for quantitative analysis of spinal cords were taken from both right and left sides for each region, therefore representing a duplicate determination apart from lamina X of spinal cord where only one measurement was taken. Brain structures were identified using the mouse brain atlas of Paxinos and Franklin (1997), and spinal cord structures were referenced to cytoarchitecture of rat spinal cord (Molander et al. 1984). The data obtained were expressed as fmol/mg tissue, considering the standard curve generated from [3H]-standards (nCi/mg) and the specific activity of [3H]CI-977 (Ci/mmol).

Statistical analysis

Data from acute antinociceptive effects of nicotine were compared by using a between-subjects two-way anova (pre-treatment and treatment as factors of variation) following by one-way anova for individual differences and post hoc comparisons (Dunnett’s test) when required. Data from cross-tolerance between nicotine and U-50,488H were compared by using a between-subjects two-way anova (chronic treatment and acute treatment as factors of variation) followed by one-way anova and post hoc comparisons (Dunnett’s test) when required. ED50 values and 95% confidence limits (CL) were determined by nonlinear regression analysis of the dose-response curves using PRISM (GraphPad, San Diego, CA, USA). Non-overlapping 95% confidence intervals were considered significantly different. Studies on the role of NMDA receptors in the development of nicotine antinociceptive tolerance were analyzed by using two-way anova with repeated measures (treatment as between-subjects factor and day as within-subjects factor), followed by one-way anova and post hoc comparisons (Tukey test) when required. Biochemical studies were analyzed by using two-way anova with repeated measures (treatment as between-subjects factor and brain region or spinal cord layer as within-subjects factor), followed by one-way anova. The level of significance was p < 0.05 in all experiments.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

KOP-r is involved in spinal acute antinociceptive responses induced by nicotine

The role of KOP-r in locomotor and antinociceptive effects induced by acute nicotine (1, 3, and 5 mg/kg, s.c.) injection was evaluated in C57BL/6 mice pre-treated with nor-BNI (3 mg/kg, i.p.) or saline. KOP-r participates in the spinal acute antinociception produced by nicotine since the pre-treatment with the kappa antagonist nor-BNI attenuated the response in the tail-immersion test (Fig. 1a). However, no modification of this effect was observed in the hot-plate test (Fig. 1b) nor in the horizontal locomotor activity (Fig. 1c). Thus, two-way anova revealed a significant effect of treatment (F(3,175) = 79.45, p < 0.0001), pre-treatment (F(1,175) = 13.33, p < 0.0001) and interaction between these two factors (F(3,175) = 3.30, p < 0.05) in the tail-immersion test. Subsequent one-way anova indicated a significant effect of nicotine treatment in mice pre-treated with nor-BNI (F(3,86) = 26.81, p < 0.0001) or saline (F(3,89) = 56.07, p < 0.0001). Nicotine induced an antinociceptive response at the doses of 3 and 5 mg/kg (p < 0.0001) in both groups of pre-treated mice as revealed by post hoc comparison. Post hoc comparison also showed a reduction of nicotine-induced antinociception in nor-BNI-pre-treated mice when compared with saline-pre-treated mice at the doses of 3 and 5 mg/kg (p < 0.05). In the hot-plate test, two-way anova revealed a significant effect of treatment (F(3,175) = 173.88, p < 0.0001), but not the effect of pre-treatment (F(1,175) = 2.58, NS) nor interaction between these two factors (F(3,175) = 1.10, NS). In horizontal locomotor activity, two-way anova showed a significant effect of treatment (F(3,117) = 172.95, p < 0.0001), but not the effect of pre-treatment (F(1,117) = 0.21, NS) nor interaction between these two factors (F(3,117) = 0.78, NS).

image

Figure 1.  Effects of pre-treatment with the KOP-r antagonist nor-BNI on acute nicotine responses. Nor-BNI reduced acute nicotine antinociception in the tail-immersion test (a) but not in the hot-plate test (b). Nor-BNI did not modify the hypolocomotor effects induced by nicotine (c). Nor-BNI (3 mg/kg, i.p.) was administered 24 h before nicotine (1, 3, and 5 mg/kg, s.c.) or saline injection. Locomotor activity was measured 5 min after acute drug administration during a 10 min period. Antinociceptive responses in the tail-immersion and hot-plate tests were evaluated 15 and 16 min, respectively, after nicotine or saline administration. Data are expressed as mean ± SEM of latency time (a and b) (n = 18–35 mice for each group) or counts (c) (n = 7–20 mice for each group) in saline and nicotine treated mice. ⋆⋆p < 0.01 when compared to saline-treated mice (Dunnett’s test). p < 0.05 when compared to saline pre-treated mice (one-way anova).

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The density of KOP-r is modified in both spinal cord and brain areas as a consequence of chronic nicotine treatment

Possible modifications in the density of KOP-r in spinal cord and brain of nicotine tolerant mice were assessed by using autoradiography of [3H]CI-977 binding. A decrease in KOP-r levels was found in all layers of the spinal cord in nicotine tolerant mice. Thus, two-way anova revealed a significant treatment effect (p < 0.05). Subsequent one-way anova showed a significant decrease in the density of KOP-r in layers I–II (p < 0.05), layers III–VI (p < 0.05) and layer X (p < 0.05) (Fig. 2a and b). However, an enhancement in the density of this opioid receptor was observed in some brain areas as a consequence of chronic nicotine treatment. Thus, two-way anova revealed a significant treatment effect (p < 0.05). Subsequent one-way anova showed a significant increase in the density of KOP-r in the caudate-putamen (p < 0.05), the motor cortex (p < 0.05), and the cingulate cortex (p < 0.01). No changes were observed in the other brain regions evaluated (Fig. 3a and b).

image

Figure 2.  KOP-r binding in several lumbar spinal cord layers (a) and representative autoradiograms (b) from saline and chronic nicotine treated mice. The density of KOP-r decreased in all layers of the spinal cord. A drawing showing the anatomical locations where KOP-r levels were analyzed is also included. The data are expressed as mean ± SEM of fmol/mg (5–6 animals per group). Autoradiograms were processed according to the conditions described in Materials and methods. p < 0.05 when compared to saline group (one-way anova). NSB, non-specific binding.

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image

Figure 3.  KOP-r binding in several brain regions (a) and representative autoradiograms (b) from saline and chronic nicotine treated mice. The density of KOP-r increased in the caudate-putamen, motor and cingulate cortex as a consequence of chronic nicotine treatment. The data are expressed as mean ± SEM of fmol/mg (5–6 animals per group). Autoradiograms were processed according to the conditions described in Materials and methods. p < 0.05; ★★p < 0.01 when compared to saline group (one-way anova). CPu, caudate-putamen; Nac, nucleus accumbens; M, motor cortex; Cg, cingulate cortex; SPT septum; CL, claustrum; HPth, hypothalamus; PAG, periaqueductal gray. NSB, non-specific binding.

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Bi-directional cross-tolerance between nicotine and U-50,488H is developed in spinal antinociceptive responses

To go further in the study of the role of KOP-r in the development of nicotine tolerance, bi-directional cross-tolerance between nicotine and U-50,488H was studied. Bi-directional cross-tolerance was achieved in antinociceptive responses in the tail-immersion test, but not in the hot-plate test nor in locomotor responses (Fig. 4). In the tail-immersion test, two-way anova showed a significant effect of chronic treatment (F(2,239) = 65.54, p < 0.0001), acute treatment (F(6,239) = 54.33, p < 0.0001) and interaction between these two factors (F(12,239) = 13.11, p < 0.0001). Subsequent one-way anova indicated a significant effect of acute treatment in mice chronically treated with saline (F(6,83) = 37.34, p < 0.0001). Post hoc comparisons showed a significant acute antinociceptive effect of nicotine (3 and 5 mg/kg: p < 0.01) (ED50 = 3.02 mg/kg) and U-50,488H (10 and 20 mg/kg: p < 0.01) in these animals. One-way anova also revealed a significant effect of acute administration in mice chronically treated with nicotine (F(6,84) = 14.98, p < 0.0001). Nicotine antinociceptive tolerance was developed in these mice as showed the significant decrease in the latency response after acute nicotine (3 and 5 mg/kg) injection (p < 0.01). In agreement, ED50 value (5.82 mg/kg) significantly increased reflecting the development of tolerance (Table 1). Interestingly, one-way anova also revealed a decrease in the antinociceptive response of U-50,488H (10 mg/kg: p < 0.05) in these mice chronically treated with nicotine indicating the development of cross-tolerance between nicotine and U-50,488H. One-way anova also indicated a significant effect of acute treatment in animals chronically injected with U-50,488H (F(6,72) = 27.14, p < 0.0001). The decrease in the intensity of the antinociceptive response after acute U-50,488H (10 and 20 mg/kg) injection in these mice revealed the development of tolerance (p < 0.01) to this effect. Moreover, cross-tolerance between nicotine and U-50,488H was achieved as revealed the lower latency time after acute nicotine injection at the doses of 3 and 5 mg/kg (p < 0.01) in mice receiving chronic U-50,488H (Fig. 4a). Indeed, ED50 value (5.01 mg/kg) was significantly higher to that of the saline-treated group (Table 1).

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Figure 4.  Antinociceptive and locomotor responses of acute nicotine (NIC) and U-50,488H (U) in mice chronically treated with saline (SAL), nicotine and U-50,488H. Bi-directional cross-tolerance was developed in the tail-immersion test (a) but not in the hot-plate test (b) nor in locomotor activity (c). Mice chronically treated with nicotine (5 mg/kg, s.c.), U-50,488H (10 mg/kg, s.c.), or saline, received acute saline, nicotine (1.5, 3 and 5 mg/kg, s.c.) or U-50,488H (5, 10, and 20 mg/kg, s.c.) injection on day 12. Locomotor activity was measured 5 min after acute drug administration during a 10 min period. The tail-immersion and the hot-plate tests were measured 15 and 16 min after acute drug injection, respectively. Data are expressed as mean ± SEM of percentage of MPE (a and b) or counts (c) in chronic saline mice (saline, n = 25; nicotine 1.5, 3 and 5 mg/kg, n = 10; U-50,488H 5, 10, and 20 mg/kg, n = 10–15), chronic nicotine mice (saline, n = 22; nicotine 1.5, 3, and 5 mg/kg, n = 10; U-50,488H 5 and 20 mg/kg, n = 10 and U-50,488H 10 mg/kg, n = 19) and chronic U-50,488H mice (saline, n = 21; nicotine 1.5, 3, and 5 mg/kg, n = 9–10; U-50,488H 5, 10, and 20 mg/kg, n = 10). ★★p < 0.01, when compared to chronic saline and acute saline mice (Dunnett’s test). ap < 0.05, aap < 0.01 when compared to chronic saline and acute nicotine mice. bp < 0.05, bbp < 0.01 when compared to chronic saline and acute U-50,488H mice (one-way anova).

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Table 1.   Nicotine ED50 values (mg/kg) after 12-day treatment with saline, nicotine, or U-50,488H
Chronic treatment groupTail-immersion (95% CL)Locomotor activity (95% CL)
  1. Mice were chronically treated with saline, nicotine or U-50,488H as described under Materials and methods. ED50 values were calculated from three doses of nicotine. *Significantly different from saline ED50 value. ED50 = 50% effective dose; CL = confidence interval.

Saline3.02 (1.66–4.38)1.06 (0.58–1.55)
Nicotine5.82 (5.35–6.28)*3.03 (1.56–4.52)*
U-50,488H5.01 (4.49–5.53)*0.88 (0–1.87)

In the hot-plate test, two-way anova indicated a significant effect of chronic treatment (F(2,239) = 50.69, p < 0.0001), acute treatment (F(6,239) = 91.42, p < 0.0001) and interaction between these two factors (F(12,239) = 16.26, p < 0.0001). Subsequent one-way anova revealed a significant effect of acute treatment (F(6,83) = 52.40, p < 0.0001) in animals chronically receiving saline. Post hoc analysis showed that acute nicotine (1.5, 3, and 5 mg/kg) and U-50,488H (5, 10 and 20 mg/kg) injection induced significant antinociceptive effects (p < 0.01). One-way anova also revealed a significant effect of acute treatment in mice chronically treated with nicotine and U-50,488H (F(6,84) = 30.96, p < 0.0001 and F(6,72) = 46.29, p < 0.0001), respectively. One-way anova demonstrated the development of tolerance since a lower antinociceptive response was found after acute injection of nicotine (1.5, 3 and 5 mg/kg: p < 0.01) and U-50,488H (10 and 20 mg/kg: p < 0.01) in mice chronically treated with nicotine and U-50,488H, respectively. However, no cross-tolerance between nicotine and U-50,488H was observed in any experimental group (Fig. 4b).

In the horizontal locomotor activity, two-way anova revealed a significant effect of chronic treatment (F(2,239) = 27.06, p < 0.0001), acute treatment (F(6,239) = 162.55, p < 0.0001) and interaction between these two factors (F(12,239) = 10.29, p < 0.0001). In saline chronic group, subsequent one-way anova showed a significant effect of acute administration (F(6,83) = 54.27, p < 0.0001). Post hoc comparisons revealed a hypolocomotor effect following nicotine (1.5, 3 and 5 mg/kg) (ED50 = 1.06 mg/kg) and U-50,488H (5, 10, and 20 mg/kg) (ED50 = 9.63 mg/kg) injection (p < 0.01). One-way anova also revealed a significant effect of acute injection in mice chronically treated with nicotine and U-50,488H (F(6,84) = 62.26, p < 0.0001 and F(6,72) = 74.61, p < 0.0001), respectively. One way anova showed the development of tolerance indicated by the reduction of the hypolocomotion observed after acute nicotine (1.5, 3, and 5 mg/kg: p < 0.01) in mice chronically treated with nicotine. In agreement, ED50 value (3.03 mg/kg) significantly increased in this group of animals (Table 1). A lower hypolocomotor effect was also found following acute administration of U-50,488H (10 mg/kg: p < 0.01) in animals chronically treated with U-50,488H. Interestingly, cross-tolerance was observed in mice chronically treated with nicotine and receiving acute injection of U-50,488H (10 mg/kg: p < 0.05), although ED50 value was similar to that of the saline treated group (9.79 mg/kg) (Fig. 4c) (Table 2).

Table 2.   U-50,488H ED50 values (mg/kg) after 12-day treatment with saline, U-50,488H, or nicotine
Chronic treatment groupLocomotor activity (95% CL)
  1. Mice were chronically treated with saline, U-50,488H or nicotine as described under Materials and methods. ED50 values were calculated from three doses of U-50,488H. ED50 = 50% effective dose; CL = confidence interval.

Saline9.63 (6.36–12.90)
U-50,488H9.87 (6.93–12.81)
Nicotine9.79 (6.30–13.29)

Dynorphin levels in the lumbar spinal cord are similar in saline and nicotine tolerant mice

Dynorphin immunoassay was carried out in the lumbar spinal cord to evaluate the possible modification of dynorphin content due to the chronic nicotine treatment. No difference in dynorphin levels was found when comparing mice chronically receiving saline or nicotine as revealed by Student’s t-test (p > 0.05, NS) (Fig. 5).

image

Figure 5.  Dynorphin A (1–17) content in the lumbar spinal cord of mice chronically treated with saline or nicotine (5 mg/kg s.c., three times a day for 12 days). Dynorphin levels in lumbar spinal cord were similar in both treatments. Data are expressed as mean ± SEM of pg/mg tissue (10 animals per group).

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NMDA receptors are not involved in the development of antinociceptive tolerance induced by chronic nicotine treatment

To study the role of NMDA receptors in the development of tolerance to nicotine-induced antinociception, mice were pre-treated with MK-801 (0.1, 0.03, and 0.01 mg/kg, i.p.) 30 min before each nicotine (5 mg/kg, s.c.) or saline daily injection. MK-801 at the dose of 0.1 mg/kg blocked the acute antinociceptive response produced by nicotine (data not shown). Pre-treatment with MK-801 (0.03 and 0.01 mg/kg) did not modify the development of nicotine tolerance indicating a lack of involvement of NMDA receptors in this process. Thus, two-way anova revealed in MK-801 (0.03 mg/kg) pre-treated mice a significant effect of day (F(6,216) = 15.83, p < 0.0001), treatment (F(3,36) = 26.27, p < 0.0001), and interaction between day and treatment (F(18,216) = 2.76, p < 0.0001). Subsequent one-way anova (treatment) showed a significant effect in both chronically nicotine treated mice pre-treated with saline (F(6,54) = 9.97, p < 0.0001) and MK-801 (0.03 mg/kg) (F(6,54) = 4.48, p < 0.001). No difference between mice chronically treated with nicotine and pre-treated with MK-801 (0.03 mg/kg) or saline was found in any day of the experiment (Fig. 6a) indicating a similar development of tolerance in both groups of animals. In MK-801 (0.01 mg/kg) pre-treated mice, two-way anova revealed a significant effect of day (F(6,186) = 25.71, p < 0.0001), treatment (F(3,31) = 11.60, p < 0.0001), and interaction between day and treatment (F(18,186) = 4.10, p < 0.0001). Subsequent one-way anova (treatment) indicated a significant effect in both chronically nicotine treated mice pre-treated with MK-801 (0.01 mg/kg) (F(6,48) = 13.31, p < 0.0001) or saline (F(6,42) = 9.97, p < 0.0001). From day 6 to 12 no difference between chronically nicotine treated mice and pre-injected with MK-801 (0.01 mg/kg) or saline was observed (Fig. 6b), indicating a similar development of tolerance in both groups of animals.

image

Figure 6.  Effects of the NMDA antagonist MK-801 in the development of tolerance to nicotine-induced antinociception. The NMDA antagonist did not modify the development of nicotine tolerance. MK-801 (0.03 and 0.01 mg/kg, i.p.) was administered 30 min before nicotine (5 mg/kg, s.c.) or saline injection three times a day for 12 days. Nociceptive threshold was evaluated 15 min after morning nicotine injection on days 1, 2, 4, 6, 8, 10, and 12 by using the tail-immersion test. Results are expressed as mean ± SEM of latency time (n = 8–10 per group). p < 0.05, ★★p < 0.01 in comparison with the respective basal values (Tukey test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In this study, we show that KOP-r is involved in the development of tolerance to the antinociceptive effects induced by nicotine. First, KOP-r participates in acute nicotine antinociception, as revealed by a decrease of nicotine responses in the tail-immersion test after the pre-treatment with the KOP-r antagonist nor-BNI. The density of KOP-r decreased in all layers of the lumbar spinal cord in nicotine tolerant mice. In addition, bi-directional cross-tolerance between nicotine and the KOP-r agonist U-50,488H was achieved in the tail-immersion test. We have also demonstrated that endogenous dynorphins, through the activation of NMDA receptors, do not play a role in the antinociceptive tolerance to nicotine. Thus, chronic nicotine treatment did not produce any change of dynorphin levels in the lumbar spinal cord, and the pre-treatment with the NMDA antagonist MK-801 did not modify the development of nicotine antinociceptive tolerance.

To date, few studies have investigated the involvement of KOP-r in nicotine pharmacological responses. The activation of KOP-r prevented the locomotor stimulation in rats chronically treated with nicotine (Hahn et al. 2000). Other report has shown that KOP-r agonists attenuated the mecamilamine-precipitated nicotine-withdrawal aversion (Ise et al. 2002). In addition, Marco et al. (2005) have described a blockade of the stimulatory effect of nicotine on adrenocortical activity by nor-BNI. However, the anxiogenic-like effects of nicotine were not modified by KOP-r antagonists (Balerio et al. 2005; Marco et al. 2005). In spite of these previous data, little is known about the role of KOP-r in nicotine antinociceptive effects. Our results suggest that KOP-r participates in the spinal acute antinociceptive effects induced by nicotine since the pre-treatment with nor-BNI reduced this nicotine response in the tail-immersion test, but not in the hot-plate test. Acute nicotine-induced hypolocomotion was also not modified by nor-BNI pre-administration. The nociceptive behaviour evaluated in the tail-immersion test is mediated through spinal responses (Martin et al. 2003). The anatomical distribution of KOP-r in the dorsal horn of the spinal cord (Mansour et al. 1995) and the spinal localization of neuronal nicotinic receptors (Damaj 2007) support this hypothesis.

It is well-documented that chronic nicotine administration induces tolerance to some of its behavioural responses including the antinociceptive effects (Grabus et al. 2005). Thus, several studies have shown antinociceptive tolerance after repeated nicotine treatment in different experimental models, such as the formalin test (Zarrindast et al. 1999), the tail-flick test (Damaj and Martin 1996; Damaj 2005), the tail-immersion test (Galeote et al. 2006), and the hot-plate test (Grabus et al. 2005). The mechanisms underlying nicotine tolerance are associated with nAChRs up-regulation and desensitization but remain unclear (Quick and Lester 2002; Robinson et al. 2007). Other processes independently of the drug-receptor interaction are also involved in the development of nicotine-induced antinociceptive tolerance as revealed by the participation of L-type calcium channels and calcium-dependent calmodulin protein kinase II (Damaj 2005). In addition, the endogenous opioid system has been proposed to participate in nicotine-induced tolerance although most of the experiments have been focused on the role of MOP-r. The antinociceptive response of nicotine was lower in morphine-tolerant mice (Zarrindast et al. 1999; Biala and Weglinska 2006) indicating the development of cross-tolerance between the two drugs. Furthermore, a decrease of met-enkephalin levels and an up-regulation of MOP-r in the rat striatum have been shown after chronic nicotine administration (Wewers et al. 1999). An increase in the functional activity of MOP-r in the lumbar spinal cord of nicotine tolerant mice has been also reported (Galeote et al. 2006). The efficacy of MOP-r activation seems to be enhanced to facilitate the maintenance of the nicotine antinociceptive effects since the lack of this receptor in knockout mice accelerated the development of nicotine tolerance (Galeote et al. 2006). In the present study, a decrease in all layers of the lumbar spinal cord of KOP-r binding was found in nicotine tolerant animals. The down-regulation of KOP-r in the spinal cord seems to contribute to the development of nicotine tolerance to its antinociceptive effects since these receptors were required in this study for a complete manifestation of the spinal acute nicotine antinociception. In addition to the decrease of KOP-r levels, the same chronic nicotine administration schedule induced an enhancement of spinal MOP-r function (Galeote et al. 2006). These biochemical changes at the level of KOP-r and MOP-r could be both related to a physiological adaptation within the endogenous opioid system leading to maintain its functional activity in the control of the homeostasis. Indeed, a similar compensatory change on both opioid receptors, i.e., decrease in functional activity of KOP-r and enhanced activity of MOP-r, has been described in the spinal cord (Narita et al. 2003a) as well as in the thalamus and periaqueductal grey (Narita et al. 2003b; Khotib et al. 2004) following repeated injection of the kappa-agonist U-50,488H. In agreement, the activation of MOP-r and KOP-r has been reported to produce different and/or opposite pharmacological effects. While MOP-r mediates the rewarding properties of drugs of abuse (Matthes et al. 1996; Roberts et al. 2000; Berrendero et al. 2002; Ghozland et al. 2002), KOP-r regulates dysphoric and aversive effects (Simonin et al. 1998; Mendizabal et al. 2006), and can counteract the nociceptive responses produced by MOP-r activation.

Our results also show the existence of bi-directional cross-tolerance between the KOP-r agonist U-50,488H and nicotine in the tail-immersion test. Thus, the antinociceptive response of U-50,488H at the dose of 10 mg/kg was reduced in mice chronically treated with nicotine, in agreement with our biochemical data showing a down-regulation of KOP-r in the spinal cord of these animals. Interestingly, nicotine antinociception was also reduced in mice receiving chronic U-50,488H. Chronic administration of U-50,488H has been reported to decrease the density and functional activity of KOP-r in the spinal cord (Bhargava et al. 1989; Narita et al. 2003a). The lower antinociceptive response induced by nicotine in U-50,488H tolerant mice further supports the important role of spinal KOP-r in the regulation of this nicotine effect. Cross-tolerance between nicotine and U-50,488H was not observed in the hot-plate test suggesting a selective interaction between these two compounds in the regulation of spinal antinociception. Indeed, previous studies have also reported that nicotine is not effective in enhancing the inhibition of the tail-flick response induced by U-50,488H when both drugs are administered intracerebroventricularly, suggesting a lack of interaction between nicotine and U-50,488H at supraspinal levels (Suh et al. 1996). On the other hand, chronic nicotine administration induced an increase of KOP-r levels in motor regions such as the caudate-putamen or the motor cortex. Previous studies have described similar results (down-regulation in the spinal cord and up-regulation in the caudate-putamen of KOP-r) following chronic treatment with U-50,488H or cocaine (Bhargava et al. 1989; Unterwald et al. 1994). The increase of KOP-r in motor regions could explain the existence of the cross-tolerance observed at the dose of 10 mg/kg of U-50,488H in the horizontal locomotor activity of mice chronically treated with nicotine.

The possible involvement of endogenous dynorphins and NMDA receptors in the development of antinociceptive tolerance produced by nicotine was also analyzed. Although cleavage of the large precursor prodynorphin results in the release of various peptide products, most of the studies involve the dynorphin A (1–17) fragment in the facilitation of nociceptive responses. Thus, the development of neuropathic pain (Xu et al. 2004) and antinociceptive tolerance to cannabinoids and opioids are associated to an up-regulation of dynorphin in spinal cord (Vanderah et al. 2000; Gardell et al. 2002). The non-opioid activity of dynorphin through the activation of NMDA receptors (Laughlin et al. 2001) has been proposed to participate in the pronociceptive effects of this endogenous peptide. Thus, an attenuation of the antinociceptive tolerance induced by chronic morphine has been reported after the pre-treatment with the NMDA receptor antagonist MK-801 (Trujillo and Akil 1991). Furthermore, a blockade of the behavioural manifestation of the WIN 55,212-2 cannabinoid antinociceptive tolerance has been also reported by the pre-administration of MK-801 (Gardell et al. 2002). In contrast with these two drugs, chronic nicotine did not enhance dynorphin A (1–17) levels in the spinal cord. Moreover, the pre-treatment with MK-801 (0.03 and 0.01 mg/kg) in mice receiving chronic nicotine did not modify the development of nicotine tolerance, indicating that NMDA receptors are not involved in this process. Differences in the mechanism of action of these drugs (excitatory effects of nicotine versus inhibitory effects of cannabinoids and opioids) could explain these opposite results. However, because of the down-regulation of spinal KOP-r in nicotine tolerant mice we cannot rule out that the remaining spinal dynorphin can activate other systems (Oka et al. 1998) to promote nicotine tolerance.

In conclusion, our results demonstrate the involvement of KOP-r in spinal antinociception induced by acute nicotine and the development of tolerance to these nicotine effects. Chronic nicotine administration was associated to a decrease in the density of KOP-r in the spinal cord and bi-directional cross-tolerance was observed between nicotine and KOP-r agonists. Endogenous dynorphins, through the activation of NMDA receptors, do not participate in the development of nicotine tolerance which should involve other mechanisms associated to the adaptive changes here reported on KOP-r. In addition, the results of the present study could have a clinical relevance since recent reports indicate that nAChRs agonists have a potential therapeutic use in pain relief as new analgesic agents (Cassels et al. 2005; Vincler 2005). The development of tolerance to this effect could represent a possible undesirable side effect, mainly when treating chronic pain, which involves long-term usage. Therefore, the clarification of the neurobiological mechanisms underlying tolerance to nicotine antinociceptive effects represents an important advance to identify new targets for improving this important side effect.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported by National Institute on Drug Abuse-National Institutes of Health Grant 1R01DA016768, Fondo de Investigación Sanitario (FIS) Grants G03/005, C03/06 and 04/1485, Generalitat de Catalunya (2002SGR00193), Spanish Ministry of Science and Technology (GEN 2003-20651-C06-04) and (BFU2004/00920/BFI), and the European Commission (IP ≠ 0J 2004/C164, Nº 005166, GENADDICT). We thank Dr Guadalupe Mengod and Dr Roser Cortés for their help in image analysis. The technical assistance of Ms. Raquel Martín is gratefully acknowledged. FB is a researcher supported by the Ramón y Cajal program of Ministry of Science and Technology.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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