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The role of gaseous neurotransmitters in the antinociceptive effects of morphine during acute thermal pain Gemma Gou, Sergi Leánez, Olga Pol
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Received date: 20 December 2013 Revised date: 29 April 2014 Accepted date: 7 May 2014 Cite this article as: Gemma Gou, Sergi Leánez, Olga Pol, The role of gaseous neurotransmitters in the antinociceptive effects of morphine during acute thermal pain, European Journal of Pharmacology, http://dx.doi.org/10.1016/j. ejphar.2014.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The role of gaseous neurotransmitters in the antinociceptive effects of morphine during acute thermal pain
Gemma Gou, Sergi Leánez and Olga Pol*
Grup de Neurofarmacologia Molecular, Institut d’Investigació Biomèdica Sant Pau & Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain.
*Corresponding author Olga Pol, PhD Grup de Neurofarmacologia Molecular Institut de Neurociències Facultat de Medicina. Edifici M2-115 Universitat Autònoma de Barcelona 08193 Barcelona, Spain. Tel: 34 619 757 054 Fax: 34 935 811 573 E-mail: [email protected]
Abstract Treatment with a carbon monoxide-releasing molecule (tricarbonyldichlororuthenium(II) dimer, CORM-2) or a classical inducible heme oxygenase (HO-1) inducer (cobalt protoporphyrin IX, CoPP) enhanced the antinociceptive effects of morphine during chronic pain but the role played by these compounds in acute thermal nociception was not evaluated. The effects of CORM-2 and CoPP treatments on the local antinociceptive actions of morphine and their interaction with nitric oxide during acute pain was evaluated by using wild type (WT), neuronal (nNOS-KO) or inducible (iNOS-KO) nitric oxide synthase knockout mice and assessing their thermal nociception to a hot stimulus with the hot plate test.
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Our results showed that the absence of nNOS or iNOS genes did not alter licking and jumping responses nor the antinociceptive effects produced by morphine indicating that the local thermal inhibitory effects produced by this drug in the absence of inflammation or injury is not mediated by the nitric oxide pathway triggered by nNOS or iNOS enzymes. Moreover, while the systemic administration of CORM-2 or CoPP inhibited licking and jumping latencies in all genotypes, these treatments only enhanced the local inhibition of jumping latencies produced by morphine in WT and nNOS-KO mice which effects were reversed by the peripheral administration of an HO-1 inhibitor. These data indicate that the co-administration of morphine with CORM-2 or CoPP produced remarkable local antinociceptive effects in WT and nNOS-KO mice and reveal that a significant interaction between carbon monoxide and nitric oxide systems occurs on the local antinociceptive effects produced by morphine during acute thermal nociception.
Keywords: Carbon monoxide, nitric oxide, morphine, antinociception, thermal nociception, pain.
1. Introduction Nitric oxide synthesized either by neuronal (nNOS) or inducible (iNOS) nitric oxide synthase mediates numerous chronic pain symptoms via the cGMP-PKG pathway activation (LaBuda et al., 2006; Schmidtko et al., 2008). Accordingly, the expression of both enzymes is up-regulated in the spinal cord and dorsal root ganglia from animals with chronic pain (DeAlba et al., 2006; Hervera et al., 2010b). Moreover, the hypersensitivity induced by nerve injury was significantly diminished in nNOS (nNOS-KO) and iNOS (iNOS-KO) knockout animals (Kuboyama et al., 2011; Hervera et al., 2012) or reversed by the administration of selective nNOS, iNOS, guanylate cyclase or PKG inhibitors (DeAlba et al., 2006; Schmidtko et al., 2008; Hervera et al., 2010a). However, the role played by nitric oxide during acute thermal nociception has not been completely evaluated.
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Carbon monoxide, another gaseous neurotransmitter, synthetized by inducible (HO-1) and constitutive heme oxygenase enzymes, also regulates nociception by the activation of the cGMP-PKG signalling pathway. However, while the over-expression of the constitutive heme oxygenase exerts a pronociceptive effect after nerve injury (Li and Clark 2003; Hervera et al., 2012), the increased expression of HO-1 exerts potent anti-inflammatory and antinociceptive effects during inflammatory and neuropathic pain (Fan et al. 2011; Hervera et al., 2012; Negrete et al., 2014). Indeed the administration of an HO-1 inducer compound, such as cobalt protoporphyrin IX (CoPP) or a carbon monoxide-releasing molecule, such as tricarbonyldichlororuthenium(II)dimer (CORM-2), a new class of chemical agents able to reproduce several biological effects of HO-1-derived carbon monoxide (Motterlini et al., 2002), inhibits chronic pain (Guillén et al., 2008; Hervera et al., 2012). But the exact contribution of carbon monoxide synthesized by HO-1, in the modulation of thermal nociception as well as their possible interaction with the nitric oxide system has not been investigated. It is well known that the local administration of morphine elicits antinociceptive effects during inflammatory and neuropathic pain which effects are produced by the activation of the peripheral nitric oxide-cGMP-PKG-ATP-sensitive K+ channels signalling pathway (Leánez et al., 2009; Cunha et al., 2010; Hervera et al. 2011). Recent studies also demonstrate that the administration of CORM-2 and CoPP enhances the local antiallodynic and antihyperalgesic effects produced by morphine during neuropathic pain (Hervera et al., 2013a), but the possible involvement of carbon monoxide synthetized by HO-1 in the thermal antinociceptive effects produced by morphine is still unknown. Therefore in wild type (WT), nNOS-KO and iNOS-KO mice, the local thermal antinociceptive effects produced by morphine administered alone or combined with the intraperitoneal administration of CORM-2 and CoPP, are evaluated. Moreover, the thermal antinociceptive effects of morphine combined with the subplantar administration of an HO-1 inhibitor, tin protoporphyrin IX (SnPP), are also assessed.
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2. Material and methods 2.1. Animals Experiments were performed in male nNOS-KO and iNOS-KO mice (C57BL/6J background) purchased from Jackson Laboratories (Bar Harbor, ME, USA) as well as in WT mice with the same genetic background (C57BL/6J) acquired from Harlan Laboratories (Barcelona, Spain). All mice weighing 21 to 25 g were housed under 12-h/12-h light/dark conditions in a room with controlled temperature (22º C) and humidity (66 %). Animals had free access to food and water and were used after a minimum of 6 days acclimatization to the housing conditions. All experiments were conducted between 9:00 AM and 5:00 PM. All experiments were carried out according to the Ethical Guidelines of the International Association for the Study of Pain and approved by the local ethical committee of our Institution (Comissió d’Ètica en l’Experimentació Animal i Humana de la Universitat Autònoma de Barcelona)
2.2. Induction of acute pain Thermal nociception to a hot stimulus was assessed by using the hot/cold-plate analgesia meter (Ugo Basile, Italy). Briefly, mice were placed into a Plexiglas cylinder (diameter, 20 cm; height, 18 cm) on a metal surface maintained at 52.5°C. The time between placement and licking of the hindpaws and jumping was recorded. To avoid tissue damage of hindpaws, the cut-off was 60 and 240 seconds, respectively. Only one test per animal was performed because repeated measures might cause profound latency changes (Mogil et al., 1999).
2.3. Experimental protocol In a first set of experiments, we evaluated the baseline response to an acute thermal stimulus in WT, nNOS-KO and iNOS-KO mice. In a second set of experiments, we investigated the inhibitory effects produced by the subplantar administration of morphine in WT, nNOS-KO and iNOS-KO mice. In a third set of experiments, the effects produced by the intraperitoneal administration of 2.5 mg/kg of CORM-2 or 1 mg/kg of CoPP alone or combined with a low dose of morphine (10 µg) or vehicle subplantarly administered were also evaluated (n = 8
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animals per group). In the last set of experiments, we evaluated the effects produced by the subplantar administration of 290 µg of SnPP alone or combined with the subplantar administration of a high dose of morphine (100 µg) (n = 8 animals per group). The doses of CORM-2, CoPP and SnPP were selected from our preliminary experiments and in accordance to other studies (Nascimento and Branco, 2007, 2009; Rosa et al, 2008, Hervera et al., 2012; 2013b; Negrete et al., 2014). The doses of morphine were selected according as the ones that produced a minimal thermal antinociceptive effect in each genotype.
2.4. Drugs CORM-2 was purchased from Sigma-Aldrich (St. Louis, MO), CoPP and SnPP from Frontier scientific (Livchem GmbH & Co, Frankfurt, Germany). Morphine hydrochloride was obtained from Alcaiber S.A. (Madrid, Spain). CORM-2, CoPP and SnPP were dissolved in dimethyl sulfoxide (DMSO; 1 % solution in saline). Morphine was dissolved in saline solution (0.9 % NaCl). All drugs were freshly prepared before use. CORM-2 and CoPP were intraperitoneally administered 3 hours before testing, in a final volume of 10 ml/kg. Morphine and SnPP were subplantarly administered 30 min before behavioural testing in a final volume of 20 µl. For each group treated with a drug the respective control group received the same volume of vehicle.
2.5. Statistical analysis Data are expressed as mean ± standard error of the mean (S.E.M.). The comparison of the basal nociceptive responses between genotypes was evaluated by using a one way ANOVA followed by the Student Newman Keuls test. For each knockout mice and behavioural evaluated the effects produced by the administration of different doses of morphine were also evaluated by using a one way ANOVA followed by the Student Newman Keuls. The comparison of the effects produced by a dose of morphine on the inhibition of licking or jumping latencies between genotypes was also performed by using a one way ANOVA followed by the Student Newman Keuls test. The evaluation of the effects produced by morphine alone or combined with CORM-2, CoPP or SnPP for each genotype and behavioural evaluated was also assessed
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by using a one way ANOVA followed by the Student Newman Keuls test. Antinociception is expressed as the percentage of maximal possible effect, where the test latencies pre (baseline) and post drug administration are compared and calculated according to the following equations, respectively: Maximal possible effect (%) = [(drug – baseline) / (cut-off – baseline)] x 100 A value of P< 0.05 was considered as a significant.
3. Results 3.1 Thermal nociception in WT, nNOS-KO and iNOS-KO mice. The latencies to display hindpaw licking or jumping in the hot plate were similar in WT, nNOSKO and iNOS-KO mice as shown in Fig. 1. For each behavioural evaluated, the one way ANOVA did not reveal significant differences between genotypes.
3.2 The local antinociceptive effects of morphine in WT, nNOS-KO and iNOS-KO mice The antinociceptive effects produced by the local administration of different doses of morphine in WT, nNOS-KO and iNOS-KO mice have been evaluated. Our results show that the subplantar administration of different doses (10-100 µg) of morphine inhibited the licking (Fig. 2A) and jumping (Fig. 2B) latencies in a dose dependent manner in WT, nNOS-KO and iNOSKO mice. In all genotypes, the one way ANOVA showed a significant effect produced by 25, 50 and 100 µg of morphine on the inhibition of licking and jumping latencies (P< 0.001), as compared to vehicle treated mice (one way ANOVA, followed by the Student Newman Keuls test). Our results also indicate that non-significant differences could be observed between genotypes when compared the effects produced by different doses of morphine on the inhibition of licking or jumping latencies.
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3.3 The effects of CORM-2 and CoPP treatments on the local antinociceptive actions of morphine in WT, nNOS-KO and iNOS-KO mice The effects of the intraperitoneal administration of 2.5 mg/kg of CORM-2 or 1 mg/kg of CoPP on the inhibition of licking (Fig. 3) and jumping (Fig. 4) latencies produced by the subplantar administration of a low dose of morphine (10 µg) or vehicle in WT (A), nNOS-KO (B) and iNOS-KO (C) mice were also investigated. In all genotypes, the intraperitoneal administration of CORM-2 or CoPP alone inhibited the licking and jumping latencies induced by a thermal stimulus (P< 0.04; one way ANOVA vs. respective control-vehicle treated mice). Our results also demonstrate that the subplantar administration of a low dose of morphine (10 µg) in CORM-2 or CoPP treated WT and nNOSKO mice significantly increased the local inhibition of jumping, but not licking, latencies produced by morphine alone (P< 0.001, one way ANOVA vs. CORM-2 or CoPP plus vehicle, morphine plus vehicle or control plus vehicle groups). In contrast, the inhibition of licking and jumping latencies produced by the subplantar administration of 10 μg of morphine in iNOS-KO mice was not altered by CORM-2 or CoPP treatments.
3.4 Effects of the HO-1 inhibitor, tin protoporphyrin IX, on the local antinociceptive actions of morphine in WT, nNOS-KO and iNOS-KO mice The effects of the subplantar (290 µg) administration of SnPP on the inhibition of licking and jumping latencies produced by the subplantar administration of a high dose of morphine (100 µg) or vehicle in WT, nNOS-KO and iNOS-KO mice were also investigated. For licking behavior, our results show that the subplantar administration of SnPP alone did not alter licking latencies as well as the local inhibitory effects produced by a high dose of morphine in all genotypes (Table 1). Indeed for each genotype, the inhibition of licking latencies produced by the subplantar administration of morphine alone or combined with SnPP was significantly different to that produced by the subplantar injection of vehicle or SnPP administered alone (P< 0.001; one way ANOVA vs. respective their vehicle or SnPP plus vehicle treated mice)
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In contrast, while the subplantar administration of SnPP alone did not alter jumping latencies induced by a thermal stimulus, their local coadministration with a high dose of morphine (100 µg) significantly decreased the local antinociceptive effects produced by morphine in WT and nNOS-KO mice (p < 0.001, one way ANOVA; group treated with morphine as compared to their respective vehicle, SnPP plus vehicle or morphine plus SnPP treated mice) but not in iNOS-KO animals (Table 2).
4. Discussion Several reports shown the beneficial effects produced by the administration of selective nNOS and iNOS inhibitors on the attenuation of inflammatory and neuropathic pain (DeAlba et al., 2006; LaBuda et al., 2006), but the role played by these enzymes on the inhibition of acute thermal nociception was not completely elucidated. The present study demonstrates that no significant differences in the licking and jumping latencies in the hot plate test were observed between WT, nNOS-KO and iNOS-KO mice. These results support the hypothesis that nitric oxide synthesized by nNOS or iNOS did not play a relevant role in response to a thermal nociception. In accordance to our data, other studies also demonstrated a preservation of acute thermal nociception in mice lacking L-guanylate cyclase or protein kinase G (Tegeder et al., 2004; Schmidtko et al., 2008). Subsequently, we evaluated the thermal antinociceptive effects produced by the subplantar administration of different doses of morphine in WT, nNOS-KO and iNOS-KO mice. Our findings showed that the peripheral administration of morphine dose-dependently enhanced the licking and jumping latencies in a similar manner in all genotypes indicating that nitric oxide, synthesized by nNOS or iNOS, is not involved in the acute antithermal effects produced by morphine. This is in contrast to the critical role played by nitric oxide on the local antinociceptive effects produced by morphine during inflammatory and neuropathic pain (Cunha et al., 2010; Hervera et al., 2011), where the local antinociceptive effects produced by morphine were significantly reduced by their local co-administration with selective nNOS, iNOS, L-guanylate cyclase or PKG inhibitors as well as in nNOS-KO mice (Cunha et al. 2010;
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Hervera et al. 2011; 2013). Therefore, the fact that the absence of nNOS or iNOS genes did not alter the local thermal antinociceptive effects produced by morphine revealed that the effects produced by this drug in the absence of an inflammatory or injury process is independent to the nitric oxide pathway triggered by nNOS or iNOS isoforms. In order to study the role played by carbon monoxide, synthesized by HO-1, in the thermal antinociceptive effects produced by morphine as well as their interaction with the nitric oxide system, the effects produced by CORM-2 and CoPP treatments, alone or combined with morphine, in WT, nNOS-KO and iNOS-KO mice were also evaluated. Our results demonstrate that the intraperitoneal administration of CORM-2 or CoPP alone inhibited licking and jumping latencies in all genotypes. In accordance, previous works also indicated that the local administration of an HO-1 inhibitor increased the nociceptive behavioral responses induced by formalin, while carbon monoxide gas inhalation or the HO-1 induction decreased the licking times induced by formalin in rodents (Nascimento and Branco, 2007; 2009; Rosa et al. 2008; Egea et al. 2009). Our data further demonstrated that CORM-2 or CoPP treatments also inhibited the licking and jumping behavioral responses induced by a thermal stimulus in nNOSKO and iNOS-KO mice. Indicating that carbon monoxide exogenously deliberated or endogenously produced by HO-1 did not require the presence of nNOS or iNOS to inhibit thermal nociception. These results support our previous findings demonstrating that CORM-2 and CoPP treatments also reduced the licking latencies induced by the subplantar administration of formalin in mice lacking nNOS or iNOS isoenzymes (Hervera et al. 2013b). Curiously, while the co-administration of CORM-2 or CoPP with morphine did not alter the local inhibition of licking latencies produced by this drug in the presence or absence of nNOS or iNOS genes, CORM-2 or CoPP treatments significantly increased the local inhibition of jumping latencies produced by a low dose of morphine in WT and nNOS-KO mice, but not in iNOS-KO mice, revealing that nitric oxide synthesized by iNOS might be involved in the enhanced inhibition of jumping latencies produced by a morphine in CORM-2 or CoPP treated mice. In addition, our results also show that while the local inhibition of licking latencies produced by morphine remains unaffected by their coadministration with SnPP (HO-1
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inhibitor), the local inhibition of jumping latencies produced by morphine was significantly decreased by the subplantar administration of SnPP in WT and nNOS-KO mice, but not in iNOS-KO animals. These findings indicate that whereas peripheral HO-1, expressed in intact paw tissues of all genotypes (Negrete et al., 2014), participates in the local inhibition of jumping latencies produced by morphine in WT and nNOS-KO mice, the local administration of morphine did not use this pathway for inhibiting licking latencies. The possible mechanism involved in the different effect produced by HO-1 on the inhibition licking and jumping responses produced by morphine could be explained by the fact that licking is the first primary noxious evoked response while jumping is an escape behavior in the hot plate. Therefore, the fact that the CORM-2 or CoPP might enhance the inhibition of jumping, but not licking, latencies produced by morphine indicated that this combination might only inhibited the most elaborate response (jumping) but not the rapid response (licking) to a painful thermal stimulus (Espejo and Mir, 1993). In summary, this study indicates that the coadministration of morphine with CORM-2 or CoPP produced remarkable local antinociceptive effects during acute pain and reveals that a significant interaction between the nitric oxide and carbon monoxide systems is taking place in the antinociceptive effects produced by morphine during acute thermal nociception.
Acknowledgments This work was supported by the Fundació La Marató de TV3 Barcelona [Grant: 070810] and Fondo de Investigación Sanitaria, Madrid [Grant: PS0900968], Spain.
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Figure legends Fig. 1. Latency to display licking or jumping responses in the hot plate test in WT, nNOS-KO and iNOS-KO mice (n=8 animals for each group). All data are presented as means ± S.E.M. Statistical analysis were determined by one-way ANOVA followed by the Student Newman Keuls test.
Fig. 2. Effects of the subplantar administration of different doses (0-100 µg) of morphine or vehicle on the inhibition of licking (A) and jumping (B) latencies in the hot plate test in WT, nNOS-KO and iNOS-KO mice are shown. Data are expressed as mean values of maximal possible effect (%) ± S.E.M. (n = 8 animals for dose). For each behavioral parameter and genotype, * P< 0.050 denotes significant differences vs. control group treated with vehicle (one way ANOVA followed by the Student Newman Keuls test).
Fig. 3. Effects of the intraperitoneal administration of 2.5 mg/kg of CORM-2 or 1 mg/kg of CoPP on the inhibition of licking produced by subplantar administration of 10 μg morphine or vehicle in the ipsilateral paw of WT (A), nNOS-KO (B) and iNOS-KO (C) mice. The effects of CORM-2 or CoPP administered alone are also shown. Data are expressed as mean values of the maximal possible effect (%) ± S.E.M. (n = 8 animals per group). For each genotype, * P< 0.050 denotes significant differences vs. control group treated with vehicle (one way ANOVA followed by the Student Newman Keuls test).
Fig. 4. Effects of the intraperitoneal administration of 2.5 mg/kg of CORM-2 or 1 mg/kg of CoPP on the inhibition of jumping produced by subplantar administration of 10 μg morphine or vehicle in the ipsilateral paw of WT (A), nNOS-KO (B) and iNOS-KO (C) mice. The effects of CORM-2 or CoPP administered alone are also shown. Data are expressed as mean values of the
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maximal possible effect (%) ± S.E.M. (n = 8 animals per group). For each genotype, * P< 0.050 denotes significant differences vs. control group treated with vehicle; + P< 0.050 denotes significant differences vs. control group treated with morphine and # P< 0.050 denotes significant differences vs. group treated with CORM-2 or CoPP plus vehicle (one way ANOVA followed by the Student Newman Keuls test).
BEHAVIOUR
GENOTYPE
Licking
WT
nNOS-KO
iNOS-KO
vehicle + vehicle
2.5 ± 2.5
3.5 ± 2.4
2.0 ± 2.1
morphine + vehicle
93.3 ± 4.3 a,b
97.0 ± 6.0 a,b
95.1 ± 5.2 a,b
SnPP + vehicle
3.6 ± 2.2
3.5 ± 2.0
3.1 ± 2.1
morphine + SnPP
92.1 ± 5.0 a,b
94.3 ± 4.1 a,b
93.7± 4.0 a,b
Table 1. Effects of the subplantar administration of 290 µg of SnPP on the inhibition of licking latencies produced by the subplantar administration of 100 μg of morphine or vehicle in the ipsilateral paw of WT, nNOS-KO and iNOS-KO mice. The effects of SnPP administered alone are also shown. Data are expressed as mean values of the maximal possible effect (%) ± S.E.M. (n = 8 animals per group). For each genotype, a P< 0.050 denotes significant differences vs. group treated with vehicle plus vehicle and
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b
P< 0.050 denotes significant differences vs. group treated with SnPP plus vehicle (one
way ANOVA followed by the Student Newman Keuls test).
BEHAVIOUR
GENOTYPE
Jumping
WT
nNOS-KO
iNOS-KO
vehicle + vehicle
3.2 ± 1.9
4.0 ± 2.2
3.8 ± 2.1
morphine + vehicle
94.5 ± 4.5 a,b,c
96.2 ± 4.8 a,b,c
95.2 ± 5.7 a,b
SnPP + vehicle
4.6 ± 2.8
5.5 ± 2.4
4.1 ± 2.1
morphine + SnPP
5.6 ± 3.8
5.2 ± 3.4
90.1 ± 6.2 a,b
Table 2. Effects of the subplantar administration of 290 µg of SnPP on the inhibition of jumping latencies produced by the subplantar administration of 100 μg of morphine or vehicle in the ipsilateral paw of WT, nNOS-KO and iNOS-KO mice. The effects of SnPP administered alone are also shown. Data are expressed as mean values of the maximal possible effect (%) ± S.E.M. (n = 8 animals per group). For each genotype, a P< 0.050 denotes significant differences vs. group treated with vehicle plus vehicle, b P< 0.050 denotes significant differences vs. group treated with SnPP plus vehicle and c P< 0.050 denotes significant differences vs. group treated with morphine plus SnPP (one way ANOVA followed by the Student Newman Keuls test).
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The role of gaseous neurotransmitters in the antinociceptive effects of morphine during acute thermal pain Gemma Gou, Sergi Leánez, Olga Pol
www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(14)00354-9 http://dx.doi.org/10.1016/j.ejphar.2014.05.004 EJP69281
To appear in:
European Journal of Pharmacology
Received date: 20 December 2013 Revised date: 29 April 2014 Accepted date: 7 May 2014 Cite this article as: Gemma Gou, Sergi Leánez, Olga Pol, The role of gaseous neurotransmitters in the antinociceptive effects of morphine during acute thermal pain, European Journal of Pharmacology, http://dx.doi.org/10.1016/j. ejphar.2014.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The role of gaseous neurotransmitters in the antinociceptive effects of morphine during acute thermal pain
Gemma Gou, Sergi Leánez and Olga Pol*
Grup de Neurofarmacologia Molecular, Institut d’Investigació Biomèdica Sant Pau & Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain.
*Corresponding author Olga Pol, PhD Grup de Neurofarmacologia Molecular Institut de Neurociències Facultat de Medicina. Edifici M2-115 Universitat Autònoma de Barcelona 08193 Barcelona, Spain. Tel: 34 619 757 054 Fax: 34 935 811 573 E-mail: [email protected]
Abstract Treatment with a carbon monoxide-releasing molecule (tricarbonyldichlororuthenium(II) dimer, CORM-2) or a classical inducible heme oxygenase (HO-1) inducer (cobalt protoporphyrin IX, CoPP) enhanced the antinociceptive effects of morphine during chronic pain but the role played by these compounds in acute thermal nociception was not evaluated. The effects of CORM-2 and CoPP treatments on the local antinociceptive actions of morphine and their interaction with nitric oxide during acute pain was evaluated by using wild type (WT), neuronal (nNOS-KO) or inducible (iNOS-KO) nitric oxide synthase knockout mice and assessing their thermal nociception to a hot stimulus with the hot plate test.
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Our results showed that the absence of nNOS or iNOS genes did not alter licking and jumping responses nor the antinociceptive effects produced by morphine indicating that the local thermal inhibitory effects produced by this drug in the absence of inflammation or injury is not mediated by the nitric oxide pathway triggered by nNOS or iNOS enzymes. Moreover, while the systemic administration of CORM-2 or CoPP inhibited licking and jumping latencies in all genotypes, these treatments only enhanced the local inhibition of jumping latencies produced by morphine in WT and nNOS-KO mice which effects were reversed by the peripheral administration of an HO-1 inhibitor. These data indicate that the co-administration of morphine with CORM-2 or CoPP produced remarkable local antinociceptive effects in WT and nNOS-KO mice and reveal that a significant interaction between carbon monoxide and nitric oxide systems occurs on the local antinociceptive effects produced by morphine during acute thermal nociception.
Keywords: Carbon monoxide, nitric oxide, morphine, antinociception, thermal nociception, pain.
1. Introduction Nitric oxide synthesized either by neuronal (nNOS) or inducible (iNOS) nitric oxide synthase mediates numerous chronic pain symptoms via the cGMP-PKG pathway activation (LaBuda et al., 2006; Schmidtko et al., 2008). Accordingly, the expression of both enzymes is up-regulated in the spinal cord and dorsal root ganglia from animals with chronic pain (DeAlba et al., 2006; Hervera et al., 2010b). Moreover, the hypersensitivity induced by nerve injury was significantly diminished in nNOS (nNOS-KO) and iNOS (iNOS-KO) knockout animals (Kuboyama et al., 2011; Hervera et al., 2012) or reversed by the administration of selective nNOS, iNOS, guanylate cyclase or PKG inhibitors (DeAlba et al., 2006; Schmidtko et al., 2008; Hervera et al., 2010a). However, the role played by nitric oxide during acute thermal nociception has not been completely evaluated.
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Carbon monoxide, another gaseous neurotransmitter, synthetized by inducible (HO-1) and constitutive heme oxygenase enzymes, also regulates nociception by the activation of the cGMP-PKG signalling pathway. However, while the over-expression of the constitutive heme oxygenase exerts a pronociceptive effect after nerve injury (Li and Clark 2003; Hervera et al., 2012), the increased expression of HO-1 exerts potent anti-inflammatory and antinociceptive effects during inflammatory and neuropathic pain (Fan et al. 2011; Hervera et al., 2012; Negrete et al., 2014). Indeed the administration of an HO-1 inducer compound, such as cobalt protoporphyrin IX (CoPP) or a carbon monoxide-releasing molecule, such as tricarbonyldichlororuthenium(II)dimer (CORM-2), a new class of chemical agents able to reproduce several biological effects of HO-1-derived carbon monoxide (Motterlini et al., 2002), inhibits chronic pain (Guillén et al., 2008; Hervera et al., 2012). But the exact contribution of carbon monoxide synthesized by HO-1, in the modulation of thermal nociception as well as their possible interaction with the nitric oxide system has not been investigated. It is well known that the local administration of morphine elicits antinociceptive effects during inflammatory and neuropathic pain which effects are produced by the activation of the peripheral nitric oxide-cGMP-PKG-ATP-sensitive K+ channels signalling pathway (Leánez et al., 2009; Cunha et al., 2010; Hervera et al. 2011). Recent studies also demonstrate that the administration of CORM-2 and CoPP enhances the local antiallodynic and antihyperalgesic effects produced by morphine during neuropathic pain (Hervera et al., 2013a), but the possible involvement of carbon monoxide synthetized by HO-1 in the thermal antinociceptive effects produced by morphine is still unknown. Therefore in wild type (WT), nNOS-KO and iNOS-KO mice, the local thermal antinociceptive effects produced by morphine administered alone or combined with the intraperitoneal administration of CORM-2 and CoPP, are evaluated. Moreover, the thermal antinociceptive effects of morphine combined with the subplantar administration of an HO-1 inhibitor, tin protoporphyrin IX (SnPP), are also assessed.
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2. Material and methods 2.1. Animals Experiments were performed in male nNOS-KO and iNOS-KO mice (C57BL/6J background) purchased from Jackson Laboratories (Bar Harbor, ME, USA) as well as in WT mice with the same genetic background (C57BL/6J) acquired from Harlan Laboratories (Barcelona, Spain). All mice weighing 21 to 25 g were housed under 12-h/12-h light/dark conditions in a room with controlled temperature (22º C) and humidity (66 %). Animals had free access to food and water and were used after a minimum of 6 days acclimatization to the housing conditions. All experiments were conducted between 9:00 AM and 5:00 PM. All experiments were carried out according to the Ethical Guidelines of the International Association for the Study of Pain and approved by the local ethical committee of our Institution (Comissió d’Ètica en l’Experimentació Animal i Humana de la Universitat Autònoma de Barcelona)
2.2. Induction of acute pain Thermal nociception to a hot stimulus was assessed by using the hot/cold-plate analgesia meter (Ugo Basile, Italy). Briefly, mice were placed into a Plexiglas cylinder (diameter, 20 cm; height, 18 cm) on a metal surface maintained at 52.5°C. The time between placement and licking of the hindpaws and jumping was recorded. To avoid tissue damage of hindpaws, the cut-off was 60 and 240 seconds, respectively. Only one test per animal was performed because repeated measures might cause profound latency changes (Mogil et al., 1999).
2.3. Experimental protocol In a first set of experiments, we evaluated the baseline response to an acute thermal stimulus in WT, nNOS-KO and iNOS-KO mice. In a second set of experiments, we investigated the inhibitory effects produced by the subplantar administration of morphine in WT, nNOS-KO and iNOS-KO mice. In a third set of experiments, the effects produced by the intraperitoneal administration of 2.5 mg/kg of CORM-2 or 1 mg/kg of CoPP alone or combined with a low dose of morphine (10 µg) or vehicle subplantarly administered were also evaluated (n = 8
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animals per group). In the last set of experiments, we evaluated the effects produced by the subplantar administration of 290 µg of SnPP alone or combined with the subplantar administration of a high dose of morphine (100 µg) (n = 8 animals per group). The doses of CORM-2, CoPP and SnPP were selected from our preliminary experiments and in accordance to other studies (Nascimento and Branco, 2007, 2009; Rosa et al, 2008, Hervera et al., 2012; 2013b; Negrete et al., 2014). The doses of morphine were selected according as the ones that produced a minimal thermal antinociceptive effect in each genotype.
2.4. Drugs CORM-2 was purchased from Sigma-Aldrich (St. Louis, MO), CoPP and SnPP from Frontier scientific (Livchem GmbH & Co, Frankfurt, Germany). Morphine hydrochloride was obtained from Alcaiber S.A. (Madrid, Spain). CORM-2, CoPP and SnPP were dissolved in dimethyl sulfoxide (DMSO; 1 % solution in saline). Morphine was dissolved in saline solution (0.9 % NaCl). All drugs were freshly prepared before use. CORM-2 and CoPP were intraperitoneally administered 3 hours before testing, in a final volume of 10 ml/kg. Morphine and SnPP were subplantarly administered 30 min before behavioural testing in a final volume of 20 µl. For each group treated with a drug the respective control group received the same volume of vehicle.
2.5. Statistical analysis Data are expressed as mean ± standard error of the mean (S.E.M.). The comparison of the basal nociceptive responses between genotypes was evaluated by using a one way ANOVA followed by the Student Newman Keuls test. For each knockout mice and behavioural evaluated the effects produced by the administration of different doses of morphine were also evaluated by using a one way ANOVA followed by the Student Newman Keuls. The comparison of the effects produced by a dose of morphine on the inhibition of licking or jumping latencies between genotypes was also performed by using a one way ANOVA followed by the Student Newman Keuls test. The evaluation of the effects produced by morphine alone or combined with CORM-2, CoPP or SnPP for each genotype and behavioural evaluated was also assessed
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by using a one way ANOVA followed by the Student Newman Keuls test. Antinociception is expressed as the percentage of maximal possible effect, where the test latencies pre (baseline) and post drug administration are compared and calculated according to the following equations, respectively: Maximal possible effect (%) = [(drug – baseline) / (cut-off – baseline)] x 100 A value of P< 0.05 was considered as a significant.
3. Results 3.1 Thermal nociception in WT, nNOS-KO and iNOS-KO mice. The latencies to display hindpaw licking or jumping in the hot plate were similar in WT, nNOSKO and iNOS-KO mice as shown in Fig. 1. For each behavioural evaluated, the one way ANOVA did not reveal significant differences between genotypes.
3.2 The local antinociceptive effects of morphine in WT, nNOS-KO and iNOS-KO mice The antinociceptive effects produced by the local administration of different doses of morphine in WT, nNOS-KO and iNOS-KO mice have been evaluated. Our results show that the subplantar administration of different doses (10-100 µg) of morphine inhibited the licking (Fig. 2A) and jumping (Fig. 2B) latencies in a dose dependent manner in WT, nNOS-KO and iNOSKO mice. In all genotypes, the one way ANOVA showed a significant effect produced by 25, 50 and 100 µg of morphine on the inhibition of licking and jumping latencies (P< 0.001), as compared to vehicle treated mice (one way ANOVA, followed by the Student Newman Keuls test). Our results also indicate that non-significant differences could be observed between genotypes when compared the effects produced by different doses of morphine on the inhibition of licking or jumping latencies.
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3.3 The effects of CORM-2 and CoPP treatments on the local antinociceptive actions of morphine in WT, nNOS-KO and iNOS-KO mice The effects of the intraperitoneal administration of 2.5 mg/kg of CORM-2 or 1 mg/kg of CoPP on the inhibition of licking (Fig. 3) and jumping (Fig. 4) latencies produced by the subplantar administration of a low dose of morphine (10 µg) or vehicle in WT (A), nNOS-KO (B) and iNOS-KO (C) mice were also investigated. In all genotypes, the intraperitoneal administration of CORM-2 or CoPP alone inhibited the licking and jumping latencies induced by a thermal stimulus (P< 0.04; one way ANOVA vs. respective control-vehicle treated mice). Our results also demonstrate that the subplantar administration of a low dose of morphine (10 µg) in CORM-2 or CoPP treated WT and nNOSKO mice significantly increased the local inhibition of jumping, but not licking, latencies produced by morphine alone (P< 0.001, one way ANOVA vs. CORM-2 or CoPP plus vehicle, morphine plus vehicle or control plus vehicle groups). In contrast, the inhibition of licking and jumping latencies produced by the subplantar administration of 10 μg of morphine in iNOS-KO mice was not altered by CORM-2 or CoPP treatments.
3.4 Effects of the HO-1 inhibitor, tin protoporphyrin IX, on the local antinociceptive actions of morphine in WT, nNOS-KO and iNOS-KO mice The effects of the subplantar (290 µg) administration of SnPP on the inhibition of licking and jumping latencies produced by the subplantar administration of a high dose of morphine (100 µg) or vehicle in WT, nNOS-KO and iNOS-KO mice were also investigated. For licking behavior, our results show that the subplantar administration of SnPP alone did not alter licking latencies as well as the local inhibitory effects produced by a high dose of morphine in all genotypes (Table 1). Indeed for each genotype, the inhibition of licking latencies produced by the subplantar administration of morphine alone or combined with SnPP was significantly different to that produced by the subplantar injection of vehicle or SnPP administered alone (P< 0.001; one way ANOVA vs. respective their vehicle or SnPP plus vehicle treated mice)
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In contrast, while the subplantar administration of SnPP alone did not alter jumping latencies induced by a thermal stimulus, their local coadministration with a high dose of morphine (100 µg) significantly decreased the local antinociceptive effects produced by morphine in WT and nNOS-KO mice (p < 0.001, one way ANOVA; group treated with morphine as compared to their respective vehicle, SnPP plus vehicle or morphine plus SnPP treated mice) but not in iNOS-KO animals (Table 2).
4. Discussion Several reports shown the beneficial effects produced by the administration of selective nNOS and iNOS inhibitors on the attenuation of inflammatory and neuropathic pain (DeAlba et al., 2006; LaBuda et al., 2006), but the role played by these enzymes on the inhibition of acute thermal nociception was not completely elucidated. The present study demonstrates that no significant differences in the licking and jumping latencies in the hot plate test were observed between WT, nNOS-KO and iNOS-KO mice. These results support the hypothesis that nitric oxide synthesized by nNOS or iNOS did not play a relevant role in response to a thermal nociception. In accordance to our data, other studies also demonstrated a preservation of acute thermal nociception in mice lacking L-guanylate cyclase or protein kinase G (Tegeder et al., 2004; Schmidtko et al., 2008). Subsequently, we evaluated the thermal antinociceptive effects produced by the subplantar administration of different doses of morphine in WT, nNOS-KO and iNOS-KO mice. Our findings showed that the peripheral administration of morphine dose-dependently enhanced the licking and jumping latencies in a similar manner in all genotypes indicating that nitric oxide, synthesized by nNOS or iNOS, is not involved in the acute antithermal effects produced by morphine. This is in contrast to the critical role played by nitric oxide on the local antinociceptive effects produced by morphine during inflammatory and neuropathic pain (Cunha et al., 2010; Hervera et al., 2011), where the local antinociceptive effects produced by morphine were significantly reduced by their local co-administration with selective nNOS, iNOS, L-guanylate cyclase or PKG inhibitors as well as in nNOS-KO mice (Cunha et al. 2010;
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Hervera et al. 2011; 2013). Therefore, the fact that the absence of nNOS or iNOS genes did not alter the local thermal antinociceptive effects produced by morphine revealed that the effects produced by this drug in the absence of an inflammatory or injury process is independent to the nitric oxide pathway triggered by nNOS or iNOS isoforms. In order to study the role played by carbon monoxide, synthesized by HO-1, in the thermal antinociceptive effects produced by morphine as well as their interaction with the nitric oxide system, the effects produced by CORM-2 and CoPP treatments, alone or combined with morphine, in WT, nNOS-KO and iNOS-KO mice were also evaluated. Our results demonstrate that the intraperitoneal administration of CORM-2 or CoPP alone inhibited licking and jumping latencies in all genotypes. In accordance, previous works also indicated that the local administration of an HO-1 inhibitor increased the nociceptive behavioral responses induced by formalin, while carbon monoxide gas inhalation or the HO-1 induction decreased the licking times induced by formalin in rodents (Nascimento and Branco, 2007; 2009; Rosa et al. 2008; Egea et al. 2009). Our data further demonstrated that CORM-2 or CoPP treatments also inhibited the licking and jumping behavioral responses induced by a thermal stimulus in nNOSKO and iNOS-KO mice. Indicating that carbon monoxide exogenously deliberated or endogenously produced by HO-1 did not require the presence of nNOS or iNOS to inhibit thermal nociception. These results support our previous findings demonstrating that CORM-2 and CoPP treatments also reduced the licking latencies induced by the subplantar administration of formalin in mice lacking nNOS or iNOS isoenzymes (Hervera et al. 2013b). Curiously, while the co-administration of CORM-2 or CoPP with morphine did not alter the local inhibition of licking latencies produced by this drug in the presence or absence of nNOS or iNOS genes, CORM-2 or CoPP treatments significantly increased the local inhibition of jumping latencies produced by a low dose of morphine in WT and nNOS-KO mice, but not in iNOS-KO mice, revealing that nitric oxide synthesized by iNOS might be involved in the enhanced inhibition of jumping latencies produced by a morphine in CORM-2 or CoPP treated mice. In addition, our results also show that while the local inhibition of licking latencies produced by morphine remains unaffected by their coadministration with SnPP (HO-1
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inhibitor), the local inhibition of jumping latencies produced by morphine was significantly decreased by the subplantar administration of SnPP in WT and nNOS-KO mice, but not in iNOS-KO animals. These findings indicate that whereas peripheral HO-1, expressed in intact paw tissues of all genotypes (Negrete et al., 2014), participates in the local inhibition of jumping latencies produced by morphine in WT and nNOS-KO mice, the local administration of morphine did not use this pathway for inhibiting licking latencies. The possible mechanism involved in the different effect produced by HO-1 on the inhibition licking and jumping responses produced by morphine could be explained by the fact that licking is the first primary noxious evoked response while jumping is an escape behavior in the hot plate. Therefore, the fact that the CORM-2 or CoPP might enhance the inhibition of jumping, but not licking, latencies produced by morphine indicated that this combination might only inhibited the most elaborate response (jumping) but not the rapid response (licking) to a painful thermal stimulus (Espejo and Mir, 1993). In summary, this study indicates that the coadministration of morphine with CORM-2 or CoPP produced remarkable local antinociceptive effects during acute pain and reveals that a significant interaction between the nitric oxide and carbon monoxide systems is taking place in the antinociceptive effects produced by morphine during acute thermal nociception.
Acknowledgments This work was supported by the Fundació La Marató de TV3 Barcelona [Grant: 070810] and Fondo de Investigación Sanitaria, Madrid [Grant: PS0900968], Spain.
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Figure legends Fig. 1. Latency to display licking or jumping responses in the hot plate test in WT, nNOS-KO and iNOS-KO mice (n=8 animals for each group). All data are presented as means ± S.E.M. Statistical analysis were determined by one-way ANOVA followed by the Student Newman Keuls test.
Fig. 2. Effects of the subplantar administration of different doses (0-100 µg) of morphine or vehicle on the inhibition of licking (A) and jumping (B) latencies in the hot plate test in WT, nNOS-KO and iNOS-KO mice are shown. Data are expressed as mean values of maximal possible effect (%) ± S.E.M. (n = 8 animals for dose). For each behavioral parameter and genotype, * P< 0.050 denotes significant differences vs. control group treated with vehicle (one way ANOVA followed by the Student Newman Keuls test).
Fig. 3. Effects of the intraperitoneal administration of 2.5 mg/kg of CORM-2 or 1 mg/kg of CoPP on the inhibition of licking produced by subplantar administration of 10 μg morphine or vehicle in the ipsilateral paw of WT (A), nNOS-KO (B) and iNOS-KO (C) mice. The effects of CORM-2 or CoPP administered alone are also shown. Data are expressed as mean values of the maximal possible effect (%) ± S.E.M. (n = 8 animals per group). For each genotype, * P< 0.050 denotes significant differences vs. control group treated with vehicle (one way ANOVA followed by the Student Newman Keuls test).
Fig. 4. Effects of the intraperitoneal administration of 2.5 mg/kg of CORM-2 or 1 mg/kg of CoPP on the inhibition of jumping produced by subplantar administration of 10 μg morphine or vehicle in the ipsilateral paw of WT (A), nNOS-KO (B) and iNOS-KO (C) mice. The effects of CORM-2 or CoPP administered alone are also shown. Data are expressed as mean values of the
15
maximal possible effect (%) ± S.E.M. (n = 8 animals per group). For each genotype, * P< 0.050 denotes significant differences vs. control group treated with vehicle; + P< 0.050 denotes significant differences vs. control group treated with morphine and # P< 0.050 denotes significant differences vs. group treated with CORM-2 or CoPP plus vehicle (one way ANOVA followed by the Student Newman Keuls test).
BEHAVIOUR
GENOTYPE
Licking
WT
nNOS-KO
iNOS-KO
vehicle + vehicle
2.5 ± 2.5
3.5 ± 2.4
2.0 ± 2.1
morphine + vehicle
93.3 ± 4.3 a,b
97.0 ± 6.0 a,b
95.1 ± 5.2 a,b
SnPP + vehicle
3.6 ± 2.2
3.5 ± 2.0
3.1 ± 2.1
morphine + SnPP
92.1 ± 5.0 a,b
94.3 ± 4.1 a,b
93.7± 4.0 a,b
Table 1. Effects of the subplantar administration of 290 µg of SnPP on the inhibition of licking latencies produced by the subplantar administration of 100 μg of morphine or vehicle in the ipsilateral paw of WT, nNOS-KO and iNOS-KO mice. The effects of SnPP administered alone are also shown. Data are expressed as mean values of the maximal possible effect (%) ± S.E.M. (n = 8 animals per group). For each genotype, a P< 0.050 denotes significant differences vs. group treated with vehicle plus vehicle and
16
b
P< 0.050 denotes significant differences vs. group treated with SnPP plus vehicle (one
way ANOVA followed by the Student Newman Keuls test).
BEHAVIOUR
GENOTYPE
Jumping
WT
nNOS-KO
iNOS-KO
vehicle + vehicle
3.2 ± 1.9
4.0 ± 2.2
3.8 ± 2.1
morphine + vehicle
94.5 ± 4.5 a,b,c
96.2 ± 4.8 a,b,c
95.2 ± 5.7 a,b
SnPP + vehicle
4.6 ± 2.8
5.5 ± 2.4
4.1 ± 2.1
morphine + SnPP
5.6 ± 3.8
5.2 ± 3.4
90.1 ± 6.2 a,b
Table 2. Effects of the subplantar administration of 290 µg of SnPP on the inhibition of jumping latencies produced by the subplantar administration of 100 μg of morphine or vehicle in the ipsilateral paw of WT, nNOS-KO and iNOS-KO mice. The effects of SnPP administered alone are also shown. Data are expressed as mean values of the maximal possible effect (%) ± S.E.M. (n = 8 animals per group). For each genotype, a P< 0.050 denotes significant differences vs. group treated with vehicle plus vehicle, b P< 0.050 denotes significant differences vs. group treated with SnPP plus vehicle and c P< 0.050 denotes significant differences vs. group treated with morphine plus SnPP (one way ANOVA followed by the Student Newman Keuls test).
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Figure 1
WT
nNOS-KO
iNOS-KO
100
Latency (sec)
80
60
40
20
0 Licking
Jumping
Figure 2
A
vehicle
100
*
*
*
*
*
*
40
20
0
WT
nNOS-KO
B
vehicle
10
25
*
100
Jumping Maximal possible effect (%)
50
* *
80
60
25
*
100
Licking Maximal possible effect (%)
10
*
50
100
*
*
*
80
* *
60
40
iNOS-KO
*
*
20
0 WT
nNOS-KO
iNOS-KO
*
Figure 3
A Licking Maximal possible effect (%)
100 80 60
40 20
0
vehicle
morphine
control
B
morphine
CORM-2
vehicle
morphine
CoPP
Licking Maximal possible effect (%)
100 80 60 40 20 0 vehicle
morphine
control
vehicle
morphine
CORM-2
vehicle
morphine
CoPP
100
Licking Maximal possible effect (%)
C
vehicle
80 60 40 20 0 vehicle
morphine
control
vehicle
morphine
CORM-2
vehicle
morphine CoPP
Figure 4
A Jumping Maximal possible effect (%)
100 80 60
#
#
40 20
0 vehicle
morphine
control
B
vehicle
morphine
CORM-2
morphine
CoPP
#
#
100
Jumping Maximal possible effect (%)
vehicle
80 60 40 20 0 vehicle
morphine
control
C
vehicle
morphine
CORM-2
vehicle
morphine
CoPP
Jumping Maximal possible effect (%)
100
80 60
40 20
0 vehicle
morphine
control
vehicle
morphine
CORM-2
vehicle
morphine
CoPP
