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P.1.g.040 Effects of CB2 receptor inverse agonists on JNK activation and pro-apoptotic Bax in mouse brain
S226
P.1.g. Basic and clinical neuroscience − Neuropharmacology
TNFa, BDNF, NR2B) and astroglial connexin markers (Cx30, Cx43). Pressure threshold values to elicit nocifensive responses (paw withdrawal and vocalization) increased progressively during amitriptyline treatment, confirming the anti-hyperalgesic action of this antidepressant in CCI rats. Although the connexin inhibitor THN01 was inactive on its own, it significantly enhanced amitriptyline-induced reduction of mechanical hyperalgesia. Thus, at the end of co-treatment, pressure threshold values in CCI rats were similar to those in na¨ıve healthy rats. On the other hand, mechanical hyperalgesia was associated with significantly upregulated levels of the microglia marker Ox42, the injured neuron marker ATF3 and the proinflammatory cytokine IL-6 in dorsal root ganglia and/or the dorsal spinal cord ipsilateral to sciatic nerve CCI, but neither amitriptyline or THN01 alone, nor the combined treatment, significantly reduced such CCI-induced effects. These results show that the connexin inhibition by THN01 enhanced the anti-hyperalgesic effect of amitriptyline in the rat CCI model. The unchanged overexpression of neuroinflammatory markers suggests that the anti-hyperalgesic effect of combined amitriptyline+THN01 treatment involved mechanisms downstream of these processes. Therefore, inhibition of connexinbased channel functions in astroglia could represent a promising and novel approach toward improving current neuropathic pain therapy. References [1] Picoli C., Nouvel V., Aubry F., Reboul M., Duchˆene A., Jeanson T., Thomasson J., Mouthon F., Charv´eriat M., 2012. Human connexin channel specificity of classical and new Gap Junction inhibitors. J. Biomol. Screen 17, 1339−47. [2] Bennett G.J., Xie Y.K., 1988. Peripheral mononeuropathy in rat that produces abnormal pain sensation like those seen in man. Pain 33, 87– 107.
P.1.g.040 Effects of CB2 receptor inverse agonists on JNK activation and pro-apoptotic Bax in mouse brain ´ G. Salort1 ° , M. Alvaro-Bartolom´ e1 , J.A. Garc´ıa-Sevilla1 of the Balearic Islands, IUNICS, Palma de Mallorca, Spain
1 University
Purpose: The drug AM630 (6-iodopravadoline) has been classified as a selective cannabinoid CB2 receptor protean ligand, which was recently shown to behave as an inverse agonist upregulating pro-apoptotic c-Jun NH2 -terminal protein kinase (JNK) and various pro-apoptotic proteins (FADD, cytochrome c and nuclear PARP cleavage) in mouse brain [1]. Therefore, the acute effects of AM630 could induce an enhanced apoptosis in brain. In comparison with AM630, the present aims were to assess the acute effects of novel CB2 inverse agonists (JTE907 and raloxifene) [2,3] regulating JNK and pro-apoptotic mitochondrial Bax, and to quantify the subchronic effects of AM630 on these apoptotic markers in mouse brain. Methods: Male CD1 Swiss mice were treated (i.p.) with drug-vehicle (2 ml/kg, n = 16−23), JWH133 (1 mg/kg, 1 h, n = 6), AM630 (1 mg/kg, 1.5 h, n = 5), JTE907 (3 and 10 mg/kg, 1.5 h, n = 8) and raloxifene (2 mg/kg, 1.5 h, n = 6). Other mice were repeatedly treated (i.p.) with AM630 (1 mg/kg, twice daily, for 7 days, n = 6) or drug-vehicle (n = 6) in parallel. The animals were killed by decapitation at the indicated times. Phosphorylated (p)
and total (t) JNK (ratio of p-JNK to t-JNK) and mitochondrial Bax were quantified in mouse brain cortex by Western blot analyses with validated antibodies, and the content of beta-actin was used as a loading control. Drug effects were expressed as % change when compared with the corresponding drug-vehicle control group. Student’s t-test was used for statistical analysis. Results: The acute administration of the CB2 agonist JWH133 (1 mg/kg) and the CB2 inverse agonist AM630 (1 mg/kg) resulted in opposite effects modulating pro-apoptotic JNK (p-JNK/JNK ratio) in mouse brain cortex (JWH133: 45% decrease, p < 0.05; AM630: 61% increase; p < 0.05). Basal JNK was also activated by the novel CB2 inverse agonists JTE907 (13−57%, p < 0.05) and raloxifene (31%, p < 0.05). Acute AM630 upregulated the content of pro-apoptotic mitochondrial Bax (66%, p < 0.05) in brain cortex. Acute raloxifene also increased cortical Bax content (22%, p < 0.05). In contrast, acute JTE907 did not alter the content of cortical Bax (10% increase, p > 0.05). Notably, subchronic (7 days) treatment with the selective CB2 inverse agonist AM630 did not upregulate JNK or Bax but instead it was associated with modest decreases of basal JNK (−28%, p > 0.05) and Bax content (−21%, p < 0.05) in mouse brain cortex. Conclusions: The CB2 receptor inverse agonist AM630 acutely upregulated pro-apoptotic JNK and mitochondrial Bax, which together with previous findings [1] could indicate the induction of abnormal brain cell death. Repeated AM630 treatment, however, induced neurochemical tolerance to JNK activation and Bax upregulation (less efficacy compared with the acute treatment) suggesting that the drug would not induce abnormal apoptosis in the brain in the long-term. Similarly to AM630, the CB2 ligands JTE907 and raloxifene appear to behave as somewhat weak inverse agonits modulating pro-apoptotic JNK and/or Bax in mouse brain. References ´ [1] Alvaro-Bartolom´ e, M., Salort, G., Garc´ıa-Sevilla, J.A., 2013 The CB2 ligand AM630 (6-iodopravadoline) increases the content and activity of pro-apoptotic markers in mouse brain. Eur Neuropsychopharmacol 23, Suppl. 2, S195. [2] Iwamura, H., Suzuki, H., Ueda, Y., Kaya, T., Inaba, T., 2001 In vitro and in vivo pharmacological characterization of JTE-907, a novel selective ligand for cannabinoid CB2 receptor. J Pharmacol Exp Therap 296, 420–425. [3] Kumar, P., Song, Z.-H., 2013 Identification of raloxifene as a novel CB2 inverse agonist. Biochem Biophys Res Comm 435, 76−81.
P.1.g.041 Experimental studies of the analgesic effect of pregabalin V. Kokova1 ° , E.G. Apostolova1 , L.P. Peychev1 1 Medical University, Farmacology and Drug Toxicology, Plovdiv, Bulgaria Purpose: Pregabalin is an alkylated analogue of GABA effective in the treatment of several disorders, including epilepsy, hyperalgesia, behavior disorders, generalized anxiety disorder and social phobia. Pregabalin exert antinociceptive effects in various experimental models of acute and chronic pain, reduce neuropathic pain in patients with diabetic neuropathy and post-herpetic neuralgia [1]. It is considered more effective in difficult pain management associated with stress [2]. The purpose of present study is to investigate the analgesic effect of pregabalin on acute models of pain on rats. Methods: Male Wistar rats, divided into 5 groups (n = 8), were treated intraperitoneally as follows: 1st group − with saline (control group) 0.1 ml/100 g bw; 2nd − with metamizole sodium
P.1.g. Basic and clinical neuroscience − Neuropharmacology
TNFa, BDNF, NR2B) and astroglial connexin markers (Cx30, Cx43). Pressure threshold values to elicit nocifensive responses (paw withdrawal and vocalization) increased progressively during amitriptyline treatment, confirming the anti-hyperalgesic action of this antidepressant in CCI rats. Although the connexin inhibitor THN01 was inactive on its own, it significantly enhanced amitriptyline-induced reduction of mechanical hyperalgesia. Thus, at the end of co-treatment, pressure threshold values in CCI rats were similar to those in na¨ıve healthy rats. On the other hand, mechanical hyperalgesia was associated with significantly upregulated levels of the microglia marker Ox42, the injured neuron marker ATF3 and the proinflammatory cytokine IL-6 in dorsal root ganglia and/or the dorsal spinal cord ipsilateral to sciatic nerve CCI, but neither amitriptyline or THN01 alone, nor the combined treatment, significantly reduced such CCI-induced effects. These results show that the connexin inhibition by THN01 enhanced the anti-hyperalgesic effect of amitriptyline in the rat CCI model. The unchanged overexpression of neuroinflammatory markers suggests that the anti-hyperalgesic effect of combined amitriptyline+THN01 treatment involved mechanisms downstream of these processes. Therefore, inhibition of connexinbased channel functions in astroglia could represent a promising and novel approach toward improving current neuropathic pain therapy. References [1] Picoli C., Nouvel V., Aubry F., Reboul M., Duchˆene A., Jeanson T., Thomasson J., Mouthon F., Charv´eriat M., 2012. Human connexin channel specificity of classical and new Gap Junction inhibitors. J. Biomol. Screen 17, 1339−47. [2] Bennett G.J., Xie Y.K., 1988. Peripheral mononeuropathy in rat that produces abnormal pain sensation like those seen in man. Pain 33, 87– 107.
P.1.g.040 Effects of CB2 receptor inverse agonists on JNK activation and pro-apoptotic Bax in mouse brain ´ G. Salort1 ° , M. Alvaro-Bartolom´ e1 , J.A. Garc´ıa-Sevilla1 of the Balearic Islands, IUNICS, Palma de Mallorca, Spain
1 University
Purpose: The drug AM630 (6-iodopravadoline) has been classified as a selective cannabinoid CB2 receptor protean ligand, which was recently shown to behave as an inverse agonist upregulating pro-apoptotic c-Jun NH2 -terminal protein kinase (JNK) and various pro-apoptotic proteins (FADD, cytochrome c and nuclear PARP cleavage) in mouse brain [1]. Therefore, the acute effects of AM630 could induce an enhanced apoptosis in brain. In comparison with AM630, the present aims were to assess the acute effects of novel CB2 inverse agonists (JTE907 and raloxifene) [2,3] regulating JNK and pro-apoptotic mitochondrial Bax, and to quantify the subchronic effects of AM630 on these apoptotic markers in mouse brain. Methods: Male CD1 Swiss mice were treated (i.p.) with drug-vehicle (2 ml/kg, n = 16−23), JWH133 (1 mg/kg, 1 h, n = 6), AM630 (1 mg/kg, 1.5 h, n = 5), JTE907 (3 and 10 mg/kg, 1.5 h, n = 8) and raloxifene (2 mg/kg, 1.5 h, n = 6). Other mice were repeatedly treated (i.p.) with AM630 (1 mg/kg, twice daily, for 7 days, n = 6) or drug-vehicle (n = 6) in parallel. The animals were killed by decapitation at the indicated times. Phosphorylated (p)
and total (t) JNK (ratio of p-JNK to t-JNK) and mitochondrial Bax were quantified in mouse brain cortex by Western blot analyses with validated antibodies, and the content of beta-actin was used as a loading control. Drug effects were expressed as % change when compared with the corresponding drug-vehicle control group. Student’s t-test was used for statistical analysis. Results: The acute administration of the CB2 agonist JWH133 (1 mg/kg) and the CB2 inverse agonist AM630 (1 mg/kg) resulted in opposite effects modulating pro-apoptotic JNK (p-JNK/JNK ratio) in mouse brain cortex (JWH133: 45% decrease, p < 0.05; AM630: 61% increase; p < 0.05). Basal JNK was also activated by the novel CB2 inverse agonists JTE907 (13−57%, p < 0.05) and raloxifene (31%, p < 0.05). Acute AM630 upregulated the content of pro-apoptotic mitochondrial Bax (66%, p < 0.05) in brain cortex. Acute raloxifene also increased cortical Bax content (22%, p < 0.05). In contrast, acute JTE907 did not alter the content of cortical Bax (10% increase, p > 0.05). Notably, subchronic (7 days) treatment with the selective CB2 inverse agonist AM630 did not upregulate JNK or Bax but instead it was associated with modest decreases of basal JNK (−28%, p > 0.05) and Bax content (−21%, p < 0.05) in mouse brain cortex. Conclusions: The CB2 receptor inverse agonist AM630 acutely upregulated pro-apoptotic JNK and mitochondrial Bax, which together with previous findings [1] could indicate the induction of abnormal brain cell death. Repeated AM630 treatment, however, induced neurochemical tolerance to JNK activation and Bax upregulation (less efficacy compared with the acute treatment) suggesting that the drug would not induce abnormal apoptosis in the brain in the long-term. Similarly to AM630, the CB2 ligands JTE907 and raloxifene appear to behave as somewhat weak inverse agonits modulating pro-apoptotic JNK and/or Bax in mouse brain. References ´ [1] Alvaro-Bartolom´ e, M., Salort, G., Garc´ıa-Sevilla, J.A., 2013 The CB2 ligand AM630 (6-iodopravadoline) increases the content and activity of pro-apoptotic markers in mouse brain. Eur Neuropsychopharmacol 23, Suppl. 2, S195. [2] Iwamura, H., Suzuki, H., Ueda, Y., Kaya, T., Inaba, T., 2001 In vitro and in vivo pharmacological characterization of JTE-907, a novel selective ligand for cannabinoid CB2 receptor. J Pharmacol Exp Therap 296, 420–425. [3] Kumar, P., Song, Z.-H., 2013 Identification of raloxifene as a novel CB2 inverse agonist. Biochem Biophys Res Comm 435, 76−81.
P.1.g.041 Experimental studies of the analgesic effect of pregabalin V. Kokova1 ° , E.G. Apostolova1 , L.P. Peychev1 1 Medical University, Farmacology and Drug Toxicology, Plovdiv, Bulgaria Purpose: Pregabalin is an alkylated analogue of GABA effective in the treatment of several disorders, including epilepsy, hyperalgesia, behavior disorders, generalized anxiety disorder and social phobia. Pregabalin exert antinociceptive effects in various experimental models of acute and chronic pain, reduce neuropathic pain in patients with diabetic neuropathy and post-herpetic neuralgia [1]. It is considered more effective in difficult pain management associated with stress [2]. The purpose of present study is to investigate the analgesic effect of pregabalin on acute models of pain on rats. Methods: Male Wistar rats, divided into 5 groups (n = 8), were treated intraperitoneally as follows: 1st group − with saline (control group) 0.1 ml/100 g bw; 2nd − with metamizole sodium