Search

The dynorphinergic system is involved in pain transmission at spinal level, where dynorphin exerts antinociceptive or pronociceptive effects, based on its opioid or non‐opioid actions. Surprisingly, little evidence is currently available concerning the supraspinal role of the dynorphinergic system in pain conditions. The present study aimed to investigate whether neuropathic pain is accompanied by prodynorphin (Pdyn) and κ‐opioid receptor (Oprk1) gene expression alterations in selected mouse brain areas. To this end, mice were subjected to chronic constriction injury of the right sciatic nerve and neuropathic pain behavioral signs were ascertained after 14 days. At this interval, a marked increase in Pdyn mRNA in the anterior cingulate cortex (ACC) and prefrontal cortex (PFC) was observed. Oprk1 gene expression was increased in the PFC, and decreased in the ACC and nucleus accumbens (NAc). No changes were observed in the other investigated regions. Because of the relationship between dynorphin and the brain‐derived neurotrophic factor, and the role of this neurotrophin in chronic pain‐related neuroplasticity, we investigated brain‐derived neurotrophic factor gene (Bdnf) expression in the areas showing Pdyn or Oprk1 mRNAs changes. Bdnf mRNA levels were increased in both the ACC and PFC, whereas no changes were assessed in the NAc. Present data indicate that the dynorphinergic system undergoes quite selective alterations involving the corticostriatal circuitry during neuropathic pain, suggesting a contribution to the negative affective component of pain. Moreover, parallel increases in Pdyn and Bdnf mRNA at cortical level suggest the occurrence of likely interactions between these systems in neuropathic pain maladaptive neuroplasticity., Dynorphinergic system undergoes selective alterations paralleled by Bdnf changes at corticostriatal level in chronic constriction injured mice.

Background Behavioral studies demonstrated that the administration of Withania somnifera Dunal roots extract (WSE), prolongs morphine-elicited analgesia and reduces the development of tolerance to the morphine’s analgesic effect; however, little is known about the underpinning molecular mechanism(s). In order to shed light on this issue in the present paper we explored whether WSE promotes alterations of μ (MOP) and nociceptin (NOP) opioid receptors gene expression in neuroblastoma SH-SY5Y cells. Methods A range of WSE concentrations was preliminarily tested to evaluate their effects on cell viability. Subsequently, the effects of 5 h exposure to WSE (0.25, 0.50 and 1.00 mg/ml), applied alone and in combination with morphine or naloxone, on MOP and NOP mRNA levels were investigated. Results Data analysis revealed that morphine decreased MOP and NOP receptor gene expression, whereas naloxone elicited their up-regulation. In addition, pre-treatment with naloxone prevented the morphine-elicited gene expression alterations. Interestingly, WSE was able to: a) alter MOP but not NOP gene expression; b) counteract, at its highest concentration, morphine-induced MOP down-regulation, and c) hamper naloxone-induced MOP and NOP up-regulation. Conclusion Present in-vitro data disclose novel evidence about the ability of WSE to influence MOP and NOP opioid receptors gene expression in SH-SY5Y cells. Moreover, our findings suggest that the in-vivo modulation of morphine-mediated analgesia by WSE could be related to the hindering of morphine-elicited opioid receptors down-regulation here observed following WSE pre-treatment at its highest concentration.

The therapeutic use of opioids is limited by the development of tolerance to the analgesic effect and the cellular and molecular mechanisms underlying this phenomenon are still not completely understood. For this reason the search for new analgesic derivatives, endowed with lower tolerance, is always an active field. The newly synthesized 14-O-Methylmorphine-6-sulfate (14-O-MeM6SU) shows high efficacy in in vitro assays and a strong analgesic action in the rat tail flick test. The aim of present work was to investigate: the analgesic effect of 14-O-MeM6SU in mouse tail-flick test; the tolerance to analgesic effect of 14-O-MeM6SU compared to morphine in mice, the effects of test compounds on glutamatergic neurotransmission by measuring spontaneous excitatory postsynaptic currents (sEPSCs) of layer V pyramidal cells from rat prefrontal cortices; and the effect of acute and chronic 14-O-MeM6SU treatments on opioid receptor gene expression in SH-SY5Y neuroblastoma cells expressing μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. 14-O-MeM6SU was 17 times more potent than morphine in analgesia and had long duration of action in analgesic dose equipotent to morphine. Mice were treated subcutaneously (s.c.) either with 200 μmol/kg morphine or with 14-O-MeM6SU (12 μmol/kg) twice daily for three days. The magnitude of tolerance or cross-tolerance indicated by the shift in antinociceptive ED50 measured was greater for morphine compared to 14-O-MeM6SU. Subsequent to behavioral testing, patch-clamp experiments in layer V pyramidal neurons of rat prefrontal cortical slices in the presence of bicuculline were performed. Both 14-O-MeM6SU (0.1 μM) and morphine (1 μM) decreased the frequency of sEPSCs, indicating reduction of glutamate release. The effect of the novel compound was reversed by the opioid receptor antagonist naloxone, indicating an opioid mediated action. In contrast, the amplitude was not affected. Finally, gene expression data showed a dose dependent down-regulation of MOP receptor after 24 h and 48 h exposure to 14-O-MeM6SU. Interestingly, no changes were detected for NOP receptor gene expression. The specific lack of this effect could be related to the lower tolerance development to analgesic effect of 14-O-MeM6SU. Furthermore, 14-O-MeM6SU displayed high intrinsic efficacy possibly an important factor in the observed effects. Further, the observed inhibition of glutamatergic signaling might be attributed also to the reduction of opioid tolerance. Based on our results the development of a new clinically important, safe analgesic agent might be possible.

Morphine is widely used for the treatment of severe acute and chronic pain, but long-term therapy rapidly leads to tolerance. Morphine effects are mediated by μ opioid receptor (MOP) activation as well as for fentanyl that, in contrast to morphine, induces less tolerance to anal- gesia. The mechanisms underlying opioid tolerance involve complex processes, such as MOP desensitization, internali- zation, and/or changes of gene expression. The develop- ment of morphine tolerance also involves adaptive changes of the anti-opioid nociceptin/orphanin FQ- nociceptin receptor system, as suggested by the reduc- tion of morphine tolerance in nociceptin opioid receptor (NOP) knockout mice. The aim of the present study was to investigate the MOP and NOP gene expression in the SH-SY5Y cells following morphine and fentanyl exposure. Results showed that cell exposure to 10 μM morphine for 5 h induced a significant decrease of MOP and NOP gene expression and that the MOP downregulation was reverted by the pretreatment with naloxone. Conversely, SH-SY5Y cells exposed to 0.1 and 1 μM fentanyl for 5 and 72 h showed a significant MOP upregulation, also reverted by naloxone pretreatment. Fentanyl induced no changes of NOP gene expression. The present findings showed a different effect by morphine and fentanyl on MOP mRNA levels that contributes to define the role of MOP gene expression changes in the mechanisms underlying the tolerance. Morphine also triggers an altered NOP-related signaling confirming that the nociceptin/orphanin FQ- nociceptin receptor system also plays a significant role in the development of morphine tolerance.

Background and Aim: Alcohol alters several neurotransmitter systems within the brain and accumulated evidences indicate the endogenous opioid system as an important target of its action. We studied, in vitro and in vivo the molecular alterations occurring in the prodynorphin gene following different exposures to alcohol. Methods: Human neuroblastoma SH-SY5Y cells were exposed to low, clearly not intoxicating, and high etha- 208 European Opioid Conference 2011, Kraków, Poland nol concentrations at different time points. Sprague Dawley rats received alcohol intragastrically trying to mimic human drinking that establishes tolerance and dependence conditions. Real-time RT-PCR was used to assess the abundance of mRNAs of interest. DNA methylation was analyzed by Methylation Specific-Real Time PCR and bisulfite-Pyrosequencing. Specific histone modifications at gene promoters were evaluated by Chromatin ImmunoPrecipitation. Results: In the cellular model we demonstrated a temporal relationship between selective chromatin modifications induced by ethanol or acetaldehyde, and changes in prodynorphin gene expression were demonstrated. In the amygdala complex of alcoholtreated rats differential changes in prodynorphin gene expression changes were observed depending on the time of exposure; consistently, we propose potential epigenetic mechanisms responsible for these alterations, at least upong short ethanol exposure. Conclusion: Our findings indicate a linkage between gene expression alterations and epigenetic modulation in prodynorphin promoter, thus adding novel information on how the opioid system can be affected by alcohol in several ways. Studies are ongoing to evaluate the chromatin remodelling in the neuroplasticity occurring in the progression of alcohol abuse. It will be also of value to study the ability of epigenetic modulators in reverting dynorphin genetic/epigenetic alterations and alcohol abuse-related behaviours. Moreover, opioid drugs already available in alcoholism treatment could also have possible epigenetic modulating properties.