The mitochondrial calcium uniporter contributes to morphine tolerance through pCREB and CPEB1 in rat spinal cord dorsal horn.

BACKGROUND: The long-term use of opioid analgesics is limited by the development of unwanted side-effects, such as tolerance. The molecular mechanisms of morphine anti-nociceptive tolerance are still unclear. The mitochondrial calcium uniporter (MCU) is involved in painful hyperalgesia, but the role of MCU in morphine tolerance has not been uncharacterised. METHODS: Rats received intrathecal injection of morphine for 7 days to induce morphine tolerance. The mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. The effects of an MCU inhibitor, antisense oligodeoxynucleotide against cyclic adenosine monophosphate response element (CRE)-binding protein (CREB) or cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in morphine tolerance were examined. RESULTS: Spinal morphine tolerance was associated with an increased expression of neuronal MCU, phospho-CREB (pCREB), and CPEB1 in the spinal cord dorsal horn. MCU inhibition increased the mechanical threshold and thermal latency, and reduced the accumulation of mitochondrial calcium in morphine tolerance. Intrathecal antisense oligodeoxynucleotide against CREB or CPEB1 restored the anti-nociceptive effects of morphine compared with mismatch oligodeoxynucleotide in von Frey test and hotplate test. Chromatin immunoprecipitation with quantitative PCR assay showed that CREB knockdown reduced the interaction of pCREB with the ccdc109a gene (encoding MCU expression) promoter and decreased the MCU mRNA transcription. RNA immunoprecipitation assay suggested that CPEB1 binds to the MCU mRNA 3' untranslated region. CPEB1 knockdown decreased the expression of MCU protein. CONCLUSIONS: These findings suggest that spinal MCU is regulated by pCREB and CPEB1 in morphine tolerance, and that inhibition of MCU, pCREB, or CPEB1 may be useful in preventing the development of opioid tolerance.

MnSOD mediated by HSV vectors in the periaqueductal gray suppresses morphine withdrawal in rats.

Morphine appears to be the most active metabolite of heroin; therefore, the effects of morphine are important in understanding the ramifications of heroin abuse. Opioid physical dependence (withdrawal response) may have very long-lasting effects on the motivation for reward, including the incubation of cue-induced drug-seeking behavior. However, the exact mechanisms of morphine withdrawal (MW) are not clear yet, and its treatment remains elusive. Periaqueductal gray (PAG) is one of the important sites in the pathogenesis of MW. Here, we used recombinant herpes simplex virus (HSV) vectors that encode the sod2 gene expressing manganese superoxide dismutase (MnSOD) to evaluate its therapeutic potential in MW. Microinjection of HSV vectors expressing MnSOD into the PAG reduced the MW syndrome. MnSOD vectors suppressed the upregulated mitochondrial superoxide, and endoplasmic reticulum stress markers (glucose-related protein 78 (GRP78) and activating transcription factor 6 alpha (ATF6α)) in the PAG induced by MW. Immunostaining showed that mitochondrial superoxide, GRP78 and ATF6α were colocalized with neuronal nuclei (a neuronal-specific marker), suggesting that they are located in the neurons in the PAG. These results suggest that overexpression of MnSOD by HSV vectors may relieve opioid dependence. This study may provide a novel therapeutic approach to morphine physical withdrawal response.

IL-4 mediated by HSV vector suppresses morphine withdrawal response and decreases TNFα, NR2B, and pC/EBPß in the periaqueductal gray in rats.

Chronic opiates induce the development of physical dependence. Opioid physical dependence characterized by withdrawal symptoms, may have very long-lasting effects on the motivation for reward, including the incubation of cue-induced drug-seeking behavior. Elucidation of the mechanisms involved in physical dependence is crucial to developing more effective treatment strategies for opioid dependence. Chronic morphine induces production of proinflammatory cytokines in regional-specific sites of the brain. Interleukin-4 (IL-4) is a prototypical anti-inflammatory cytokine that globally suppresses proinflammatory cytokines. Here, we used recombinant herpes simplex virus vector S4IL4 that encode mouse il4 gene to evaluate the therapeutic potential of IL-4 in naloxone-precipitation morphine withdrawal (MW). One week after microinjection of the vector S4IL4 into the PAG LacZ or mouse IL-4 immunoreactivity in the vlPAG was visualized. ELISA assay showed that vector S4IL4 into the PAG induced the expression of IL-4. S4IL4 blunted the morphine withdrawal syndrome. S4IL4 suppressed the upregulated TNFα, NR2B and pC/EBPß in the PAG induced by MW. These results show that inhibition of proinflammatory factor in the PAG suppressed MW. This study may provide a novel therapeutic approach to morphine physical withdrawal symptoms.

Identification of small molecular compounds and fabrication of its aqueous solution by laser-ablation, expanding primordial cartilage.

OBJECTIVE: The discovery of small molecular compounds that expand cartilage is needed. We searched for small molecular compounds that expand cartilage or enhance the actions of bone morphogenetic proteins (BMPs) on cartilage. DESIGN: Metatarsal primordial cartilage explants prepared from 14.5 days postcoitum (d.p.c.) mouse embryos were organ-cultured in the presence or absence of BMPs and/or 4-(5-Benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate (BPIB) and its related molecules. The perichondrium was removed from some of the cartilage explants by partial digestion with collagenase. BPIB aqueous solution was prepared by fragmenting BPIB crystals in water with laser irradiation and then added to cartilage explants in organ culture. RESULTS: We found that small molecular compounds, BPIB, available as SB431542 from Sigma and its related molecules, expand primordial cartilage explants in organ culture. These molecules are transforming growth factor-ß (TGF-ß) inhibitors, and the addition of excess TGF-ß reduced cartilage expansion induced by these molecules. The co-administration of BPIB and BMPs synergistically expanded cartilage explants. Removal of the perichondrium abolished BIPB-induced cartilage expansion but not BMP-induced cartilage-expansion, suggesting that BPIB, but not BMPs, expands cartilage through the perichondrium. Furthermore, we used the laser-ablation technique to generate BPIB aqueous solution in the presence of 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) without the use of hazardous dimethyl sulfoxide (DMSO). The laser-ablation-generated BPIB aqueous solution was more stable, expanded cartilage explants more effectively than BPIB colloidal solution prepared with DMSO, and synergistically enhanced BMP-induced cartilage expansion. CONCLUSIONS: A small molecular compound, BPIB, expands primordial cartilage explants. A BPIB aqueous solution was created by laser-ablation without using DMSO and proved to be biologically active.