Morphine modulation of the ubiquitin-proteasome complex is neuroprotective.
BACKGROUND
Over the past several decades, there is a growing need for the development of neuroprotective compounds, e.g, those that can prevent neural death. It was proposed that nitric oxide (NO), when induced by morphine, would produce neuroprotection in a human neuroblastoma cell line when tested concomitantly with compounds that produce intracellular oxidative stress and neuroinflammation.


METHODS
NO involvement in intracellular protein degradation controlled by the ubiquitin-proteasome complex was examined. Experiments were performed examining the following: a) neural cell viability and morphology; b) gene specific mRNA levels via semi-quantitative RT-PCR; c) protein levels via Western blotting; d) enzymatic activity via fluorogenic substrate-cleaving assays; and lastly, NO release via the Apollo 4000 real-time amperometric detector.


RESULTS
Morphine induces the production of NO in human neuroblastoma cells, which can be blocked by naloxone and the cNOS inhibitor L-NAME. Rotenone, which induces oxidative stress and increases the expression of the proteasomal catalytic X subunit, causes the cells to die and morphine inhibits this process via NO. Rotenone also increases the activity of the 20S proteasome, whereas morphine alone or in the presence of rotenone caused a decrease in the activity of the 20S proteasome. Morphine decreases the expression of the immunoproteasome catalytic subunit LMP7 in response to inflammatory stimulation, demonstrating that morphine's neuroprotective action does not apply to only oxidative stress. Morphine significantly increases free ubiquitin, suggesting that morphine is inducing neuroprotection by reducing the amount of oxidized proteins targeted for degradation.


CONCLUSIONS
Significant neuroprotection on the cellular and molecular levels was demonstrated and serves as a foundation for future work concerning the development of novel ligands for morphine's mu3 opiate receptor in an effort to prevent cellular death associated with neurodegenerative diseases.
Rambhia
Suraj
S
Neuroscience Research Institute, State University of New York Old Westbury, Old Westbury NY 11568, USA.
Mantione
Kirk J
KJ
Stefano
George B
GB
Cadet
Patrick
P
D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009020:Morphine / Q000494:pharmacology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009270:Naloxone / Q000494:pharmacology; D009443:Neuritis / Q000517:prevention & control; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000037:antagonists & inhibitors / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects*; D012402:Rotenone / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000187:drug effects*
D004791:Enzyme Inhibitors; D018696:Neuroprotective Agents; D025801:Ubiquitin; D012402:Rotenone; D009569:Nitric Oxide; D009270:Naloxone; D009020:Morphine; D052248:Nitric Oxide Synthase Type I; D043743:Ubiquitin-Protein Ligase Complexes; D046988:Proteasome Endopeptidase Complex; D019331:NG-Nitroarginine Methyl Ester