Receptor Activation is Not the Main Rescue Mechanism of Morphine in Peroxynitrite-Induced Death of Human Neuroblastoma SH-SY5Y Cells / 대한마취과학회지

BACKGROUND: In the present study, we examined the effect of morphine on NO- and peroxynitrite-induced cell death using a human neuroblastoma SH-SY5Y cell line which abundantly expresses micro, delta and K-opioid receptors. METHODS: The cultured cells were pretreated with morphine (100 micrometer) and exposed to 3-morpholinosydnonimine (SIN-1, 1mM). Agarose gel electrophoresis of DNA was done with the extracts from SH-SY5Y cells. The cells were treated with selective ligands for opioid receptor subtypes and with PI3-kinase inhibitors. Cell damage was assessed by using an MTT assay. Spectrophotometric absorption spectra were measured from the mixture of morphine (100 micrometer) plus peroxynitrite (1 mM) at room temperature. RESULTS: SIN-1 treated cells showed the occurrence of a specific form of chromosomal DNA fragmentation which pretreatment with morphine inhibited. The selective ligands for opioid receptor subtypes, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO, micro-opioid receptor agonist), [D-Pen2,5] enkephalin (DPDPE, delta-opioid receptor agonist) and U-69593 (K-opioid receptor agonist) at a concentration of 10 micrometer did not prevent the cell death induced by SIN-1. Naloxone (20 micrometer) hardly antagonized the effect of morphine in SIN-1-induced cell death. The PI3-kinase inhibitors Wortmannin and LY294002 did not inhibit the action of morphine on apoptotic cell death. In the measurements of spectrophotometric absorption spectra, the peak of the absorbance of the mixture of morphine plus peroxynitrite at 295 300 nm disappeared three minutes after mixing. CONCLUSIONS: The present study showed that morphine protected the human neuroblastoma cell line,SH-SY5Y, from peroxynitrite-induced apoptotic cell death. However, it is suggested that the protective action of morphine is not via the activation of opioid receptors and/or the PI3-kinase pathway but possibly via direct chemical reaction.

Morphine Protects Peroxynitrite-induced Cell Death in Primary Rat Neonatal Astrocytes / 대한마취과학회지

BACKGROUND: Astrocytes, representing a major non-neuronal cell population in the central nervous system (CNS), contain opioid receptors and are actively involved in several brain functions. This study is designed to evaluate the effects by which morphine contributes to cytotoxicity of nitric oxide (NO) species including NO and peroxynitrite (ONOO(-)) in primary astrocytes isolated from the cerebral cortexes of 1 - 2 day Sprague-Dawley rats. METHODS: The cultured cells were pretreated with morphine and exposed to 3-morpholinosydnonimine (SIN-1) which simultaneously generates NO and superoxide, thus possibly forming peroxynitrite. The cell damage was assessed by using an MTT (methylthizol-2-yl-2, 5-diphenyl, tetrazolium bromide) assay. Morphological nuclear changes of the cells after exposure to SIN-1 for 24 hours was evaluated by using 4', 6-diamidino-2-phenylindole (DAPI) staining. RESULTS: Morphine significantly protected primary rat astrocytes in a dose-dependent manner from the death mediated by sodium nitroprusside (SNP), a donor of nitric oxide, and SIN-1. Moreover, it was found that naloxone antagonized the protective effect of morphine on SIN-1-induced cell death, revealed as apoptosis by the occurrence of morphological nuclear changes characteristic of apoptosis. Morphine also inhibited the nuclear condensation of SIN-1-treated cells, however the action of morphine was antagonized by pretreatment of naloxone. The protective role of morphine on SIN-1-induced cytotoxicity was inhibited by DL-Buthionine-[S, R]-sulfoximine (BSO). Furthermore, the effects of morphine on SIN-1-induced cytotoxicity were blocked by pretreatment of Gi protein inhibitor, pertussis toxin, and phosphoinositide 3-kinase (PI3 kinase) inhibitors, Wortmannin and LY294002. CONCLUSIONS: These results suggest that morphine may protect primary rat astrocytes from NO species via the signaling cascades involving G-protein and PI3-kinase, and possibly regulates the anti-oxidant, glutathione (GSH).

Effects of Morphine on Free Radical-induced Apoptosis of Human Neuroblastoma SH-SY5Y Cells / 대한마취과학회지

BACKGROUND: The effect of opioids on nitric oxide (NO)- and peroxynitrite-induced neuronal cell death is largely unknown. In the present study, we examined the effect of morphine on NO- and peroxynitrite-induced cell death using a human neuroblastoma SH-SY5Y cell line, which abundantly expresses micro, delta, kappa-opioid receptors. METHODS: The cultured cells were pretreated with morphine and exposed to 3-morpholinosydnonimine (SIN-1) that simultaneously generates NO and superoxide, thus possibly forming peroxynitrite. The cell damage was assessed by using MTT assay and crystal violet staining. Morphological nuclear changes and enzymatic evidences of apoptosis of the cells after exposure to SIN-1 for 24 hours were evaluated by using 4', 6-diamidino-2-phenylindole (DAPI) staining and the measurement of pro-apoptotic protease (caspase-3) activity, respectively. Levels of reduced glutathion (GSH) were measured by monochloronimane (MCB) assay. RESULTS: Pretreatment of SH-SY5Y with morphine significantly inhibited the apoptotic cell death. Morphine also inhibited SIN-1-induced caspase-3 (pro-apoptotic protease) activity in a dose-dependent manner. However, naloxone (20 microM) could not antagonize completely the effect of morphine in SIN- 1-induced cell death. Pre-administered GSH and N-acetylcysteine (NAC) have been found to protect SIN-induced apoptosis, and the neuroblastoma cells treated with morphine had significantly elevated the levels of GSH. CONCLUSIONS: The present study shows that morphine protects the human neuroblastoma cell line SH- SY5Y from peroxynitrite-induced apoptotic cell death through elevated GSH levels. The protective actionof morphine seems to be associated with inhibition of the apoptotic pathway. However, it is suggested that morphine protects the cells possibly via other unknown mechanisms in addition to the activation of opioid receptors.