Sachiko Todoroki

Melatonin induces γ-glutamylcysteine synthetase mediated by activator protein-1 in human vascular endothelial cells

Abstract In the present study, we show that melatonin induces the expression of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting enzyme of glutathione (GSH) synthesis, in ECV304 human vascular endothelial cells. One micromolar melatonin induced the expression of γ-GCS mRNA followed by an increase in the concentration of GSH with a peak at 24 h. An electrophoretic mobility shift assay showed that melatonin stimulates the DNA-binding activity of activator protein-1 (AP-1) as well as retinoid Z receptor/retinoid receptor-related orphan receptor α (RZR/RORα). ECV304 cells transiently transfected with a plasmid containing the γ-GCS promoter-luciferase construct showed increased luciferase activity when treated with melatonin. The melatonin-dependent luciferase activity was found in the γ-GCS promoter containing AP-1 site. The luciferase activity mediated by AP-1 was repressed in the promoter containing RZR/RORα site. In addition, cell cycle analysis showed that melatonin increases the number of cells in the G0/G1 phase; however, treatment of the cells with buthionine sulfoximine, a specific inhibitor of γ-GCS, abolished the effect of melatonin on the cell cycle, suggesting induction of cell arrest by melatonin requires GSH. As conclusion, induction of GSH synthesis by melatonin protects cells against oxidative stress and regulates cell proliferation.

Ropivacaine inhibits neurite outgrowth in PC-12 cells.

Peripheral nerve injury often leads to neuropathic pain, which might involve sympathetic postganglionic nerve fiber sprouting in the dorsal root ganglion. Recent studies suggest the effectiveness of ropivacaine in blocking neuropathic pain. To determine if ropivacaine affects sympathetic sprouting, we used pheochromocytoma (PC-12) cells, which differentiate into neurons on exposure to nerve growth factor (NGF). PC-12 cells were precultured in the presence of 50 ng/mL of NGF for 4 days. Neurite outgrowth was quantified as neurite extension after 24-, 48-, and 72-h exposure to ropivacaine at doses ranging from 10 to 200 μg/mL. Ropivacaine inhibited the neurite outgrowth in a dose-dependent manner. The inhibitory effect of ropivacaine was completely reversible because the NGF-stimulated neurite outgrowth was recovered to control level after washing out ropivacaine. Ropivacaine, therefore, may exert its therapeutic action on neuropathic pain, at least in part, by suppressing sympathetic sprouting.

Phosphatidylinositol responses are involved in the vascular effects of thiamylal and fentanyl

Although thiobarbiturates potentiate, and fentanyl attenuates peripheral vasoconstriction, the intracellular mechanism involved in this phenomenon is not clear. Because smooth muscle contraction induced by α1-adrenoceptor agonists is mediated by the phosphatidylinositol (PI) response, this study was carried out to clarify if thiamylal and fentanyl affect the norepinephrineinduced PI response in rat aortic slices. Rat aortic slices were incubated in Krebs-Henseleit solution containing 5 mM LiCl, [3H]myo-inositol, and varying concentrations of thiamylal or fentanyl. The PI response was stimulated by 0.09 μM (ED50) norepinephrine (NE). The [3H]inositoI monophosphate (IP1) was separated from [3H]myo-inositol by column chromatography and counted with a liquid scintillation counter. The basal IP1, accumulation was not affected by thiamylal and fentanyl. Norepinephrine-induced IP1 accumulation was potentiated by thiamylal at concentrations of 10 μM and 100 μM. Norepinephrine-induced IP1 accumulation was attentuated by 1 μM and 10 μM fentanyl. The results suggest that thiamylal stimulates the NE-induced PI response, which potentiates the vasoconstriction, and fentanyl attentuates NE-induced PI response, which would attenuate the vasoconstriction.RésuméOn connaît mal le mécanisme intracellulaire qui fait que le thiamylal potentialise la vasoconstriction périphérique et que le fentanyl l’atténue. Comme la constriction du muscle lisse induite par les agonistes α1-adrénergiques dépend de la réponse du phosphatidylinositol (PI), cette étude vise à vérifier sur des tranches d’aorte de rat si le thiamylal et le fentanyl affectent la réponse du PI induite par la norépinéphrine (NE). Cellesci sont incubées dans une solution de Krebs-Henseleit contenant 5 mM de LiCl, du [3H] myo-inositol et différentes concentrations de thiamylal ou de fentanyl. La réponse du PI est provoquée par 0,09 μM (ED50) de NE. Le [3H] inositol monophosphate (IP1) est séparé du [3H] myo-inositol par Chromatographie sur colonne anionique et mesuré avec un compteur à scintillation liquide. L’accumulation d’IP1 initiale n’est pas affectée par le thiamylal et le fentanyl. L’accumulation d’IP1 induite par la NE est potentialisée par le thiamylal à des concentrations de 10 μM et de 100 μM. L’accumulation d’IP1 induite par la NE est atténuée par 1 μM et 10 μM de fentanyl. Ces résultats suggèrent que le thiamylal stimule la réponse induite par la NE, laquelle potentialise la vasoconstriction, et que le fentanyl atténue la réponse de l’IP1 induite par la NE, laquelle pourrait atténuer la vasoconstriction.