| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2005: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Human Leukemic T cell lines CCRF-CEM and MOLT-3 were used.
1.Effects of small interfering RNA (siRNA) on Ca^<2+> signaling in T cells.
Transfection of anti-M_3, anti-M_5 and anti-α7 small interfering RNA significantly down-regulated respective mRNA expression, while no changes were observed in gene expression of other mAChR subtypes or nAChR subunits. Ca^<2+> signals evoked by oxotremorine-M (Oxo-M), a non-selective mAChR agonist, were reduced by anti-M_3 or anti-M_5 siRNA. Ca^<2+> signals evoked by nicotine were reduced by anti-α7 siRNA. These findings indicate that M_3, M_5 mAChR and α7 nAChR subtypes play major roles in Ca^<2+> signals to acetylcholine in T cells, and suggest that these receptors are involved in regulation of immune function.
2.Effects of anti-CD11a monoclonal antibody on lymphocytic cholinergic activity in T cells.
Anti-CD11a mAb significantly increased both the ACh content of MOLT-3 cells and its release into the culture medium, whereas simvastatin had no effect on ACh content or its release. On the other hand, simvastatin completely blocked the enhancement of ACh content and release evoked by anti-CD11a mAb.
Similarly, anti-CD11a mAb significantly up-regulated ChAT mRNA expression, whereas simvastatin had no effect on the expression of ChAT mRNA. But when added to the culture medium along with anti-CD11a mAb, simvastatin completely blocked anti-CD11a mAb-induced ChAT mRNA expression. Likewise, simvastatin significantly diminished anti-CD11a mAb-induced expression of M_5 mAChR mRNA. By contrast, neither simvastatin nor anti-CD11a mAb had any effect on expression of M_3 or M_4 mAChR mRNA.
Consistent with its effects on ChAT expression and ACh content and release, anti-CD11a mAb significantly enhanced ChAT-catalyzed ACh synthesis in MOLT-3 cells, as compared to control. And consistent with all of the results summarized above, simvastatin alone did not affect basal ChAT activity, but did abolish the anti-CD11a mAb-induced enhancement of that activity.
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