Screening and characterization of new regulation factor of Ca^<2+> release channels

Ca^<2>释放通道新调控因子的筛选与表征

• 批准号: 12470014
• 负责人: FURUKAWA Ken-ichi
• 金额: $ 2.75万
• 依托单位: Hirosaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470014/
• 关键词: intracellular calcium; muscle contraction; calcium release; bioactive natural products; リアノジン受容体; Ca遊離; 機械的刺激; L6細胞

1. Screening of novel bioactive substances acting on the calcium release channel.a. ZT-B, a novel bioactive substance obtained from dinoflagellate, caused a sustained contraction of the aorta in an external Ca^<2+>-dependent manner, suggesting Ca^<2+> influx from extracellular space plays an important role in the contraction. However, it was suggested that Ca^<2+>-independent fraction of the contraction was due to the Ca^<2+> release from intracellular store sites. We are now investigating the site of action of ZT-B related to the induction of Ca^<2+> release from sarcoplasmic reticulum of smooth muscle cells.b. Vitisin C, a novel plant oligostilbene from Vitis plants, dose-dependently inhibited the contractile responses of endothelium-intact rabbit thoracic aorta. These inhibitory effects were abolished in the presence of N^G-nitro-L-arginine methyl ester (L-NAME, 300 μM), a potent inhibitor of nitric oxide synthase. Vitisin C significantly enhanced the ^<45>Ca^<2+> influx, which was … More inhibited by nifedipine (10 μM), an L-type Ca^<2+> channel blocker. In the presence of SK&F96365, a receptor-operated Ca^<2+> channel blocker, the ^<45>Ca^<2+> influx induced by vitisin C was not affected. These results suggest that vitisin C evokes endothelium-dependent vasorelaxation through enhancing nitric oxide release, which was facilitated by Ca^<2+> influx into endothelial cells via nifedipine-sensitive Ca^<2+> channels.2. Intracellular Ca^<2+> oscillationPGI_2 evoked intracellular Ca^<2+> oscillation in ligament cells from patients with ossification of posterior longitudinal ligament of the spine. Some relevance of Ca^<2+> release from intracellular Ca^<2+> store site to the oscillation was suggested, because cyclopiazonic acid, a potent inhibitor for endoplasmic reticulum Ca^<2+>-pumping ATPase diminished the Ca^<2+> oscillation. Cyclic AMP is a second messenger produced by PGI_2 stimulation and an inhibitor for cAMP-dependent protein kinase markedly reduced the oscillation. The phosphorylation site would appear to be located on the regulatory protein of the Ca^<2+> release channel.3. Expression of regulatory proteins of Ca^<2+> release channelsL6 muscle cell line can differentiates into a myotube muscle cell phenotype. To assess whether mechanical stress affects the differentiation process, cyclic stretch was applied to L6 cells attached to a deformable silicon chamber and expression of proteins related to excitation-contraction coupling was investigated. Differentiation into myotube was accelerated by cyclic stretch in L6 cells than cells in static culture. Furthermore, it has been revealed that proteins related to the regulation of Ca^<2+> release channel such as calsequestrin and triadin appeared earlier than cell in static culture. There seems to be a correlation between the appearance of these proteins and the differentiation of the contractile machinery in muscle cells including Ca^<2+> signaling system. Less

1. 作用于钙释放通道的新型生物活性物质的筛选ZT-B是一种从鞭毛藻中获得的新型生物活性物质，它以外部Ca^<2+>依赖的方式引起主动脉持续收缩，表明Ca^<2+>从细胞外空间流入在收缩中起重要作用。然而，我们认为Ca^<2+>无关的部分收缩是由于Ca^<2+>从细胞内储存位点释放。我们现在正在研究ZT-B的作用部位与诱导Ca^<2+>从平滑肌细胞肌浆网释放有关。葡萄素C是一种从葡萄属植物中提取的新型植物低聚二苯乙烯，其剂量依赖性地抑制了内皮完整兔胸主动脉的收缩反应。这些抑制作用在一氧化氮合酶抑制剂N^ g -硝基- l -精氨酸甲酯（L-NAME, 300 μM）存在下被消除。Vitisin C显著增强Ca^< 45>Ca^<2+>内流，l型Ca^<2+>通道阻滞剂硝苯地平（10 μM）对其抑制作用较弱。在受体操作的Ca^<2+>通道阻滞剂SK&F96365存在时，vitisin C诱导的Ca^< 45>Ca^<2+>内流不受影响。这些结果表明，vitisc通过促进一氧化氮的释放而引起内皮依赖性血管松弛，这是由Ca^<2+>通过硝苯地平敏感的Ca^<2+>通道流入内皮细胞促进的。pgi_2可引起脊柱后纵韧带骨化患者韧带细胞内Ca^<2+>振荡。细胞内Ca^<2+>储存位点的Ca^<2+>释放与振荡有一定的相关性，因为环吡唑酸是内质网Ca^<2+>泵送atp酶的有效抑制剂，可减少Ca^<2+>振荡。环AMP是PGI_2刺激产生的第二信使，是camp依赖性蛋白激酶的抑制剂，可显著降低振荡。磷酸化位点可能位于Ca^<2+>释放通道的调节蛋白上。Ca^<2+>释放通道调节蛋白的表达使sl6肌细胞分化为肌管肌细胞表型。为了评估机械应力是否影响分化过程，我们对附着在可变形硅腔上的L6细胞进行了循环拉伸，并研究了兴奋-收缩耦合相关蛋白的表达。与静态培养相比，循环拉伸使L6细胞向肌管的分化加快。此外，在静态培养中，与Ca^<2+>释放通道调节相关的蛋白如calsequestrin和triadin出现的时间早于细胞。这些蛋白的出现似乎与肌细胞包括Ca^<2+>信号系统在内的收缩机制的分化有关。少

项目成果

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Sasamura, S., Furukawa, K.-I., Shiratori, M., Motomura, S., Ohizumi, Y.: "Antisense-Inhibition of Plasma Membrane Ca^<2+> Pump Induces Apoptosis in Vascular Smooth Muscle Cells"JAPANESE JOURNAL OF PHARMACOLOGY. 90. 164-172 (2003)

Sasamura, S.、Furukawa, K.-I.、Shiratori, M.、Motomura, S.、Ohizumi, Y.：“血浆膜 Ca^<2 > 泵的反义抑制诱导血管平滑肌细胞凋亡”日语

Sasamura, S., Furukawa, K.-I., Shiratori, M., Motomura, S., O hizumi, Y.: "Antisense-Inhibition of Plasma Membrane Ca2+ Pump Induces Apoptosis in Vascular Smooth Muscle Cells"JAPANESE JOURNAL OF PHARAMACOLOGY. 90. 164-172 (2002)

Sasamura, S.、Furukawa, K.-I.、Shiratori, M.、Motomura, S.、O hizumi, Y.：“血浆膜 Ca2 泵的反义抑制诱导血管平滑肌细胞凋亡”日本药理学杂志

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Kuniyasu, A., Kawano, S., Hirayama, Y., Ji, Y.-H., Hu, K., Ohkura, M., Furukawa, K.-I., Ohizumi, Y., Hiraoka, M., Nakayama, H.: "A new scorpion toxin (BmK-PL) stimulates Ca^<2+>-release channel activity of the skeletal-muscle ryanodine receptor by an indi

Kuniyasu, A.、Kawano, S.、Hirayama, Y.、Ji, Y.-H.、Hu, K.、Ohkura, M.、Furukawa, K.-I.、Ohizumi, Y.、Hiraoka, M.