Survey of regulatory roles of protein dephosphorylation process in cell motility and trans-membrane ion movements

蛋白质去磷酸化过程在细胞运动和跨膜离子运动中的调节作用调查

• 批准号: 07670052
• 负责人: TAKAI Akira
• 金额: $ 1.41万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670052/
• 关键词: protein phosphatases; protein phosphorylation; CFTR; okadaic acid; tautomycin; enzyme inhibitors; ion channel; smooth muscle; 蛋白質リン酸化

During the term of this project we carried out physiological and biochemical experiments chiefly in smooth and cardiac muscles. Part of the results have been published (see the reprint included in the pamphlet attached :(1) Okadaic acid (OA) is a potent inhibitor of protein phosphatase 1 and 2A (PP1 and PP2A) having a higher affinity to PP2A than to PP1. In smooth muscle preparations with intact plasma membrane, OA reversibly inhibits contraction and myosin light-chain (MLC) phosporylation. Another PP inhibitor, tautomycin, which exhibits a higher affinity PP1 than PP2A is known to enhance contraction and myosin light chain phosphorylation. In the present experiments we have observed that OA strongly inhibits this contractile effect of tautomycin. The MLC phosphorylation was also decreased by addition of OA but about 10% of MLC were still phosphorylated even when contraction was completely inhibited by OA.These results indicate that there are several steps toward smooth muscle contract … More ion which is suppressed by protein phosphorylation.(2) In smooth muscle cells isolated from bovine ciliary body we have studied effects of carbachol on the membrane potential and current using the whole-cell clamp technique. We have demonstrated the existence of a non-selective cation channel which is activated by muscarinic receptors belonging to the M3 subtype. This channel, which admits Ca^<2+>, may serve at least partly as a Ca^<2+> entry required for sustained contraction.(3) In isolated myocyte of guinea-pig ventricle, we have examined the effect of anthracene-9-carboxylicacid (9AC), aCl^- channel inhibitor, on the CFTR channel. We have shown that the activating effect of isoprenaline or forskolin on the CFTR is reversibly enhanced and prolonged in the presence of 9AC.We have also shown that 9AC inhibits a fraction of intracellular p-nitrophenyl phosphatase activity, which is insensitive to known phosphatase inhibitors, such as OA,tartaric acid or bromotetramisole.(4) We have developed a new assay method for protein phosphatase inhibitors which utilizes firefly bioluminescence. The methods is especially suitable for quantitative analysis of inhibitors of PP2A. Less

在这个项目的任期内，我们主要采用光滑和心脏肌肉进行了物理和生化实验。结果的一部分已经发表（请参阅所附的小册子中包含的重印：（1）冈田酸（OA）是蛋白质磷酸酶1和2A（PP1和PP2A）的潜在抑制剂，其与PP2A具有更高亲和力的蛋白质抑制剂，而不是在pp1上。已知的另一个PP抑制剂，比PP2A具有更高的亲和力PP1，可以增强收缩和肌球蛋白轻链phosporrylation。当收缩完全被OA抑制时。这些结果表明，有几个步骤，肌肉收缩……更多的离子被蛋白质磷酸化所抑制。（2）在从牛睫状体中分离出的平滑肌细胞中，我们研究了卡巴醇对膜电位的效果，并使用了整个细胞板技术的效果。属于Ca^<2+>的M3亚型。在9AC存在下，CFTR上的异丙肾上腺素或Forskolin的CFTR可逆增强和延长。我们还表明，9AC抑制了一小部分细胞内吡咯并磷酸酶的活性，这对已知的磷酸酶抑制剂不敏感，例如OA，tartaric Acid Oromotetralamisole（4）新的。使用萤火虫生物发光的抑制剂

项目成果

Bolton,T.B.(編): "Smooth Muscle Excitation" Academic Press,London, 527 (1996)

Bolton，T.B.（编辑）：“平滑肌兴奋”学术出版社，伦敦，527（1996）

Bolton, T. B. (編): "Smooth Muscle Excitation" Academic Press, London, 527 (1996)

Bolton, T. B.（编辑）：“平滑肌兴奋”学术出版社，伦敦，527 (1996)

SUGIYAMA et al: "Molecular shape analysis and activity of fautomycin,a protein phosphatase inhibitor" Bioorganic and Medicinal Chemistry Letters. 6. 3-8 (1996)

SUGIYAMA 等人：“蛋白磷酸酶抑制剂福托霉素的分子形状分析和活性”生物有机和药物化学快报。

FUKAMI et al.: "Activity of smooth muscle protein phosphatases 1 and 2A in rabbit basilar artery in vasospasm" Stroke. 26. 2321-2327 (1994)

FUKAMI 等人：“血管痉挛时兔基底动脉中平滑肌蛋白磷酸酶 1 和 2A 的活性”中风。