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

在这个项目期间，我们主要在平滑肌和心肌中进行了生理和生化实验。部分结果已发表（见附小册子中的转载内容：（1）冈田酸（OA）是蛋白磷酸酶1和2A（PP1和PP2A）的有效抑制剂，对PP2A的亲和力高于对PP1的亲和力。在具有完整质膜的平滑肌制剂中，OA可逆地抑制收缩和肌球蛋白轻链（MLC） 磷酸化。另一种 PP 抑制剂，tautomycin，其对 PP1 的亲和力高于 PP2A，已知可增强收缩和肌球蛋白轻链磷酸化。在本实验中，我们观察到 OA 强烈抑制互变霉素的这种收缩作用。添加 OA 也降低了 MLC 磷酸化，但即使添加 OA，仍有约 10% 的 MLC 被磷酸化。 OA完全抑制了收缩。这些结果表明，平滑肌收缩有几个步骤被蛋白质磷酸化抑制。(2)在从牛睫状体分离的平滑肌细胞中，我们使用全细胞钳技术研究了卡巴胆碱对膜电位和电流的影响。我们已经证明存在被毒蕈碱受体激活的非选择性阳离子通道 属于M3亚型。该通道允许Ca^<2+>进入，可能至少部分充当持续收缩所需的Ca^<2+>进入。(3)在离体豚鼠心室肌细胞中，我们研究了aCl^-通道抑制剂蒽-9-羧酸(9AC)对CFTR通道的影响。我们已经证明了其激活作用 在 9AC 存在下，CFTR 上的异丙肾上腺素或毛喉素可逆性增强并延长。我们还表明，9AC 抑制部分细胞内对硝基苯基磷酸酶活性，该活性对已知的磷酸酶抑制剂（如 OA、酒石酸或溴四咪唑）不敏感。（4）我们开发了一种新的蛋白磷酸酶测定方法 利用萤火虫生物发光的抑制剂。该方法特别适用于PP2A抑制剂的定量分析。少

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。