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Pathogenesis of cerebral vasospasm, particularly phosphorylation of contractile and cytoskeletal proteins

脑血管痉挛的发病机制，特别是收缩蛋白和细胞骨架蛋白的磷酸化

基本信息

批准号：
06454424
负责人：
TANI Eiichi
金额：
$ 1.92万
依托单位：
Hyogo College of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454424/
关键词：
vasospasm myosin light chain calponin phosphorylation protein phosphatase 1 protein phosphatase 2A フォスファターゼ-1 フォスファターゼ-2A リン酸化ミオシン軽鎖

项目摘要

Subarachnoid hemorrhage often leads a longterm narrowing of cerebral artery called vasospasm. The present study examines two potential features of contractile system regulation in the basilar artery in vasospasm and in vasocontraction ; phosphorylation of myosin light chain (MLC) and calponin (CaP). Vasospasm was produced in the canine or rabbit basilar artery by a two-hemorrhage method. Vasocontraction was induced by local application of KCl or serotonin to the canine or rabbit basilar artery after a transclival exposure. The control animals were treated with saline instead of fresh blood. The phosphorylation of MLC was analyzed by pyrophosphate polyacrylamide gel electrophoresis (PPi PAGE), whereas that of CaP by immunoprecipitation followed by immunoblotting. Serine/threonine protein phosphatase (PP) activities were assayd with the use of [^<32>P]-labelled phosphorylase a as a substrate. The PPi PAGE showed three myosin bands in the spastic groups as well as in the KCl and serotonin … More groups, suggesting that MLC in vasospasm is phosphorylated by myosin light chain kinase (MLCK) but not by protein kinase C (PKC), because the pattern of smooth muscle myosin resolved by PPi PAGE is modified by the differential phosphorylation of MLC by MLCK and PKC ; namely, occurrence of three myosin bands in the MLCK-mediated phosphorylation and a single band in the PKC-mediated phosphorylation. In immunoprecipitation analysis, CaP was reduced in level significantly only in the spastic group. The phosphorylation of CaP on serine and threonine residues by immunoprcipitation and immunoblot analysis was increased significantly in the spastic group but not changedin the KCl and serotonin groups. Mean activities of PP1 in myofibrillar extract and PP2A in cytosolic extract were significantly decreased in the spastic group, and there was no evidence of significant changes of PP1 and PP2A in KCl and serotonin groups. Since PP1 catalyzes the dephosphorylation of MLC and CaP while PP2A catalyzes the dephosphorylation of CaP,the signisicant decrease in activities of PP1 and PP2A in vasospasm may result in continued phosphorylation of not only MLC but also CaP to increase contractility of basilar artery. In addition, the phosphorylation of CaP on tyroine residues was also significantly kincreased only in the spastic group, but its significance is unknown at present. Less

蛛网膜下腔出血常导致脑动脉长期狭窄，称为脑血管痉挛。本研究探讨了两个潜在的特点，收缩系统调节基底动脉血管痉挛和血管收缩;磷酸化的肌球蛋白轻链（MLC）和钙调蛋白（CaP）。用二次出血法在犬或兔基底动脉内造成血管痉挛。血管收缩是由局部应用KCl或5-羟色胺的犬或兔的基底动脉后translival曝光。对照组动物用生理盐水代替新鲜血液处理。MLC的磷酸化通过焦磷酸聚丙烯酰胺凝胶电泳（PPi PAGE）分析，而CaP的磷酸化通过免疫沉淀随后通过免疫印迹分析。用[~ 1 P]标记的磷酸化酶α作底物测定丝氨酸/苏氨酸蛋白磷酸酶（PP）活性<32>。PPi PAGE显示痉挛组、KCl组和5-羟色胺组肌球蛋白均为三条带 ...更多信息组，表明MLC在血管痉挛是磷酸化的肌球蛋白轻链激酶（MLCK），但不是由蛋白激酶C（PKC），因为模式的平滑肌肌球蛋白解析PPi PAGE修改MLC的MLCK和PKC的差异磷酸化;即，发生三个肌球蛋白带MLCK介导的磷酸化和一个单一的带在PKC介导的磷酸化。在免疫沉淀分析中，仅痉挛组的CaP水平显著降低。免疫沉淀和免疫印迹分析显示，痉挛组丝氨酸和苏氨酸残基上的CaP磷酸化水平显著升高，而KCl和5-羟色胺组无明显变化。肌原纤维提取物中的PP 1和胞浆提取物中的PP 2A的平均活性在痉挛组中显著降低，并且没有证据表明KCl和5-羟色胺组中的PP 1和PP 2A的显著变化。由于PP 1催化MLC和CaP的去磷酸化，而PP 2A催化CaP的去磷酸化，因此，在血管痉挛中PP 1和PP 2A活性的显著降低可能导致不仅MLC而且CaP的持续磷酸化，从而增加基底动脉的收缩力。此外，酪氨酸残基上的CaP磷酸化也仅在痉挛组显著增加，但其意义目前尚不清楚。少