ROLE OF PHOSHOLIPASE D IN SIGNAL TRANSDUCTION AND ACTIVATION MECHANISM IN RABBIT PERITONEAL NEUTROPHILS

磷脂酶D在兔腹膜中性粒细胞信号转导和激活机制中的作用

• 批准号: 04680186
• 负责人: KANAHO Yasunori
• 金额: $ 1.41万
• 依托单位: TOKYO INSTITUTE OF TECHNOLOGY (1993)Gifu University (1992)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04680186/
• 关键词: Phospholipase D; Neutrophils; Signal transduction; Permeabilized cells; GTP-bnding protein; Calmodulin; Cytosol; Calcium; 好中球; スタウロスポリン; 2、3ージホスホグリセリン酸; GTP結合蛋白質; 酵素放出反応; 活性酸素

It has recetly been reported that, in vaious types of mammalian cells, many agonists activates phospholipase D thrugh receptors on plasma membranes. The mechanism f the enzyme activation is nt yet been clarified. To investigate the activation mechanism f phospholipase D in rabbit peritoneal neutrophls, permeabilized neutrophils and cytosol prepared from rat brain were reconstituted and the phoshlipase D activation was investigated under various conditions.[I] Phosholipase D ctivation in reconstituted system containing permeabilized neutrohils and cytosol - After prelabelng 1-omicron-alkyl-phoshatidylcholine in rabbit peritoneal neutrophils with [^3H]lyso platelet-activating factor at 37ﾟC for 2 hr, the cells were permeabilized with 1 U/ml of streptolysin Oat 37ﾟC for 20 mn to remove cytosol. The permeabilized cells were incubated in the presence of 1% ethanol under vaius conditions, then [^3H]phsphatidylethanl (PEt), an unambiguous product of phospholipase D, were determined. When cyto … More sol prepared from rat brain were reconstituted with permeabilized cells, GTPgammaS/Ca^<2+>/Mg-ATP significantly stimulted [^3H]PEt formation. The stimulation was not observed in the absence of cytosol. Since phospholipase D locates on plasma membranes, these results suggest that phospholipase D activation requires cytosolic factor(s).[II] Phospholipase D activation factor : Since it is likely that a Ca^<2+>-dependent factor is, at least in part, involved in phospholipase D activation in rabbit peritoneal neutrophils it was investigated whether or not calmodulin (CaM) is involved in phospholipase D activation in the reconstituted system described above. CaM-free cytosol was prepared by flufenazine-immobilized affinity column chromatography. CaM bound to the column as eluted with EGTA slution. In permeabilized neutrophils reconstituted with CaM-free cytosol, GTPgammaS/Ca^<2+>/Mg-ATP stimulated phospholipase D to much less extent than in the reconstitute dsystem with cytosol. When CaM euted frm the column was added to the reconstituted system with CaM-free cytosol, phospholipase D activation by GTPgammaS/Ca^<2+>/Mg-ATP was maredly augmented. The urified CaM from bovine brain cytosl had the same effect with CaM eluted from the affinity column. From these results, it is suggested that CaM ia, at least in part, involved in phospholipase D activation as a cytosolic factor in rabbit peritoneal neutrophils. Less

近年来有报道，在各种类型的哺乳动物细胞中，许多激动剂通过质膜上的受体激活磷脂酶D。酶的激活机制尚未阐明。为探讨磷脂酶D在兔腹腔中性粒细胞中的活化机制，将大鼠脑组织制备的透化中性粒细胞和胞浆重组，并在不同条件下研究磷脂酶D的活化。[I]磷脂酶D在含有透性化嗜中性粒细胞和胞质溶胶的重建系统中的激活-在37 ℃下用[^3H]溶血血小板激活因子预标记兔腹膜中性粒细胞中的1-omicron-烷基-磷脂酰胆碱2小时后，用1 U/ml的链球菌溶血素燕麦在37 ℃下透性化细胞20分钟以除去胞质溶胶。将透化细胞在1%乙醇存在的条件下孵育，然后测定磷脂酶D的明确产物[^3H]磷脂酰乙醇（PEt）。当细胞 ...更多信息 从大鼠脑中制备溶胶与透化细胞重组，GTP γ S/Ca^2+/Mg-ATP显著刺激[^3H]PEt的形成。在不存在胞质溶胶的情况下未观察到刺激。由于磷脂酶D位于质膜上，这些结果表明磷脂酶D的激活需要胞浆因子。[II]磷脂酶D激活因子：由于Ca^<2+>依赖性因子可能至少部分参与兔腹膜中性粒细胞中磷脂酶D的激活，因此研究了钙调蛋白（CaM）是否参与上述重构系统中磷脂酶D的激活。通过氟非那嗪固定化亲和柱层析制备无钙调素的胞浆。当用EGTA洗脱时，CaM结合到柱上。在用无CaM的胞质溶胶重建的透化中性粒细胞中，GTP γ S/Ca^2+/Mg-ATP对磷脂酶D的刺激程度远低于用胞质溶胶重建的系统。当从柱中提取的CaM加入到无CaM的胞质中时，GTP γ S/Ca^<2+>/Mg-ATP对磷脂酶D的激活作用显著增强。从牛脑细胞浆中纯化的钙调素与从亲和柱上洗脱的钙调素具有相同的效果。从这些结果，它表明，CaM ia，至少部分，参与磷脂酶D激活作为一种胞质因子在兔腹膜中性粒细胞。少

项目成果

Hiroyuki Kanoh: "Pertussis toxin-insensitive G protein mediates carbachol activation of phospholipase D in rat pheochromocytoma PC12 cells" J.Neurochem.59. 1786-1794 (1992)

Hiroyuki Kanoh：“百日咳毒素不敏感 G 蛋白介导大鼠嗜铬细胞瘤 PC12 细胞中磷脂酶 D 的卡巴胆碱激活”J.Neurochem.59。

Yasunori Kanaho: "Calcium rather than protein kinase C is the major factor to activate phospholipase D in fMLP-stimulated rabbit peritoneal neutrophils" J.Immunol.149. 622-628 (1992)

Yasunori Kanaho：“在 fMLP 刺激的兔腹膜中性粒细胞中，钙而不是蛋白激酶 C 是激活磷脂酶 D 的主要因素”J.Immunol.149。

金保 安則: "実験医学" 羊土社, 7 (1993)

金安康典：《实验医学》Yodosha，7 (1993)

金保 安則、ほか: "神経研究の進歩:神経系の細胞骨格・細胞内情報伝達系" 医学書院, 532 (1992)

Yasunori Kaneyasu 等：“神经研究进展：神经系统的细胞骨架和细胞内信息传递系统” Igaku Shoin，532 (1992)

Hiroyuki Kanoh: "Requirement of adenosine5'-triosphate and Ca^<2+> for guanosine 5'-triphosphate-binding proteinmediated phospholipase D activation in rat pheochroocytoma PC12 cells" Neuros.Lett. 151. 146-149 (1993)

Hiroyuki Kanoh：“大鼠嗜铬细胞瘤PC12细胞中鸟苷5-三磷酸结合蛋白介导的磷脂酶D激活需要5-三磷酸腺苷和Ca 2+ ”Neuros.Lett。