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

它收到的是，在哺乳动物类型的哺乳动物细胞中，许多激动剂激活质膜上的磷脂酶D。酶激活的机理尚未阐明。为了研究兔腹膜嗜中性粒细胞中F磷脂酶D的激活机制，重组了从大鼠脑中制备的透化的中性粒细胞和胞质醇，并在各种条件下研究了Phoshlipase d激活。 1-兔腹膜嗜中性粒细胞中的1-分子 - 烷基 - 苯基丁酰胆碱，在37°C时在[^3H] lyso lyso血小板激活因子2小时，用1 U/mL链霉菌素OAT 37 c透过20 mn的细胞以去除细胞溶质。在VAIUS条件下在1％乙醇的存在下孵育透化细胞，然后确定[^3H] phsphatidylethanl（PET），这是磷脂酶D的明确产物。当Cyto…从大鼠大脑中制备的更多溶胶被用透化细胞重组时，gtpgammas/ca^<2+>/mg-atp显着刺激了[^3H] PET的形成。在没有细胞质的情况下未观察到刺激。由于磷脂酶D位于质膜上，因此这些结果表明，磷脂酶D活化需要胞质因子（S）。[ii]磷脂酶D活化因子：由于Ca^<2+> - 很可能与磷酸酶D的磷酸酶D活化有关，这可能是ca^<2+> - 依赖性因子是否涉及磷酸脂肪酶d活化，这是否涉及中性含量属于磷酸磷脂的含量。上述重构系统中的磷脂酶D激活。通过氟替当嗪 - 毫米化亲和色谱柱色谱法制备无凸轮的细胞质。凸轮与圆柱结合，并用Egta slute洗脱。在透化的中性粒细胞中，与无凸轮细胞质的GTPGAMMAS/CA^<2+>/mg-ATP重组相比，刺激磷脂酶D的程度要比与细胞质的重建系统相比。当Cam Euted Frm用无CAM的细胞质添加到重构系统中时，GTPGAMMAS/Ca^<2+>/mg-ATP激活磷脂酶D激活。来自牛脑细胞质的尿液凸轮具有相同的效果，而从亲和力柱中洗脱了凸轮。从这些结果中可以提出，至少部分地，CAM IA参与了磷脂酶D活化作为兔腹膜嗜中性粒细胞中的胞质因子。较少的

项目成果

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。

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。

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)

H.Kanoh et al.: "Requirement of ATP and Ca^<2+> for GTP-binding protein-mediated phospholipase D activation in rat pheochromocytoma PC 12 cells." Neurosci.Lett.(in press).

H.Kanoh等人：“大鼠嗜铬细胞瘤PC 12细胞中GTP结合蛋白介导的磷脂酶D激活需要ATP和Ca 2+ ”。