Immunohistochemical study for localization of G-protein and P-glycoprotein in the inner ear

内耳 G 蛋白和 P 糖蛋白定位的免疫组织化学研究

• 批准号: 07671847
• 负责人: SAITO Takehisa
• 金额: $ 1.41万
• 依托单位: Fukui Medical School
• 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-07671847/
• 关键词: P-glycoprotein; G-protein; Immunohistochemistry; Western-blot; Inner ear; Blood-inner ear barrier; 血液-内耳関門

1.Patterns of immunostaining in the guinea pig cochlea using anti-G-protein (Gsalpha, Gialpha, Goalpha) antibodies were compared. Strongly positive staining against Gsalpha, Gialpha and Goalpha were observed in the outer hair cells, inner hair cells and stria vascularis. Immunoreactivity in the spiral ligament was light. In the cochlear nerve and spiral ganglionic fibers, immunoreactivity against Goalpha was strong but those against Gsalpha, Gialpha were light. Western blot analysis in cochlear tissue with the same antibodies used in the immunohistochemistry demonstrated a predominant 40 and 45 kDa proteins corresponding to the molecular mass of Gsalpha, 41 kDa protein for Gialpha and 39 kDa for Goalpha. From the present study, it was clarified that G-protein was abundant in the hair cells and stria vascularis and suggested that G-protein playd an important role in signal transduction of the cochlea.2.Expression of P-glycoprotein (P-gp) was detected in the guinea pig inner ear by immunohistochemical and Western immunoblot analyzes using mouse anti-P-gp monoclonal antibody C219. P-gp was found only in the capillary endothelial cells of the cochlea and vestibule. The pattern of P-gp immunostaining in the inner ear was similar to that of the brain. By Western immunoblot analysis, P-gp of 170 kDa was detected at a high level in isolated brain capillaries and at low levels in whole brain, lateral wall tissue of the cochlea, vestibular tissue and cochlear nerve in the modiolus. The present investigation suggested that P-gp expression in inner ear capillary endothelial cells might play an important role in the blood-inner ear barrier by acting as an extrusion pump.

1.比较使用抗G蛋白（Gsalpha、Gialpha、Goalpha）抗体对豚鼠耳蜗的免疫染色模式。在外毛细胞、内毛细胞和血管纹中观察到针对 Gsalpha、Gialpha 和 Goalpha 的强阳性染色。螺旋韧带的免疫反应性较弱。在耳蜗神经和螺旋神经节纤维中，针对Goalpha的免疫反应性较强，而针对Gsalpha、Gialpha的免疫反应性较弱。使用免疫组织化学中使用的相同抗体对耳蜗组织进行蛋白质印迹分析，结果显示主要的 40 和 45 kDa 蛋白质对应于 Gsalpha 的分子量，Gialpha 的分子量为 41 kDa，Goalpha 的分子量为 39 kDa。本研究阐明G蛋白在毛细胞和血管纹中含量丰富，提示G蛋白在耳蜗的信号转导中发挥重要作用。2.P-糖蛋白(P-gp)的表达使用小鼠抗 P-gp 单克隆抗体 C219 通过免疫组织化学和蛋白质免疫印迹分析在豚鼠内耳中检测到。 P-gp 仅在耳蜗和前庭的毛细血管内皮细胞中发现。内耳中 P-gp 免疫染色的模式与大脑中的相似。通过Western免疫印迹分析，在离体脑毛细血管中检测到170 kDa的P-gp高水平，在全脑、耳蜗侧壁组织、前庭组织和蜗轴耳蜗神经中检测到低水平。目前的研究表明，内耳毛细血管内皮细胞中的 P-gp 表达可能通过充当挤压泵而在血液-内耳屏障中发挥重要作用。