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

1.使用抗G蛋白（Gsalpha，Gialpha，Goalpha）抗体在豚鼠耳蜗中的免疫染色模式进行了比较。在外毛细胞、内毛细胞和血管纹中观察到抗Gsalpha、Gialpha和Goalpha的强阳性染色。螺旋韧带的免疫反应较轻。在耳蜗神经和螺旋神经节纤维中，Goalpha的免疫反应较强，而Gsalpha、Gialalpha的免疫反应较弱。在耳蜗组织中用免疫组织化学中使用的相同抗体进行的Western印迹分析表明，主要的40和45 kDa蛋白对应于Gsalpha的分子量，41 kDa蛋白对应于Gialpha，39 kDa蛋白对应于Goalpha。结果表明，耳蜗毛细胞和血管纹中存在丰富的G蛋白，G蛋白在耳蜗信号转导中起重要作用。2.应用免疫组化和Western免疫印迹法检测豚鼠内耳P-糖蛋白（P-gp）的表达。P-gp仅见于耳蜗和前庭的毛细血管内皮细胞。P-gp在内耳的免疫染色模式与脑相似。通过Western免疫印迹分析，170 kDa的P-gp在分离的脑毛细血管中以高水平检测到，而在全脑、耳蜗外侧壁组织、前庭组织和蜗轴中的耳蜗神经中以低水平检测到。提示P-gp在内耳毛细血管内皮细胞中的表达可能在血-内耳屏障中起着重要的泵作用。

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.