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

1.比较了抗G蛋白(Gsalpha、Gipha、GoAlpha)抗体在豚鼠耳蜗内的免疫染色模式。Gsalpha、GiAlpha和GoAlpha在外毛细胞、内毛细胞和血管纹中呈强阳性染色。螺旋韧带免疫反应较弱。在耳蜗神经和螺旋神经节纤维中，对GoAlpha的免疫反应较强，而对Gsalpha和Gipha的免疫反应较弱。免疫组织化学中相同抗体的Western印迹分析表明，Gsalpha的分子质量分别为40 kDa和45 kDa，Gipha为41 kDa，GoAlpha为39 kDa。结果表明，G蛋白在毛细胞和血管纹中含量丰富，提示G蛋白在耳蜗信号转导中起重要作用。2.用小鼠抗P-gp单抗C219进行免疫组织化学和免疫印迹分析，检测到P-gp在豚鼠内耳中的表达。P-gp仅见于耳蜗部和前庭的毛细血管内皮细胞。内耳P-gp免疫组织化学染色与脑内P-gp免疫反应相似。免疫印迹分析显示，P-gp在离体脑毛细血管中呈高水平表达，在全脑、耳蜗侧壁组织、前庭组织和蜗神经中呈低水平表达。提示P-gp在内耳毛细血管内皮细胞中的表达可能通过作为一种挤出泵在血-内耳屏障中发挥重要作用。

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.