淀粉样β蛋白和自由基对爪赡卵母细胞表达的大鼠脑谷氨酸受体功能的影响

EFFECTS OF FREE RADICALS AND AMYLOID β PROTEIN ON THE CURRENTS OF EXPRESSED RAT RECEPTORS IN XENOPUS OOCYTES

目的 :探讨淀粉样 β蛋白 (Aβ1-4 0 )和自由基对爪蟾卵母细胞表达的大鼠脑谷氨酸受体 (GluR)功能的影响。方法 :采用Promega试剂盒提取 3月龄大鼠脑组织总RNA和mRNA ,并将 5 0nl(5 0ng)的mRNA显微注射到每个爪蟾卵母细胞。注射后的卵母细胞在 (19± 1)℃条件下以改良的巴氏液孵育进行受体表达。这些表达的受体的激活电流采用双电极电压钳位技术记录。超氧阴离子自由基 (SAFRs)和Aβ1-4 0 于记录前 12h、2 4h、96h分别加入孵育液。结果 :爪赡卵母细胞可以表达出M型ACh、谷氨酸、多巴胺、GABA和 5 羟色胺受体。Aβ1-4 0 对表达的GluR有抑制效应 ,其程度依作用时间和浓度而异。 2 0nmol/LAβ1-4 0 作用 2 4h对表达的GluR功能无影响。与SAFRs共同孵育时 ,2 0nmol/LAβ1-4 0 作用 12h即对表达的GluR功能有明显影响 ,6 0nmol/LAβ1-4 0 作用 12h即对表达的GluR有明显抑制效应 (降低 2 1% ,P <0 .0 5 ) ,而 6 0nmol/LAβ1-4 0 作用 2 4h下降达 5 2 %。维生素E对这些效应有拮抗作用。结论 :自由基和Aβ对GluR具有抑制效应 ,该效应能被维生素E拮抗。

Aim: To investigate the effects of free radicals(FRs) and amyloid β protein(Aβ 1-40 ) on the functions of expressed neurotransmitter receptors(NRs) from rat brains in Xenopus oocytes. Methods: Total RNA and Messenger RNA(mRNA) was prepared from 3 month old Wistar rat brain tissues with Promega kits and microinjected into maturated Xenopus oocytes(stage Ⅴ Ⅵ) with 50 nl (50 ng) for each oocyte for receptor expression and their currents were recorded with double electrode voltage clamp technique. Superoxide anion free radicals(SAFRs) and Aβ 1-40 was added 12 h, 24 h, 96 h to incubation solution before recording. Results: The results showed that oocytes expressed mACh, glutamate, dopamine, serotonin and γ aminobutyric acid receptors. The current characteristics of these receptors were inward currents carried by chloride ion with their equibrilium potentials close to -22 mV. Aβ 1-40 and free radicals had a kind of inhibitory effect on the expressed GluR. When treated with 60 nmol/L Aβ 1-40 over 24 h, the currents of GluR significantly decreased(25% off, P <:0.01). When oocytes were co treated with 60 nmol/L Aβ 1-40 and SAFRs over a period of 12 h, the currents of glutamate receptor significantly decreased(21% of P <0.05), and the decreased percentage reached 52% over 24h co treated with 60 nmol/L Aβ 1-40 and SAFRs. Vitamin E had partial antagonistic effect against these effects. Conclusion: The result suggests that Aβ has a kind of inhibitory effects upon glutamate receptor, which is similar to those of free radicals. Their effects can be antagonized by vitamin E. This implies that Aβ may play roles via inhibiting receptor function in pathophysiology of Alzheimer's disease.