Effects of free radicals and amyloid β protein on the currents of expressed rat receptors in Xenopus oocytes

Objective To investigate the effects of free radicals (FRs) and amyloid β protein 1 40 (Aβ 1 40 ) on the functions of expressed neurotransmitter receptors (NRs) 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 (stages Ⅴ Ⅵ) with 50?nl (50?ng) for each oocyte The microinjected oocytes were incubated with modified Bath's solution at 19 0℃±1 0℃ for receptor expression and their currents were recorded with double electrode voltage clamp technique Superoxide anion free radicals (SAFRs) were produced via a reaction system (HPX/XO) with hypoxanthine (HPX, 0 05?mol/L) and xanthine oxidase (XO, 0 1?U/L) In order to observe the effects of Aβ and SAFRs on the expressed glutamate receptor, HPX/XO and Aβ 1 40 were added to incubation solution at 12?h, 24?h and 96?h before recording Results The results showed that the oocytes expressed functional NRs originating from rat brain tissues These NRs included muscarinic acetylcholine (mACh), glutamate (Glu), dopamine (DA), serotonin (5 HT) and γ aminobutyric acid (GABA) The current characteristics of expressed receptors were inward currents carried by chloride ion with their equibrilium potentials close to -22?mV The extent of effect on the current of expressed glutamate receptor from rat brain was different among different Aβ concentrations and incubation times Aβ 1 40 at a concentration of 20?nmol/L had little effect on the currents of expressed rat brain glutamate receptors up to 24?h of incubation period; but the currents of glutamate receptor were significantly decreased (25% off, P <0 01) in the treatment of 60?nmol/L Aβ 1 40 over 24?h Moreover, when 20?nmol/L Aβ 1 40 was co incubated over 12?h with SAFRs produced by the reaction system of HPX/XO, it was found that the currents of expressed rat brain glutamate receptors had been changed markedly When the 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% off, P <0 05), and the decreased percentage reached 52% over 24?h co treatment with 60?nmol/L Aβ 1 40 and SAFRs In addition, vitamin E had a partial effect against this inhibitory effect Conclusion The results suggest that Aβ has a kind of inhibitory effect upon the current of the glutamate receptor, similar to the effects of free radicals The effects can be antagonized by vitamin E These imply that Aβ may play a role via inhibiting receptor function in the pathophysiology of Alzheimer's disease

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功能的影响。方法 采用Promega试剂盒提取 3月龄大鼠脑组织mRNA ,并将 50nl (50ng)的mRNA显微注射到成熟的每个爪蟾卵母细胞进行受体表达。表达的受体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 等在记录前的 12h、2 4h、96h分别加入孵育液。结果 注射到爪蟾卵母细胞的鼠脑mRNA可以表达出M型ACh、谷氨酸等受体 ,这些受体的激活电流特征是由氯离子载流的内向电流。Aβ1 4 0 对表达的谷氨酸受体有抑制效应 ,其程度依作用时间和浓度不同而不同。 2 0nmol/LAβ1 4 0 作用 2 4小时对表达的谷氨酸受体功能无影响 ,SAFRs共孵育时 2 0nmol/LAβ1 4 0 作用 12小时即对表达的谷氨酸受体功能有明显影响。与SAFRs共同孵育时 6 0nmol/LAβ1 4 0 作用 12小时即对表达的谷氨酸受体有明显抑制效应 (降低 2 1% ,P <0 0 5) ,而作用 2 4小时时下降达 52 %。维生素E对这些效应有拮抗作用。结论 自由基和Aβ对谷氨酸受体具有抑制效应 ,该效应能被维生素E拮抗。