Huang F, Li W, Zhang B, Cui X, Han Z, Fang Z, Cai S, Yin L, Wang L
Department of Neurobiology, Institute of Gerontology and Geriatrics, Chinese PLA General Hospital and Postgraduate Medical School, Beijing 100853, China.
Chin Med J (Engl). 2001 Mar;114(3):244-7.
To investigate the effects of free radicals (FRs) and amyloid beta protein 1-40 (A beta 1-40) on the functions of expressed neurotransmitter receptors (NRs) in Xenopus oocytes.
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 degrees C +/- 1.0 degree C 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 A beta and SAFRs on the expressed glutamate receptor, HPX/XO and A beta 1-40 were added to incubation solution at 12 h, 24 h and 96 h before recording.
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 gamma-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 A beta concentrations and incubation times. A beta 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 A beta 1-40 over 24 h. Moreover, when 20 nmol/L A beta 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 A beta 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 A beta 1-40 and SAFRs. In addition, vitamin E had a partial effect against this inhibitory effect.
The results suggest that A beta 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 A beta may play a role via inhibiting receptor function in the pathophysiology of Alzheimer's disease.
研究自由基（FRs）和β淀粉样蛋白1 - 40（Aβ1 - 40）对非洲爪蟾卵母细胞中表达的神经递质受体（NRs）功能的影响。
使用Promega试剂盒从3月龄Wistar大鼠脑组织中提取总RNA和信使RNA（mRNA），并将其以每枚卵母细胞50 nl（50 ng）的量显微注射到成熟的非洲爪蟾卵母细胞（V - VI期）中。将显微注射后的卵母细胞在19.0℃±1.0℃下用改良的巴氏溶液孵育以进行受体表达，并用双电极电压钳技术记录其电流。通过含有次黄嘌呤（HPX，0.05 mol/L）和黄嘌呤氧化酶（XO，0.1 U/L）的反应体系（HPX/XO）产生超氧阴离子自由基（SAFRs）。为了观察Aβ和SAFRs对表达的谷氨酸受体的影响，在记录前12小时、24小时和96小时将HPX/XO和Aβ1 - 40添加到孵育溶液中。
结果表明，卵母细胞表达了源自大鼠脑组织的功能性NRs。这些NRs包括毒蕈碱型乙酰胆碱（mACh）、谷氨酸（Glu）、多巴胺（DA）、5 - 羟色胺（5 - HT）和γ - 氨基丁酸（GABA）。表达受体的电流特征是由氯离子携带的内向电流，其平衡电位接近 - 22 mV。不同Aβ浓度和孵育时间对大鼠脑源性表达谷氨酸受体电流的影响程度不同。浓度为20 nmol/L的Aβ1 - 40在孵育24小时内对大鼠脑源性表达谷氨酸受体的电流影响较小；但在60 nmol/L Aβ1 - 40处理24小时后，谷氨酸受体电流显著降低（降低25%，P < 0.01）。此外，当20 nmol/L Aβ1 - 40与HPX/XO反应体系产生的SAFRs共同孵育12小时以上时，发现大鼠脑源性表达谷氨酸受体的电流发生了明显变化。当卵母细胞在12小时内用60 nmol/L Aβ1 - 40和SAFRs共同处理时，谷氨酸受体电流显著降低（降低21%，P < 0.05），在60 nmol/L Aβ1 - 40和SAFRs共同处理24小时后，降低百分比达到52%。此外，维生素E对这种抑制作用有部分拮抗作用。
结果表明，Aβ对谷氨酸受体电流具有一种抑制作用，类似于自由基的作用。这些作用可被维生素E拮抗。这意味着Aβ可能在阿尔茨海默病的病理生理学中通过抑制受体功能发挥作用。
