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) i n Xenopus oocytes.Methods Total RNA and messenger RNA (mRNA) was prepared from 3-month-old Wistar rat br ain tissues with Promega kits and microinjected into maturated Xenopus oocyt es (stages Ⅴ-Ⅵ) with 50 nl (50 ng) for each oocyte. The microinjec ted oocy tes were incubated with modifiedBath’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 (HPXXO) 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 e xpressed 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 ra t brain tissues. These NRs included muscarinic acetylcholine (mACh), glutamate (Glu), dopamine (DA), serotonin (5-HT) and γ-aminobutyric acid (GABA). T he c urrent characteristics of expressed receptors were inward currents carried by ch loride ion with their equibrilium potentials close to -22 mV. The extent of ef fect on the current of expressed glutamate receptor from rat brain was different among different Aβ concentrations and incubation times. Aβ(1-40) at a c oncentration of 20 nmol/L had little effect on the currents of expressed rat br ain glutamate receptors up to 24 h of incubation period; but the currents of gl utamate receptor were significantly decreased (25% off, P<0.01) in the trea tment 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 syste m of HPXXO, it was found that the currents of expressed rat bra in glutamate rec eptors had been changed markedly. When the oocytes were co-treated with 60 nm ol/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 perce ntage reached 52% over 24 h co-treatment with 60 nmol/L Aβ(1-40) and SA FRs. In addition, vitamin E had a partial effect against this inhi bitory 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 c an be antagonized by vitamin E. These imply that Aβ may play a role via inhibi ting receptor function in the pathophysiology of Alzheimer’s disease.