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

Abstract

Objective: 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.

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 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.

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 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.

Conclusion: 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.