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

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.