EFFECT OF DITHIOTHREITOL ON LIPID-PEROXIDATION INDUCED MODIFICATION OF NMDA RECEPTOR IN FETAL GUINEA-PIG BRAIN

The present study examines if the NMDA receptor modification induced b y lipid peroxidation is mediated through its redox site and is therefo re reversible by dithiothreitol (DTT) by performing [H-3]MK-801 bindin g in the fetal guinea pig brain. P2 membrane fractions were prepared f rom fetal guinea pig brains and were peroxidized in vitro by 100 muM a scorbate and 25 muM ferric chloride for 20 min. Control and peroxidize d membranes were then incubated with 100 muM DTT for 30 min at 37-degr ees-C. [H-3]MK-801 binding was performed in DTT treated and untreated membranes in the presence of 100 muM each of glutamate and glycine. In addition, to study the glutamate- and glycine-dependent activation, [ H-3]MK-801 binding was determined in the absence (basal) and presence (activated) of glutamate and glycine. B(max) (number of binding sites) and K(d) (affinity) of the binding sites were used as indices of NMDA receptor modification and its reversibility by DTT. After lipid perox idation, the K(d) value increased from 4.44 +/- 0.12 in control to 10. 39 +/- 1.78 nM (P < 0.01) suggesting decreased affinity following lipi d peroxidation. Following treatment with DTT, there was no significant change in K(d), but B(max) was significantly (P < 0.007) decreased in the peroxidized membrane. This suggests that DTT did not improve the affinity of the NMDA receptor of the lipid peroxidized membrane but ma y have a deleterious effect by reducing the number of binding sites. H owever, in the control membrane DTT significantly increased the affini ty (P < 0.004) and the B(max) (P < 0.01) of the NMDA receptors. Lipid peroxidation decreased the glutamate- and glycine-dependent activation by 65% as compared to control. Treatment with DTT further decreased t he activation by 50% in the peroxidized membrane, while increased by 6 0% in the control membrane which may be due to modification of the glu tamate and glycine sites or the ion channel properties of the NMDA rec eptor by DTT. The data suggest that the modification of the NMDA recep tor and its modulatory sites by lipid peroxidation is a complex proces s, is not reversible by DTT and is therefore not mediated through its redox site. We conclude that DTT further modifies the NMDA receptor an d its modulatory sites and/or its ion channel in the lipid peroxidized membrane.