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Effect of dithiothreitol on lipid peroxidation induced modification of NMDA receptor in fetal guinea pig brain
R Goel1, O P Mishra, M D Papadopoulos
1Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia 19104.
Abstract:
The present study examines if the NMDA receptor modification induced by lipid peroxidation is mediated through its redox site and is therefore reversible by dithiothreitol (DTT) by performing [3H]MK-801 binding in the fetal guinea pig brain. P2 membrane fractions were prepared from fetal guinea pig brains and were peroxidized in vitro by 100 microM ascorbate and 25 microM ferric chloride for 20 min. Control and peroxidized membranes were then incubated with 100 microM DTT for 30 min at 37 degrees C. [3H]MK-801 binding was performed in DTT treated and untreated membranes in the presence of 100 microM each of glutamate and glycine. In addition, to study the glutamate- and glycine-dependent activation, [3H]MK-801 binding was determined in the absence (basal) and presence (activated) of glutamate and glycine. Bmax (number of binding sites) and Kd (affinity) of the binding sites were used as indices of NMDA receptor modification and its reversibility by DTT. After lipid peroxidation, the Kd value increased from 4.44 +/- 0.12 in control to 10.39 +/- 1.78 nM (P < 0.01) suggesting decreased affinity following lipid peroxidation. Following treatment with DTT, there was no significant change in Kd, but Bmax 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 may have a deleterious effect by reducing the number of binding sites. However, in the control membrane DTT significantly increased the affinity (P < 0.004) and the Bmax (P < 0.01) of the NMDA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Lipid peroxidation decreases NMDA receptor affinity. Dithiothreitol (DTT) did not reverse this effect in peroxidized membranes but reduced binding sites, while improving controls.
Area of Science:
- Neuroscience
- Biochemistry
- Pharmacology
Background:
- Lipid peroxidation can alter protein function.
- NMDA receptors are crucial for synaptic plasticity.
- The redox site of NMDA receptors is a potential target for modification.
Purpose of the Study:
- To investigate if lipid peroxidation-induced NMDA receptor modification is redox-mediated.
- To determine the reversibility of these modifications by dithiothreitol (DTT).
Main Methods:
- In vitro lipid peroxidation of fetal guinea pig brain membranes.
- [3H]MK-801 binding assays to measure NMDA receptor activity.
- Assessment of Bmax and Kd values before and after DTT treatment.
Main Results:
- Lipid peroxidation significantly decreased NMDA receptor affinity (increased Kd).
- DTT treatment did not restore affinity in peroxidized membranes.
- DTT significantly reduced the number of binding sites (Bmax) in peroxidized membranes.
- DTT enhanced both affinity and Bmax in control membranes.
Conclusions:
- NMDA receptor modification by lipid peroxidation is not readily reversible by DTT.
- DTT may have detrimental effects on peroxidized NMDA receptors.
- DTT positively impacts NMDA receptor function in non-peroxidized conditions.