Neurochemical studies of rat glutamate and aspartate receptors

Sharif, Najam Arfeen (1981) Neurochemical studies of rat glutamate and aspartate receptors. University of Southampton, Doctoral Thesis.

Abstract

The major afferent and efferent excitatory projections in the mammalian CNS are likely to utilise the dicarboxylic amino-acids, glutamate (glu) and aspartate (asp) as transmitters. By the application of a radioreceptor labelling technique I have investigated the interaction of L-3H-glu and L-3H-asp with specific binding sites on purified cerebellar synaptic membranes. Substantial enhancement of specific 3H-glu binding was achieved by subjecting membranes to mild sonication and/or preincubation at 37°C/ 30 min. followed by rigorous washing procedures. Re-addition of postincubation supernatant to control membranes reduced binding, suggesting the possible existence of endogenous inhibitory agent(s). Binding of 3H-glu to fresh membranes was saturable and involved a single class of high affinity, (Kd = 359 nM;Bmax = 117 pmol/mg), non-interacting receptor sites with a pharmacological specificity similar to that reported for electrophysiological studies. L-3H-glu receptor binding was virtually abolished upon cold-storage of membranes and this denaturation could not be prevented by cryoprotectants. Lyophilisation, however, not only preserved but greatly augmented the binding capacity, this stability being conferred for at least one month. Specific chemical and enzymic perturbation of presumed glu-receptors and lipids on fresh cerebellar membranes greatly modified 3H-glu binding to the effect that the integrity of the glycoproteolipid complex is crucial for maintenance of binding properties. Of the purine nucleotides only the guanine derivatives specifically inhibited 3H-glu binding (CGMP > GTP > guanosine). Kinetic studies conducted in the presence of these nucleotides revealed the inhibitory mechanism to be primarily one of reducing receptor affinity without significantly altering receptor number. Tire specific and saturable interaction of L-3H-asp with cerebellar membranes was optimal at pH 7.1 and at 37°C. Eadie-Hofstee analysis of binding isotherms revealed homogeneity of binding sites, less numerous and of a lower affinity than sites that bind 3H-glu, and which displayed no co-operativity. Binding was reversible and displaced readily by putative agonists and antagonists with a profile of activity thatdiffered from glu-receptor pharmacology, and was markedly influenced by cations. Lyophilisation also afforded protection against cold-lability of 3H-asp binding, but only an increased affinity was apparent following this treatment. The effects of specific striatal lesions on 3H-glu binding; and the developmental changes in glutamatergic function accompanying maturation of rat cerebellum were investigated and are discussed.

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