Neurochemical,pharmacological, and developmental studies on cerebellar receptors for dicarboxylic amino acids |
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Authors: | N A Sharif P J Roberts |
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Institution: | (1) Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 21201 Baltimore, Maryland;(2) Department of Physiology and Pharmacology, Southampton University, S09 3TU Southampton, UK;(3) Present address: Department of Biochemistry, University of Nottingham, Queen's Medical Centre, NG7 2UH Nottingham, UK |
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Abstract: | Specific binding ofl-3H]glutamate (3H]Glu) andl-3H]asparate (3H]Asp) to cerebellar membranes represented a time-, temperature- pH- and protein-dependent interaction which was both saturable and reversible. Binding sites for both radioligands appeared maximally enriched in synaptosomal fractions isolated by gradient centrifugation. Kinetically derived dissociation constant (K
off/K
on=K
d) for 3H]Glu binding to this fraction indicated high-affinity (443 nM). Competition experiments employing analogs of excitatory amino acids, including new antagonists, helped identify binding sites for 3H]Glu and 3H]Asp as receptors with differential pharmacological, specificities. Membrane freezing reduced numbers of both receptor types, but binding activity could be recovered partially by incubation at 37°C. Glu receptors exhibited a pronounced deleterious sensitivity to thiol modifying reagents andl-Glu (50–1000 M) provided protection, against these compounds during co-incubation with cerebellar membranes. It is suggested that cold storage may induce partially reversible receptor inactivation by promoting sulfhydryl group/bond modification. Rat cerebellar glutamatergic function (endogenous Glu content, Glu uptake and receptor sites) exhibited an apparent ontogenetic peak between days 8–12 postpartum with a plateauing profile from day 30 to adulthood. The accelerated development (days 8–12) coincides with the first demonstrable Glu release and kainic acid neurotoxicity, as described previously. |
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