Biochemical studies on the dopamine receptor in the central nervous system

Senior, Katharine Ann (1984) Biochemical studies on the dopamine receptor in the central nervous system. University of Southampton, Doctoral Thesis.

Abstract

In this study two novel series of rigid dopamine analogues were tested for dopamine-like activity using three model systems. Those tetrahydronaphthalenol derivatives without amine or N-methyl group substitution were equipotent with dopamine in stimulating striatal adenylate cyclase activity and locomotor activity but less potent in displacing [³H]-sulpiride binding. This series of compounds corresponds to the α-rotameric form of dopamine. Of the isochroman series only those derivatives corresponding to the β-rotameric form of dopamine were active. Again these compounds had similar activity in behavioural and adenylate cyclase models to dopamine and less activity against [³H]-sulpiride binding. The binding of the atypical neuroleptic [³H]-sulpiride to rate striatal membranes has been well documented. It has been reported that sulpiride displaces dopaminergic ligands with different potencies in different species. In this study [³H]-sulpiride binding in rat and pig striatal tissue was compared. [³H]-sulpiride was found to bind to pig striatal tissue in a saturable and stereospecific manner, with a similar affinity to that found in rat striatal tissue. The pharmacological profile of this site was comparable in both animals as was the sodium dependency of the binding. Therefore it appears that the [³H]-sulpiride binding site in the pig striatum is similar to that in the rat. A novel dopamine antagonist zetidoline was tested for its ability to displace [³H]-sulpiride binding and was found to be equipotent in both rat and pig striata. Guanine nucleotides were shown to reduce agonist affinity for the [³H]-sulpiride binding site in pig striatal tissue. Millimolar concentrations of the sulfhydryl group alkylating agent N-ethylmaleimide (NEM) were found to inhibit sulpiride binding in rat and pig striatal membranes in a dose dependent manner whilst the disulfide bond reducing agent dithiothreitol (DTT) had no effect. Inhibition by NEM could be protected against by preincubation with dopamine agonists and antagonists which were active in displacing [³H]-sulpiride binding. Compounds which were inactive in displacing [³H]-sulpiride binding were unable to protect against the effect of NEM. This implies that a sulfhydryl group plays a role in the binding of [³H]-sulpiride. Micromolar concentrations of NEM and millimolar concentrations of DTT were found to reduce the affinity of agonists for the [³H]-sulpiride binding site in a similar manner to guanine nucleotides. These results provide further evidence that guanine nucloetide regulatory proteins are involved in regulating [³H]-sulpiride binding. Preliminary studies have shown that [³H]-NEM can be used to specifically and irreversibly label the sulpiride binding site. (D68636/86)

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