A comparative study of dopamine receptors in the mammalian central nervous system

Holden-Dye, Linda Mary (1986) A comparative study of dopamine receptors in the mammalian central nervous system. University of Southampton, Doctoral Thesis.

Abstract

Using the standard radioligand binding technique and the selective D2 dopamine receptor antagonist sulpiride, this study identified [³H]-sulpiride binding sites in pig striatal membranes with the characteristics of depamine receptors. These sites showed marked similarities to those identified in rat striatal membranes, both in affinity (K_{D = 9.1 nM, rat; K}_D = 6.8 nM, pig) and in pharmacological profile (r = 0.96, p < 0.001), although binding capacity in pig striata was approximately half that in rat (B_{max = 426 fmol/mg protein, rat; B}_max = 231 fmol/mg protein, pig). [³H]-sulpiride binding in both species was Na⁺ dependent, susceptible to the sulphydryl group alkylating agent N-ethylmaleimide (NEM), and insensitive to the disulphide group reducing agent dithiothreitol (DTT). Agonist affinity at the [³H]-sulpiride binding site in pig striatal membranes was decreased by 100 μM Gpp(NH)p, 20 μM NEM, heat treatment and 3 mM dithioerythritol (DTE) whilst antagonist affinity was unchanged by these treatments. The effects of Gpp(NH)p, 20 μM NEM and heat treatment were apparently non-additive. This was interpreted as indicating that these treatments may affect agonist affinity in a common fashion by disrupting the link between the site labelled by [^3H]-sulpiride, presumably a D2 receptor, and a regulatory guanyl-nucleotide binding protein. Evidence was provided to support the contention that [^3H]-sulpiride and [^3H]-spiroperidol label a common population of receptors in pig striatal membranes. There was no significant difference between the binding capacities for both ligands and there was a significant correlation between the pharmacological profiles at the two sites (r = 0.82, p < 0.05). This study describes a technique for the co-solubilisation of dopamine D1 and D2 receptors as identified by [^3H]-piflutixol and [^3H]-spiroperidol respectively, using the non-denaturing zwitterionic detergent CHAPS. Agonist affinity for both sites was decreased by the solubilisation procedure. The addition of 100 μM Gpp(NH)p further decreased the affinity of the agonist ADTM for the [³H]-spiroperidol binding site in the soluble fraction, indicating that sites labelled by [³H]-spiroperidol may be associated with a regulatory guanyl-nucleotide binding protein in the soluble fraction. The binding site labelled by [³H]-piflutixol in the soluble fraction fulfilled all the criteria for labelling a D1 receptor binding site, except that it lost its high affinity for the benzazepines SK & F38393 and SCH23390 upon solubilisation. This effect was most marked for the selective D1 antagonist SCH23390. This may reflect a conformational change induced by the solubilisation procedure or the loss of a component, essential for its high affinity, upon solubilisation. The investigations described here will advance studies aimed at characterising dopamine receptor proteins at the molecular level. (D72234/87)

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