European Journal of Pharmacology, 144 (1987) 163-171 163 Elsevier EJP 50035 Dopamine D-2 receptors in canine brain: ionic effects on [3Hlneuroleptic binding Keith R. Jarvie a, Hyman B. Niznik 2 and Philip Seeman L • University of Toronto, Medical Sciences Building, Departments of 1 Pharmacology and 2 Medicine, Toronto, Ontario, Canada M5S 1A8 Received 27 May 1987, revised MS received 10 September 1987, accepted 15 September 1987 In order to determine the effects of monovalent cations on the binding of 3H-neuroleptics to canine striatal dopamine D-2 receptors, a study of the effects of ions on the binding of two 3H-neuroleptics from different drug classes was undertaken. Dopamine D-2 receptors are selectively labeled in a sodium-sensitive manner by the benzamide ligand [ 3H]YM-09151-2. In the presence of 120 mM NaCI, [3 H]YM-09151-2 had a dissociation constant of 55 pM and a maximal receptor density of 34 pmol/g of tissue. In the absence of NaCI, the dissociation constant was 440 pM with no change in the receptor density. The binding of [3H]YM-09151-2 (52 pM) was increased by 700% with 150 mM Na ÷, 440% with 500 mM Li + and 290% with 500 mM K +. The ion concentrations producing half-maximal increases in binding were 4 mM Na ÷, 8.5 mM Li + and 115 mM K +. The ionic strength control, N-methyl-D-gluca- mine, did not increase [3H]YM-09151-2 binding. [3H]Spiperone binding was much less affected by monovalent cations. Analysis of saturable binding showed that these changes were due to changes in binding affinity, independent of changes in receptor density. The sulfhydryl alkylating agent, N-ethylmaleimide, inactivated [3H]YM-09151-2 binding. Sodium ions at 120 mM protected approximately 40% of the susceptible sites while lithium and potassium ions (120 mM) did not. The anion exchange blocker, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS), has been shown to be capable of blocking the effect of Na + on the binding of agonists to a2-adrenoceptors. Pretreatment with DIDS had no effect on the ability of Na + to alter [3H]YM-09151-2 binding. Consistent with their actions on other benzamide ligands, monovalent cations are capable of affecting the interaction of the potent benzamide [3H]YM- 09151-2 with the canine striatal dopamine D-2 receptor. The insensitivity to N-methyl-D-glucamine and extent of stimulation by low millimolar Na + concentrations indicate that this effect is not solely due to ion strength. D-2 dopamine receptors; Ion modulation; Neuroleptics; Benzamides (substituted) 1. In~oduction The subdivision of brain dopamine receptors into two distinct categories has been supported by studies which have examined both the binding characteristics of selective ligands and the effects of agonists and antagonists on the activation and inactivation of the enzyme adenylate cyclase. As such, dopamine D-1 receptors have been defined as being those dopamine receptors which upon * To whom all correspondence should be addressed. agonist occupation stimulate adenylate cyclase (Spano et al., 1978; Kebabian and Calne, 1979) and which bind [3H]thioxanthenes (Hyttel, 1978; Left et al., 1985) and the benzazepine SCH 23390 (Hyttel, 1983; Billard et al., 1984). Conversely, dopamine D-2 receptors have been defined as being those associated with the inhibition of adenylate cyclase (McDonald et al., 1984; Onali et al., 1984) and which bind drugs of the butyrophenone and substituted benzamide classes (Leysen et al., 1978; Fleminger et al., 1983). Substituted benzamide ligands bind to dopa- mine D-2 receptors in a sodium-sensitive manner 0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)