Mechanisms of inhibitory effects of zinc and cadmium ions on agonist binding to adenosine A1 receptors in rat brain.

The dose-dependent inhibition of zinc and cadmium ions of agonist binding to A1 adenosine receptors in rat brain is prevented by histidine and cysteine, respectively. In the present study, the possible different mechanisms of Zn2+ and Cd2+ inhibitions were examined. The effects of Zn2+ and Cd2+ on equilibrium binding parameters of the agonists N6-cyclohexyl-[2,8-3H]-adenosine ([3H]CHA) or chloro-N6-cyclopentyl-adenosine ([3H]CCPA) and the antagonist cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) were compared with those effects of reagents or binding conditions which altered histidyl or cysteinyl residues of the A1 receptor. Zn2+ pretreatment did not change A1 agonist or antagonist affinity, but did reduce the Bmax. The inhibitory effects of Zn2+ pretreatments were also maintained after several membrane washings. Diethylpyrocarbonate, a histidine-specific alkylating reagent, behaved like zinc ions: pretreatment with A1 agonist protected the histidyl residues of the [3H]CHA binding site against modification by Zn2+, while the modification of the protonation state of the nitrogen of the imidazole group of histidines by changing pH indicated that the interactions of Zn2+ with the histidyl residues were feasible with their unprotonated form. These findings suggest the formation of coordination bonds between Zn2+ and histidines critical for [3H]CHA or [3H]DPCPX binding, which may prevent the ligand interaction with the specific sites without modifying the binding kinetics of radioligand to the non-chelated recognition sites. Cd2+ pretreatment reduced the [3H]CCPA affinity, but did not modify the affinity of the antagonist [3H]DPCPX, the Bmax remaining unaffected. As with cadmium effects, the oxidation of the thiol group of cysteine by dithionitrobenzoic acid (DTNB) reduced [3H]CCPA affinity without changing the number of binding sites. The reducing reagent dithiothreitol, which alone was unable to modify [3H]CCPA binding, overcame the inhibiting effects of both Cd2+ and DTNB. These findings suggest that cadmium ions may oxidize SH groups of cysteines localized on the A1 receptor molecule or a cysteine localized in the region of G(i)alpha subunit involved in the coupling with receptors. This mechanism can justify potential conformational modifications of the receptor molecule producing the decrease in affinity.