Effects of heavy metal cations, sulfhydryl reagents and other chemical agents on striatal D 2 dopamine receptors

Abstract

To investigate aspects of the biochemical nature of the membrane-bound D 2 dopamine receptor, rat striatal homogenates were pretreated with heavy metal cations and a variety of other chemical agents, and their effects on D 2 receptor density were subsequently determined using a standard [ 3H]spiperone binding assay. Preincubation of striatal membranes in the presence of 3 mM Mn 2+, Fe 2+, Co 2+, EDTA, or ascorbate enhanced subsequently measured stereospecific binding of [ 3H]spiperone compared to control (e.g. control: B max = 140 mfoles/mg protein, K D = 0.21 nM; Mn 2+-treated: B max = 253 fmoles/mg protein, K D = 0.20 NM). Another group of metal cations, is that Zn 2+, Cd 2+, Cu 2+, Hg 2+ and CH 3Hg +, all of which have significant −SH reactivity, as well as the −SH alkylating agent N-ethylmaleimide (NEM), caused a decrease in the specific binding sites. Pretreatment with 3mM Cd 2+ or Cu 2+ resulted in a 40–60% reduction in the subesequently measured stereospecific binding of [ 3H]-spiperone, whereas 1 mM Hg 2+ or 3 mM NEM completely abolished specific [ 3Hspiperone binding. The effect of Hg 2+ could not be reversed by washing the membranes, nor by further incubation of the membranes in the presence of excess EDTA or 2,3-dimercapto-1-propanesulfonic acid (DMPS). Further incubation in the presence of 3 mM dithioerythritol (DTE) resulted in the regeneration of about 40% of lost sites. Agents which enhanced receptor density, such as Mn 2+ or EDTA, could not antagonize the effect of Hg 2+, nor could the mercury-chelating agent DMPS, when added to crude homogenates prior to Hg 2+. Ascorbate protected 25–35% of specific binding sites by virtue of its ability to reduce Hg 2+ to insoluble Hg +. Only 3 mM DTE afforded afforded complete protection against 1 mM Hg 2+. Prior formation of the spiperone/receptor complex also demonstrated considerable ability to protect receptors from destruction by Hg 2+. Preincubation of striatal membranes in the presence of 0.5 mM spiperone protected about 80% of sites from the subsequent addition of 1 mM Hg 2+. A major conclusion of these studies is that one or more free -SH groups on or adjacent to the active site may be a requirement for specific antagonist binding to the membrane-bound D 2 receptor. Occlusion of these -SH groups by sulfhydryl reagents results in partial to complete abolition of subsequently measured specific 3H-antagonist binding. Only agents which can regenerate free -SH groups, such as DTE, are able to induce any recovery in specific binding sites. Other substances, such as Mn 2+, EDTA and ascorbate which, on their own, cause an enhanced density of specific antagonist binding sites are generally ineffective in preventing the action of sulfhydryl binding agents. Their enhancing effect is probably due to the protection of receptors from normal degradation during incubation and to an improved recovery of receptor sites after centrifugation procedures.