Effects of heavy metal cations, sulfhydryl reagents and other chemical agents on striatal D2 dopamine receptors.
To investigate aspects of the biochemical nature of the membrane-bound D2 dopamine receptor, rat striatal homogenates were pretreated with heavy metal cations and a variety of other chemical agents, and their effects on D2 receptor density were subsequently determined using a standard [3H]spiperone binding assay. Preincubation of striatal membranes in the presence of 3 mM Mn2+, Fe2+, Co2+, EDTA, or ascorbate enhanced subsequently measured stereospecific binding of [3H]spiperone compared to control (e.g. control: Bmax = 140 fmoles/mg protein, KD = 0.21 nM; Mn2+-treated: Bmax = 253 fmoles/mg protein, KD = 0.20 nM). Another group of metal cations, that is Zn2+, Cd2+, Cu2+, Hg2+ and CH3Hg+, all of which have significant -SH reactivity, as well as the -SH alkylating agent N-ethylmaleimide (NEM), caused a decrease in specific binding sites. Pretreatment with 3 mM Cd2+ or Cu2+ resulted in a 40-60% reduction in the subsequently measured stereospecific binding of [3H]spiperone, whereas 1 mM Hg2+ or 3 mM NEM completely abolished specific [3H]spiperone binding. The effect of Hg2+ 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 Mn2+ or EDTA, could not antagonize the effect of Hg2+, nor could the mercury-chelating agent DMPS, when added to crude homogenates prior to Hg2+. Ascorbate protected 25-35% of specific binding sites by virtue of its ability to reduce Hg2+ to insoluble Hg+. Only 3 mM DTE afforded complete protection against 1 mM Hg2+. Prior formation of the spiperone/receptor complex also demonstrated considerable ability to protect receptors from destruction by Hg2+. Preincubation of striatal membranes in the presence of 0.5 mM spiperone protected about 80% of sites from the subsequent addition of 1 mM Hg2+. 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 D2 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.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002104:Cadmium / Q000494:pharmacology; D002413:Cations, Divalent; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D003342:Corpus Striatum / Q000378:metabolism*; D004492:Edetic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008628:Mercury / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D008854:Microscopy, Electron; D051381:Rats; D011919:Rats, Inbred Strains; D011954:Receptors, Dopamine / Q000187:drug effects / Q000378:metabolism*; D013134:Spiperone / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D002413:Cations, Divalent; D005296:Ferrous Compounds; D008670:Metals; D011954:Receptors, Dopamine; D013439:Sulfhydryl Reagents; D002104:Cadmium; D003035:Cobalt; D008345:Manganese; D013134:Spiperone; D003300:Copper; D004492:Edetic Acid; D008628:Mercury; D005033:Ethylmaleimide; D001205:Ascorbic Acid