title: Characterization of [3H]hemicholinium-3 binding associated with neuronal choline uptake sites in rat brain membranes.
abstract: Hemicholinium-3 (HCh-3) is a potent and specific inhibitor of the high-affinity choline transport process (HAChT) localized on cholinergic neurons. In this study, the specific binding of [3H]HCh-3 (120 Ci/mmol) was characterized in crude synaptic membranes prepared from rat brain. The binding of [3H]HCh-3 to forebrain membranes was saturable, reversible and specific with an apparent Kd under optimal conditions of 35 nM and a Bmax of 56 fmol/mg protein. The potency of various HAChT inhibitors correlated with their apparent affinities for the specific [3H]HCh-3 binding site. The specific binding of [3H]HCh-3 exhibited an uneven regional distribution in the adult rat brain that corresponded to the activity of the HAChT in these regions. Transsection of the fornix, which causes a degeneration of the septal hippocampal cholinergic pathway, resulted in comparable reductions of the specific [3H]HCh-3 binding and the specific activity of choline acetyltransferase, a presynaptic marker for cholinergic terminals in the hippocampal formation; the lesion did not affect the specific activity of glutamic acid decarboxylase, a presynaptic marker for GABAergic neurons within the hippocampus. Maximal binding occurred in the presence of 200 mM NaCl: potassium, lithium, rubidium and calcium substituted poorly for sodium; and bromide, fluoride, iodide, sulfate and phosphate were less effective anions than chloride. Increasing concentrations of NaCl increased the affinity of the site for [3H]HCh-3 with no significant effect on the maximal number of sites; the enhancement of affinity was due to a selective slowing of the rate of dissociation of the ligand from its binding site. These findings indicate that [3H]HCh-3 binds to the carrier site mediating the HAChT on cholinergic neurons; thus, this radioligand may be a useful probe for investigating this presynaptic component (HAChT) of cholinergic neurons.
mesh_terms: D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D001921:Brain / Q000378:metabolism*; D002794:Choline / Q000378:metabolism*; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D006426:Hemicholinium 3 / Q000378:metabolism* / Q000494:pharmacology; D006624:Hippocampus / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D009409:Nerve Crush; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011950:Receptors, Cholinergic / Q000032:analysis* / Q000187:drug effects; D012965:Sodium Chloride / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism
chemical_list: D011950:Receptors, Cholinergic; D006426:Hemicholinium 3; D012965:Sodium Chloride; D002795:Choline O-Acetyltransferase; D002794:Choline