title: Characterization of [3H]vesamicol binding in rat brain preparations.
abstract: The binding of (1)-[3H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P2 fraction from the rat cerebral cortex reached equilibrium within 4 min at 37 degrees C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P2 fractions from the cerebral cortex (Kd: 21 nM and 980 nM), striatum (Kd: 28 nM and 690 nM), and cerebellum (Kd: 22 nM and 833 nM). High affinity Bmax values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity Bmax values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC50: 17 nM) and efficaciously (over 90%). Both high affinity and low affinity Bmax values for [3H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by purified synaptosomes, crude membranes and P2 fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [3H]vesamicol binding. Isoosmotic replacement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [3H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville 32610-0267.
authors.forename: E M
authors.initials: EM
authors.lastname: Meyer
authors.forename: S O
authors.initials: SO
authors.lastname: Bryant
authors.forename: R H
authors.initials: RH
authors.lastname: Wang
authors.forename: R J
authors.initials: RJ
authors.lastname: Watson
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism / Q000648:ultrastructure; D003342:Corpus Striatum / Q000378:metabolism; D007038:Hypotonic Solutions; D008297:Male; D010880:Piperidines / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013572:Synaptic Vesicles / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism; D014316:Tritium
chemical_list: D007038:Hypotonic Solutions; D010880:Piperidines; D014316:Tritium; C006189:vesamicol