Characterization of [3H]Vesamicol binding in rat brain preparations |
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Authors: | Edwin M Meyer Sean O Bryant Ruei-Hsin D Wang R Jason Watson |
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Institution: | (1) Department of Pharmacology and Therapeutics, University of Florida College of Medicine, P.O. Box 100267, HSC, 326100267 Gainesville, FL |
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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°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 puriffied 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 replaement 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. |
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Keywords: | Vesamicol binding primary neuronal cultures rat brain preparations |
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