Abstract
The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2.2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 ± 58 fmol/mg protein and a KD of 0.14 ± 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 μM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3–30 μM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3–30 μM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.
Footnotes
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Send reprint requests to: Dr. William A. Hewlett, Department of Psychiatry, A-2207, Vanderbilt University Medical Center, Nashville TN 37232.
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↵1 This work was supported by the Obsessive-Compulsive Disorder/Tourette Program and the Department of Psychiatry, Vanderbilt University School of Medicine (Nashville, TN). We also acknowledge the Neuropharmacology and Drug Development Program under the direction of Dr. L. Brady (Division of Neuroscience and Behavioral Science, National Institute of Mental Health) for generous support in underwriting the cost of the NOVASCREEN assays.
- Abbreviations:
- CNS
- central nervous system
- DAIZAC
- desamino-3-iodo-(S)-zacopride
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- 5-HT
- 5-hydroxytryptamine
- Received April 22, 1998.
- Accepted August 5, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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