Improvement of 5-HT3 receptor binding assay: enhancement of specific [3H]quipazine binding with Triton X-100-treated membranes from rat cortex.
The 5-hydroxytryptamine (5-HT)3 receptor binding assay using [3H]quipazine was examined. It was impossible to obtain specific [3H]quipazine binding with the membrane fractions from rat cortex prepared by the usual procedure. When the membranes were pretreated with detergent Triton X-100, the ratio of specific [3H]quipazine binding markedly increased, depending upon the concentration of Triton X-100 in the range of 0.01-0.1% (w/v). At a concentration of more than 0.05%, the specific binding reached a maximum of 55 to 60% of the total binding. The specific [3H]quipazine binding to the Triton X-100-treated membranes was reversible and was potently inhibited by several 5-HT3 antagonists, while 5-HT1, 5-HT2 receptor antagonists and other receptor-specific ligands had no effect on the binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.62 nM and Bmax of 97 fmol/mg protein. Thus, the Triton X-100-treated membranes retained the characteristics of 5-HT3 binding sites, making it possible to use [3H]quipazine for a 5-HT3 receptor binding assay with a high ratio of specific binding.
Une
T
T
Department of Pharmacology, Dainippon Pharmaceutical Co., Ltd., Osaka, Japan.
Furukawa
K
K
Komiya
M
M
D000818:Animals; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000187:drug effects; D007700:Kinetics; D008297:Male; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011814:Quipazine / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011985:Receptors, Serotonin / Q000378:metabolism*; D014316:Tritium
D011985:Receptors, Serotonin; D014316:Tritium; D011092:Polyethylene Glycols; D011814:Quipazine; D017830:Octoxynol