caa a22 4500 46907941X CHVBK 20180323132945.0 cr unu---uuuuu 170328e19920401xx s 000 0 eng 10.1007/BF00176611 doi (NATIONALLICENCE)springer-10.1007/BF00176611 Characterization of nicotinic acetylcholine receptors on cultured bovine adrenal chromaffin cells using modified l -[3H]nicotine binding assay [Elektronische Daten] [Ken Lee, Soichi Miwa, Kunio Koshimura, Akihiro Ito] Summary: To characterize the properties of nicotinic acetylcholine receptors (nAChRs) in autonomic ganglia, we examined l-[3H]nicotine binding to membrane fraction prepared from cultured bovine adrenal chromaffin cells, using a modified filtration method. Binding of l-[3H]nicotine to non-treated glass fiber filters interfered with the detection of specific binding to the membrane fraction. Presoaking glass fiber filters in 3% or higher concentrations of polyethyleneimine (PEI) solution (sixty times higher than earlier used concentration) for at least 5 h could reduce the binding of l-[3H]nicotine to the filters to the background level. Specific l-[3H]nicotine binding to the membrane fraction was detected only when the membrane fraction was prepared in Ca2+- and Mg2+ (EDTA, EGTA and protease inhibitors were added)-free buffer. Specific binding of l-[3H]nicotine was saturable and reversible. Both computer program and Scatchard analysis revealed a single class of high affinity binding sites with an average Kd of 8.9 nM and a Bmax of 42.5 fmol/mg protein. The Hill coefficient was 0.98. In inhibition studies, both cholinergic agonists (carbachol and l-nicotine) and ganglionic agonists (lobeline and 1,1-dimethyl-4-phenylpiperazinium iodide) were much effective in inhibiting l-[3H]nicotine binding, whereas both neuromuscular blocking (α-bungarotoxin and d-tubocurarine) and ganglionic blocking agents were less effective. These results suggest that high affinity nicotinic binding sites on adrenal chromaffin cells are nAChRs of the ganglion-type, which have properties different from nAChRs on the neuromuscular junction but similar to nAChRs in the brain. Springer-Verlag, 1992 Chromaffin cell nationallicence l -[3H]Nicotine nationallicence Nicotinic acetylcholine receptor nationallicence Binding assay nationallicence Lee Ken Department of Pharmacology, Kyoto University Faculty of Medicine, 606, Kyoto, Japan aut Miwa Soichi Department of Pharmacology, Kyoto University Faculty of Medicine, 606, Kyoto, Japan aut Koshimura Kunio Department of Pharmacology, Kyoto University Faculty of Medicine, 606, Kyoto, Japan aut Ito Akihiro Department of Neurosurgery, Kyoto University Faculty of Medicine, 606, Kyoto, Japan aut Naunyn-Schmiedeberg's Archives of Pharmacology Springer-Verlag 345/4(1992-04-01), 363-369 0028-1298 345:4<363 1992 345 210 https://doi.org/10.1007/BF00176611 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00176611 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lee Ken Department of Pharmacology, Kyoto University Faculty of Medicine, 606, Kyoto, Japan aut NATIONALLICENCE 700 1- Miwa Soichi Department of Pharmacology, Kyoto University Faculty of Medicine, 606, Kyoto, Japan aut NATIONALLICENCE 700 1- Koshimura Kunio Department of Pharmacology, Kyoto University Faculty of Medicine, 606, Kyoto, Japan aut NATIONALLICENCE 700 1- Ito Akihiro Department of Neurosurgery, Kyoto University Faculty of Medicine, 606, Kyoto, Japan aut NATIONALLICENCE 773 0- Naunyn-Schmiedeberg's Archives of Pharmacology Springer-Verlag 345/4(1992-04-01), 363-369 0028-1298 345:4<363 1992 345 210 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer