title: Pharmacological properties of nicotinic acetylcholine receptors on adrenal chromaffin cells and their modification by hypoxic exposure.
abstract: To characterize the properties of nicotinic acetylcholine receptors (nAChRs) in autonomic ganglia, we examined specific 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 detection of specific binding to the membrane fraction. Presoaking the glass fiber filters in 3% or greater concentrations of polyethyleneimine solution (sixty times higher than the reported concentration) for more than 5 h could reduce the binding of L-[3H]nicotine to the filters to background level. Furthermore, specific L-[3H]nicotine binding to the membrane fraction was detected only when the membrane fraction was prepared in buffer containing no divalent cations such as Ca2+ and Mg2+ (EDTA and EGTA were added) and protease inhibitors. Specific binding of L-[3H]nicotine was saturable and reversible. Scatchard analysis revealed a single class of high affinity binding sites with an average Kd of 8.7 nM and a Bmax of 43.2 fmol/mg protein. Specific binding was sensitive to cholinergic agonists (carbamylcholine and L-nicotine) and ganglionic stimulating agents (lobeline and DMPP), but was resistant to neuromuscular blocking agents (alpha-bungarotoxin, d-tubocurarine) and hexamethonium. These results suggest that high-affinity nicotine binding sites on adrenal chromaffin cells are nAChRs of ganglion-type, which have properties different from nAChRs on the neuromuscular junction but similar to nAChRs in the brain.
authors.affiliation: Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Miwa
authors.forename: K
authors.initials: K
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Narumiya
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D015687:Cell Hypoxia; D002478:Cells, Cultured; D002838:Chromaffin System / Q000166:cytology / Q000378:metabolism*; D009538:Nicotine / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000145:classification / Q000378:metabolism*
chemical_list: D011978:Receptors, Nicotinic; D009538:Nicotine