Characterization of nicotinic acetylcholine receptors on cultured bovine adrenal chromaffin cells using modified L-[3H]nicotine binding assay.
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 Ca(2+)-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 (alpha-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.
Lee
K
K
Department of Pharmacology, Kyoto University Faculty of Medicine, Japan.
Miwa
S
S
Koshimura
K
K
Ito
A
A
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002412:Cations; D002417:Cattle; D002478:Cells, Cultured; D004759:Enterochromaffin Cells / Q000737:chemistry* / Q000187:drug effects; D005725:Ganglia, Autonomic / Q000737:chemistry / Q000187:drug effects; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D009538:Nicotine / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000302:isolation & purification*
D002412:Cations; D011978:Receptors, Nicotinic; D009538:Nicotine