ISSN:
1432-1912
Keywords:
Chromaffin cell
;
l-[3H]Nicotine
;
Nicotinic acetylcholine receptor
;
Binding assay
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00176611
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