Pharmacological properties of nicotinic acetylcholine receptors on adrenal chromaffin cells and their modification by hypoxic exposure

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.