Identification of putative high affinity nicotinic receptors on cultured cortical neurons

Primary cell cultures containing predominantly neurons or glia were prepared from fetal rat cerebral cortex. The presence of nicotinic receptor sites in neuronal cultures was indicated by the specific binding of L-[3H]nicotine to cell membrane preparations. No binding was observed with membrane preparations derived from glial cell cultures. Binding to neuronal membranes was saturable, reversible and stereoselective. Scatchard analysis revealed a single class of high affinity sites with a Kd of 3 nM and an average maximum number of binding sites of 25 fmol/mg of protein. The affinity of the sites was the same as that in adult cortical tissue, but the maximum number of sites was 25% of adult levels. The time course of binding exhibited complex kinetics that were consistent with the conversion of the sites to a high affinity state. The apparent equilibrium dissociation constant calculated from the kinetic rate constants for association (0.014 min-1 nM-1) and dissociation (0.03 min-1) was 2 nM, in good agreement with the results of equilibrium binding studies. In general, the pharmacological specificity of the sites, as judged from inhibition binding studies, was similar to that in adult brain. Nicotinic agonists were the most potent competitive inhibitors of [3H]nicotine binding and antagonists were the least effective. The D-isomer of nicotine was about 30-fold less potent than the L-isomer. The results show that cortical neurons contain high affinity nicotinic binding sites and that the properties of these sites are similar to those attributed to putative nicotinic cholinergic receptors in adult rat brain tissue.