Characterization of nicotinic receptors in immortalized hippocampal neurons

Nicotinic acetylcholine receptors, particularly nicotinic alpha-bungarotoxin (alpha-BGT) receptors, are present in relatively high concentrations in rat hippocampus. Because of the difficulties encountered in studying receptors using primary cells in culture, especially for biochemical work, we investigated the possibility of using an immortalized cell line from embryonic rat hippocampus (H19-7). RNase protection assays show that alpha 4, alpha 7 and beta 2 neuronal nicotinic receptor subunit mRNAs are present in differentiated but not undifferentiated H19-7 cells, while alpha 2, alpha 3, alpha 5 and beta 3 subunit mRNAs were not detectable under either condition. In line with these results, the present data demonstrate that the H19-7 cells express cell surface nicotinic alpha-BGT binding sites, which were maximal after seven days of differentiation in culture. The receptors were saturable, of high affinity (Kd = 1.30 nM and Bmax = 11.70 fmol/10(5) cells) and had a pharmacological profile similar to that observed for CNS alpha-BGT receptors. On the other hand, although alpha 4 and beta 2 neuronal nicotinic subunit mRNAs were present in differentiated H19-7 cells, no [3H]cytisine binding was observed. Because immortalized cell lines have the advantage that they provide a limitless supply of cells as compared to primary cell cultures, but yet are not malignant in origin, the present results may suggest that the H19-7 immortalized hippocampal cell line represent a useful CNS model system for examining alpha-BGT nicotinic receptors.