Characterization of human alpha 4 beta 2 neuronal nicotinic receptors stably expressed in SH-EP1 cells

These studies characterized human alpha4 beta2 neuronal nicotinic receptors stably expressed in a human epithelial cell line (SH-EP1). Receptors in tr ansfected SH-EP1-h alpha4 beta2 cells were functional, as determined by inc reases in intracellular Ca2+ in response to a nicotine stimulus. Nicotine i ncreased Fura-2 fluorescence in a concentration-dependent manner with an ap parent EC50 of 2.4 muM, a response that was blocked by the specific antagon ist mecamylamine. When cells were incubated in 50 nM nicotine for 24 hours, the Ca2+ response inactivated by 44%, an effect that recovered within 24 h ours. SH-EP1-h alpha4 beta2 cells expressed a single class of high affinity binding sites for [H-3]cytisine with a Kd of 0.63 +/-0.08 nM and a Bmax of 6797 +/- 732 femtomoles/mg protein. Incubation of cells with 50 nM nicotin e for 24 hours increased the Bmax by 45% without changing affinity, a conce ntration-dependent effect with an EC50 of 58.6 nM. The nicotine-induced up regulation was reversible, and control values were achieved within 24 hours . Results indicate that SH-EP1-h alpha4 beta2 cells may be a good model sys tem to study regulation of human alpha4 beta2 receptors, the most abundant nicotinic receptor subtype in brain.