title: Characterization of human alpha4beta2 neuronal nicotinic receptors stably expressed in SH-EP1 cells.
abstract: These studies characterized human alpha4beta2 neuronal nicotinic receptors stably expressed in a human epithelial cell line (SH-EP1). Receptors in transfected SH-EPI-halpha4beta2 cells were functional, as determined by increases in intracellular Ca2+ in response to a nicotine stimulus. Nicotine increased Fura-2 fluorescence in a concentration-dependent manner with an apparent EC50 of 2.4 microM, a response that was blocked by the specific antagonist mecamylamine. When cells were incubated in 50 nM nicotine for 24 hours, the Ca2+ response inactivated by 44%, an effect that recovered within 24 hours. SH-EP1-halpha4beta2 cells expressed a single class of high affinity binding sites for [3H]cytisine with a Kd of 0.63 +/- 0.08 nM and a Bmax of 6,797 +/- 732 femtomoles/mg protein. Incubation of cells with 50 nM nicotine for 24 hours increased the Bmax by 45% without changing affinity, a concentration-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-EPI-halpha4beta2 cells may be a good model system to study regulation of human alpha4beta2 receptors, the most abundant nicotinic receptor subtype in brain.
authors.affiliation: Department of Pharmacology and Therapeutics University of South Florida College of Medicine, Tampa 33612-4799, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Pacheco
authors.forename: T E
authors.initials: TE
authors.lastname: Pastoor
authors.forename: R J
authors.initials: RJ
authors.lastname: Lukas
authors.forename: L
authors.initials: L
authors.lastname: Wecker
mesh_terms: D000470:Alkaloids / Q000378:metabolism; D001392:Azocines; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D016257:Fura-2; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008464:Mecamylamine / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009538:Nicotine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D009994:Osmolar Concentration; D011807:Quinolizines; D011978:Receptors, Nicotinic / Q000378:metabolism*; D013997:Time Factors; D015854:Up-Regulation; D000093843:Quinolizidine Alkaloids
chemical_list: D000470:Alkaloids; D001392:Azocines; D002118:Calcium; D016257:Fura-2; D008464:Mecamylamine; D009538:Nicotine; D011807:Quinolizines; D011978:Receptors, Nicotinic; C403531:nicotinic receptor alpha4beta2; C004712:cytisine; D000093843:Quinolizidine Alkaloids