Agonist Regulation of Rat 3 4 Nicotinic Acetylcholine Receptors Stably Expressed in Human Embryonic Kidney 293 Cells

Effects of agonists on rat 3 4 nicotinic acetylcholine receptors expressed in KX 3 4R2 cells [human embryonic kidney 293derived cells] were studied. The potencies of seven agonists varied over a 7000-fold range, with a rank order of epibatidine A85380 cytisine 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) nicotine acetylcholine carbachol. The efficacies of all of the agonists studied here were similar except for DMPP, which seemed to be a partial agonist compared with nicotine and acetylcholine. Nicotine and carbachol desensitized the receptors in a timeand concentration-dependent manner. The EC50 values for nicotine and carbachol to desensitize the receptors during a 60-min exposure were 3 and 51 M, respectively, indicating that these agonists are more potent at desensitizing the receptors than at activating them. The function of the receptors recovered from agonist-induced desensitization rapidly and almost completely. The half-time for recovery of function from desensitization after a 60-min treatment with nicotine increased with the concentration of nicotine used to desensitize the receptors. In contrast, no such concentration dependence for time to recovery of function was found when carbachol was used to desensitize the receptors. We propose that this difference may be due to the cell permeability of nicotine, allowing it to enter and be sequestered inside of cells and then slowly diffuse out to maintain receptor desensitization. After a 5-day exposure to 100 M nicotine, the receptors were completely desensitized, but receptor function recovered to 83% of control values with a half-time of about 10.5 min. Although the number of nicotinic receptor binding sites measured with ( )-[H]epibatidine was increased during the chronic treatment with nicotine, no increase in function was detected. Neuronal nicotinic receptors are expressed throughout the CNS and peripheral nervous system. These receptors are composed of and subunits and exist as subtypes defined by their particular subunit composition. Nine different subunits ( 2– 10) and three different subunits ( 2– 4) have been identified in vertebrates to date, indicating significant structural diversity among these receptor subtypes. The range of functional properties and pharmacological characteristics among these receptors is primarily a direct reflection of this structural diversity. One putative nicotinic receptor is the 3 4 subtype, composed of 3 and 4 subunits. This subtype or a close variant of it may be one of the major nicotinic receptors in some autonomic ganglia (Conroy and Berg, 1995; Wong et al., 1995; Poth et al., 1997), sensory ganglia (Flores et al., 1996), and adrenal gland (Campos-Caro et al., 1997), as well as in several important regions of the CNS (Mulle et al., 1991; Winzer-Serhan and Leslie, 1997; Zoli et al., 1998; Quick et