Effects of Prolonged Nicotinic Ligand Exposure on Function of Heterologously Expressed, Human α4β2- and α4β4-Nicotinic Acetylcholine Receptors

Abstract

Effects of prolonged nicotinic ligand exposure on the function of human ␣4␤2-and ␣4␤4-nicotinic acetylcholine receptor (nAChR) subtypes were studied using receptors heterologously expressed in SH-EP1 human epithelial cells. Magnitudes of acute, nAChR-mediated, specific 86 Rb ϩ efflux responses to 1 mM carbamylcholine were reduced after pretreatment with specific nAChR ligands in effects that depended on pretreatment drug dose, duration of drug pretreatment, and duration of drug-free recovery. Fifty percent inhibition of ␣4␤2-nAChR function following 5 min of recovery occurred after 1 min of pretreatment with 1 mM nicotine but also after 1-h pretreatment at 10 nM nicotine. Seventy-five percent loss in function persisted 1 h after drug removal following 15 min or more of exposure to 1 mM nicotine. However, functional recovery was nearly complete after 1 h in drug-free medium following 1 min to 24 h pretreatment with 0.1 to 1 M nicotine, i.e., in the range of smoker plasma nicotine levels. ␣4␤4-nAChR was similarly sensitive to persistent inactivation by prolonged nicotine exposure. Carbamylcholine exhibited slightly lower persistent inactivation potency than nicotine at both ␣4␤2-and ␣4␤4-nAChR. The nAChR antagonist, mecamylamine, exhibited persistent inactivation potency and efficacy similar to nicotine at ␣4␤2-nAChR but had a reduced effect on ␣4␤4-nAChR. These studies illustrate persistent inactivation of human ␣4␤2-or ␣4␤4-nAChR induced by prolonged exposure to nicotine and show that other ligands induce nAChR persistent inactivation in a subtype-specific manner.

Withdrawal from Chronic Nicotine Administration Impairs Contextual Fear Conditioning in C57BL/6 Mice

“…Furthermore, evidence from pharmacokinetic studies of nicotine (for review, see Benowitz et al, 1989;Henningfield and Keenan, 1993;Mathieu-Kia et al, 2002) suggests that periods of abstinence (i.e., when an individual attempts to quit smoking) could cause some desensitized nAChRs to recover function. Consistent with this suggestion, Gentry et al (2003) demonstrated that desensitized nAChRs can recover function in the absence of nicotine in vitro. These receptor-level changes could account for the behavioral tolerance demonstrated by mice treated chronically with nicotine and for deficits in contextual fear conditioning demonstrated by mice withdrawn from chronic nicotine in the present study.…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…In addition to numerous studies conducted with native nAChRs, nicotine-induced upregulation has also been examined extensively in heterologous expression systems, including both Xenopus oocytes [267,268] and cultured cell lines [267,[269][270][271][272][273][274][275]. Such studies have helped to confirm that nicotineinduced upregulation is a post-transcriptional event and that it may occur by a mechanism consistent with nicotine acting as a molecular chaperone [276].…”

Regulation of the Distribution and Function of [125I]Epibatidine Binding Sites by Chronic Nicotine in Mouse Embryonic Neuronal Cultures

“…The apparent decrease in receptor function produced by chronic ligand exposure may arise from ligand-induced persistent desensitization or inactivation of nAChRs (Peng et al, 1994;Gentry et al, 2003). However, it is also possible that the population of receptors that are increased by nicotine treatments remain in intracellular compartments such that receptors at the plasma membrane are unaltered or even reduced.…”

“…Furthermore, primary neurons in culture chronically treated with nicotine up-regulate nAChRs (Bencherif et al, 1995;Nashmi et al, 2003;Lomazzo et al, 2011;Govind et al, 2012). Few studies have addressed the resulting functionality of the receptors expressed on neurons in primary culture after prolonged agonist exposure, although nAChR function has been measured after chronic nicotine exposure in cells expressing ␣4␤2-nAChRs heterologously (Peng et al, 1994;Gopalakrishnan et al, 1996;Buisson et al, 2000;Gentry et al, 2003).…”

“…Chronic nicotine exposure induces up-regulation of nAChRs in mice (Marks et al, 1983), rats (Schwartz and Kellar, 1983), transfected oocytes (Fenster et al, 1999), stably transfected fibroblasts (Peng et al, 1994(Peng et al, , 1997Bencherif et al, 1995;Warpman et al, 1998;Whiteaker et al, 1998;Gentry et al, 2003), and embryonic neurons in culture (Bencherif et al, 1995;Dá vila-García et al, 1999;Nashmi et al, 2003;Lomazzo et al, 2011;Govind et al, 2012). [ 3 H]nicotine binding sites are also increased in smokers.…”