Chronic Nicotine Exposure Differentially Affects the Function of Human a3, a4, and a7 Neuronal Nicotinic Receptor Subtypes

Because chronic exposure to nicotine and nicotinic drugs might both activate and desensitize nicotinic acetylcholine receptors (AChRs), we sought to determine whether prolonged exposure to nicotine concentrations encountered in tobacco users differentially affects electrophysiological properties of major subtypes of human neuronal nicotinic AChRs. Xenopus laevis oocytes were injected with subunit cRNAs encoding (1) homomeric a7 AChRs, (2) heteromeric a4b2 AChRs and (3) heteromeric a3 AChRs formed from combinations of a3, b2, b4 and a5 cRNAs. Acute activation required micromolar concentrations of nicotine. Chronic exposure to submicromolar concentrations of nicotine irreversibly inactivated many a4b2 AChRs and a7 AChRs but inhibited a3 AChRs much less. Thus, although a3 AChRs are present in the brain in much smaller amounts than are a4b2 AChRs or a7 AChRs, a3 AChRs in brain and autonomic ganglia may be able to play a relatively large role in acute responses to endogenous ACh or subsequent doses of nicotine after chronic exposure to nicotine. The behavioral effects of nicotine may typically reflect the sustained inhibition of a4b2 AChRs and a7 AChRs in combination with the residual susceptibility of a3 AChRs and perhaps some other AChR subtypes for acute activation. Tolerance for nicotine exhibited by tobacco users may reflect the long-term irreversible functional inactivation of a4b2 AChRs and a7 AChRs produced by chronic exposure to nicotine. Nicotine acting at neuronal nicotinic AChRs is the primary component of tobacco that drives its habitual use (Benowitz, 1996). It has been hypothesized that smoking a cigarette results in a rapid bolus of nicotine that activates the mesolimbic dopaminergic system producing pleasure and reward, that nicotine slowly builds to a low steady concentration which causes both reversible desensitization and long-term inactivation of AChRs as well as increases in the amounts of some AChR subtypes and that smokers medicate themselves with nicotine to regulate their AChR response (Collins and Marks, 1996; Dani and Heinemann, 1996; Wonnacott et al., 1996). Nicotine and nicotinic drugs could be important in some neurological diseases because it has been shown that a substantial decrease in nicotinic AChRs is characteristic of both Alzheimer’s and Parkinson’s disease (Lange et al., 1993; Whitehouse et al., 1988). Epidemiological studies also indicate that smoking may be protective against Parkinson’s disease and, to a lesser extent, Alzheimer’s disease (Morens et al., 1995). In Parkinson’s disease, there is loss of dopamine due to the degeneration of the substantia nigra. It is known that presynaptic AChRs can modulate the release of dopamine (Wonnacott et al., 1996) and that nicotine can be neuroprotective against excitotoxicity (Akaike et al., 1994). These results suggest mechanisms by which nicotine might be protective against Parkinson’s disease and by which nicotinic drugs might be therapeutic. The effects of nicotine in several other diseases suggest that nicotinic AChRs may be involved in some way in their pathology or therapy. For example, nicotine from transdermal patches is effective in reducing the severity of Tourette’s syndrome (Dursun et al., 1994). As another example, it has been reported that a7 AChRs may be responsible for an attentional deficit that may be a predisposing genetic factor for schizophrenia, that a7 AChRs are reduced in brains of schizophrenia patients and that schizophrenia patients may smoke heavily to self-medicate with nicotine (Freedman et al., 1997). Along with the synchronized activation of AChRs by a rapid bolus of nicotine, long-term application of this agonist can lead to inactive states of these AChRs, some of which are readily reversible and others of which are not (Collins and Marks, 1996; Dani and Heinemann, 1996; Hsu et al., 1996a; Lester and Dani, 1994; Lukas, 1991). An understanding of Received for publication May 30, 1997. 1 This work was supported by grants to J.L. from the National Institutes of Health, The Smokeless Tobacco Research Council and the Muscular Dystrophy Association. 2 These two authors contributed equally to this work. ABBREVIATIONS: ACh, acetylcholine; AChR, acetylcholine receptor. 0022-3565/97/2832-0675$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 283, No. 2 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 283:675–683, 1997 675 at A PE T Jornals on N ovem er 9, 2017 jpet.asjournals.org D ow nladed from the effects of chronic exposure to nicotine on various AChR subtypes might provide better insights into mechanisms of nicotine dependence, tolerance and withdrawal and into the effects of medication with nicotine or nicotinic drugs. An AChR subtype with the subunit stoichiometry (a4)2 (b2)3 is thought to account for most of the high affinity nicotine binding in brain (Anand et al., 1991; Flores et al., 1992; Lindstrom, 1996; Wada et al., 1989). Nearly equal amounts of a subtype thought to have an (a7)5 subunit stoichiometry are found in brain (Alkondon and Albuquerque., 1993; Anand et al., 1993; Couturier et al., 1990; Del Toro et al., 1994; Lindstrom, 1996; Schoepfer et al., 1990; Seguela et al., 1993). The a7 AChRs are also often found in peripheral ganglion neurons, which also express a mixture of a3 AChR subtypes (Conroy and Berg, 1995). The a3 AChRs are found in brain, although in lower amounts than a4b2 AChRs or a7 AChRs (Wada et al., 1989). The a3 forms functional AChRs in combination with b2 or b4 subunits (Gerzanich et al., 1995; Papke, 1992), and a5 subunits assemble efficiently with both combinations (Wang et al., 1996). Presumably many subtypes of AChRs are expressed in discrete populations of neurons performing particular functional roles. Many of the AChRs in brain are thought to be located presynaptically and have been implicated in facilitating release of transmitters including ACh, dopamine, glutamate and g-aminobutyric acid (Collins and Marks, 1996; Gray et al., 1996; Lena and Changeux, 1997; McGehee and Role, 1995; Wonnacott et al., 1996). Chronic exposure to nicotine has been shown to differentially affect both the amount and function of neuronal AChR subtypes. Chicken a4b2 AChRs expressed in Xenopus laevis oocytes or a permanently transfected cell line were shown to double in amount when chronically exposed to nicotine (Peng et al., 1994). The EC50 for upregulation was 0.2 mM, essentially equal to the concentration of nicotine typically found in the serum of smokers (Benowitz et al., 1990). The upregulation was due to a decrease in the rate of destruction of these AChRs resulting from an inactive conformation of these AChRs (Peng et al., 1994). Chronic exposure to high concentrations of nicotine not only reversibly desensitized these AChRs but also permanently inactivated some of them (Peng et al., 1994). Similarly, human a4b2 AChRs in a permanently transfected cell line were upregulated by chronic nicotine exposure, but the amount of ACh-induced ion flux per AChR was decreased (Gopalakrishnan et al., 1996). The a7 AChRs and the mixture of a3 AChRs expressed by the human neuroblastoma cell line SH-SY5Y increased by 30% and 600%, respectively, in response to chronic exposure to nicotine but only when extremely high concentrations were used (Peng et al., 1997). In a comparison of the electrophysiological responses to a 48-hr exposure to nicotine of rat a4b2 AChRs expressed in X. laevis oocytes, it was found that nanomolar concentrations of nicotine eliminated most a4b2 AChR function, whereas micromolar concentrations of nicotine blocked only 50% to 60% of rat a3b2 AChR responses (Hsu et al., 1996a). This study compares both the shortand long-term effects of chronic nicotine treatment on electrophysiological function of cloned human a4b2, a3b2, a3b2a5, a3b4, a3b4a5, a3b2b4a5, and a7 subunit combinations expressed in X. laevis oocytes. It examines the concentration and time dependence of the responses of these AChR subtypes to acute activation by nicotine and both reversible desensitization and permanent inactivation caused by chronic exposure to nicotine.