The opioid antagonist naltrexone inhibits activity and alters expression of α7 and α4β2 nicotinic receptors in hippocampal neurons: implications for smoking cessation programs
Introduction
Cigarette smoking-induced diseases, including cardiovascular disorders, chronic obstructive pulmonary disease, and certain types of malignant tumors, are among the leading causes of death worldwide (see Jacobs et al., 1999). Extensive evidence has now accumulated on the health and cost benefits of smoking cessation (see Rose, 1996, Thompson and Hunter, 1998 and references therein). However, currently available cigarette smoking cessation programs are far from being optimal, particularly because no more than 10–30% of cigarette smokers who attend these programs remain abstinent at the end of the first year (reviewed in Rose, 1996).
Nicotine is a primary cause of tobacco dependence. It provides positive reinforcement that leads to high self-administration rates, and it causes negative reinforcement in the form of withdrawal symptoms when withheld after continuous use (Thompson and Hunter, 1998). The withdrawal syndrome associated with abstinence from cigarette smoking may be accounted for by nicotine-induced up-regulation of nicotinic receptors (nAChRs) in the central nervous system (CNS) (Dani and Heinemann, 1996). Additionally, the reinforcing properties of nicotine appear to be mediated by release of dopamine and, possibly, peptide opioids in the mesolimbic system (Davenport et al., 1990, Picciotto et al., 1998). Thus, clinical trials have investigated the effectiveness of dopaminergic and opioid antagonists in smoking cessation programs (reviewed in Pomerleau, 1998, Rose, 1996).
Most clinical trials, with a few exceptions (Brauer et al., 1999, Wong et al., 1999), have indicated that naltrexone, a well known competitive narcotic antagonist used in the maintenance treatment of acute opioid intoxication, is a beneficial co-adjuvant therapy in cigarette smoking cessation programs (Hutchison et al., 1999). A clinical study using a single dose of naltrexone reported a small, albeit significant reduction in the perceived difficulty of abstaining during a 24-h period of smoking deprivation, and no changes on behavior or satisfaction from smoking in ad libitum cigarette smokers (Sutherland et al., 1995). Other clinical studies reported that scheduled administration of naltrexone has significant effects on cigarette smoking behavior (Covey et al., 1999, Lerner et al., 1998, Wewers et al., 1998). It alters: (i) latency to the first cigarette of the day, (ii) plasma nicotine levels, (iii) number of cigarettes smoked per day, and (iv) satisfaction during ad libitum smoking (Wewers et al., 1998).
The mechanisms by which naltrexone affects cigarette smoking behavior are unclear. If the only action of naltrexone was to block opioid receptors, and endogenous opioids released by nicotine were involved in aspects of smoking behavior, one would expect that treatment of cigarette smokers with naltrexone would precipitate a withdrawal syndrome. A possible alternative is that naltrexone's effects on smoking behavior are mediated by changes in function and/or expression of nAChRs in the CNS. To date, there have been no studies directed at analyzing the effects of naltrexone on neuronal nAChRs. However, naltrexone is known to interact directly with nAChRs in skeletal muscles; at sub-micromolar concentrations, it increases the function of muscle nAChRs, and at micromolar concentrations, it acts as an open-channel blocker on muscle nAChRs (Le Dain et al., 1991, Madsen and Albuquerque, 1985, Oliveira et al., 1987).
The present study was undertaken to investigate the effects of naltrexone on activity and expression of α7 and α4β2 nAChRs — the two major subtypes of nAChRs in the CNS, and to determine whether naltrexone alters nicotine-induced changes in neuronal nAChR expression. To this end, the patch–clamp technique was applied to hippocampal neurons in slices and cultures. Evidence is provided that naltrexone blocks activity and increases expression of α7 nAChRs in hippocampal neurons. Naltrexone also blocks α4β2 nAChR activity, and at concentrations that do not affect nAChR function naltrexone inhibits nicotine-induced up-regulation of non-α7 (presumably α4β2) nAChRs in hippocampal neurons. These findings further our understanding of the means by which naltrexone is an effective adjuvant in cigarette smoking cessation programs.
Section snippets
Cell cultures and slice preparation
Primary cultures of neurons harvested from the hippocampus of 16–19-day-old rat fetuses were prepared as described previously (Alkondon and Albuquerque, 1993). Experiments were performed on hippocampal neurons cultured for 7–40 days. Hippocampal slices (250 μm thickness) were obtained with a vibratome (Leica Microsystems, Wetzlar, Germany) from 15–30-day-old Sprague–Dawley rats and placed in a chamber containing artificial cerebral-spinal fluid (ACSF), which was continuously bubbled with a
Naltrexone blocks α7 nAChRs in hippocampal neurons in cultures and in slices
In cultured hippocampal neurons, acetylcholine (ACh, 300 μM; 1-s pulses) evoked whole-cell currents that were characterized as type IA currents if they decayed to the baseline within the agonist pulse (Fig. 1(A)). These rapidly decaying currents, which were recorded from ∼85% of the neurons sampled, resulted from activation of α7 nAChRs, because they were sensitive to blockade by the α7 nAChR-selective antagonist methyllycaconitine (MLA, 1 nM; data not shown) and could be evoked by the α7
Discussion
This study demonstrates that naltrexone blocks the activity and alters the expression of α7 and α4β2 nAChRs in hippocampal neurons. The mechanisms by which naltrexone exerts these effects and the relevance of these findings to the effectiveness of this opioid antagonist in smoking cessation programs are discussed below.
Acknowledgements
This work was supported by United States Public Health Science grants NS25296 and ES05730 (for E.X.A.). The authors would like to thank Mr Benjamin Cumming, Mrs Barbara Marrow, and Ms Mabel Zelle for their technical assistance.
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