Sensitivity of neuronal nicotinic acetylcholine receptors to the opiate antagonists naltrexone and naloxone: receptor blockade and up-regulation

doi:10.1016/j.bmcl.2004.01.004

Bioorganic & Medicinal Chemistry Letters

Volume 14, Issue 8, 19 April 2004, Pages 1879-1887

https://doi.org/10.1016/j.bmcl.2004.01.004 Get rights and content

Abstract

In HEK293 cells stably expressing α4β2 nAChRs, naltrexone, but not naloxone, blocked α4β2 nAChRs via an open-channel blocking mechanism. In primary hippocampal cultures, naltrexone inhibited α7 nAChRs up-regulated by nicotine, and in organotypic hippocampal cultures naltrexone caused a time-dependent up-regulation of functional α7 nAChRs that was detected after removal of the drug. These results indicate that naltrexone could be used as a smoking cessation aid.

Introduction

Drug addiction is an illness where drug-seeking behavior dominates the motivation of an individual. The pharmacological actions of different drugs in the central nervous system (CNS) remain the major determinants of addiction. Numerous studies have demonstrated that the addictive properties of many drugs are primarily the result of changes in the rewarding pathways and memory circuits in the brain.¹

Prototypic addictive drugs are diverse chemicals, including opiates (e.g., morphine and heroin) and nicotine, which interact with distinct receptors in the brain. For instance, the pharmacological effects of opioids are derived from their complex interactions with three opioid receptor types, μ, δ and κ.² Likewise, the neurological effects of nicotine, the psychoactive substance in tobacco, arise from its interactions with various nicotinic receptor (nAChR) subtypes in the brain, particularly the α7 and the α4β2 nAChRs.³ However, the rewarding effects of all of these drugs result from a common change in the CNS, that is, a significant increase in the mesolimbic dopaminergic activity.⁴ The craving-seeking behavior induced by addictive drugs, on the other hand, appears to be due to multiple alterations in memory circuits in the prefrontal cortex, amygdala, hippocampus and dorsal striatum, all of which receive innervation from the mesolimbic dopaminergic system.⁵

The chemical and neuronal networks in the brain allow for considerable reciprocal interactions among the various neurotransmitter systems. Consequently, drugs acting on neuromodulatory systems, such as the cholinergic and the opioid systems, modify each other's effects. For instance, the opioid agonist heroin is known to increase the number of cigarettes smoked⁶ and nicotine can block morphine-induced analgesia.⁷ This potential interaction between the nicotinic and opioid systems and the success and substantial safety of opioid antagonists in the treatment of opioid addiction led to clinical trials designed to investigate the effects of the opioid antagonists naltrexone and naloxone on smoking behavior.

Unfortunately, results from these trials were not consistently positive. For example, Karras and Kane⁸ showed a positive correlation between a single subcutaneous naloxone dose and smoking reduction. In contrast, Nemeth-Coslett and Griffiths⁹ reported that intramuscular injections of naloxone had no effect in smoking. The different naloxone administration routes could have explained the opposite outcomes. Naltrexone has been more thoroughly investigated in cigarette smoking cessation programs. Three double-blind and placebo controlled studies10, 11, 12 compared nicotine abstinence after naltrexone administration. Wewers and colleagues¹⁰ showed that a number of indicators of nicotine addiction were significantly reduced by naltrexone. Others11, 12 reported that naltrexone did not influence cigarette consumption or withdrawal symptoms after tobacco abstinence. Two other studies investigated co-administration of naltrexone and nicotine replacement therapy.13, 14 Both groups concluded that naltrexone influenced the efficacy of nicotine replacement therapy. However, Hutchison and colleagues¹³ reported that concomitant nicotine skin patch and oral administration of naltrexone were additive in reducing smoking urge and decreasing nicotine withdrawal, whereas Brauer et al.¹⁴ found a negative association between naltrexone and skin nicotine patches. Differences in naltrexone dosages could have accounted for the discrepant results.

It is not clear that the usefulness of naltrexone or naloxone in the treatment of nicotine addiction results exclusively from physiological interactions between the opioid and nicotinic cholinergic systems in the brain. Earlier studies have demonstrated that naltrexone can interact directly with muscle nAChRs.15, 16 Recent studies from this laboratory also reported that naltrexone acts as a non-competitive antagonist of α7 nAChRs and causes up-regulation of these receptors in primary hippocampal cultures.¹⁷ The present study was designed to examine the effects of naltrexone and naloxone on α4β2 nAChRs, the most prevalent neuronal nAChR in the brain, and to analyze the time-dependent effects of naltrexone on α7 nAChR activity in hippocampal neurons in primary and organotypic cultures.

Section snippets

Characterization of α4β2 nAChRs stably transfected into HEK 293 cells

HEK 293 cells stably expressing α4β2 nAChRs were cultured onto polylysine-coated coverslips and used 2–5 days after plating.¹⁸ Agonist-evoked currents were recorded from either isolated cells or small cell aggregates under the whole-cell mode of the patch-clamp technique.¹⁹ Agonist application to cell aggregates resulted in currents that had larger amplitudes than those recorded from isolated cells, thus facilitating measurements of current amplitudes particularly at low agonist concentrations.

Naltrexone and naloxone effects on α4β2 nAChRs

A previous study carried out in cultured hippocampal neurons demonstrated that naltrexone blocks non- competitively α7 nAChRs with an IC₅₀ of 30 μM.¹⁷ That same study indicated that higher concentrations of naltrexone were needed to block α4β2 nAChRs in hippocampal neurons.¹⁷ Here, the mechanism of inhibition of α4β2 nAChRs by naltrexone was determined using the HEK293 cells. In these experiments, nicotinic currents were evoked by 100 μM ACh. The low- and the high-affinity receptors contributed

Naltrexone blocks nicotine induced up-regulation of α7 nAChRs: Consequences for smoking cessation therapy

Up-regulation of nAChRs is considered to be one of the mechanisms involved in initiation and maintenance of nicotine addiction, and craving for smoking during quitting attempts.³¹ A previous study from this laboratory demonstrated that a 1-h exposure of primary hippocampal cultures to clinically relevant concentrations of nicotine (10 or 30 nM) causes significant up-regulation of functional α7 nAChRs.¹⁷ Thus, experiments were designed to investigate whether naltrexone could modify expression

Naltrexone up-regulates functional nAChRs in hippocampal organotypic cultures

Normal preparation procedures for primary neuronal cultures involve destruction of the physiological synaptic patterns. In addition, the primary cultures used in the present study were obtained from embryonic animals. Thus, organotypic hippocampal cultures were used to investigate the effects of naltrexone in a more physiological preparation. Organotypic hippocampal cultures were obtained using a modification of the procedure of Stoppini et al.⁴¹ Briefly, 8–11-day-old Sprague–Dawley rats, kept

Acknowledgements

The authors gratefully acknowledge the excellent technical assistance of Mrs. Barbara Marrow and Mrs. Mabel A. Zelle. This work was supported by grants from USPHS NS41671, University of Maryland School of Medicine Other Tobacco Related Diseases Research Grant and Janssen Pharmaceutical Research Foundation (to EXA).

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Sensitivity of neuronal nicotinic acetylcholine receptors to the opiate antagonists naltrexone and naloxone: receptor blockade and up-regulation

Abstract

Introduction

Section snippets

Characterization of α4β2 nAChRs stably transfected into HEK 293 cells

Naltrexone and naloxone effects on α4β2 nAChRs

Naltrexone blocks nicotine induced up-regulation of α7 nAChRs: Consequences for smoking cessation therapy

Naltrexone up-regulates functional nAChRs in hippocampal organotypic cultures

Acknowledgements

Neurobiol. Learn. Mem.

Gen. Pharmacol.

Pharmacol. Biochem. Behav.

Drug Alcohol Depend.

Neuron

Psychopharmacology (Berl.)

Eur. J. Pharmacol.

Life Sci.

Psychopharmacology (Berl.)

Psychopharmacology (Berl.)

Addiction

Psychopharmacology (Berl.)

Psychopharmacology (Berl.)

Psychopharmacology (Berl.)

FEBS Lett.

Eur. J. Pharmacol.

Neuropharmacology

J. Pharmacol. Exp. Ther.

Pflügers. Arch

Methods Neurosci.

Neurosci. Methods

J. Neurosci.

Mol. Pharmacol.