Dextromethorphan and Its Metabolite Dextrorphan Block a 3 b 4 Neuronal Nicotinic Receptors 1 , 2

Dextromethorphan (DM), a structural analog of morphine and codeine, has been widely used as a cough suppressant for more than 40 years. DM is not itself a potent analgesic, but it has been reported to enhance analgesia produced by morphine and nonsteroidal anti-inflammatory drugs. Although DM is considered to be nonaddictive, it has been reported to reduce morphine tolerance in rats and to be useful in helping addicted subjects to withdraw from heroin. Here we studied the effects of DM on neuronal nicotinic receptors stably expressed in human embryonic kidney cells. Studies were carried out to examine the effects of DM on nicotine-stimulated whole cell currents and nicotine-stimulated Rb efflux. We found that both DM and its metabolite dextrorphan block nicotinic receptor function in a noncompetitive but reversible manner, suggesting that both drugs block the receptor channel. Consistent with blockade of the receptor channel, neither drug competed for the nicotinic agonist binding sites labeled by [H]epibatidine. Although DM is approximately 9-fold less potent than the widely used noncompetitive nicotinic antagonist mecamylamine in blocking nicotinic receptor function, the block by DM appears to reverse more slowly than that by mecamylamine. These data indicate that DM is a useful antagonist for studying nicotinic receptor function and suggest that it might prove to be a clinically useful neuronal nicotinic receptor antagonist, possibly helpful as an aid for helping people addicted to nicotine to refrain from smoking, as well as in other conditions where blockade of neuronal nicotinic receptors would be helpful. Dextromethorphan (DM) is structurally closely related to levorphanol, codeine, and morphine, but unlike these opiates it has low affinity for opiate receptors and is not considered to be addictive. Nevertheless, it shares with most opiates the ability to suppress cough and has been used as an effective antitussive drug for more than 40 years. Although DM appears to produce little analgesia by itself, it has recently been shown to attenuate tolerance and/or enhance analgesia produced by morphine (Elliott et al., 1994; Mao et al., 1996) and nonsteroidal anti-inflammatory drugs (Price et al., 1996). Furthermore, DM has been reported to reduce morphine dependence in rats (Mao et al., 1996) and possibly to be useful in treating addicted subjects withdrawing from heroin (Koyuncuoglu and Saydam, 1990). In addition, DM has been shown to have neuroprotective effects in models of glutamate neurotoxicity (Choi, 1987; Choi et al., 1987). Some of these diverse effects may be related to the ability of DM and/or its demethylated major metabolite dextrorphan (DP) to block N-methyl-D-aspartate (NMDA) receptor channels (Church et al., 1985); however, there is one report that indicates that DM might also block nicotinic receptors in PC12 cells (Yamamoto et al., 1992). We have recently stably expressed and characterized a neuronal nicotinic receptor comprised of a3 and b4 subunits in HEK-293 cells (Xiao et al., 1998). This stably transfected cell line, designated KXa3b4R2, expresses a high density of nicotinic receptors that can be labeled by [H]epibatidine ([H]EB) and that pass Rb in response to stimulation by the agonists acetylcholine, nicotine, cytisine, and epibatidine. The function of these receptors is blocked by the nicotinic antagonists mecamylamine, d-tubocurarine, hexamethonium, and dihydro-b-erythroidine (Xiao et al., 1998). These cells provide an excellent model system in which to examine drug effects on the a3b4 nicotinic receptor. Here, we have examined and compared the effects of DM and DP, their structural analogs, and several other drugs that block nicotinic receptor and/or NMDA receptor channels, on nicotine-stimulated receptor function in these cells. Experimental Procedures Materials and Drugs. Tissue culture medium, serum, and antibiotics were purchased from Life Technologies (Gaithersburg, MD). Received for publication November 12, 1999. 1 Supported by National Institutes of Health Grant DA06486 (K.J.K.) and a grant from Algos Pharmaceutical Corp. 2 Portions of this work have appeared in abstract form: Hernandez et al. (1998) Soc Neurosci Abstr 24:86. ABBREVIATIONS: DM, dextromethorphan; DP, dextrorphan; [H]EB, [H]epibatidine; NMDA, N-methyl-D-aspartate. 0022-3565/00/2933-0962$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 293, No. 3 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 293:962–967, 2000 /2283/824336 962 at A PE T Jornals on M ay 0, 2017 jpet.asjournals. org D ow nladed from [H]EB and RbCl were supplied by NEN (Boston, MA). Chemicals were purchased from Fisher Scientific Co. (Fairlawn, NJ). (2)-Nicotine, DM, DP, and mecamylamine were purchased from Sigma Chemical Co. (St. Louis, MO). MK-801 was purchased from Research Biochemicals International (Natick, MA), and phencyclidine was provided by the National Institute on Drug Abuse. All other chemicals were reagent grade. Cell Culture. KXa3b4 cells were grown as described previously (Xiao et al., 1998) in minimum essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, 100 mg/ml streptomycin, and 0.7 mg/ml of geneticin. Cells were maintained at 37°C with 5% CO2 in a humidified incubator. Rb Efflux Assay. The function of nicotinic acetylcholine receptors expressed in the KXa3b4 cells was measured using a Rb efflux assay (Lukas and Cullen, 1988; Xiao et al., 1998). Briefly, 1-ml aliquots of cells in growth medium were plated onto 24-well plates coated with poly(D-lysine). The plated cells were grown at 37°C for 16 to 18 h until reaching 90 to 100% confluence. On the day of the experiment, the growth medium was aspirated and the cells were incubated in fresh medium containing 2 mCi/ml RbCl for 4 h at 37°C. After this loading procedure, the medium was aspirated and the cells were washed three times with 1-ml aliquots of buffer (15 mM HEPES, 140 mM NaCl, 2 mM KCl, 1 mM MgSO4, 1.8 mM CaCl2, and 11 mM glucose at pH 7.4) to remove Rb free in the medium. After washing, 1 ml of buffer with or without the drugs under study was added to each well, and the cells were incubated for 2 min. The incubation buffer was then collected, after which the cells were lysed in 0.1 N NaOH. The radioactivity in the buffer samples and cell lysates was measured by liquid scintillation counting. The total Rb loaded into the cells (after washing) was calculated as the sum of the buffer samples and the cell lysates from each well, and the amount of Rb efflux was then expressed as a percentage of the total Rb loaded (fractional release). Stimulated efflux was defined as the difference between efflux in the presence and absence of nicotine (i.e., total minus basal efflux). The maximum Rb efflux, found at a nicotine concentration of ;300 mM or higher, was ;45% of the amount loaded and was independent of the amount of Rb loaded into the cell. In studies to determine the inhibition of nicotinestimulated Rb efflux by the drugs under study, data were expressed as a percentage of control values measured with 100 mM