Introduction

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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 3 and 4 subunits in HEK-293 cells (Xiao et al., 1998). This stably transfected cell line, designated KX34R2, expresses a high density of nicotinic receptors that can be labeled by [³H]epibatidine ([³H]EB) and that pass ⁸⁶Rb⁺ 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--erythroidine (Xiao et al., 1998). These cells provide an excellent model system in which to examine drug effects on the 34 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

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Materials and Drugs. Tissue culture medium, serum, and antibiotics were purchased from Life Technologies (Gaithersburg, MD). [³H]EB and ⁸⁶RbCl were supplied by NEN (Boston, MA). Chemicals were purchased from Fisher Scientific Co. (Fairlawn, NJ). ()-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. KX34 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 µg/ml streptomycin, and 0.7 mg/ml of geneticin. Cells were maintained at 37°C with 5% CO₂ in a humidified incubator.

⁸⁶Rb⁺ Efflux Assay. The function of nicotinic acetylcholine receptors expressed in the KX34 cells was measured using a ⁸⁶Rb⁺ 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 µCi/ml ⁸⁶RbCl 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 ⁸⁶Rb⁺ 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 ⁸⁶Rb⁺ 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 ⁸⁶Rb⁺ efflux was then expressed as a percentage of the total ⁸⁶Rb⁺ 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 ⁸⁶Rb⁺ efflux, found at a nicotine concentration of ~300 µM or higher, was ~45% of the amount loaded and was independent of the amount of ⁸⁶Rb⁺ loaded into the cell. In studies to determine the inhibition of nicotine-stimulated ⁸⁶Rb⁺ efflux by the drugs under study, data were expressed as a percentage of control values measured with 100 µM nicotine.

Radioligand Binding Assay. Nicotinic receptor binding sites in membrane homogenates from KX34 cells were measured using [³H]EB, which binds with high affinity to the 34 receptors (Xiao et al., 1998). DM or DP at concentrations from 1 to 100 µM competed against 300 pM [³H]EB for receptors in KX34 cell membranes. Ligand binding was measured as described previously (Xiao et al., 1998). Briefly, cultured cells at >90% confluence were harvested in 50 mM Tris-HCl buffer (pH 7.4) and homogenized with a Polytron homogenizer. The homogenates were centrifuged at 35,000g for 10 min and the pellets were washed twice with fresh buffer. Membrane pellets were resuspended in fresh buffer and aliquots equivalent to approximately 55 µg of protein were incubated with [³H]EB for 4 h at 24°C in a final volume of 2.5 ml. Bound and free radioligand were separated by vacuum filtration through Whatman GF/C filters pretreated with polyethylenimine. The radioactivity bound to the filter was determined by liquid scintillation counting. Nonspecific binding was determined in the presence of 300 µM ()-nicotine. Specific binding was defined as the difference between total binding and nonspecific binding.

Electrophysiology. Ionic currents in whole-cell configuration were measured using the patch-clamp technique and a fast drug delivery system. Cells were maintained at a holding potential of 60 mV. Briefly, cells coated onto coverslips were positioned in a recording chamber (1-ml volume) and perfused with Ringer's solution (120 mM NaCl, 3.1 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 26 mM NaHCO₃, 1 mM K₂HPO₄, and 5 mM glucose) at 24°C. The solution was continuously bubbled with 5% CO₂ and 95% O₂ to maintain the pH at 7.4. Cells were visually identified using an upright microscope (Axioskop; Carl Zeiss, Jena, Germany). A gravity-fed Y-tubing system was placed within 100 µm of the cell under investigation, and a complete change of solution surrounding the cell was achieved in less than 1 s. The pipette solution contained 145 mM CsCl, 1 mM MgCl₂, 2 mM ATP, 1 mM EGTA, and 10 mM HEPES (pH was adjusted to 7.2 with CsOH).

	Results

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Effects of DM and DP on Nicotinic Receptors. The effects of DM and its metabolite DP on 34 nicotinic receptors were examined first in assays that measure nicotine-stimulated ⁸⁶Rb⁺ efflux through the nicotinic receptor channel. In these cells, nicotine stimulates ⁸⁶Rb⁺ efflux with an EC₅₀ of 28 µM, and 100 µM nicotine stimulates a near maximal ⁸⁶Rb⁺ efflux response, which is approximately 8 times basal efflux (Xiao et al., 1998). Both DM and DP blocked this receptor function stimulated by 100 µM nicotine in a concentration-related manner, with IC₅₀ values of approximately 9 and 30 µM, respectively (Fig. 1 and Table 1). We also examined the effect of DM on nicotinic receptors in whole-cell patch-clamp studies. Nicotine stimulates a large inward current in these cells (Fig. 2a). Consistent with its effect on nicotine-stimulated ⁸⁶Rb⁺ efflux, DM at a concentration of 100 µM nearly completely blocked the nicotine-activated current (Fig. 2b). After an 8-min drug washout period, a substantial fraction of the nicotine-stimulated receptor function returned (Fig. 2c). However, the function had not fully recovered compared with the initial response to nicotine (compare Fig. 2, a and c), suggesting either that some residual DM remained in the preparation, or that the receptor was partially desensitized at this time after the prior exposure to nicotine.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Inhibition by DM and DP of nicotine-stimulated 86Rb+ efflux from KX34 cells. Cells were loaded with 86RbCl as described in Experimental Procedures and then stimulated with 100 µM nicotine in the presence of increasing concentrations of DM or DP. The amount of 86Rb+ efflux was expressed as a percentage of 86Rb+ loaded (fractional release). Basal release, measured in the absence of nicotine, was approximately 5% and was subtracted. Control release stimulated by 100 µM nicotine in the absence of DM or DP was approximately 35% of the loaded 86Rb+. The IC50 values for DM and DP are the mean and S.E. of three independent experiments, each measured in quadruplicate.

View this table: [in this window] [in a new window]   TABLE 1 IC50 values of antagonists for inhibition of nAChR function in KX34 cells 86Rb+ efflux was measured as described in Experimental Procedures. IC50 values and Hill coefficients (nH) were calculated from the Hill equation. Values shown are the mean ± S.E. of three experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Inhibition by DM of nicotine-stimulated whole-cell currents in KX34 cells. Cells were maintained at a holding potential of 60 mV. a, whole-cell currents elicited by application of 10 µM nicotine alone. b, simultaneous application of nicotine and 100 µM DM. c, partial recovery of response to second application of nicotine after an 8-min drug washout period. The experiments shown are representative of four independent experiments.

Mode of Nicotinic Receptor Block by DM and DP. To determine the type of pharmacological block produced by DM and DP, the effects of these drugs on the concentration-response relationship for nicotine-stimulated ⁸⁶Rb⁺ efflux were examined. As shown in Fig. 3a, at concentrations near their IC₅₀ values both DM and DP decreased the maximum nicotine-stimulated response without significantly shifting the EC₅₀ for nicotine in the concentration-response curves. This result is consistent with a noncompetitive pharmacological block and suggests that DM and DP block the nicotinic receptor channel without necessarily binding to the agonist recognition site. To assess this further, we examined the ability of these drugs to compete for 34 receptor agonist binding sites labeled by [³H]EB in membrane homogenates from these cells. Neither DM nor DP competed effectively for the 34 receptor agonist binding sites labeled by [³H]EB; for example, at a concentration of 100 µM, DM and DP inhibited [³H]EB binding by no more than 20% (Fig. 3b).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Mechanism of nicotinic receptor inhibition by DM and DP. a, inhibition of nicotine stimulated 86Rb+ efflux from KX34 cells by DM and DP. Dose-response relationships for nicotine-stimulated 86Rb+ efflux from KX34 cells in the absence and presence of 10 µM DM or 25 µM DP. Data are expressed as a percentage of the maximal stimulation by nicotine. Both DM and DP reduced the maximum effect of nicotine to stimulate 86Rb+ efflux without significantly affecting the EC50 of nicotine for stimulation. This experiment is representative of three independent studies each measured in quadruplicate. Inset, dose-response curve for nicotine-stimulated 86Rb+ efflux from KX34 cells expressed as a percentage of 86Rb+ loaded. The basal release, approximately 5%, has been subtracted. b, effects of DM and DP on [3H]EB binding in KX34 cells. Neither DM nor DP at concentrations up to 100 µM competed effectively against 300 pM [3H]EB for receptor binding sites on KX34 cell membranes.

Recovery of Receptor Function after Exposure to DM or mecamylamine. To examine the reversibility and time course of recovery of receptor function after exposure to DM, cells were exposed to 10 µM DM for 30 min or for 24 h before being prepared for measurement of nicotine-stimulated ⁸⁶Rb⁺ efflux. The preparation of the cells after drug exposure included four washes with HEPES buffer over a 7-min period, including one 5-min wash period, to effectively remove any drug free in solution. Despite this washing procedure, the maximum nicotine-stimulated ⁸⁶Rb⁺ efflux was still decreased by approximately 40 and 60% in cells exposed to DM for 30 min and 24 h, respectively (Fig. 4a). For comparison, the effect of mecamylamine, a well established nicotinic receptor channel blocker, was measured in parallel under the same conditions. Mecamylamine is more potent than DM at blocking the 34 receptors in these cells (see Table 1), but in contrast to the effects of DM, the 7-min washing procedure nearly completely reversed the effects of both the 30-min and the 24-h exposure to 10 µM mecamylamine (Fig. 4a).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Reversibility of blockade of nicotine-stimulated function by DM and mecamylamine. a, cells were preincubated with 10 µM DM or 10 µM mecamylamine (Mec) for 30 min or 24 h before being washed four times over a 7-min period. Immediately after the washing procedure, nicotine-stimulated 86Rb+ efflux was measured. b, cells were preincubated with 10 µM DM for 4 or 5 days and then washed four times over a 7-min period or allowed to recover in media for an additional 2 or 24 h, as indicated, before measurements of nicotine-stimulated 86Rb+ efflux.

Comparison of DM and DP to Structural Analogs of Opiates and Other Receptor Channel Blockers. Both DM and DP are structural analogs of opiates; therefore, we measured the nicotinic receptor blocking activity of two other opiates, codeine and hydrocodone, both of which retain the methoxy group at the 3 position and the N-methyl group at position 17 of the more potent DM. Although both opiates appeared to be capable of blocking nicotine-stimulated ⁸⁶Rb⁺ efflux, they were much less potent than DM or DP; at a concentration of 100 µM, codeine and hydrocodone blocked efflux by less than 20 and 30%, respectively (Fig. 5).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Inhibition of nicotine-stimulated 86Rb+ efflux by DM, DP, codeine, and hydrocodone. KX34 cells were loaded with 86RbCl and then stimulated with 100 µM nicotine in the absence or presence of 100 µM of each of the drugs shown, as described under Experimental Procedures. Values are the mean ± S.E. from three independent experiments, each measured in quadruplicate.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Comparison of the inhibition of nicotine-stimulated 86Rb+ efflux by DM, DP, and several other ligand-gated ion channel blockers. KX34 cells were loaded with 86RbCl and then stimulated with 100 µM nicotine in the absence or presence of each one of the drugs shown at the indicated concentrations. Data are representative of three independent experiments, each measured in quadruplicate. See Table 1 for IC50 values for each drug.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The studies presented here demonstrate that both DM and its metabolite DP block 34 nicotinic receptors noncompetitively. The noncompetitive nature of the blockade and the observation that neither DM nor DP competes for the agonist binding site strongly suggest that these drugs bind within and block the receptor channel. DM, in particular, is a relatively potent nicotinic receptor blocker, with an IC₅₀ of less than 10 µM in assays that measured ⁸⁶Rb⁺ efflux stimulated by 100 µM nicotine; consistent with this potency, DM nearly completely blocked whole cell currents stimulated by 10 µM nicotine. The apparent potency of DM at 34 receptors is similar to that of phencyclidine and about 9-fold lower than that of mecamylamine, which is one of the most potent blockers of these receptors reported so far (Fig. 6 and Table 1; see also Xiao et al., 1998). Both DP and MK-801 are approximately 3-fold less potent than DM at blocking 34 receptors. MK-801 has been found previously to block muscle nicotinic receptors (Ramoa et al., 1990; Amador and Dani, 1991) as well as neuronal types of nicotinic receptors (Halliwell et al., 1989; Ramoa et al., 1990; Briggs and McKenna, 1996).

Interestingly, whereas the receptor blockade by mecamylamine was completely reversed by the standard washing conditions used here (four washes over 7 min), DM appeared to require a longer washout period for complete reversal of its effects. Thus, after exposure of cells to DM for 30 min or 24 h, followed by our standard washing procedure, receptor function was still inhibited by 40 to 60% compared with controls. However, the receptor function returned to control values within 2 h after removing the drug, even after a 5-day exposure to DM. One possible explanation for these results is that DM may bind to a site deep within the receptor channel and, even though the affinity of DM for its binding site may be lower than that of mecamylamine, it may not exit the channel as easily.

As a consequence of the relatively slow recovery from exposure to DM, it is uncertain whether the diminished response to nicotine seen in the whole cell patch-clamp studies 8 min after exposure of cells to DM plus nicotine (Fig. 2c) was due to residual blockade by DM or desensitization of the receptor after exposure to nicotine. In other studies, we found that the 34 receptors in these cells do, in fact, desensitize during acute exposure to nicotine and that the half-time for recovery of their function after a 5-min exposure to nicotine is approximately 8 min (E. L. Meyer, Y. Xiao, and K. J. Kellar, in preparation).

Although DM and DP are structurally similar to many opioid drugs, they do not share certain key pharmacological properties of the opioids. Thus, DM and DP are not by themselves potent analgesics, and they are not usually associated with addictive behavior. DM, however, does appear to potentiate analgesia produced in rodents by both opiate drugs (Elliott et al., 1994; Mao et al., 1996) and nonsteroidal anti-inflammatory drugs (Price et al., 1996). In addition, DM, like codeine and other opiates, is a good cough suppressant; but interestingly, although the opiate receptor antagonist naloxone blocks the antitussive effect of codeine, it does not block that of DM (Reisine and Pasternak, 1996). Thus, cough suppression probably involves more than one kind of mechanism, including one that might be mediated by a nicotinic receptor. Interestingly, nicotinic receptors have recently been found in human and rodent bronchial epithelial tissue (Zia et al., 1997; Maus et al., 1998), a location that could make them a ready target of DM in its cough suppression action.

The ability of DM to enter the central nervous system and to block neuronal nicotinic receptors could contribute to its actions to both potentiate analgesic activity and suppress cough, either centrally or peripherally. In addition, recent studies from Rose and colleagues (Rose et al., 1994, 1998) have suggested that nicotinic receptor antagonists, such as mecamylamine, when combined with the nicotine transdermal patch, may be useful as adjuncts treating nicotine addiction. Because DM produces a sustained but reversible noncompetitive block of neuronal nicotinic receptors, it too could complement the activity of other smoking cessation approaches, such as nicotine replacement therapy and bupropion. In this regard, it should be noted that there has been a long period of experience with DM as a cough suppressant (>40 years) and that it has a high safety index (Bem and Peck, 1992). Furthermore, because the complex behavioral effects of nicotine may involve downstream transsynaptic actions of excitatory amino acids at NMDA receptors (Shoaib and Stolerman, 1992; Shoaib et al., 1994), the concurrent blockade of both nicotinic and NMDA receptors by DM and DP may be additive in producing beneficial pharmacological effects. In this regard, it should be pointed out that the combined plasma concentrations of DM and DP vary widely, but can reach 1 µM after a single 30-mg oral dose (Capon et al., 1996); however, the drug is often taken 6 or more times per day and often at higher doses. Furthermore, the brain level of a lipophilic drug like DM may be significantly higher than its plasma level and, in any case, the pharmacological effects of a noncompetitive blocker may be greater than would be predicted by its blood level because it is not competing with an endogenous agonist. In addition, as shown here, the effects of DM appear to reverse relatively slowly.

In conclusion, DM and DP are noncompetitive blockers of neuronal nicotinic receptors composed of 34 subunits. The blockade produced by DM is reversible but is sustained beyond the time typically needed to remove drugs from the incubation media, suggesting that DM binds to a site deep inside the channel or otherwise finds a depot away from the surrounding media, perhaps within the cell. The blockade of neuronal nicotinic receptors by DM and its metabolite DP may contribute to their clinical utility as cough suppressants, as well as to their potential use as analgesic boosters and for smoking cessation therapy.