Introduction

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Mecamylamine is widely used as a broad-spectrum antagonist of neuronal nicotinic acetylcholine (ACh) receptors (nAChRs) and, despite its common usage, questions still exist as to its precise specificity and mechanisms of action. The present study evaluates the effects of mecamylamine on a wide range of nAChRs and additionally evaluates whether the stereoisomers of this drug augment the specificity or efficacy for desired therapeutic effects.

In the context of therapeutics, mecamylamine (Inversine) was developed and characterized by Merck (Darmstadt, Germany) as a ganglionic blocker with clinically significant hypotensive actions (Stone et al., 1956). Unique characteristics of mecamylamine, including exceptional oral efficacy, rapid onset, long duration of action, and nearly complete absorption from the gastrointestinal tract, made mecamylamine, at that time, a more desirable antihypertensive medication than the existing ganglionic blockers (Baer et al., 1956).

Despite mecamylamine's efficacy in the treatment of hypertension, its side effects resulting from broad parasympathetic inhibition at antihypertensive doses reduced its popularity in the treatment of essential hypertension. However, in addition to its peripheral ganglionic blocking actions, mecamylamine crosses the blood-brain barrier and functions as a cholinergic antagonist specific to neuronal nicotinic receptor subtypes (Varanda et al., 1985; Martin et al., 1989, 1993; Banerjee et al., 1990). Because mecamylamine readily blocks most of the physiological, behavioral, and reinforcing effects of nicotine, it has become the most commonly used of all neuronal nAChR-specific inhibitors in basic nicotine research (Martin et al., 1989, 1993) and has been reported to be effective as an aid to smoking cessation (Tennant et al., 1984; Rose et al., 1994, 1998).

Since nicotine has both agonist and antagonistic properties, nicotinic receptor antagonists, such as mecamylamine, may also be useful for a wide range of nicotine-responsive neuropsychiatric disorders. For example, mecamylamine, like nicotine, has also been reported to possibly augment neuroleptic therapy of Tourette's syndrome (Sanberg et al., 1998). In addition, growing evidence suggests that nicotinic receptor activation may also be involved with the actions of other drugs of abuse, including psychostimulants and alcohol. It has been reported that mecamylamine prevents the induction of locomotor sensitization to amphetamine in mice (Karler et al., 1996), and low doses of mecamylamine were recently found to reduce cue-induced craving in human cocaine abusers (Reid et al., 1999). Furthermore, several preclinical studies have reported that mecamylamine reduces the behavioral and reinforcing effects of alcohol in animals (Blomqvist et al., 1996, 1997; Ericson et al., 1998; Nadal et al., 1998).

The present study investigates the selectivity of mecamylamine and its stereoisomers by using the genes cloned from the nervous system, which code for nicotinic acetylcholine receptor subunits. The nAChRs are known to be formed by the association of five monomeric subunits. The neuronal nAChR gene family include nine proteins designated as  subunits (2-10) and three proteins designated as  subunits (2-4) (Papke, 1993; Elgoyhen et al., 1994, 2001). Except for 7 subunits that may form functional homomeric receptors, most nicotinic receptors found in the mammalian central nervous system require at least one type of  and one type of  subunit to form functional nAChR channels. Neuronal nAChRs are both structurally and phylogenetically related to the nicotine receptors of the neuromuscular junction. The mature muscle-type nAChRs contain two 1 subunits, one 1 subunit, one  subunit, and one  subunit (Mishina et al., 1986).

This study specifically evaluated the efficacy and potency of highly pure R-()-mecamylamine and S-(+)-mecamylamine on human 34, 42, 32, and 7 receptors expressed in Xenopus oocytes and compared their activity to that of racemic mecamylamine. The voltage dependence and reversibility of inhibitory activity was also investigated. Since prior research in this lab suggested that a residual inhibition of receptors by mecamylamine is still present after a 5-min wash period (Webster et al., 1999), it was hypothesized that the persistence of block (an indirect indication of the dissociation rate) might prove to be an important distinguishing feature of the stereoisomers. In addition, the report of finding "interesting differences" between the actions of mecamylamine stereoisomers in assays measuring neuromuscular transmission (Schoenenberger et al., 1986) prompted our investigation of the inhibitory actions of these stereoisomers at adult-type mouse muscle nAChRs expressed in Xenopus oocytes.

	Materials and Methods

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Chemicals. Fresh acetylcholine (Sigma, St. Louis, MO) stock solutions were made daily in Ringer's solution and diluted. Racemic-(±)-mecamylamine (N-2,3,3-tetramethylbicyclo[2.2.1]heptan-2-amine) and the stereoisomers were supplied by Layton Biosciences (Menlo Park, CA). All other chemicals for electrophysiology were obtained from Sigma (St. Louis, MO).

Preparation of RNA and Expression in Xenopus Oocytes. Mature (>9 cm) female Xenopus laevis African toads (Nasco, Ft. Atkinson, WI) were used as a source of oocytes. Before surgery, frogs were anesthetized by placing the animal in a 1.5 g/l solution of MS222 (3-aminobenzoic acid ethyl ester) for 30 min. Eggs were removed from an incision made in the abdomen. The incisions were disinfected with gentamicin, sutured with 4-0 gut, and the animals were allowed to recover from the anesthesia in a humid environment. Postoperative animals were kept in isolation tanks and checked daily before return to the colony.

Electrophysiology. Oocyte recordings were made with a Warner Instruments (Hamden, CT) OC-725C oocyte amplifier and RC-8 recording chamber interfaced to a Macintosh personal computer. Data were acquired using Labview software (National Instruments, Austin, TX) and filtered at a rate of 6 Hz. Oocytes were placed in the recording chamber with a total volume of about 0.6 ml and perfused at room temperature by frog Ringer's solution (115 mM NaCl, 2.5 mM KCl, 10 mM HEPES, pH 7.3, 1.8 mM CaCl₂) containing 1 µM atropine to inhibit potential muscarinic responses. A Mariotte flask filled with Ringer's solution was used to maintain a constant hydrostatic pressure for drug deliveries and washes. Current electrodes were filled with a solution containing 250 mM CsCl, 250 mM CsF, and 100 mM EGTA and had resistances of 0.5 to 2 M. Voltage electrodes were filled with 3 M KCl and had resistances of 1 to 3 M.

Experimental Protocols and Data Analysis. Current responses to drug application were studied under two-electrode voltage clamp at a holding potential of 50 mV unless otherwise noted. Holding currents immediately before agonist application were subtracted from measurements of the peak response to agonist. All drug applications were separated by a wash period of 5 min unless otherwise noted. At the start of recording, all oocytes received two initial control applications of ACh. Subsequent drug applications were normalized to the second ACh application to control for the level of channel expression in each oocyte. The second application of control ACh was used to minimize the affects of rundown that occasionally occurred after the initial ACh-evoked response. To measure residual inhibitory effects, an experimental application of ACh with inhibitor or of inhibitor alone was followed by a second application of ACh alone and compared with the preapplication control ACh response. Means and standard errors (S.E.M.) were calculated from the normalized responses of at least four oocytes for each experimental concentration.

	Results

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Inhibition of nAChR Subtypes by Mecamylamine Stereoisomers. Racemic mecamylamine and its stereoisomers were tested for their ability to inhibit the ACh-evoked responses of 42, 34, 32, and 7 type receptors expressed in Xenopus oocytes. Raw data traces showing the effect of 10 µM mecamylamine stereoisomers on these receptor subtypes are shown in Fig. 1. To prevent confounding the effect of changing both ACh and mecamylamine concentrations during the rising phase of the evoked responses, cells were first pre-equilibrated with mecamylamine before ACh was applied in the continuing presence of mecamylamine (see Materials and Methods). As shown in Fig. 1, 10 µM of either mecamylamine stereoisomer produced significant inhibition of the nAChR subtypes. Additionally, for each of the  subunit-containing receptor subtypes, there was a marked decrease in subsequent control responses to ACh when it was applied 5 min after exposure to mecamylamine. A summary of the residual inhibition produced by 10 µM mecamylamine is shown in Fig. 2. The residual inhibition was greatest for 2-containing receptors and least for 7 receptors. There was no stereo-selectivity apparent in the 5-min recovery data. The time dependence of recovery is considered further below.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effect of mecamylamine (Mec) on neuronal nAChR. A, representative traces showing the effect of the two mecamylamine stereoisomers on ACh-evoked currents in 42-expressing oocytes. The traces illustrate initial control responses to 30 µM ACh alone (application indicated by black bar) and then the application of 30 µM ACh in the presence of 10 µM mecamylamine (open bar). B, representative traces showing the effect of the two mecamylamine stereoisomers (10 µM) on currents evoked by 100 µM ACh in 34-expressing oocytes. C, representative traces showing the effect of the two mecamylamine stereoisomers (10 µM) on currents evoked by 30 µM ACh in 32-expressing oocytes. D, representative traces showing the effect of the two mecamylamine stereoisomers (10 µM) on currents evoked by 300 µM ACh in 7-expressing oocytes. In each panel, the third trace in each row shows the response to a control ACh application 5 min after the application of mecamylamine.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Residual effects of mecamylamine (Mec) on ACh control responses. Control ACh responses 5 min after the coapplication of 10 µM mecamylamine to 42 receptors (A), 34 receptors (B), 32 receptors (C), and 7 receptors (D). Control ACh concentrations used for these receptor subtypes were 30 µM, 100 µM, 30 µM, and 300 µM, respectively. Each bar represents the average normalized responses of four to seven cells.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Concentration-response relationships for the effects of mecamylamine (Mec) and its stereoisomers on neuronal nAChR. A, concentration-response relationship for mecamylamine's inhibition of the peak currents of 42 receptors when 30 µM ACh was coapplied with mecamylamine at the indicated concentrations. Data from each oocyte were normalized to that cell's response to 30 µM ACh alone. B, concentration-response relationship for mecamylamine's inhibition of the peak currents of 34 receptors when 100 µM ACh was coapplied with mecamylamine at the indicated concentrations. Data from each oocyte were normalized to that cell's response to 100 µM ACh alone. C, concentration-response relationship for mecamylamine's inhibition of the peak currents of 32 receptors when 30 µM ACh was coapplied with mecamylamine at the indicated concentrations. Data from each oocyte were normalized to that cell's response to 30 µM ACh alone. D, concentration-response relationship for mecamylamine's inhibition of the peak currents of 7 receptors when 300 µM ACh was coapplied with mecamylamine at the indicated concentrations. All cells were pre-equilibrated with mecamylamine, at the indicated concentration, for 10 s before the coapplication of agonist with antagonist. Each point represents the average normalized response of four to seven cells.

The Selectivity of Mecamylamine for Neuronal nAChR. To confirm the selectivity of mecamylamine for neuronal nAChR over muscle-type nAChR, we also examined the effects of R-()-mecamylamine and S-(+)-mecamylamine on adult-type 11 receptors, using mouse cDNA clones. Mecamylamine could produce a transient inhibition of muscle-type receptor responses with a potency roughly comparable with that for the inhibition of 42 receptors (Fig. 4 and Table 1). There was a tendency for R-()-mecamylamine to produce more inhibition of muscle-type receptors than S-(+)-mecamylamine; however the difference was only significant at the 1 µM concentration (unpaired t test p < 0.05). In contrast to the inhibition of  subunit-containing neuronal receptors, the inhibition of muscle receptors was fully reversed after a 5-min wash (Fig. 4B).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of mecamylamine (Mec) on ACh-evoked currents in oocytes expressing the 11 subunits of the adult muscle-type nAChR. A, concentration-response relationship for mecamylamine's inhibition of the peak currents of muscle-type receptors when 30 µM ACh was coapplied with mecamylamine at the indicated concentrations. Data from each oocyte were normalized to that cell's response to 3 µM ACh alone. All cells were pre-equilibrated with mecamylamine at the indicated concentration for 10 s before the coapplication of agonist with antagonist. Each point represents the average normalized response of at least four cells. B, amplitude of 3 µM ACh control responses obtained 5 min after the application of ACh and mecamylamine at the indicated concentrations. In contrast to the residual inhibition seen with neuronal nAChR receptors, the inhibition of muscle-type receptors was essentially fully reversible.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   NMDA receptors containing NR1 and NR2b subunits. Effect of mecamylamine stereoisomers on NMDA receptor currents. Oocytes expressing NR1 and NR2b subunits were tested for the effects of S-(+)-mecamylamine and R-()-mecamylamine at the indicated concentrations. NMDA receptor responses were activated by 10 µM glutamate (glut.) + 10 µM glycine (gly.), and data are normalized to the cells' initial responses to glutamate and glycine. A, solid bars represent the effect of mecamylamine on peak current amplitudes during the coapplication. Open bars represent the net charge calculated during the coapplication response. Hatched bars represent the peak amplitude of control glutamate/glycine responses after a 5-min wash. Data represent the average values for at least four cells under each condition with either S-(+)-mecamylamine (left) or R-()-mecamylamine (right). B, raw data from an experiment testing the effect of 100 µM S-(+)-mecamylamine and R-()-mecamylamine on NMDAR1 + NMDAR2b receptors. Note the large rebound currents occurring with the washout of mecamylamine, consistent with the relief of open channel block. In order to control for the intrinsic variability of the control responses, statistical analyses were based on t test comparisons between experimental data and repeated glutamate controls (i.e., sequential responses to the applications of 10 µM glutamate + 10 µM glycine). The peak amplitude of the repeated NMDA receptor controls were 0.958 ± 0.062 of the previous control responses, and the area of the repeated NMDA receptor controls were 1.042 ± 0.007 of the previous controls. *p < 0.05, **p < 0.01, and ***p < 0.001. Unmarked bars are not significantly different from corresponding glutamate controls at the 0.05 level.

Recovery Time Course. Since significant residual inhibition was detected after a 5-min washout for all neuronal nAChR other than 7, we conducted recovery time course experiments in which we obtained an initial inhibition of greater than 50% with the coapplication of 10 µM R-()-mecamylamine or S-(+)-mecamylamine and ACh at the control concentration (see above), and then followed the recovery of response with control ACh applications repeated at 5-min intervals. As shown in Fig. 6, for 2-containing receptors, recovery from mecamylamine-induced inhibition seemed to follow simple exponential kinetics. For 32 receptors, both R-()-mecamylamine and S-(+)-mecamylamine had time constants of recovery of about 33 ± 4 min, which was similar to the time constant of recovery for S-(+)-mecamylamine at 42. The  for R-()-mecamylamine on 42 receptors was somewhat faster (23 ± 1.2 min). Note that although inhibition of 42 receptors during the initial coapplication was not significantly different between cells treated with R-()-mecamylamine or S-(+)-mecamylamine, at each time point represented in Fig. 6A there was significantly less residual inhibition for the cells treated with R-()-mecamylamine than with S-(+)-mecamylamine (p < 0.05 at 5 and 25 min, and p < 0.01 at other time points).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   The recovery of ACh control responses. Cells were monitored for their responses to control applications of ACh at 5-min intervals after 10 µM mecamylamine (Mec) was coapplied with ACh on: A, 42 receptors; B, 32 receptors; and C, 34 receptors. Data from each oocyte were normalized to that cell's response to ACh alone. Each point represents the average normalized response of at least four cells.

Competition Studies. To determine whether mecamylamine produced inhibition of neuronal nAChR through a mechanism that is noncompetitive with ACh, we conducted ACh concentration-response studies of 42 and 34 receptors in the absence or presence of either S-(+)-mecamylamine or R-()-mecamylamine (Table 2). Data were analyzed both in terms of peak currents and in terms of the net charge during the entire evoked response (see Materials and Methods). Data were normalized to the peak or net charge of corresponding control ACh responses. As shown in Fig. 7, both forms of analysis indicate that the inhibition produced by the mecamylamine stereoisomers was not surmounted by increasing the ACh concentration and that the relative amount of inhibition produced by a fixed concentration of mecamylamine was relatively constant over a wide range of ACh concentrations.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Competition studies. A, ACh concentration response curves for 42 receptors were determined with ACh alone or ACh coapplied with 3 µM S-(+)-mecamylamine or R-()-mecamylamine. Data were initially normalized to the 30 µM ACh responses obtained in the same cells and then scaled by the ratio of 30 µM ACh control responses to the maximal ACh responses, obtained with 3 mM ACh. Each point represents the average normalized response of at least four cells. Data in the left plot show the effect of mecamylamine (Mec) on peak currents. Data in the right plot show the effect of mecamylamine on the net charge during a coapplication of ACh and mecamylamine, normalized to the net charge in the responses to ACh alone. B, ACh concentration-response curves for 324 receptors were determined with ACh alone or ACh coapplied with 3 µM S-(+)-mecamylamine or R-()-mecamylamine. Data were initially normalized to the 100 µM ACh responses obtained in the same cells and then scaled by the ratio of the 100 µM ACh control responses to the maximal ACh responses, obtained with 3 mM ACh. Each point represents the average normalized response of at least four cells. Data in the left plot show the effect of mecamylamine on peak currents. Data in the right plot show the effect of mecamylamine on the net charge during a coapplication of ACh and mecamylamine, normalized to the net charge in the responses to ACh alone.

Voltage Dependence of Inhibition. We evaluated the voltage dependence of inhibition by R-()-mecamylamine and S-(+)-mecamylamine in coapplication experiments. Specifically, cells were held at either 40 mV or 90 mV and tested for their responses to control concentrations of ACh (see above). After a 5-min wash, mecamylamine was coapplied with ACh. Cells were then washed for 5 min and tested again for their response to a second control ACh application. Cells were held at the indicated holding potential throughout the entire procedure. Through the use of a coapplication protocol rather than a preincubation procedure (as with the concentration-response curve experiments described above), we can evaluate both the voltage dependence of the onset of inhibition (Fig. 8, t = 0 data) and the voltage dependence of recovery, presumably representing the off-rates of the drugs (Fig. 8, t = 5 data). Note that the data in Fig. 8 represent the effects of mecamylamine on amplitude of the peak currents relative to the amplitude of the peak currents of control applications of ACh before mecamylamine was applied. The mecamylamine concentrations tested in these coapplication experiments were 10 µM for 7 receptors, 5 µM for 42 and 32 receptors, and 1 µM for 34 receptors. As shown in Fig. 8 (A and B), there was significant voltage dependence for the off-rate of both R-() and S-(+)-mecamylamine with 42 and 34 receptors (p < 0.01). Significant effects of voltage (p < 0.01) were detected in the t = 0 peak responses only for S-(+)-mecamylamine on 7 (Fig. 8C) and R-()-mecamylamine on 34 (Fig. 8D). With 32 receptors, only the off-rate of S-(+)-mecamylamine showed significant voltage dependence (p < 0.01), although the off-rate of R-()-mecamylamine showed a trend toward significance (p = 0.0505). In general, these results suggest that the binding site for mecamylamine may be deep enough into the membrane's electric field to slow the dissociation of mecamylamine when the cell is hyperpolarized.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Voltage dependence of mecamylamine (Mec)-induced inhibition of peak currents. Mecamylamine was coapplied with agonist at either 40 or 90 mV. Inhibition was calculated relative to initial control ACh responses and measured during the coapplication (t = 0) or after a 5 min wash (t = 5). Concentrations of mecamylamine used were: A, 5 µM for 42 receptors; B, 1 µM for 34 receptors; C, 5 µM for 32 receptors; and D, 10 µM for 7 receptors. Each point represents measurements from at least four cells. Columns marked with a double asterisk were determined to be significantly different from the corresponding 40 mV control data at p < 0.01 by unpaired t tests.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   Voltage dependence of mecamylamine (Mec)-induced inhibition of net charge during the coapplication of ACh and either S-(+)-mecamylamine or R-()-mecamylamine. Mecamylamine was coapplied with agonist at either 40 or 90 mV. Inhibition of net charge was calculated relative to the net charge in the initial control ACh responses. Representative traces are shown to the right of the bar graphs. Drug applications were 12 s in duration and correspond to the bars. Note that the rapidly desensitizing 7 receptor-mediated responses are shown at an expanded time scale. In all cases, the response obtained in the presence of mecamylamine is shown as the thin line contained within the thick line that corresponds to the control ACh response obtained 5 min before the ACh/mecamylamine coapplication. The concentrations of mecamylamine used were: A, 5 µM for 42 receptors; B, 1 µM for 34 receptors; C, 5 µM for 32 receptors; and D, 10 µM for 7 receptors. Each point represents measurements from at least four cells. Analysis was conducted on the same responses that were represented in Fig. 8 in terms of peak amplitude. However, more significant effects of voltage were revealed when the data were analyzed in terms of net charge rather than peak currents. Columns marked with a double asterisk were determined to be significantly different from the corresponding 40 mV control data at p < 0.01 by unpaired t tests.

	Discussion

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Most previous in vitro studies investigating mecamylamine's mode of action have found its inhibitory activity to be voltage-dependent, which suggests that it functions as an open channel blocker (Ascher et al., 1979; Varanda et al., 1985; Fieber and Adams, 1991). Thus, mecamylamine is generally thought of as a noncompetitive antagonist producing inhibition by binding to sites other than the agonist activation site (Francis and Papke, 1996). However, Ascher et al. (1979) also found that inhibition of parasympathetic ganglia of the rat was lessened when the drug was coapplied at high agonist concentrations, an observation that is more consistent with a competitive mechanism of inhibition. Moreover, Bertrand et al. (1990) reported that the inhibitory effects of mecamylamine on heterologously expressed chick 42 receptors was voltage independent, yet present only after coapplication with an agonist. Previous research by this lab suggests that mecamylamine's inhibition of rat 34 receptors is voltage dependent, which is consistent with open channel block (Webster et al., 1999). Moreover, analysis of point mutations indicated that residues at the 6' position within the  subunit TM2 domain may be important for mecamylamine's inhibitory properties (Webster et al., 1999).

In a recent study investigating the effects of mecamylamine on human AChRs expressed in Xenopus oocytes, Chavez-Noriega et al. (1997) found that a single concentration of mecamylamine (3 µM) produced approximately 50% inhibition of 22, 42, and 7 receptors and between 75 and 90% inhibition at 34, 32, 44, and 24 receptors. However, these findings were difficult to interpret since it was not clear which of four possible nicotinic agonists were used for each subunit combination tested, and only a single concentration of mecamylamine was investigated.

Our results indicate that for  subunit-containing receptors, the effects of mecamylamine are long-lived and use-dependent. Although the slow reversibility of mecamylamine's effects is most apparent in our recovery studies, our concentration/response analyses primarily measure the onset of inhibition over a relatively brief period of activation. This implies that our IC₅₀ estimates, which are based only on a single period of activation during which inhibition accumulates, are not likely to reflect equilibrium IC₅₀ values that, if feasible to measure, would be considerably lower. It may be the case that previous reports, which have suggested higher potency for inhibition by mecamylamine, may have been affected by the accumulation of inhibition with the repeated activation of the receptors. A similar consideration could account for why our apparent IC₅₀ values for  subunit-containing neuronal receptors do not differ as much as might be expected from the apparent values for muscle-type receptors. Specifically, since the off-rate of mecamylamine from muscle-type receptors is probably at least 10 times faster than for the neuronal  subunit-containing receptors, our IC₅₀ measurements should approximate the equilibrium IC₅₀ values for muscle receptors, while underestimating the equilibrium IC₅₀ of the  subunit-containing neuronal receptors. Likewise, for the same reason, the rapid reversibility of mecamylamine's block of 7 receptors would be consistent with the relatively small effects of mecamylamine on neuronal 7-type receptors (Frazier et al., 1998).

In general, previous studies of mecamylamine have used a racemic mixture comprising the optical isomers R-()-mecamylamine and S-(+)-mecamylamine hydrochloride. The few previous studies aimed at investigating the pharmacology of these two isomers have generally found little or no difference in potency or efficacy. For example, Stone et al. (1962) compared the effects of S-(+)-mecamylamine hydrochloride with racemic mecamylamine hydrochloride on nicotine-induced convulsions and pupil dilation in mice and found essentially no significant differences between the two compounds. They concluded that "optical isomerism does not play a significant role in determining the degree of activity". Suchocki et al. (1991) also investigated the actions of R-()-mecamylamine and S-(+)-mecamylamine hydrochloride in assays measuring nicotine-induced depression of spontaneous locomotor activity and antinociception. They found that both optical isomers had similar potency in blocking the antinociception caused by nicotine, whereas the potency of the S-(+)-mecamylamine isomer in blocking the nicotine-induced depression of spontaneous locomotor activity could not be determined because S-(+)-mecamylamine, like nicotine, also induced depression of spontaneous locomotor activity. Despite some evidence for agonist activity of the S-isomer of mecamylamine, these investigators also concluded that optical isomerism does not play a significant role in determining the inhibitory activity of mecamylamine. However, Schoenenberger et al. (1986) reported finding "interesting differences" between the actions of R-()-mecamylamine and S-(+)-mecamylamine hydrochloride in assays measuring neuromuscular transmission, but the details regarding these differences were not described nor ever published.

The present findings indicate that while the various neuronal nAChR differ in their sensitivity to inhibition by mecamylamine and its stereoisomers, there seems to be little difference between S-(+)-mecamylamine and R-()-mecamylamine in terms of IC₅₀ values for a given receptor subtype. However, there appeared to be some significant differences in the off-rates for the mecamylamine isomers from the receptors. Specifically, S-(+)-mecamylamine appears to dissociate more slowly from 42 and 34 receptors than does R-()-mecamylamine.

Additionally, adult (11) muscle-type receptors appeared to be somewhat more sensitive to R-()-mecamylamine than to S-(+)-mecamylamine. This differential sensitivity of muscle receptors to the mecamylamine stereoisomers may have important clinical implications. For example, Tennant et al. (1984) found that "weakness" was one of the most intolerable side effects of mecamylamine when used as an aid to smoking cessation. In addition, in a recent double-blind placebo-controlled study investigating the safety of mecamylamine as a monotherapy for the treatment of Tourette's disorder, weakness was the most common adverse experience reported by patients treated with mecamylamine. Patients were asked each week of the 8-week treatment study if they felt "fatigue or tiredness" (along with 113 other potential side effects). Twenty-eight percent (8 of 29) of patients in the mecamylamine group experienced this side effect versus 9% (3 of 32) in the placebo group (Silver et al., 2000). Taken together, the combination of an increased inhibitory effect on neuronal receptors and a decreased effect on muscle-type receptors, would suggest that in chronic (i.e., therapeutic) application, S-(+)-mecamylamine might be preferable to R-()-mecamylamine, especially in terms of equilibrium inactivation of neuronal receptors, and with decreased side effects associated with neuromuscular transmission.

Our competition experiments indicate that the mecamylamine stereoisomers are noncompetitive inhibitors of neuronal nAChR. This is consistent with our data that indicates that the inhibition produced by the mecamylamine isomers is voltage dependent, which is consistent with previous findings from this lab (Webster et al., 1999), as well as others (Ascher et al., 1979; Varanda et al., 1985; Fieber and Adams, 1991; Giniatullin et al., 2000).

Although the kinetics of recovery suggest a single exponential process for 2-containing receptors, the inhibition of 34 receptors seems more complex. Recovery of 34 receptors occurred in two phases, one phase of which was too slow to resolve over 30 min. This would suggest either multiple sites of action or an inhibition-dependent allosteric conversion of receptors to a long-lived inactive state.

Our data provide an indication that S-(+)-mecamylamine may be a somewhat more desirable inhibitor of neuronal nAChR than R-()-mecamylamine. However, a number of issues deserve further investigation. Recent in vivo studies suggest that mecamylamine has unique pharmacological effects at relatively low doses. For example, low (0.1 mg/kg s.c.), but not higher, doses of mecamylamine were found to attenuate the plasma corticosterone response to stress in rats (Newman et al., 2000) and improve executive cognitive function in aged primates (Terry et al., 1999). It would therefore be of interest to look at the effects of mecamylamine and its stereoisomers on neuronal receptors containing the 5 subunits, because these may better model the properties of some ganglionic and brain-type receptors (Conroy et al., 1992; Vernallis et al., 1993; Conroy and Berg, 1998). Additionally, other differences are likely to exist between the receptors expressed in oocytes and those found in vivo, such as additional complex subunit arrangements, post-translational modifications, etc. These factors might also impact the sensitivity of native neuronal receptors to mecamylamine.

Although some previous reports have suggested that mecamylamine's effects in the brain may involve NMDA receptors (O'Dell and Christensen, 1988; McDonough and Shih, 1995), our data indicate that at least for the rat subtype consisting of NR1 and NR2b subunits, inhibitory effects of mecamylamine would be small and transient.

In conclusion, our results suggest that the new therapeutic potentials that have been proposed to exist for the central nervous systemactive nicotinic antagonists may be better realized if we take advantage of the stereochemistry of mecamylamine to target more selectively the nicotinic receptors of the brain. Also, our studies of mecamylamine and its stereoisomers illustrate that there still may be much to learn from old drugs when we approach them with modern methodology and the refined perspective of molecular neurobiology.