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2-Fluoro-3-(4-nitro-phenyl)deschloroepibatidine Is a Novel Potent Competitive Antagonist of Human Neuronal 42 nAChRs

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (G.R.A., M.I.D., B.R.M.); and Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, North Carolina (F.I.C.)

Received December 19, 2005; accepted February 27, 2006

	Abstract

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A patch-clamp technique in a whole-cell configuration was used to examine the functional activity of recently developed 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs on two major subtypes of neuronal nicotinic acetylcholine receptors (nAChRs), 42 and 34, that predominate in the central and peripheral nervous systems, respectively. These epibatidine analogs have been shown previously to possess high binding affinity to 42 but not to 7 nAChRs and to inhibit nicotine-induced analgesia in behavioral pain tests. The 2-fluoro-3-(4-nitro-phenyl)deschloroepibatidine (4-nitro-PFEB) exhibited the most pronounced antagonist activity among these analogs when tested electrophysiologically on 42 nAChRs. It inhibited acetylcholine (ACh)-induced currents in a concentration-dependent manner with an IC₅₀ value of 0.1 µM and produced complete inhibition at 1 µM concentration. 4-Nitro-PFEB at 0.1 µM concentration produced a 4-fold rightward shift in the ACh concentration-response curve without altering maximum ACh-induced response. This inhibitory effect of 4-nitro-PFEB was voltage- and use-independent and was partially reversible at its 1 µM concentration. The rise and decay kinetics of ACh-induced currents was not altered in the presence of 4-nitro-PFEB. In contrast to 42 nAChRs, this compound did not affect 34 nAChR-mediated currents at 1 µM (IC₅₀ 63.9 µM). Overall, these functional data agree with previous binding and behavioral findings and suggest collectively that 4-nitro-PFEB is the most effective and selective antagonist of 42 versus 34 and 7 nAChRs among the tested analogs, acting on 42 nAChR through a competitive mechanism with a potency 17-fold higher than that of dihydro--erythroidine.

2-Fluoro-3-(substituted phenyl)deschloroepibatidine analogs (Fig. 1) were obtained by replacement of the 2-chloro atom present in epibatidine by fluorine and addition of a 3-phenyl or a 3- or 4-substituted phenyl group to the pyridine ring bind. They bind with high affinity to 42 (the K_i values varied from 9 to 87 pM) but not to 7 nAChRs (Carroll et al., 2004). With the exception of 3-fluoro-PFEB, they antagonized nicotine-induced antinociceptive effects in the hot-plate test with potencies 2 to 4 times higher than that of mecamylamine, an nAChR subtype-nonselective blocker (Papke et al., 2001). In contrast to mecamylamine (Damaj et al., 1995), they failed to block nicotine-induced hypothermia.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Structure of epibatidine (left) and 2-fluoro-3-(substituted phenyl) deschloroepibatidine analogs (right). Substituents in the 4 and 3 positions of the phenyl group are indicated for six analogs and correspond in the descriptions of X and Y, respectively.

Herein, we report that 4-nitro-PFEB is a potent competitive antagonist of neuronal nAChRs that selectively inhibits 42-mediated currents with an IC₅₀ value of 0.1 µM (17-fold more potent than DHE). In 42 nAChRs, the inhibitory effect of 4-nitro-PFEB caused a shift of the ACh concentration-response curve typical for competitive antagonist, was both voltage- and use-independent, was not accompanied by alteration in the current kinetics, and was more pronounced after pre-exposure of the cell to the analog.

	Materials and Methods

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Cell Transfection and Culture. Stably transfected HEK 293 and SH-EP1 cells expressing rat 34 and human 42 neuronal nAChRs, respectively, were prepared as described previously (Zhang et al., 1999; Eaton et al., 2003). Both cell lines were maintained at 37°C with 5% CO₂ in the incubator. Growth medium for HEK 293 cells was minimum essential medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Transfection was conducted by the calcium phosphate method. The stably transfected cell line was raised in selective growth medium containing 0.7 mg/ml geneticin (Invitrogen, Carlsbad, CA). Growth medium for SH-EP1 cells was Dulbecco's modified Eagle's medium with high glucose supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 8 mM L-glutamine, 1 mM sodium pyruvate, and 0.25 µg/ml amphotericin (all from Invitrogen). Transfection was conducted by the electroporation method. This stably transfected cell line was raised in selective medium containing 0.5 mg/ml zeocin (Invitrogen) and 0.4 mg/ml hygromycin B (Roche Diagnostics, Indianapolis, IN). Reverse transcription-polymerase chain reaction analysis was used to confirm expression of nAChR subunit messages in the cells, and immunoprecipitation-Western analyses using solubilized membrane samples from transfected cells clearly indicated that subunits were expressed as a protein and assembled together. Control experiments excluded the possible activation of muscarinic ACh receptors by ACh application in both cell lines.

Difference in species of the nAChRs used in this study (rat 34 and human 42) is due to current unavailability of these nAChR subtypes that would be functional and expressed in the cells at a sufficiently high level. Rat and human nAChR subunits share 82 to 95% sequence identity, and when they are present in neuronal nAChR receptors of the same subunit composition, they provide numerous similarities between their properties (Chavez-Noriega et al., 1997, 2000; Zhang et al., 1999; Albuquerque et al., 2000; Xiao and Kellar, 2004).

Whole-Cell Current Recording. Functional expression of nAChRs was evaluated in the whole-cell configuration of the patch-clamp technique using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The patch electrodes, pulled from borosilicate glass capillaries (Sutter Instrument Company, Novato, CA), had a resistance of 2.5 to 3.5 M when filled with internal solution containing 110 mM Tris-phosphate dibasic, 28 mM Tris base, 11 mM EGTA, 2 mM MgCl₂, 0.1 mM CaCl₂, and 4 mM Na-ATP (pH adjusted to 7.3 with Tris base) (Wu et al., 2004). In some cells, 85% of electrode resistance was compensated electronically so that the effective series resistance in the whole-cell configuration was accepted when less than 20 M. Stably transfected HEK and SH-EP1 cells were studied for 2 to 3 days after plating the cells on the 15-mm round plastic coverslips (Thermanox; Nalge Nunc, Napierville, IL). Generation of voltage-clamp protocols and acquisition of the data were carried out using pCLAMP 9.0 software (Molecular Devices). Sampling frequency was 5 kHz and current signals were filtered at 5 or 10 kHz before digitization and storage. All experiments were performed at room temperature (22-25°C).

Application of Drugs and Perfusion System. Cells plated on coverslips were transferred to an experimental chamber mounted on the stage of an inverted microscope (Olympus IX50; Olympus Corporation, Tokyo, Japan) and were bathed in a solution containing 140 mM NaCl, 3 mM KCl, 2 mM MgCl₂, 25 mM D-glucose, 10 mM HEPES, and 2 mM CaCl₂ (pH adjusted to 7.4 with Tris base). The experimental chamber was constantly perfused with control bathing solution (1-2 ml/min). The amplitude and time course of currents mediated by neuronal nAChRs is highly dependent on the speed of drug application. The high-speed solution exchange system HSSE-2 (ALA Scientific Instruments, Westbury, NY) is able to switch rapidly between control and four test solutions delivered through two output tubes which face each other at 90° in the same plane. Under optimal conditions, the delay in switching between solutions is 10 ms. Data presented herein were obtained through subtraction from the leak current.

Data Analysis. The peak amplitude, the rise time (10-90%), and the exponential decay time constant () of the whole-cell currents were determined using the pCLAMP 9.0 program. EC₅₀,IC₅₀, and n_H values were determined with the Origin 5.0 program (OriginLab Corp., Northampton, MA). IC₅₀ values correspond to the concentration of inhibiting agent causing a 50% reduction in the current evoked by a pulse of ACh near the EC₅₀ value (20 µM for 42 and 100 µM for 34 nAChRs). The ACh-evoked currents in the presence of the analog were measured at -80 mV and normalized to the amplitude of the current elicited by ACh alone. Values were plotted against the concentrations of the inhibitor on a logarithm scale and fitted with an equation: y = 1/(1 + (IC₅₀/[analog])^nH), where n_H is the Hill coefficient.

To determine EC₅₀ values, ACh-induced responses were recorded at -80 mV in the absence or presence of the tested analog and were normalized to the amplitude of the current elicited by ACh alone at its saturating concentration (1 mM). Values were plotted against the concentration of ACh on a logarithm scale and fitted with an equation: y = 1/(1 + (EC₅₀/[ACh])^nH), where [ACh] is the ACh concentration, EC₅₀ is the concentration of ACh eliciting a half-maximal response, and n_H is the Hill coefficient. A similar approach was used to evaluate agonist effect of 4-nitro-PFEB in 34 nAChRs.

Results are presented as the mean ± S.E.M. for the number of cells (n) or as averaged means. Where appropriate, Student's t test for paired data were used, and values of P 0.05 were regarded as significant.

Drugs. ACh chloride, DHE, and salts were purchased from Sigma-Aldrich (Atlanta, GA). Six different 2-fluoro-3-(substituted phenyl)deschloroepibaitidine analogs (Fig. 1) were synthesized as reported previously (Carroll et al., 2004).

	Results

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ACh-induced whole-cell currents were elicited by pulse application (200 ms) of ACh on SH-EP1 or HEK 293 cells stably expressing human 42 or rat 34 nAChRs, respectively. In agreement with previous studies (Zhang et al., 1999; Wu et al., 2004), our control experiments indicated that the 42 nAChRs (EC₅₀ 23 µM) were more sensitive to ACh than 34 (EC₅₀ 101 µM).

Inhibitory Potency of the Analogs on 42 and 34 nAChRs. Based on the previously reported potent antagonist activity of 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs examined in nicotine-induced analgesia tests (Carroll et al., 2004) and evidence that the antinociceptive effect of nicotine occurs via activation of neuronal nAChRs (Marubio et al., 1999; Bitner et al., 2000), the potency of the 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs in inhibiting the neuronal nAChR activity of these two nAChR subtypes was determined. The cell under recording was exposed to an EC₅₀ concentration of ACh and 30 s later to ACh at the same concentration in the presence of various concentrations of the analog. When the inhibitory effect of the analog was reversible, two more concentrations were tested on the same cell. Except for the 3-fluoro-PFEB, the peak amplitude of ACh-induced currents was decreased by epibatidine analogs more effectively in 42 than in 34 nAChRs. Different potencies of the analogs for each receptor subtype are presented in Fig. 2, A and B. The IC₅₀ values and their ratios for 42 and 34 nAChRs and Hill coefficients for the analogs are summarized in Table 1. Comparison of IC₅₀ values for six 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs in the two nAChRs subtypes revealed that 4-nitro-PFEB induced half-maximal inhibition of 42 nAChR currents at a lower concentration (0.1 µM) than the other five analogs (Fig. 2A). In contrast, in 34 nAChRs the 4-nitro-PFEB was less potent as an antagonist than the other analogs, with a maximal inhibitory effect of 82 ± 1.9% and an IC₅₀ value of 63.9 µM (Fig. 2B). Thus, 4-nitro-PFEB analog was 639-fold more effective as an antagonist in 42 than in 34 nAChRs.

View larger version (30K): [in this window] [in a new window]   Fig. 2. The inhibitory effect of 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs on 42 and 34 nAChRs. Concentration-response relationships for the epibatidine analogs and DHE are presented for 42 and 34 nAChRs in A and B, respectively. The peak amplitude of ACh (EC50)-evoked currents was taken in each cell to normalize the peak amplitude of the currents, evoked in the presence of the analogs at different concentrations. The inhibitory potency of the analogs is compared with that of DHE. The concentrations of ACh used in 42 and 34 nAChRs were 20 and 100 µM, respectively. The curves were fitted to the Hill equation. Symbols and bars represent the mean ± S.E.M. of results obtained from three to eight cells for each point. Examples of inhibitory effect of 4-nitro-PFEB on ACh (EC50)-induced currents in cells expressing 42 nAChRs at 0.1 (C) and 1 µM (D) concentrations. E, inhibition induced by 1 µM DHE in a cell expressing 42 nAChRs. F, in 34 nAChR-expressing cells, coapplication of 1 µM 4-nitro-PFEB with ACh did not affect the peak amplitude of ACh(EC50)-induced control response. Holding potential, -80 mV.

View this table: [in this window] [in a new window]   TABLE 1 Potency of tested 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs and DHE for inhibition of ACh-induced currents in human 42 and rat 34 nAChRs

Figure 2, C and D, illustrate typical ACh-induced currents recorded from two 42 nAChR-expressing cells voltage-clamped at -80 mV in the absence and presence of 0.1 or 1 µM 4-nitro-PFEB that suppressed the amplitude of the ACh-induced response by half or almost completely, respectively. Pre-exposure (30 s) of four cells to 0.1 µM 4-nitro-PFEB resulted in a strong enhancement of the inhibitory effect of the 4-nitro-PFEB on 42 nAChR activity (new coverslip with the cells was used each time). Potency of the 4-nitro-PFEB in inhibition of human 42 nAChR activity was compared with that of DHE under similar experimental conditions. The IC₅₀ value for DHE in human 42 nAChRs was determined as 1.7 µM (Fig. 2A), being 13-fold lower than that for rat 34 nAChRs (22 µM) (Fig. 2B). Less than 50% inhibition occurred in the presence of 1 µM DHE (Fig. 2E), and an increase of the DHE concentration up to 10 µM suppressed the current by 80% (Fig. 2A). In contrast to 42, ACh-induced current mediated by 34 receptors was not affected in the presence of 1 µM 4-nitro-PFEB (Fig. 2F) and was inhibited only by 22 ± 8% at its 10 µM concentration (Fig. 2B).

Control experiments were performed to test the 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs for possible agonist activity when the cells were examined first for the presence of nAChR functional expression, followed by the application of each analog depicted in Fig. 1. No substantial current activation was elicited at either 1 or 10 µM concentrations of the analogs at 42 nAChRs. The agonist effect of 4-nitro-PFEB, the most potent 42 antagonist, is shown in a representative cell in Fig. 3A. In 34 nAChRs, the analogs applied alone at a 10 µM concentration induced some current activation when expressed as a percentage of the current induced by 100 µM ACh and averaged (n = 4-5 cells) 4.6, 1.3, 4.8, 1.8, 31.8, and 6.0% for 3-fluoro-, 4-fluoro-, 3-chloro-, 4-chloro-, 4-nitro-, and 3-nitro-PFEBs, respectively. At 1 µM, 4-nitro-PFEB induced 10% of the half-maximal ACh-induced whole-cell response in 34 nAChRs. Detailed examination of the effect of the 4-nitro-PFEB by itself on 34 nAChRs revealed that the compound behaved as a weak partial agonist with an intrinsic activity of 23.7 ± 1.8%, when normalized to that produced by the full agonist ACh at 1 mM concentration, and revealed an EC₅₀ value of 3.7 µM (Fig. 3B). It is important to note that at concentrations 1 µM, the agonist effect of 4-nitro-PFEB on 34 nAChRs was negligible.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Testing of 4-nitro-PFEB for possible agonist activity. A, current traces recorded from the same representative cell expressing 42 nAChRs in the presence of 20 µM ACh (top) or 10 µM 4-nitro-PFEB (bottom). B, dose-response curves for ACh and 4-nitro-PFEB tested on 34 nAChRs. For each compound, data points indicate mean ± S.E.M. of the peak current amplitudes (n = 4-3), normalized to that induced by 1 mM ACh (holding potential, -80 mV). The curves were fitted to the Hill equation. The EC50 and nH values for ACh and 4-nitro-PFEB were 101 µM and 2.5 and 3.7 µM and 1.8, respectively.

Mechanism of Action of the 4-Nitro-PFEB at an 42 nAChR.

View larger version (16K): [in this window] [in a new window]   Fig. 4. The mechanism underlying the inhibitory action of 4-nitro-PFEB in 42 nAChRs. Effect of the 4-nitro-PFEB on the concentration-response relationship for ACh-evoked currents. In each cell, the peak amplitude of the currents evoked by 1 mM ACh was used to normalize the peak amplitude of currents evoked by other concentrations of ACh in the presence or absence of 4-nitro-PFEB. Each symbol represents the mean ± S.E.M. (total n = 21). The EC50 and nH values for ACh were 23 versus 106 µM, and 0.84 versus 1.2, in the absence and presence of 0.1 µM 4-nitro-PFEB, respectively.

Effect of the 4-Nitro-PFEB on the Kinetics of ACh-Induced Currents in 42 nAChRs.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Effect of 4-nitro-PFEB on the rise time and decay of ACh-induced currents in 42 nAChRs. Top inset, superimposed and normalized recordings evoked by the application of ACh (20 µM) in the absence and the presence of 0.1 µM 4-nitro-PFEB to a cell held at -80 mV. The bar graph shows a comparison of the rise time and decay time constants of the ACh-induced currents evoked in the absence () and presence () of 4-nitro-PFEB. Results are presented as means ± S.E.M. (n = 8-9).

Reversibility of Inhibition and Its Use and Voltage Dependence. The reversibility of 4-nitro-PFEB inhibition in 42 nAChRs was tested with a subsequent application of ACh to the cell. The experiment shown on Fig. 6A was performed on a cell exposed to 1 µM 4-nitro-PFEB. The inhibition was in part reversible so that at the sixth pulse of 20 µM ACh in 2.5 min, ACh induced a response of 25% (n = 3) of its initial magnitude. The magnitude of the current did not increase further after four ACh applications. Longer washout experiments were complicated by the limitations of maintaining cells under excellent recording conditions. After inhibition with 0.1 µM 4-nitro-PFEB, it was possible to achieve full recovery in the ACh-induced responses after two to four ACh pulses (n = 4).

View larger version (29K): [in this window] [in a new window]   Fig. 6. Reversibility, use dependence, and voltage dependence of the inhibitory effect of 4-nitro-PFEB in 42 nAChRs. A, ACh-induced currents as a control (left), after registration of stable inhibition in the presence of 1 µM 4-nitro-PFEB (center), and during washout of the analog (right). The ACh-induced currents shown on the right were recorded in a time interval of 25 s, and, after being normalized in their peak amplitude to that of control ACh-induced response, were plotted versus timing (holding potential, -80 mV). B, use dependence of the inhibitory effect of 4-nitro-PFEB in 42 nAChRs. 4-Nitro-PFEB inhibition was not use-dependent. Five consequent pulses of ACh (20 µM, 200 ms) were delivered to the representative cell every 20 s, and then, 30 s after the last ACh pulse, were repeated in 20-s intervals on the same cell in the presence of 0.1 µM 4-nitro-PFEB analog. C, inhibitory effect of 4-nitro-PFEB on the amplitude of ACh-induced currents in 42 nAChRs at various holding potentials. Current traces were evoked by the application of 20 µM ACh () or 20 µM ACh plus 0.1 µM 4-nitro-PFEB () to a representative cell held at various potentials of -100, -80, -60, -40, and -20 mV (shown as insets) and plotted versus the corresponding holding potential (left). The relationship between the holding potential and the ratio of the amplitude of the currents evoked in the presence of the analog to ACh alone at the corresponding holding potential is summarized for four cells on the right. Symbols and bars in C represent the mean ± S.E.M.

The 42 nAChR-expressing cells were also examined with respect to use-dependence of the inhibitory effect of 4-nitro-PFEB (Fig. 6B). As expected, in three cells, there was no progressive change in the inhibition when the ACh-induced pulses in the presence of 0.1 µM 4-nitro-PFEB were repeated at least five consecutive times in 20-s intervals. These data demonstrate that the inhibitory effect of 4-nitro-PFEB was not use-dependent.

Analysis of the voltage dependence of the effect of 4-nitro-PFEB on the peak amplitude of the 42 nAChR-mediated current favored the notion that 4-nitro-PFEB acts as a competitive antagonist at this nAChR subtype. Currents evoked by 200-ms pulses of ACh (20 µM) were recorded from 42 nAChR-expressing cells in the absence and presence of 4-nitro-PFEB as the holding potential was changed from -100 to -20 mV in 20-mV steps. Due to a strong rectification of the outward currents at positive holding potentials typical for 42 nAChRs, the analysis was performed only at this range of the holding potentials. The data were combined by normalizing all of the responses in the presence of 4-nitro-PFEB relative to the peak amplitude of the control ACh-induced currents at the same holding potential (Fig. 6C, left). The ratio of the peak amplitude evoked by ACh in the presence of the analog to the amplitude of the current evoked by ACh alone did not change significantly at the holding potentials from -100 to -20 mV (Fig. 6C, right, n = 4). Thus, the reduction by the analog of the peak ACh-induced currents in 42 nAChRs was voltage-independent.

	Discussion

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Our results demonstrate that 2-fluoro-3-(4-nitro-phenyl-)deschloroepibatidine acts as a potent competitive antagonist at 42 nAChRs, which give rise to the majority of nicotinic responses in the central nervous system. Based on the IC₅₀ values obtained in this study, the 4-nitro-PFEB seemed to be 17-fold more potent in inhibiting 42 nAChR-mediated currents than DHE, which is currently known as one of the most potent competitive antagonists of 42 nAChRs, and 639-fold more potent in inhibiting 42 than 34 nAChRs.

Higher potency of 4-nitro-PFEB than of the other 2-fluoro-3-(substituted phenyl)deschloroepibatidine analogs in inhibiting ACh-induced currents in 42 nAChRs is consistent with its higher affinity in binding assays (K_i 9 pM) and more pronounced antagonist effect of nicotine-induced analgesia (AD₅₀ 0.12 mg/kg) (Carroll et al., 2004). This analgesia, as measured in the hot-plate test, occurs at the supraspinal level and has been shown to be mediated by 42 nAChRs (Marubio et al., 1999). On the other hand, the 3-fluoro-PFEB, which produced only 20% inhibition of ACh-induced currents at 10 µM concentration, exhibited little analgesia in the hot-plate test (20% at 1 mg/kg) and bound with lower affinity than the other five analogs to nAChR receptors in the rat brain (K_i 87 pM) (Carroll et al., 2004). 3-Fluoro-PFEB was the most effective in inhibiting ACh-evoked currents in 34 nAChRs. There was also consistency between the functional potency order of the other four analogs with their binding and behavioral data: the 4-fluoro- and 4-chloro-PFEBs had similar IC₅₀ values of 0.4 µM and possessed somewhat lower binding affinity (K_i values were 29 and 44 pM, respectively) than the 4-nitro-PFEB in the rat brain. They also had similar AD₅₀ values in the hot-plate test (0.23 and 0.26 mg/kg) that were higher than that for 4-nitro-PFEB. 3-Chloro- and 3-nitro-PFEBs were less effective than 4-fluoro- and 4-chloro-PFEBs in inhibition of ACh-induced currents (IC₅₀ values were 0.8 and 0.7 µM, respectively). Likewise, they possessed lower affinity in the binding assays (K_i values were 73 and 53 pM, respectively), and the AD₅₀ value in the hot-plate test was higher (0.45 mg/kg) for 3-chloro-PFEB. However, no correlation was observed between the hot-plate test (AD₅₀ 0.13 mg/kg), receptor affinity, and functional data for 3-nitro-PFEB.

The inhibitory potency of 4-nitro-PFEB was compared under similar experimental conditions with that of another routinely used competitive antagonist of the 42 nAChRs, DHE, that has a K_i value of 14.3 nM for nicotinic receptors in the rat brain (Damaj et al., 1995).The AD₅₀ value for DHE for blocking nicotine antinociception in the tail-flick test was 0.45 mg/kg (Damaj et al., 1995) compared with the AD₅₀ of 0.003 mg/kg for 4-nitro-PFEB. The IC₅₀ values for these two competitive antagonists obtained under similar experimental conditions indicated that 4-nitro-PFEB was 17-fold more potent than DHE in inhibition of 42 nAChR activity. Indeed, 1 µM concentration of 4-nitro-PFEB was much more effective in inhibiting ACh-induced current in 42 nAChRs than a similar concentration of DHE. It was necessary to increase the DHE concentration to 10 µM to achieve a similar inhibition, as induced by 1 µM 4-nitro-PFEB. The IC₅₀ value for DHE determined in our study using whole-cell recordings from SH-EP1 cells stably expressing human 42 nAChRs (1.7 µM) corresponds well to values reported for human 42 nAChRs using ⁸⁶Rb⁺ efflux in different expression systems, such as SH-EP1 cells (1.5 µM) (Eaton et al., 2003) or HEK 293 cells (1.9 µM) (Gopalakrishnan et al., 1996). This IC₅₀ value is 13-fold lower than the IC₅₀ value for DHE for rat 34 nAChRs expressed in HEK 293 cells (22 µM), which is very similar to that (23 µM) for rat 34 nAChRs expressed in Xenopus oocytes (Harvey and Luetje, 1996). Similar to the partial recovery after the methyllycaconitine effect in hippocampal nicotinic receptors (Alkondon et al., 1992), the 4-nitro-PFEB (1 µM) inhibition of 42 nAChRs was reversible only in part after a 4-min washout, whereas after 10 µM DHE inhibition, the recovery was complete in the same time frame (data not shown), possibly because of slow receptor/4-nitro-PFEB dissociation.

Studies of the concentration-response relationship for ACh-evoked currents in the absence and presence of 0.1 µM analog (IC₅₀ concentration) demonstrated that the analog increased the EC₅₀ value of ACh from 23 to 106 µM, whereas the maximal responsiveness of the 42 receptors for ACh was not affected by 4-nitro-PFEB, and the inhibitory effect of the analog on current amplitude was more pronounced after pre-exposure of the cell, suggesting that the analog may also act on 42 nAChR channels that are not opened. Furthermore, the reduction of the peak amplitude of ACh-induced currents in the presence of 4-nitro-PFEB was voltage-independent, suggesting that the analog does not interact with sites located inside the ion channel pore. The fact that the reduction of the ACh-induced current amplitude was not accompanied by an alteration of the rise time was probably due rather to the limitations in the speed of the application system, whereas the absence of the effect on the decay kinetics of the currents suggests that 4-nitro-PFEB does not affect desensitization of the receptor. Together, our findings support the notion that 4-nitro-PFEB is a competitive antagonist of 42 nAChRs. It is important to mention that it lacked significant agonistic activity at 42 nAChRs.

4-Nitro-PFEB also exhibits neuronal nAChR selectivity. 4-Nitro-PFEB bound with high affinity to 42 but not to 7 neuronal nAChRs in the rat brain (Carroll et al., 2004). In contrast to its ability to inhibit 42 nAChRs, a much higher concentration was required to inhibit ACh-induced currents in 34 nAChRs, and only 60% inhibition was achieved at a 100 µM concentration. Although 4-nitro-PFEB exhibited weak partial agonist activity in 34 nAChRs, it is important to stress that at the 1 µM concentration, at which 4-nitro-PFEB induced almost complete inhibition of 42 nAChR activity, it did not markedly inhibit ACh-induced currents in 34 nAChRs or evoke substantial activation of 34 nAChRs.

Considering the current lack of 42 nAChR subtype-selective competitive antagonists and the heterogeneity of nAChR subtypes in a single neuron (Azam et al., 2002), the novel compound 2-fluoro-3-(4-nitro-phenyl)deschloroepibatidine may serve as a pharmacological tool for specific isolation of responses mediated by native neuronal nAChRs containing the 42 subunit combination (Dani et al., 2004). Because of high binding affinity of 4-nitro-PFEB to 42 nAChRs (Carroll et al., 2004), this compound may serve as a guide for the development of additional 42 subtype-selective probes.

A subtype-specific neuronal nAChR antagonist has properties suggesting that it can be used as a therapeutic agent. 42 nAChRs are expressed in a high density in the ventral tegmental area, substantia nigra, and nucleus accumbens, which are believed to play a central role in the reinforcing effect of nicotine (Wooltorton et al., 2003). 42 nAChRs are localized on pre- or postsynaptic sites and presynaptically modulate dopamine release (Zhou et al., 2001). Nicotine-induced up-regulation of 42 AChRs (Picciotto et al., 1995; Vallejo et al., 2005) on presynaptic dopamine-releasing terminals probably leads to enhanced presynaptic depolarization and an increased release of dopamine. In support of this premise, self-administration of nicotine is reduced in 2 subunit knockout mice (Picciotto et al., 1998). Another supportive finding is that the antidepressant bupropion, which affects neuronal nAChRs and dopamine/norepinephrine transporters, is used clinically for smoking cessation (Slemmer et al., 2000; Ross and Williams, 2005). In contrast to bupropion-induced inhibition of neuronal nAChRs, which is not subtype-selective (34>42) (Alkondon and Albuquerque, 2005), 4-nitro-PFEB is a potent competitive selective antagonist of 42 versus 7 and 34 nAChRs. Therefore, this compound may serve as a valuable investigative agent for further exploring the role of 42 nAChRs in nicotine dependence. Although it remains to be established how effective antagonists will be in the treatment of nicotine dependence, it is anticipated that 4-nitro-PFEB will not readily act at peripheral neuronal nAChR function (De Biasi, 2002), thereby decreasing potential side effects.

	Acknowledgements

 
Human 42 in SH-EP1 cells were generously provided by Dr. R. Lukas (Barrow Neurological Institute, Phoenix, AZ) and rat 34 in HEK 293 cells Dr. K. Kellar from (Georgetown University, Washington, DC).

	Footnotes

 
This work was supported by National Institute on Drug Abuse grants P50-DA05274 and R01-DA12001.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.021782.

ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; DHE, dihydro--erythroidine; 4-nitro-PFEB, 2-fluoro-3-(4-nitro-phenyl)deschloroepibatidine; HEK, human embryonic kidney; ACh, acetylcholine; A-186253, 2-chloro-3-(4-chloro-phenyl)-5-((S)-1-methyl-pyrrolidin-2-ylmethoxy)-pyridine.

Address correspondence to: Dr. Galya Abdrakhmanova, Assistant Professor, Department of Pharmacology and Toxicology, Virginia Commonwealth University, 1112 E. Clay Street, P.O. Box 980524, Richmond, VA 23298. E-mail: gabdrakhmano{at}mail1.vcu.edu

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