Introduction

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Although the primary mechanism by which TCAs relieve the symptoms of depressive illness appears to involve the inhibition of neurotransmitter reuptake, TCAs have additional effects on a variety of ion channels (Choi et al., 1992; Ogata et al., 1989; Schauf et al., 1975). For example, TCAs block Na⁺ channels in cardiac tissues in a fashion similar to the action of local anesthetics (Barber et al., 1991; Nattel, 1987), an effect that may contribute to their alteration in cardiac conduction and dysrhythmogenic actions. In addition to their antidepressant actions, TCAs have been known to exert an analgesic effect (Paoli et al., 1960) that is independent of the antidepressant action (Panerai et al., 1990). Acute administration of TCAs can exert an analgesic effect (Bromm et al., 1986; Coquoz et al., 1991; Poulsen et al., 1995), which may be useful in postoperative pain management (Tiengo et al., 1987). Long-term administration of the TCAs amitriptyline and desipramine diminishes pain in patients with diabetic neuropathy (Max et al., 1987, 1992) and other chronic pain conditions (Magni, 1991; Watson et al., 1982). Because the effects of TCAs on neuronal Na⁺ currents are less well described, we conducted a series of experiments to determine the sensitivity of neuronal Na⁺ channels to TCAs, and we compared their potency with that of the two atypical antidepressants, trazodone and fluoxetine, which differ greatly in analgesic potency. Bovine adrenal chromaffin cells, a well-characterized model for neuronal electrophysiology and secretion, were used to study the alteration of voltage-gated Na⁺ current (I_Na) and modulation of Na⁺ channel inactivation by antidepressants. In addition, the use-dependent Na⁺ channel blockade by amitriptyline was characterized in isolated DRG neurons. A preliminary account of this work has been reported (Pancrazio et al., 1996).

	Methods

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Bovine adrenal chromaffin cells were generously supplied by Dr. Y. I. Kim (Department of Biomedical Engineering, University of Virginia, Charlottesville, VA) and were isolated according to a previously reported method (Greenberg and Zinder, 1982) with modifications (Creutz et al., 1987). Neonatal (P8-P12) rat DRG neurons were isolated by a method modified from McLean et al. (1988). Briefly, after dissection from the perispinal tissue, ganglia were minced and incubated in 2.5 mg/ml trypsin (Sigma Chemical, St. Louis, MO) supplemented with 2 mg/ml type I collagenase (Sigma Chemical) for 30 min at 37°C. Cells were triturated five to eight times and resuspended in Dulbecco's modified Eagle's medium (GIBCO BRL, Gaithersburg, MD) with 10% fetal calf serum (Hyclone Laboratories, Logan, UT), 50 U/ml penicillin and 50 µg/ml streptomycin (Sigma Chemical). Cells were plated onto poly-L-lysine-coated glass coverslips and maintained in an incubator at 37°C in 5% CO₂/95% air. Best results for voltage-clamp experiments were obtained from cells within 2 days of isolation, before extensive processes developed.

For measurement of I_Na in bovine adrenal chromaffin cells, the external bathing solution contained (in mM) 141 NaCl, 5 KCl, 0.2 CaCl₂, 1 CoCl₂, 1 MgCl₂ and 10 HEPES, adjusted to pH 7.4 with 1 M NaOH. The patch pipette solution contained (in mM) 120 CsCl, 20 tetraethylammonium chloride, 1 CaCl₂, 11 EGTA-CsOH, 11 HEPES and 5 MgATP, adjusted to pH 7.3 with 1 M HCl. For voltage-clamp measurements from DRG neurons, the external recording solution contained (in mM) 10 or 30 NaCl, 130 or 150 tetraethylammonium chloride, 1 MgCl₂, 0.2 CaCl₂, 1 CoCl₂ and 10 HEPES, adjusted to pH 7.4 with 1 M CsOH. The patch pipette solution for these experiments contained (in mM) 100 CsCl, 2.5 MgCl₂, 10 EGTA, 30 CsOH, 40 HEPES, 2 MgATP and 0.3 NaGTP, adjusted to pH 7.3 with 1 M CsOH. For current clamp measurements from DRG neurons, the external recording solution contained (in mM) 140 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂ and 10 HEPES, adjusted to pH 7.4 with 1 M NaOH. The pipette solution contained (in mM) 140 KCl, 0.5 EGTA-KOH, 5 MgATP and 5 HEPES, adjusted to pH 7.3 with 1 M HCl. Using a gravity-feed perfusion system, complete solution exchange was accomplished within 2 sec, and measurements were typically made 120 sec after solution application. Amitriptyline was obtained from Sigma Chemical. Desipramine, doxepin and trazadone were obtained from Research Biochemicals (Natick, MA). Fluoxetine was provided by Eli Lilly (Indianapolis, IN). For our detailed mechanistic studies, we focused on amitriptyline as a model TCA.

Standard patch-clamp methods were used as described by Hamill et al. (1981). Voltage-clamp or current-clamp measurements were taken 4 to 6 min after initiation of the whole-cell recording configuration using the Axopatch 200 (Axon Instruments, Foster City, CA) patch-clamp amplifier. Patch electrodes were fabricated from KIMAX-51 borosilicate glass (American Scientific, Charlotte, NC) with a two-stage micropipette puller and heat-polished with a microforge and had resistances of 1.5 M when filled with internal solution. Unless otherwise noted, cells were voltage-clamped at 80 mV and the commonly used P/n approach (n = 4) was used to estimate leakage and capacitive current. Whole-cell currents were filtered at 5 kHz with a four-pole Bessel low-pass filter and digitized at 20 kHz. Current records were analyzed offline using a custom program capable of preparing I-V relations and nonlinear curve fitting (Pancrazio, 1993) or with Sigmaplot for Windows version 2 (Jandel, San Rafael, CA). For steady-state activation and inactivation estimates, data were fit to the Boltzmann function, f(V):

(1)

where V_n is the potential when f(V) is equal to 0.5 and k_n is the slope factor.

Where appropriate, data are presented as mean percentage of control ± S.E.M. and the number of cells tested (n). The rates of recovery from inactivation and the onset of use-dependent blockade were fit to exponential equations using Sigmaplot. For the analysis of concentration-dependence and rate of recovery from inactivation, standard errors derived from the fitted data were used to test for significant differences. Statistical significance of a drug effect was determined using paired Student's t test with P < .05 considered significant.

	Results

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As initially reported by Fenwick et al. (1982), bovine adrenal chromaffin cells express a rapidly activating and inactivating I_Na, which in our experiments reached a peak of 845 ± 450 pA (mean ± S.D., n = 54) in response to a step depolarization of 10 to +10 mV from a holding potential of 80 mV. The addition of amitriptyline to the bathing solution induced a marked decrease in I_Na over a range of test potentials (fig. 1A) within 30 sec of drug application. In the presence of 20 µM amitriptyline, peak I_Na fell by 51 ± 2% (mean ± S.E.M., n = 13 cells). The time to peak (t_P) of I_Na was not affected by antidepressant treatment; for example, t_P at 0 mV under control conditions and in the presence of amitriptyline was 1.3 ± 0.1 and 1.2 ± 0.1 msec (n = 5), respectively. Figure 1B summarizes the peak I_Na triggered by test potentials ranging from 50 to +60 mV from a holding potential of 80 mV under control, 20 µM amitriptyline-treated and recovery conditions. Although there appeared to be no obvious shift in the voltage dependence of activation based on the I/V relation, steady-state activation was estimated by calculation of the peak conductance, G_Na(V), at each test potential, V, according to Ohm's law:

(2)

where E_Na is the apparent reversal potential based on the I-V curve. G_Na(V) was then fit to the Boltzmann function, which under control conditions yielded k_n = 6.3 ± 0.5 mV (n = 10) and V_n = 9.1 ± 3.0 mV. Amitriptyline altered neither k_n nor V_n, suggesting a lack of effect on the voltage dependence of Na⁺ channel activation.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Inhibition of INa from a representative bovine adrenal chromaffin cell by the TCA amitriptyline. A, INa waveforms shown were elicited by step potentials to 20, 10 and 0 mV from a holding potential of 80 mV. The dotted lines indicate the zero transmembrane current level. B, Peak INa as a function of test potential indicates no obvious shift in the voltage-dependence of activation with the application of 20 µM amitriptyline.

The suppression of I_Na by amitriptyline was concentration dependent and also characteristic of the TCAs doxepin, desipramine and nortriptyline (fig. 2). The concentration dependence of amitriptyline-induced blockade was fitted to a logistic equation:

(3)

where K_D is the concentration for half-maximal blockade, [D] is the drug concentration and n_H is the slope factor (or Hill coefficient). For amitriptyline, K_D and n_H were estimated to be 20.2 µM and 1.2, respectively, consistent with the notion of a single binding site. Although the suppression of I_Na was a shared property of the TCAs examined, the atypical antidepressants trazodone and fluoxetine differed in their ability to inhibit I_Na. Trazodone (100 µM) induced only a minor blockade of I_Na to 80 ± 2% (n = 4) of control (fig. 3A). In contrast, 20 µM fluoxetine reversibly depressed I_Na to 38 ± 4% (n = 5) of control (fig. 3B), a level comparable to that induced by amitriptyline, while the onset of I_Na blockade was somewhat slower than that of amitriptyline and the other TCAs. Figure 4 illustrates the comparative time course of amitriptyline and fluoxetine-induced blockade on I_Na measured from the same chromaffin cell. Cells were repetitively pulsed from 80 to 0 mV every 4 sec, a rate slow enough to permit complete recovery from inactivation (determined by pilot studies) but fast enough to allow resolution of onset and recovery from blockade. Both phases were well described by a single exponential decay function with on and off time constants _ON and _OFF, respectively. For 20 µM amitriptyline, _ON was 16 ± 3 sec (n = 4), and _OFF was 26 ± 4 sec. In contrast, the time course for blockade with 20 µM fluoxetine was significantly slower: _ON was 126 ± 24 sec (n = 4) and _OFF was 54 ± 7 sec. Unlike amitriptyline, the onset of I_Na suppression was preceded by a delay of 45 ± 13 sec (n = 4), while recovery began immediately on removal of fluoxetine from the recording chamber.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Concentration dependence of INa inhibition by tricyclic and atypical antidepressants. Data are reported as mean ± S.E.M. of percentage of control peak INa triggered from a holding potential of 80 mV. For amitriptyline (), KD and nH, which correspond to the concentration for half-maximal blockade and the number of binding sites, respectively, were estimated to be 20.2 µM and 1.2.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Differential effects of atypical antidepressants on INa in two typical chromaffin cells. Although 100 µM trazodone exerted only a minor effect, 20 µM fluoxetine produced a level of blockade similar to that of the TCAs.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Comparative time course of the onset of and relief from blockade by equivalent concentrations of antidepressants in the same chromaffin cell. Amitriptyline (20 µM) produced a relatively fast onset and recovery of INa blockade, while the time course for fluoxetine (20 µM) was relatively slower with an initial delay. The onset of blockade was fitted to A0 + A1exp(t/ON), and the recovery phase was fitted to B0 + B1[1  exp(t/OFF)].

Time-dependent Na⁺ channel inactivation was assessed by fitting the inactivating phase of I_Na to a single exponential decay function of time, I_Na = A·exp(t/_H), where A is the current amplitude and _H is the time constant of inactivation. Amitriptyline (20 µM) had no effect on the time course of inactivation for test potentials ranging from 10 to +40 mV. For example, _H was 1.5 ± 0.2 and 1.4 ± 0.2 msec (n = 5) under control and amitriptyline-treated conditions, respectively, in response to a voltage step to 0 mV. Although the TCAs did not change the inactivation rate, steady-state inactivation was markedly altered (fig. 5A). To estimate steady-state inactivation, peak I_Na was triggered by a test pulse to 0 mV from prepulse potentials, 5 sec in duration, from 95 to +40 mV. For one set of experiments, data were normalized and fit to the Boltzmann function, yielding control values for k_n of 6 ± 1 mV (n = 5) and for V_n of 58 ± 2 mV. Amitriptyline (20 µM) shifted V_n toward hyperpolarized potentials by 15 ± 1 mV (n = 5), while it exerted no observable effect on k_n. This effect was readily reversible and concentration dependent (fig. 5B). To gain further insight into the mechanisms of amitriptyline-induced blockade of I_Na inactivated state, the rate of recovery from inactivation was examined. This was accomplished using two voltage steps to +10 mV separated by a repolarizing interpulse to 80 mV for durations ranging from 120 msec to 6 sec. Recovery from inactivation was estimated by the ratio of peak I_Na evoked by the second pulse (I_Na,2) to peak I_Na triggered by the initial pulse (I_Na,1). The results plotted in figure 6 show that amitriptyline delayed recovery of a major fraction of I_Na from inactivation. Under both control and amitriptyline-treated conditions, I_Na,2/I_Na,1 was well fit as a biexponential process:

(4)

where A_F and _RF are the amplitude and time constant for a fast component, and A_S and _RS are the amplitude and time constant for a slow component. As shown in figure 6, 10 µM amitriptyline markedly increased _RS by 10-fold. To assess this in another manner, depression of the peak I_Na by amitriptyline was estimated using lower holding potentials (V_H) in which more channels would be inactivated. For V_H of 70, 60 and 50 mV, the EC₅₀ was estimated to be 14, 7 and 3 µM amitriptyline.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   The effect of antidepressants on steady-state inactivation of INa. Chromaffin cells were held at a prepulse voltage ranging from 95 to 40 mV for 5 sec before evoking a test pulse to 0 mV. Currents were not corrected for leak and capacitive artifact; instead, leakage current was considered negligible at 0 mV. For each cell, the peak INa triggered after each prepulse voltage was normalized to the peak INa and fit to the Boltzmann function with parameters describing the voltage for half steady-state inactivation, Vn, and the slope factor, kn. A, Steady-state inactivation from a cell before (), during exposure to 20 µM amitriptyline () and after recovery (). B, Concentration-dependence of amitriptyline of the shift in Vn, and effect of 20 µM concentration of the other drugs examined.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Amitriptyline decreases the rate of recovery from inactivation. A dual-pulse voltage-clamp protocol, which had an interpulse voltage of 80 mV, 20 msec to 6 sec in duration, was applied to chromaffin cells. The ratio of INa elicited by the second pulse to INa triggered by the first pulse, INa,2/INa,1 was fitted to 1  AF exp(t/RF)  AS exp(t/RS). Amitriptyline (10 µM) increased the slower time constant of recovery (RS), suggesting that it stabilizes an inactivated state of the channel.

To verify Na⁺ channel inhibition in the DRG neurons by amitriptyline, voltage-clamp measurements were taken using an external recording solution with the Na⁺ concentration decreased from 141 mM to 10 or 30 mM to ensure suitable clamp conditions. DRG neurons can express two types of Na⁺ channels (Roy and Narahashi, 1992), which may account for the differential response of neurons to sustained depolarizing current injections under current-clamp conditions (Elliott and Elliott, 1993). As shown in figure 7, both slow and fast I_Na, measured from two different neurons, appeared to exhibit a similar range of sensitivity to amitriptyline. Overall, DRG peak I_Na fell to 48 ± 4% of the control amplitude with application of 20 µM amitriptyline, a level similar to that of chromaffin cell I_Na.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Direct demonstration of INa inhibition in rat DRG neurons by 20 µM amitriptyline. The slow (A) and fast (B) INa waveforms shown were both elicited by step potentials to 10 mV from a holding potential of 80 mV from two different neurons. Although there appears to be two kinetically distinct forms of INa in DRG neurons, consistent with previous work (Elliott and Elliott, 1993; Roy and Narahashi, 1992), amitriptyline appeared to have similar blocking efficacy.

To assess the effect of the slowed recovery from inactivation in generating use-dependent blockade, DRG neurons were voltage-clamped and depolarized from 80 mV to 10 mV for 20 msec at a rate of 10 Hz. The results of a 10-Hz stimulus for 14 pulses is shown in figure 8, and the greater use-dependence in 10 µM amitriptyline is evident from the tracings of I_Na with subsequent depolarizations. The peak current of the nth depolarization (I_Na,n) could be described as an exponential function of the number of depolarizations following the initial peak current after rest (I_Na,1):

(5)

where f is percent of the peak I_Na that decreased with repetitive depolarization, and b is the rate constant of decline expressed as number of depolarizations. The solid lines in figure 8C are least-squares fits to equation 5 for the experimental traces in A and B. In the five cells studied in this manner, physiological use-dependence was evident in that control peak I_Na,1 decreased from 5.8 ± 1.2 to 5.1 ± 1.2 nA with the repetitive 10-Hz depolarizations, a 14 ± 6% decline. In 10 µM amitriptyline, mean I_Na,1 was 4.6 ± 1.4 nA and decreased to a steady-state of 3.4 ± 1.3 nA, a significantly greater decrease of 35 ± 7% (P = .012, paired t test). Rate constants b for control and in the presence of 10 µM amitriptyline, were 7.7 ± 1.3 and 7.6 ± 0.5 depolarizations, respectively. The resting block of I_Na,1 caused by amitriptyline was to 73 ± 8% of control, an effect identical to that observed in chromaffin cells, but the steady-state depression by 10 µM amitriptyline at 10 Hz was to 56 ± 11% of the control steady-state I_Na at 10 Hz (P = .008, paired t test). Because fluoxetine did not show as marked a shift in steady-state inactivation, we examined its effect on use-dependent decline in I_Na by using a pulse protocol identical to that described in figure 8. In five experiments in DRG cells, the resting block of I_Na,1 caused by 10 µM fluoxetine was to 51 ± 12% of control, while the steady-state use-dependent depression at 10 Hz was to 40 ± 13% of the control steady-state I_Na at 10 Hz (P = .031, paired t test). Despite a somewhat greater block of I_Na,1 compared with amitriptyline, the use-dependent effect of fluoxetine appeared less prominent at 10 Hz.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   The use-dependent decline in current in peak INa under control conditions and after the application of 10 µM amitriptyline. P/4 subtraction was not used. A 20-msec depolarization from 80 to 10 mV was applied at a rate of 10 Hz in solution containing 30 mM Na+. A and B, Currents in response to the first, fourth, eighth and fourteenth depolarizations for control and with amitriptyline. C, Peak INa values from the experimental traces in A and B are plotted for 14 consecutive depolarizations. The curves fit to the peak currents are defined by the equation: INa,n = INa,1 (1  f{1f) exp[(1  n)/b]}), where INa,1 is the peak INa of the first impulse (n = 1). For control and amitriptyline, the fractional use-dependent decline of current (f) was 2.2% and 44.7%, respectively, and the rate constant b was 7.4 and 8.9 depolarizations, respectively.

To determine whether amitriptyline inhibition of neuronal Na⁺ channels would alter physiological processes, we examined AP behavior and I_Na in neonatal rat DRG neurons. Current-clamped neurons exhibited resting potentials ranging from 55 to 65 mV, consistent with previous work (Caviedes et al., 1990; Elliott and Elliott, 1993), and all-or-nothing APs in response to depolarizing current injections of +200 to +800 pA. In 4 of 7 neurons, sustained depolarizing current injection triggered bursts of 5.6 ± 0.8 Aps/stimulus of 200 msec in duration, as shown in figure 9A. The application of 20 µM amitriptyline reversibly decreased the number of APs evoked by the same depolarizing stimulus to 55 ± 4% of control. The inhibition followed the pattern illustrated in figure 9A, which is consistent with a use-dependent inhibition induced by amitriptyline. In the remaining neurons, that displayed only a single AP with current injection, a series of current steps were applied to assess changes in excitability. Although amitriptyline failed to alter the voltage response to a hyperpolarizing current injection of 30 pA (fig. 9B), effects consistent with decreased membrane excitability were observed with depolarizing current pulses. Larger depolarizing current steps than those shown (typically >400 pA) did trigger APs, which were virtually indistinguishable between control and amitriptyline-treated conditions.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Amitriptyline inhibits action potential activity in isolated rat neonatal DRG neurons. A, The DRG neuron was current-clamped and pulsed with a depolarizing current of +400 pA. In the presence of 20 µM amitriptyline (right), AP amplitude of the repetitively firing neuron was significantly attenuated. B, Another DRG neuron, which failed to show repetitive firing, was pulsed by current steps from 30, +70, +170 and +270 pA. The application of 20 µM amitriptyline decreased neuronal excitability with no apparent change in the hyperpolarizing response.

	Discussion

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In addition to the blockade of the cardiac Na⁺ channel, the TCAs inhibit the neuronal Na⁺ channel but with a far lower potency. The calculated neuronal Na⁺ channel K_D value of 20 µM for amitriptyline is similar to that for desipramine, 9 µM, previously reported in Myxicola giant axon (Schauf et al., 1975). Amitriptyline (10-30 µM) reduced AP amplitude and maximum rate of depolarization (dV/dt_max) in crayfish giant axon (Wang et al., 1981), while TCAs inhibited ²²Na⁺ influx in bovine chromaffin cells with IC₅₀ values of 10 to 17 µM, levels similar to the present study (Arita et al., 1987). Amitriptyline also has been shown to inhibit neurotransmitter release from rat striatal brain slices via blockade of Na⁺ channels (Ishii and Sumi, 1992). Ogata et al. (1989) directly demonstrated a voltage-dependent reduction of I_Na from neuroblastoma cells by 3 µM imipramine, another TCA. In these neurally derived cells, the concentrations of amitriptyline and other TCAs required to cause >50% depression of I_Na is ~10 to 50 times higher than the 0.4 to 3.2 µM concentration of amitriptyline that is required for an equivalent effect in cardiac myocytes (Barber et al., 1991) and Purkinje fibers (Nattel, 1987) when stimulation rates are increased above 3 Hz.

Our results provide a more complete description of TCA action on steady-state inactivation and demonstrate the apparent stabilization by amitriptyline of an inactivated state that requires a more sustained repolarization to revert to an available closed state. Figure 6 predicts that after a repolarization for 80 to 100 msec, approximately twice as many channels will be inactivated in the presence of amitriptyline as in the control setting. Stabilization of the inactivated state by the antidepressants results in use-dependent blockade of Na⁺ channels, as evidenced in the DRG experiments with 10-Hz stimulation. Such rapid stimulation increased the depression of I_Na by 10 µM amitriptyline from 27% for the initial depolarization to 44% for the steady-state 10 Hz I_Na.

Based on competition experiments in myocytes, Barber et al. (1991) suggested that amitriptyline and lidocaine compete for the same LA binding site, recently shown to reside in amino acids present on the transmembrane segment S6 of domain IV of the alpha subunit (Ragsdale et al., 1994). It is noteworthy that the action of amitriptyline and the other TCAs is similar to the LAs in a number of regards. First, amitriptyline shifts the steady-state inactivation curve in the hyperpolarizing direction, just as the LAs. Second, amitriptyline depresses I_Na in a frequency-dependent manner, while the degree of use-dependent inhibition may be somewhat less for the neuronal than the myocardial Na⁺ channels. Third, the cardiac Na⁺ channel is more sensitive than the neuronal type to blockade by amitriptyline. Likewise, the LA concentration required for equivalent inhibition in nerve is typically 10-fold greater than that for myocardium if one compares studies of LA depression of I_Na or action potential rate of depolarization (dV/dt) in myocardium vs. nerve. However, amitriptyline must have a 20- to 200-fold greater affinity for the LA binding site than do the LAs themselves if one compares the effective concentrations for Na⁺ channel or conduction blockade. It is noteworthy that TCAs have a tertiary amine group connected by a three carbon chain to a large aromatic moiety, while LAs have a tertiary amine connected to an aromatic group by a chain of two carbons and an amide (or ester) linkage.

The clinical relevance of such neuronal Na⁺ channel inhibition is unclear. There is considerable evidence demonstrating the analgesic effects of tricyclic (Hameroff et al., 1984; Max et al., 1992; Valverde et al., 1994) and certain atypical (Max et al., 1992; Rani et al., 1996) antidepressants under differing clinical conditions of chronic pain. For example, the TCA doxepin has been shown to effectively treat migraine headache (Mørland et al., 1979) and lower back pain (Hameroff et al., 1982, 1984), and fluoxetine is a useful analgesic against rheumatic pain (Rani et al., 1996). Although the ability of all antidepressants to inhibit the reuptake of neurotransmitters may underlie their effectiveness against chronic pain, the usefulness of antidepressants in the treatment of acute pain appears to be primarily limited to the TCAs, suggesting a mechanism of acute analgesic activity independent from the antidepressant mechanism of action. The demonstrated voltage- and frequency-dependent inhibition of Na⁺ channels by TCAs, particularly amitriptyline, are properties that appear to be necessary properties of Na⁺ channel modulators with analgesic efficacy (Tanelian and Brose, 1991). In view of the use-dependent effect on Na⁺ channels, sensitized neurons that fire repetitive APs or are partially depolarized would be predicted to be more susceptible.

Several observations raise the possibility that Na⁺ channels may be involved in the usefulness of TCAs in the treatment of pain. All TCAs have the same effect on neuronal Na⁺ channels, whereas the effects of the atypical antidepressants on Na⁺ channels varies. A high concentration of trazodone, with little apparent clinical efficacy in the treatment of acute pain (Davidoff et al., 1987), had negligible effect on I_Na. Conversely, fluoxetine has questionable usefulness in pain management (Messiha, 1993) but can inhibit I_Na at concentrations comparable to the TCAs. Important differences between the blockade by fluoxetine and the TCAs is the use-dependence of this activity. Unlike amitriptyline, fluoxetine appears to cause less use-dependent inhibition of current, which might make its physiological effects less prominent.

Amitriptyline has also been administered intrathecally and found to augment the action of narcotics, an effect attributed to alteration in monamine reuptake (Eisenach and Gebhart, 1995). However, an analgesic dose of drug may achieve concentrations sufficient to mediate some effects via Na⁺ channel blockade. In sheep, 5 mg of amitriptyline injected into the CSF (20-25 ml) gives an estimated concentration of >500 µM and has been found to yield a CSF concentration of 1.3 ± 0.6 µg/ml (4.8 µM) after 2 hr, with higher concentrations present in the gray (5.9 ± 2.7 µg/g of tissue) and white (2.9 ± 1.5 µg/g of tissue) matter near the site of injection (Cerda et al., 1997). A decrease in blood pressure in the animals with acute intrathecal amitriptyline is similar to changes observed with local anesthetics in blocking sympathetic outflow from the spinal cord.

The concentrations of antidepressants used in this study are far higher than clinically relevant plasma concentrations. In patients receiving daily doses of 75 to 300 mg of amitriptyline, plasma steady-state concentrations range from 0.3 to 0.9 µM (Baldessarini, 1985). However, the plasma binding (>90%) is such that the free drug concentration in plasma is much lower. On the other hand, antidepressants do accumulate in the brain in concentrations 20-fold greater than in plasma (Karson et al., 1993), but the free drug concentration available for action at central neuronal Na⁺ channels is unclear.

Antidepressants block a variety of other ion channels, including the Ca²⁺-activated K⁺ channels, which are inhibited by amitriptyline with a K_D value of 40 µM (Kamatchi and Ticku, 1991). Amitriptyline also inhibits voltage-gated K⁺ channels in rat superior cervical ganglion cells with a K_D value of 12 µM (Wooltorton and Mathie, 1995). Studies have reported the sensitivity of L-type Ca⁺⁺ channels to antidepressant inhibition (Antkiewicz-Michaluk et al., 1991; Choi et al., 1992; Schwaninger et al., 1995). In mouse DRG neurons, the K_D value of imipramine blockade of L-type calcium channel is 30 µM (Choi et al., 1992). It is striking that the concentration of TCAs that inhibits Ca⁺⁺ and K⁺ channels is only slightly greater than that for neuronal Na⁺ channels. The significance of antidepressant effects of these ion channels to clinical effects is presently unclear; however, the potential exists for synergistic action due to combined inhibition of Na⁺ and Ca⁺⁺ channels.