INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

It is now well established that nitric oxide (NO), at levels similar to those associated with inflammation, can reversibly block action potential conduction (Redford et al. 1997). Demyelinated axons are especially susceptible to NO-induced conduction block (Redford et al. 1997). The mechanism(s) responsible for this action of NO, however, has not been delineated. One possibility is that NO/NO-related activity might alter neuron electrical excitability by modulating Na⁺ channels.

NO, produced by the exogenous NO donor papa-NONOate, has been shown to block both tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na⁺ currents in baroreceptor neurons (Li et al. 1998). In contrast, the NO donors sodium nitroprusside and S-nitroso-N-acetyl-DL-penicillamine (SNAP) have been shown to increase the amplitude of the inactivation-resistant, persistent TTX-S Na⁺ currents in hippocampal neurons by 60-80%, without significantly affecting the fast TTX-S Na⁺ currents in these cells (Hammarstrom and Gage 1999). In both types of neurons, the mechanism of action for NO has been suggested to be S-nitrosylation of Na⁺ channels. These results suggest that depending on cell type, NO may modulate Na⁺ currents differently.

Small C-type dorsal root ganglion (DRG) neurons, which play a role in nociception, express distinct rapidly inactivating (fast) TTX-S and slowly-inactivating (slow) TTX-R Na⁺ currents (Caffrey et al. 1992; Kostyuk et al. 1981; Roy and Narahashi 1992) that are attributable to different Na⁺ channels (Akopian et al. 1996; Black et al. 1996; Sangameswaran et al. 1996). In addition, a distinct persistent TTX-R Na⁺ current, attributed to sodium channel Na_v1.9 (NaN), has been described in small DRG neurons (Cummins et al. 1999). Nitric oxide synthase (NOS) is upregulated following axotomy in small C-type DRG neurons, and in these injured neurons, NO acts as an autocrine regulator of Na⁺ currents (Renganathan et al. 2000). In this study, we examined the effects of NO donors on these three types of Na⁺ currents in C-type DRG neurons, the fast TTX-S, slow TTX-R, and persistent TTX-R Na⁺ currents. We also examined the mechanism of action of NO donors on these Na⁺ currents.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal care

Experiments were carried out in accordance with National Institutes of Health guidelines for the care and uses of laboratory animals. 6-wk-old Sprague-Dawley female rats (120 g) were used for the study. Animals were housed under a 12 h light-dark cycle in a pathogen-free area with free access to water and food at the Veterans Affairs Medical Center (VAMC), West Haven.

Sciatic nerve axotomy

Axotomy of the sciatic nerve was performed as previously described (Waxman et al. 1994). Animals were anesthetized with ketamine/xylazine (38/5 mg/kg ip). The right sciatic nerve was exposed, and a tight ligature was placed around the sciatic nerve near the sciatic notch proximal to the pyriform ligament. The nerve was transected immediately distal to the ligature, and the proximal nerve stump was fit into a silicone cuff to avoid nerve regeneration. The cuff contained 4 µl of hydroxystilbamine methanesulfonate (4% wt/vol in distilled water; Molecular Probes, Eugene, OR) for retrograde labeling of axotomized neurons. The fluorescent label clearly identified neurons whose axons were transected. The incision was sutured and the animal was allowed to recover. The animals were studied 7-14 days after sciatic nerve ligation.

Culture of DRG neurons

Rats were rendered unconscious by exposure to CO₂ and decapitated. L₄ and L₅ DRG were freed from their connective sheaths in sterile calcium-free saline solution. Cell cultures were prepared as described by Renganathan et al. (2000). Briefly, the L₄ and L₅ DRG ganglia were harvested, treated with collagenase and papain, dissociated in DMEM and Ham's F12 medium supplemented with 10% fetal bovine serum, and plated on polyornithine- and laminin-coated glass coverslips. Neurons were placed in a 5% CO₂-95% air incubator at 37°C and, 1 h after isolation, were fed with fresh medium. Na⁺ current properties were investigated 16-24 h after isolation. DRG neurons displayed only short (<10 µM) axon processes during the short period of culture, facilitating voltage clamp.

Electrophysiological recordings

Coverslips were mounted in a flow-through chamber continuously perfused with bath-external solution (see following text). Sodium currents were recorded from C-type (<30 µM diam) L₄ and L₅ DRG neurons using the whole cell patch-clamp technique with an Axopatch-200B amplifier (Axon Instruments, Foster City, CA) using standard techniques. Membrane capacitance of the DRG neurons used for electrophysiological experiments was 20.86 ± 0.89 pF (n = 88). For currents >20 nA, we switched to the 50 M feedback-resistor ( = 0.1), which can pass 200 nA. When Na⁺ current amplitudes of >100 nA associated with series resistance errors were observed, Na⁺ concentration in the bath solution was reduced to 20 mM to decrease the Na⁺ driving force (Fig. 2, C and D). Micropipettes (0.4-0.6 M) were pulled from borosilicate glasses (Boralex) with a Flaming Brown P80 micropipette puller and polished on a microforge and coated with a mixture of three parts of finely shredded parafilm and one part each of light and heavy mineral oil (Sigma, St. Louis, MO) to reduce capacitance. Series resistance was 0.74 ± 0.04 M (n = 72). Capacity transients were cancelled, and series resistance was compensated (90%) as necessary. The pipette solution contained (in mM) 140 CsF, 1 EGTA, 10 NaCl, and 10 HEPES, pH 7.3 and adjusted to 310 mOsmol/l with glucose. The bath solution contained (in mM) 140 NaCl, 3 KCl, 1 MgCl₂, 1 CaCl₂, 0.1 CdCl₂, and 20 HEPES, pH 7.3, adjusted to 320 mOsmol/l with glucose. CdCl₂ was used to block Ca²⁺ currents. The pipette potential was zeroed before seal formation, and voltages were not corrected for liquid junction potential. Leakage current was digitally subtracted on-line using hyperpolarizing control pulses, applied before the test pulse, of one-sixth test pulse amplitude (-P/6 procedure). Whole cell currents were filtered at 5 kHz and acquired at 50 kHz using Clampex 8.03 software (Axon Instruments). For current density measurements, membrane currents were normalized to membrane capacitance, which was calculated as the integral of the transient current in response to a brief hyperpolarizing pulse from 120 mV (holding potential) to 130 mV. Experiments were performed at room temperature (21°-25°C) unless otherwise noted.

Conductance was determined as I_p/(V_R  V), where I_p is the peak inward current, V_R is the reversal potential, and V is the test pulse voltage. V_R was determined by fitting the normalized Na⁺ current voltage (I/I_max) relationships to the Boltzmann equation
where V_0.5 is the voltage for half-maximal activation in mV, V is the test pulse voltage, k is the corresponding slope factor, and G_i is a scaling factor with the dimensions of a conductance.

Normalized conductance (G/G_max) was fit with a single Boltzmann relationship of the form
where V is the test pulse voltage, V_0.5 is the voltage for half-maximal activation in mV, and k is the corresponding slope factor.

Steady-state inactivation curves were measured using 500-ms prepulses to the indicated potentials followed by a test pulse to 0 mV for fast and slow Na⁺ currents. For persistent Na⁺ current, the test pulse was 50 mV, where no activation of slow Na⁺ current occurs. Peak test pulse current was plotted as a function of prepulse potential, normalized and fit with a single Boltzmann function
where V_pp is the prepulse potential, V_h is the mid-point potential, and k_h is the corresponding slope factor.

Fast and slow Na⁺ channel slow inactivation

Voltage dependence of steady-state slow inactivation was studied using a sequence of 1-min prepulse protocols: 130, 0, 120, 10, 110, 20, 100, 30, 90, 40, 80, 50, 70, and 60 mV, followed by a 50-ms pulse to 130 mV to remove fast inactivation. The fraction of slow inactivated Na⁺ currents was measured by a test pulse to 20 mV. At the end of each slow inactivation pulse sequence, neurons were held at 130 mV for 30 s to ensure complete recovery from inactivation and avoid accumulation of inactivation. Peak test pulse current was plotted as a function of prepulse potential, normalized and fit with a single Boltzmann function
where V_pp is the prepulse potential, V_h is the mid-point potential, and k_h is the corresponding slope factor. Slow inactivation of persistent Na⁺ current was not studied due to run-down (see RESULTS).

Separation of fast and slow Na⁺ currents using prepulse inactivation

We used prepulse inactivation to separate the fast Na⁺ currents in DRG neurons that showed fast and slow Na⁺ currents. We did not attempt to isolate fast Na⁺ currents in DRG neurons that showed fast, slow, and persistent currents due to the similarity in voltage dependence of steady-state inactivation of fast and persistent Na⁺ currents. Prepulse inactivation takes advantage of differences in inactivation properties of fast and slow Na⁺ currents (Cummins and Waxman 1997; McLean et al. 1988; Roy and Narahashi 1992). At hyperpolarized prepulse potentials (130 to 50 mV), fast and slow Na⁺ currents are available to open; however at depolarized potentials (50 to 10 mV), only slow Na⁺ currents are available to open (Table 1). Subtraction of slow Na⁺ currents obtained at approximately 50 mV prepulse potential, from currents obtained at more hyperpolarized prepulse potentials (130 to 50 mV), which elicit both fast and slow Na⁺ currents, yield fast Na⁺ currents. In some studies where the effect of papa-NONOate on fast Na⁺ currents was investigated, axotomized C-type DRG neurons, most of which express only fast Na⁺ channels, were used (Cummins and Waxman 1997). The inhibition of fast Na⁺ currents by papa-NONOate in DRG neurons isolated from control noninjured and axotomized DRG neurons was similar.

Separation of slow and persistent TTX-R Na⁺ currents using prepulse inactivation

Prepulse inactivation takes advantage of the differences in the inactivation properties of the slow and persistent Na⁺ currents, which are both TTX resistant (Cummins et al. 1999). TTX (300 nM) was included in the bath solution to isolate slow and persistent Na⁺ currents from fast Na⁺ currents (which are eliminated by 300 nM TTX). Na⁺ currents were evoked from a holding potential of 130 mV to test pulses ranging from 100 to 60 mV. Persistent Na⁺ current was obtained by subtracting the current obtained following a 50 mV prepulse (500-ms duration), which elicits only slow Na⁺ current, from the current obtained with more hyperpolarizing prepulse (130 mV), which elicits both slow and persistent Na⁺ currents (Table 1).

Preparation of NO donors, NO scavengers, and chemicals

Stock solutions (300 mM) of papa-NONOate (Alexis Biochemicals, San Diego, CA) were prepared in 0.1 N NaOH just before the NO donor was applied to cells and were diluted to desired concentration within the bath solution. Solutions were prepared in cold 0.1N NaOH to maintain stability. Buffer concentration (20 mM) ensured that the maximum NO donor concentration used, 10 mM, did not change the pH of the bath solution. SNAP (Alexis Biochemicals) was dissolved in DMSO. Hemoglobin was used as a NO scavenger. A selective cGMP-dependent protein kinase inhibitor, CG-PKI (Promega, Madison, WI), was dissolved in pipette solution for intracellular application at a final concentration of 360 µM. TTX was dissolved in distilled water, and aliquots were stored at 4°C until used. 8-bromo-cGMP was purchased from Research Biochemicals International (Natick, MA). All other chemicals were purchased from Sigma Chemical.

Statistics

All results are expressed as means ± SE. Statistical significance was evaluated using Student's t-test and P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Earlier studies from our laboratory demonstrated that axotomy upregulates nNOS, and NO acts as an autocrine regulator of Na⁺ currents in C-type DRG neurons (Renganathan et al. 2000). In the present study, we investigated the mechanism of action of NO, using NO donors, on the three separable Na⁺ currents that can be recorded in C-type DRG neurons.

Fast, slow, and persistent Na⁺ currents are observed in C-type DRG neurons

Fast, slow, and persistent Na⁺ currents studied here are similar to those previously described in C-type DRG neurons (Caffrey et al. 1992; Cummins and Waxman 1997; Cummins et al. 1999; Elliott and Elliott 1993; Rizzo et al. 1994; Roy and Narahashi 1992; Rush et al. 1998). In this paper, we refer to TTX-S fast-inactivating (Fig. 1), TTX-R slowly inactivating (Fig. 2) and TTX-R persistent Na⁺ currents (Fig. 4) as "fast," "slow," and "persistent," respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Nitric oxide (NO) donor papa-NONOate blocks fast Na+ currents in C-type dorsal root ganglion (DRG) neurons. Stepwise depolarizations of C-type DRG neurons from a holding potential of 130 mV to test potentials between 100 and 60 mV elicited inward and outward Na+ currents with the reversal potential close to 50 mV. A: fast Na+ currents measured under control conditions 5 min after attaining the whole cell configuration. B: the same neuron displays a time-dependent current potentiation 30 min after attaining the whole cell configuration. C: fast Na+ currents measured in another neuron before (C) and after a 25-min exposure to 5 mM papa-NONOate (D), illustrating papa-NONOate-mediated fast Na+ current inhibition. E: current-voltage relationship of fast Na+ currents measured 5 min after attaining the whole cell configuration and before papa-NONOate addition (, n = 16), 30 min after attaining the whole cell configuration (, n = 8), and after a 25-min exposure to 5 mM papa-NONOate (, n = 8). Peak currents observed at various potentials before and after 5 mM papa-NONOate exposure were normalized to peak current observed at 20 mV before papa-NONOate addition. Fast Na+ current inhibition was measured from axotomized and intact DRG neurons that express only fast Na+ currents. F: conductance-voltage relationship (,  and ) and steady-state voltage dependence of inactivation (,  and ) of fast Na+ currents measured 5 min ( and , n = 16) and 30 min after attaining the whole cell configuration ( and , n = 8) and after a 25-min exposure to 5 mM papa-NONOate (30 min after attaining the whole cell configuration; , , n = 8) were determined and fit with a Boltzmann equation.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Papa-NONOate blocks slow Na+ currents. Slow Na+ currents were evoked in the presence of 300 nM TTX 5 min (A) and 30 min after attaining the whole cell configuration from the same neuron. C: slow Na+ currents measured in another neuron (expressing only slow Na+ current) before (C) and after a 25-min exposure to 5 mM papa-NONOate (D). Due to larger current amplitude (100 nA), Na+ concentration in bath solution was decreased to 20 mM to reduce the current amplitude and the associated voltage error. Slow Na+ currents in these neurons were elicited by test pulses between 100 and 35 mV from a holding potential of 130 mV. Slow Na+ currents measured in 20 mM Na+ concentration in bath solution were averaged to illustrate the current-voltage relationship. E: current-voltage relationship of slow Na+ currents measured 5 (, n = 16) and 30 min after attaining the whole cell configuration (, n = 8) and after a 25-min exposure to 5 mM papa-NONOate (30 min after attaining the whole cell configuration; ,, n = 8). F: conductance-voltage relationship (, , and ) and steady-state voltage dependence of inactivation (, , and ) of Na+ currents measured 5 min ( and , n = 16) and 30 min after attaining the whole cell configuration ( and , n = 8), and after a 25-min exposure to 5 mM papa-NONOate (, , n = 8) were determined and fit with a Boltzmann equation.

Papa-NONOate blocks fast Na⁺ currents

Figure 1A displays fast Na⁺ current elicited from a representative DRG neuron, 5 min after attaining the whole cell configuration. Because the maximal effects of papa-NONOate were seen at ~30 min (Figs. 1D and 3A) and a time-dependent hyperpolarizing shift in voltage-dependent activation and inactivation has been reported in Na⁺ currents of neuroblastoma cell line N1E115 (Renganathan et al. 1995), we examined the time-dependent changes in Na⁺ current properties in DRG neurons under control conditions. A time-dependent potentiation (~30% increase in peak current amplitude) of fast Na⁺ currents was typically observed (Fig. 1B). Figure 1, C and D, illustrates Na⁺ currents recorded before and after a 25-min exposure to 5 mM papa-NONOate in another neuron. Papa-NONOate was applied 5 min after attaining whole cell configuration; thus after 25 min of exposure to papa-NONOate, the cell has been in whole cell configuration for 30 min, permitting a direct comparison with Fig. 1, A and B. Na⁺ current inhibition by papa-NONOate was estimated by calculating the ratio of current measured after 25 min of papa-NONOate exposure (Fig. 1D) to the current measured before papa-NONOate addition (Fig. 1C). This method of determining papa-NONOate-mediated inhibition of Na⁺ current utilizes each neuron as its own control; it may underestimate current block because it does not correct for the time-dependent increase in Na⁺ currents in control (untreated) neurons.

View larger version (17K): [in this window] [in a new window]   Fig. 3. A: time course of NO-mediated block of fast and slow Na+ currents. Fast Na+ currents were measured from axotomized DRG neurons as described in Fig. 1 (, , , , n = 5). Slow Na+ currents were evoked from DRG neurons as described in Fig. 2 (, , , , n = 5). The percentage of inhibition at 0.01 (, ), 0.1 (, ), 1 (, ), and 5 mM (, ) papa-NONOate concentrations was determined by normalizing the peak current amplitude at 20 mV observed at different time points to the peak current value at 20 mV observed at 0 min. B: concentration dependence of inhibition of fast and slow Na+ currents in response to papa-NONOate. Fast and slow Na+ current inhibition were estimated by normalizing the peak current amplitude at 20 mV obtained in the presence of papa-NONOate to peak current amplitude at 20 mV obtained in the absence of pap-NONOate. Points for 0.01, 0.1, 0.5, and 5 mM represent mean ± SE values obtained from 5 to 8 C-type DRG neurons. One and 2 mM papa-NONOate-mediated block of fast and slow Na+ currents were measured in 3 DRG neurons (*). The best fit to fast and slow Na+ current dose response curves for papa-NONOate yielded a similar IC50 = ~0.75 mM.

Figure 1E displays the averaged current-voltage relationship from DRG neurons expressing only fast Na⁺ currents measured 5 min () and 30 min after attaining the whole cell configuration (), and 25 min after the addition of 5 mM papa-NONOate (). Papa-NONOate (5 mM) decreased the fast Na⁺ current amplitude to 0.22 ± 0.03 nA/pF (n = 8) from a value of 1.00 ± 0.15 nA/pF (n = 16) observed before papa-NONOate application (Fig. 1E, ). Both inward and outward fast Na⁺ currents were inhibited in a monotonic and continuous voltage-dependent manner, i.e., there was no apparent voltage dependence in papa-NONOate-mediated block of fast Na⁺ currents.

We monitored voltage-dependent activation and inactivation of fast Na⁺ currents to determine whether papa-NONOate blocks Na⁺ currents by modulating these parameters. As seen in Table 2, time-dependent hyperpolarized shifts in voltage-dependent activation and inactivation was observed for fast Na⁺ currents under control (no papa-NONOate) conditions. Figure 1F shows activation curves for control neurons 5 min () and 30 min after attaining the whole cell configuration (), together with the activation curve after 25 min exposure to papa-NONOate (). Similar curves are shown for steady-state inactivation. There was no significant difference between the V_1/2 and k values for activation and inactivation for papa-NONOate-treated (Fig. 1F,  and ) and untreated neurons ( and ) at a similar time (30 min) after attaining whole cell configuration (Table 2). Thus papa-NONOate-mediated fast Na⁺ current block is not due to hyperpolarized shift in the V_1/2 for inactivation and/or changes in the slope values.

Papa-NONOate blocks slow Na⁺ currents

Two TTX-R Na⁺ currents, slow and persistent, are expressed in small DRG neurons. The slow Na⁺ current in small DRG neurons is produced by the Na_v1.8 (SNS) sodium channel isoform (Akopian et al. 1999), while the persistent TTX-R Na⁺ current is produced by the Na_v1.9 Na⁺ channel (Cummins et al. 1999). While ~80% of the neurons studied with TTX produced slow TTX-R Na⁺ current, the persistent TTX-R Na⁺ current was seen in ~50% of the neurons. In these DRG neurons, when held at 130 mV and depolarized to membrane potentials ranging from 100 to 60 mV, a persistent TTX-R current (the noninactivating currents seen at the end of the depolarizing test pulses) was elicited at low depolarizing test pulses (Fig. 2A). Papa-NONOate-mediated block of slow Na⁺ currents is described here, and block of persistent Na⁺ currents is described in the next section.

Figure 2A shows recordings from a representative DRG neuron displaying slow and persistent Na⁺ currents in the presence of 300 nM TTX 5 min after obtaining whole cell configuration. An increase of ~30% in slow TTX-R Na⁺ current was seen in these neurons 30 min after attaining the whole cell configuration (Fig. 2B). In contrast, a run-down in persistent Na⁺ current was observed. The potentiation of slow Na⁺ currents was associated with a hyperpolarized shift in activation and inactivation, similar to the fast Na⁺ currents (Table 1, bottom row, control). Figure 2, C and D, illustrates a typical cell expressing only slow Na⁺ currents measured before and after a 25-min exposure to 5 mM papa-NONOate. Papa-NONOate decreased the slow Na⁺ current amplitude to 0.21 ± 0.05 nA/pF (n = 8) from a value of 1.10 nA/pF (n = 16) observed before papa-NONOate addition (Fig. 2E, , P < 0.01). Both inward and outward slow Na⁺ currents were inhibited in a monotonic and continuous voltage-dependent manner.

Figure 2E presents the averaged current-voltage relationship of slow TTX-R Na⁺ currents measured 5 min () and 30 min after attaining whole cell configuration (), and after a 25-min exposure to 5 mM papa-NONOate (). Some of these neurons expressed persistent Na⁺ currents in addition to slow Na⁺ currents, hence the averaged current-voltage relationship display a hump (due to the persistent Na⁺ current) (see Cummins et al. 1999) between 80 and 40 mV (Fig. 2E, ) and in the activation curve (Fig. 2F, ). Time-dependent hyperpolarized shifts in voltage-dependent activation and inactivation were observed for slow Na⁺ currents under control (no papa-NONOate) conditions (Table 2). There is no significant difference between the V_1/2 and slope values for activation and inactivation for papa-NONOate-treated (Fig. 2F, , ) and untreated neurons ( and ) studied 30 min after attaining whole cell configuration. These results suggest that papa-NONOate-mediated block of slow Na⁺ currents is not due to a hyperpolarized shift in voltage-dependent inactivation.

Time course and concentration dependence of fast and slow Na⁺ current inhibition

To determine the time course and concentration dependence of fast Na⁺ current inhibition, we used axotomized C-type DRG neurons, 80% of which express only fast Na⁺ currents (whereas only 25% of control C-type DRG neurons display only fast Na⁺ currents). Inhibition of fast and slow Na⁺ currents began immediately after the addition of papa-NONOate and was maximal at ~30 min after addition of papa-NONOate (Fig. 3A). Increasing concentration of papa-NONOate, i.e., 0.01, 0.1, 1, 5 mM papa-NONOate, produced an increase in Na⁺ current inhibition. The time course of Na⁺ current inhibition could be fit with a single exponential with time constants of 16, 21, 15, and 18 min, for 0.01, 0.1, 1, and 5 mM papa-NONOate concentrations and was similar for fast and slow Na⁺ currents (Fig. 3A). The similarity in time course of Na⁺ current inhibition at different concentrations of papa-NONOate probably reflects the similar time course of NO release at different concentrations. The time course of Na⁺ current inhibition closely matches the NO production by papa-NONOate reported by Gbadegesin et al. (1999), who observed that under conditions similar to electrophysiological measurements, i.e., pH 7.3 and 25°C, NO production by papa-NONOate rises to a peak in 10 min. Papa-NONOate (0.01, 0.1, 0.5, 1, 2, and 5 mM) was used to determine concentration dependence of the fast and slow Na⁺ current inhibition that exhibited IC₅₀ at 0.75 mM (Fig. 3B,  and ). Papa-NONOate-mediated block of fast Na⁺ currents () was determined from control noninjured DRG neurons displaying fast Na⁺ currents and from axotomized DRG neurons displaying only fast Na⁺ currents. No significant difference in inhibition was observed between the two groups of neurons. The IC₅₀ values for papa-NONOate inhibition of fast and slow Na⁺ currents in DRG neurons were similar to the inhibition of baroreceptor fast and slow Na⁺ currents (Li et al. 1998). Run-down in persistent Na⁺ current precluded study of persistent Na⁺ current inhibition time course (see following text).

Papa-NONOate blocks persistent Na⁺ currents

Persistent TTX-R currents in DRG neurons activate at 80 mV and manifest as noninactivating currents at the end of depolarizing test pulses (Fig. 2A). Run-down in persistent Na⁺ current was observed after attaining whole cell configuration and persistent Na⁺ current was greatly attenuated or undetectable in most cells by 15-20 min after attaining whole cell configuration (Fig. 2B). The opposing time-dependent changes, i.e., rundown in persistent Na⁺ currents and an increase in fast and slow Na⁺ currents, suggests that the channels may be modulated by distinct intracellular pathways. To control for the possibility that these pathways are altered during cellular dialysis by the patch electrode, persistent, fast, and slow current densities were measured from neurons that were not exposed to papa-NONOate and from neurons that were exposed to 5 mM papa-NONOate in the culture medium at 37°C for 15 min (Figs. 4 and 5). In these experiments, neurons were exposed to papa-NONOate in culture medium, but papa-NONOate was not present in the external bath solution when these neurons were patch clamped, and current densities were measured within 4 min after attaining whole cell configuration. These conditions minimized changes in the intracellular milieu during the course of cell dialysis by the patch electrode.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Separation of persistent Na+ currents from slow Na+ currents. Persistent Na+ currents were measured in the presence of 300 nM TTX and separated from slow Na+ currents by a subtraction protocol as described in METHODS. A: persistent Na+ currents from a small DRG neuron not exposed to papa-NONOate is displayed. B: DRG neuron exposed to 5 mM papa-NONOate for 15 min in the culture medium display smaller persistent Na+ current amplitude. C: current-voltage relationship of persistent Na+ currents measured from control neurons (, n = 5) and 5 mM papa-NONOate-treated neurons (, n = 5). D: conductance-voltage relationship (, ) and steady-state voltage dependence of inactivation (, ) of Na+ currents measured from control neurons (, n = 5) and 5 mM papa-NONOate-treated neurons (, n = 5) were determined and fit with a Boltzmann equation.

View larger version (21K): [in this window] [in a new window]   Fig. 5. A: papa-NONOate reduces fast, slow, and persistent Na+ current densities. Fast, slow, and persistent Na+ current densities were estimated in control neurons and in neurons preincubated with 5 mM papa-NONOate for 15 min at 37°C in culture medium by voltage clamping at room temperature. Neurons were used within 10 min to avoid reversibility associated with washout. Papa-NONOate was not present in the external bath solution when these neurons were voltage clamped. Fast, slow, and persistent Na+ current densities were obtained by dividing the peak current by cell capacitance (B). Data are given as means ± SE. *P < 0.05 compared with control neurons.

Figure 4A illustrates persistent Na⁺ current measured from a control neuron that was not exposed to papa-NONOate. Figure 4B illustrates persistent Na⁺ current measured from neuron that was exposed to 5 mM papa-NONOate in the culture medium at 37°C for 15 min. Because the time course of NO release is faster at 37°C than at 24°C (Keefer et al. 1996), the neurons were exposed for 15 min instead of 30 min. Figure 4C represents the averaged current-voltage relationship of persistent TTX-R Na⁺ currents measured from control neurons (, n = 5) and 5 mM papa-NONOate-treated neurons (, n = 5). Papa-NONOate-mediated block of persistent Na⁺ currents was not voltage dependent, similar to the block of fast and slow Na⁺ currents. The midpoint potential and slope values for voltage-dependent activation for persistent Na⁺ currents from control (Fig. 4D, ) and papa-NONOate-treated neurons (Fig. 4D, ) are 57.18 ± 1.82 mV, 5.57 ± 0.90 mV/e-fold and 55.68 ± 2.48 mV and 6.59 ± 1.31 mV/e-fold, respectively. Due to overshoot observed between 130 and 80 mV in steady-state inactivation of control and treated neurons, Boltzmann fit was not used to determine the midpoint potential or slope values. However, experimental values for voltage-dependent inactivation curves for control (Fig. 4D, ) and papa-NONOate-treated neurons (Fig. 4D, ) were similar and indicate that voltage-dependent inactivation does not play a role in papa-NONOate-mediated block of persistent Na⁺ current.

As seen in Fig. 5A, the magnitude of the Na⁺ current block by papa-NONOate was similar for fast, slow, and persistent Na⁺ currents. TTX-R-persistent current amplitude was 2.34 ± 1.06 nA (n = 45 from 3 different preparations) in papa-NONOate-treated neurons, significantly smaller than the TTX-R-persistent current amplitude, 17.33 ± 3.84 nA, in untreated neurons (n = 21 neurons from 3 different preparations; P < 0.0001). The persistent Na⁺ current density was 1.13 ± 0.27 nA/pF in untreated neurons (Fig. 5A, n = 21 neurons from 3 different preparations) and was reduced to 0.16 ± 0.10 nA/pF (n = 45 from 3 different preparations, P < 0.001) in papa-NONOate-exposed DRG neurons. Fast Na⁺ current density, normalized to capacitance, was 1.20 ± 0.21 nA/pF in control neurons (n = 30 from 3 different preparations), while it was 0.20 ± 0.05 nA/pF (n = 24 from 3 different preparations) in papa-NONOate-treated neurons (Fig. 5A; P < 0.001). Slow Na⁺ current density was 1.25 ± 0.16 nA/pF in control neurons (n = 30, from 3 different preparations) and was significantly reduced to 0.26 ± 0.05 nA/pF (Fig. 5A) in papa-NONOate-treated neurons (n = 24, from 3 different preparations; P < 0.001). These results demonstrate that exposure of DRG neurons to papa-NONOate in culture medium blocks persistent as well as fast and slow Na⁺ currents. The decrease in fast, slow, and persistent current density is not due to a decrease in the capacitance of the papa-NONOate-exposed neurons because the capacitance of the control and papa-NONOate-exposed neurons were not significantly different (Fig. 5B). These results suggest that dialysis of intracellular solution does not alter the outcome of NO-modulated inhibition of Na⁺ currents. These results also suggest that papa-NONOate can inhibit fast, slow, and persistent Na⁺ currents in DRG neurons at physiological resting potential.

It is possible that other unanticipated effects, including direct effects on Na⁺ channels by the byproducts of papa-NONOate, could be a mechanism for the block of Na⁺ currents. However, SNAP, whose byproducts are different from papa-NONOate (Keefer et al. 1996), at 1 mM concentration blocked fast Na⁺ currents in DRG neurons to ~50% (Fig. 6A, n = 5), similar to papa-NONOate. Fast Na⁺ current density was reduced from 1.12 ± 0.16 to 0.48 ± 0.14 nA/pF in the presence of SNAP. A time-dependent shift in voltage dependence of activation and inactivation of fast Na⁺ currents was observed in SNAP-treated neurons (Fig. 6B, n = 5), as seen in papa-NONOate-treated DRG neurons and untreated DRG neurons (Fig. 1F). Slow and persistent Na⁺ currents were reduced to 0.51 ± 0.1 and 0.48 ± 0.15 nA/pF by 1 mM SNAP from 0.99 ± 0.24 and 1.2 ± 0.28 nA/pF, respectively. These results strongly suggest that NO and NO-related byproducts, but not the byproducts associated with the NO donor decomposition, block Na⁺ currents.

View larger version (16K): [in this window] [in a new window]   Fig. 6. S-nitroso-N-acetyl-DL-penicillamine (SNAP), a different class of NO donor, blocks fast Na+ currents similar to papa-NONOate. Fast Na+ currents were measured from axotomized DRG neurons that expressed only fast Na+ currents. A: current-voltage relationship of fast Na+ currents measured 5 min after attaining the whole cell configuration (, n = 5) and after a 25-min exposure to 1 mM SNAP (, n = 5). B: conductance-voltage relationship (, , n = 5) and steady-state voltage dependence of inactivation (, , n = 5) of fast Na+ currents before (, ) and after a 25-min exposure to 1 mM SNAP (, ) were determined as mentioned in METHODS.

Effect of NO scavenger hemoglobin on papa-NONOate block of Na⁺ currents

On adding 5 µM NO scavenger hemoglobin together with 5 mM papa-NONOate, fast, slow, and persistent Na⁺ current density increased from 1.07 ± 0.04, 0.99 ± 0.08, and 1.15 ± 0.09 nA/pF to 1.28 ± 0.12, 1.18 ± 0.25, and 1.22 ± 0.18 nA/pF, respectively (n = 4). The increase in current density is similar to the time-dependent potentiation seen in control conditions. These results suggest that NO scavenger hemoglobin at 5 µM concentration prevented papa-NONOate (5 mM)-mediated block of fast, slow, and persistent Na⁺ currents. The prevention of papa-NONOate-mediated block of Na⁺ currents by hemoglobin provides further evidence that NO or NO products play a role in Na⁺ current inhibition.

Na⁺ currents are reversibly blocked by NO donors

NO donor inhibition of fast Na⁺ currents was reversed by washout (Fig. 7A, n = 5). The time course of washout was slower than the onset of inhibition by NO donor. The hyperpolarized shift in steady-state voltage-dependent activation and inactivation in control and NO-donor-treated neurons did not reverse after the wash-out (Fig. 7B, n = 5), further strengthening the idea that NO block of fast Na⁺ currents is not due to the shift in voltage-dependent inactivation and activation. NO-donor-mediated inhibition of slow and persistent Na⁺ currents was similarly reversible. On exposure to 5 mM papa-NONOate, slow and persistent Na⁺ current density decreased to 0.25 ± 0.12 and 0.16 ± 0.1 nA/pF, respectively. But the reduction was reversed to 1.04 ± 0.30 and 1.00 ± 0.3 nA/pF (n = 12) after three washes with the culture medium for 10 min. These results suggest that NO reversibly modulates fast, slow, and persistent Na⁺ currents in C-type DRG neurons.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Papa-NONOate-mediated block of fast Na+ currents is reversible. A: fast Na+ current inhibition was measured from axotomized DRG neurons that express only fast Na+ currents. Inhibition of fast Na+ currents by 5 mM papa-NONOate addition (closed arrow) was reversed by washout (open arrow) of the 5 mM papa-NONOate (n = 5). Results from 3 neurons are given. B: conductance-voltage relationship (, , and ) and steady-state voltage dependence of inactivation (, , and ) of fast Na+ currents before ( and ) after a 25-min exposure to 5 mM papa-NONOate ( and ) and after complete reversal of inhibition ( and ) were determined as mentioned in METHODS.

NO signaling pathway

Many of the cellular effects of NO are mediated by soluble guanyl cyclase (GC) and cGMP (Garthwaite and Boulton 1995; Moncada et al. 1991). To determine whether the inhibition of Na⁺ currents by papa-NONOate in C-type DRG neurons was mediated by cGMP and cGMP-dependent protein kinase, Na⁺ currents were recorded from DRG neurons before (Fig. 8A, ) and after a 20-min exposure to the membrane permeable cGMP analogue 8-bromo-cGMP (2 mM; Fig. 8A, ). 8-bromo-cGMP did not block fast Na⁺ currents (n = 5). In fact, the amplitude of the fast Na⁺ currents increased to 1.25 ± 0.09 nA/pF after 8-bromo-cGMP treatment from 1.02 ± 0.10 nA/pF observed in control conditions (P < 0.05). These results suggest that NO-mediated block of fast Na⁺ currents is not mediated by guanyl cyclase or cGMP. A time-dependent shift in voltage dependence of activation and inactivation was observed as seen in control DRG neurons (Fig. 8B, ,  and , ). The density of slow and persistent Na⁺ currents after DRG neurons were exposed to 8-bromo-cGMP for 20 min was 1.37 ± 0.22 and 0.93 ± 0.1 nA, respectively, not significantly different from control values. These results suggest that cGMP does not mediate NO inhibition of slow or persistent Na⁺ currents in C-type DRG neurons.

View larger version (39K): [in this window] [in a new window]   Fig. 8. NO blocks Na+ currents by S-nitrosylation. A: 8-bromo-cGMP, a membrane permeable analogue of cGMP does not block fast Na+ currents. Fast Na+ current inhibition was measured from axotomized DRG neurons that express only fast Na+ currents. Fast Na+ currents measured in the absence of 8-bromo-cGMP, 5 min after attaining the whole cell configuration (, n = 5). 8-bromo-cGMP at 2 mM concentration was applied through the bath solution and incubated for 20 min and Na+ currents were elicited (, n = 5). B: the steady-state voltage-dependent activation (, , n = 5) and inactivation (, , n = 5) before (,  after (, ) exposure to 2 mM 8-bromo-cGMP is shown. C: intracellular application of 360 µM cG-PKI does not prevent 1 mM papa-NONOate-mediated block of Na+ currents. cG-PKI was diluted into pipette solution before attaining whole cell configuration. Fast Na+ current inhibition was measured from axotomized DRG neurons that express only fast Na+ currents. Fast Na+ currents were elicited from DRG neuron before (, n = 5) and after a 25-min exposure to 1 mM papa-NONOate (, n = 5). D: the steady-state voltage-dependent activation (, , n = 5) and inactivation (, , n = 5) before (, ) and after ( and ) exposure to 1 mM papa-NONOate is shown. E: pretreatment of DRG neurons with the alkylating agent N-ethylmaleimide (NEM, 2 mM) prevents papa-NONOate-mediated block of Na+ currents. Fast Na+ current inhibition was measured from axotomized DRG neurons that express only fast Na+ currents. The alkylating agent NEM was applied to DRG neurons via bath solution. Recordings were made in neurons incubated for 10 min before (, n = 10) and after the addition of NEM and 1 mM papa-NONOate (, n = 10). For comparison, the inhibition of Na+ currents by 1 mM papa-NONOate is shown (). F: the steady-state voltage-dependent activation (, , n = 10) and inactivation (, , n = 10) before (, ) and after exposure to NEM and 1 mM papa-NONOate (, ) is shown. The steady-state voltage-dependent activation () and inactivation () after exposure to only 1 mM papa-NONOate is also shown for comparison.

In the second set of experiments, a peptide inhibitor selective for cGMP-dependent protein kinase (cG-PKI, 360 µM) (Colbran et al. 1992) was included in the intracellular solution. Under these conditions, 1 mM papa-NONOate still inhibited fast (Fig. 8C) Na⁺ currents by 50% (n = 5), a magnitude of inhibition similar to that observed in the absence of cG-PKI. Fast and slow Na⁺ currents were reduced to 0.45 ± 0.17 and 0.52 ± 0.22 nA/pF, respectively, under these conditions. The hyperpolarized shift in voltage-dependent activation and inactivation was not significant (Fig. 8D). These results suggest that NO donor actions on fast and slow Na⁺ currents are independent of GC and cGMP signaling pathways. Due to persistent Na⁺ current rundown in whole cell configuration, we could not examine the effect of cG-PKI on papa-NONOate inhibition of persistent Na⁺ currents.

A guanylyl cyclase and cGMP-independent signaling pathway via modification of sulfhydryl groups, S-nitrosylation, has been proposed to mediate the actions of NO and NO-related species in some tissues (Bolotina et al. 1994; Broillet and Firestein 1996; Campbell et al. 1996; Stamler et al. 1997). The alkylating agent NEM covalently modifies sulfhydryl groups and prevents S-nitrosylation. To determine whether papa-NONOate-mediated Na⁺ current block was caused by S-nitrosylation, NEM (2 mM) was added to the bath solution 10 min prior to exposure of neurons to papa-NONOate. The inhibitory effect of 1 mM papa-NONOate on fast Na⁺ currents (Fig. 8E, , shown here for comparison) was abolished after the neurons had been treated with NEM (Fig. 8E; , n = 10, P < 0.02). No significant difference between the V_1/2 and the slope values for activation and inactivation of fast Na⁺ currents were observed for untreated neurons (Fig. 8F,  and ), papa-NONOate-treated (), and NEM and papa-NONOate-treated neurons ( and ). Preincubation of neurons with NEM also prevented papa-NONOate-mediated block of slow and persistent Na⁺ currents (data not shown). NEM by itself did not block slow and persistent Na⁺ currents. Taken together, these results suggest that NO or NO byproducts act via modification of sulfhydryl groups located in fast, slow, and persistent Na⁺ channels or a closely associated protein.

(Ultra)slow inactivation of fast and slow Na⁺ channels

Slow inactivation or ultraslow inactivation is a process that is different from the classical fast inactivation described by Hodgkin and Huxley (1952) in Na⁺ channels (Ruff et al. 1988; Vilin et al. 1999; Wang and Wang 1997). Slow inactivation has been shown to cause a hyperpolarized shift in steady-state voltage dependence of TTX-insensitive Na⁺ current inactivation (Ogata and Tatebayashi 1992). The transition into a slow inactivated state requires prolonged depolarization or long bursts of action potentials (Cummins and Sigworth 1996; Richmond et al. 1998). Slow inactivation therefore can affect membrane excitability and firing properties (Fleidervish et al. 1996; Ruff et al. 1988; Sawczuk et al. 1997). In a study on voltage-dependent Na⁺ currents in nodose ganglion neurons, Bielefeldt et al. (1999) reported that NO enhances slow inactivation. To test the possibility that NO-mediated inhibition of Na⁺ currents in DRG neurons is due to a transition of Na⁺ channels into a slow inactivated state by NO, we investigated slow inactivation of fast and slow Na⁺ currents before and after the addition of papa-NONOate. Slow inactivation is measured by extended bouts of pulsing and long recording durations (~30 min). Because the time course of NO donor action on fast and slow Na⁺ currents is ~30 min, slow inactivation in DRG neurons was not measured from the same neuron before and after exposure to NO donor but measured from two groups of neurons. One group of neurons not exposed to papa-NONOate was used as control and the other group of neurons, which was exposed to 5 mM papa-NONOate in the culture medium for 15 min, was used to test the effect of NO donor on slow inactivation. Because the time course of NO release is faster at 37 than at 24°C (Keefer et al. 1996), neurons were exposed for 15 min instead of 30 min. Due to persistent current run-down (~15 min), only fast and slow Na⁺ channel slow inactivation were investigated.

Fast Na⁺ channel slow inactivation was measured in axotomized C-type DRG neurons, most of which express only fast Na⁺ channels (Cummins and Waxman 1997). To check whether these neurons co-expressed slow Na⁺ channels, each neuron was tested using 500 ms prepulse duration to 50-mV prepulse. Neurons that expressed slow Na⁺ currents were not used. Slow Na⁺ channel slow inactivation was studied in nonaxotomized DRG neurons, which express both fast and slow Na⁺ channels, in the presence of 300 nM TTX to block fast Na⁺ channels.

Figure 9A shows the voltage dependence of fast Na⁺ channel slow inactivation before (, n = 9) and after 15-min exposure to 5 mM papa-NONOate (, n = 9). Fast Na⁺ channel current density before and after exposure to 5 mM papa-NONOate were 1.12 ± 0.18 and 0.29 ± 0.05 nA/pF (P < 0.05). The V_1/2 and the slope for control neurons were 71.26 ± 2.59 mV and 17.36 ± 2.05 mV/e-fold change. For neurons that were exposed to 5 mM papa-NONOate, these values were 70.44 ± 4.00 mV and 14.74 ± 2.48 mV/e-fold. These results suggest that NO does not inhibit fast Na⁺ channels by facilitating the transition to slow inactivation.

View larger version (17K): [in this window] [in a new window]   Fig. 9. The protocol used to measure slow inactivation is shown at the top of the figure. Slow inactivation was measured by holding the neuron at 130 mV for 30 s to eliminate both fast and slow inactivation; prepulse of 60 s at various membrane potentials, 130 to 0 mV, was applied to induce slow inactivation; a 50 ms pulse to 130 mV selectively recovered fast inactivation before the test pulse; the amplitudes of sodium currents elicited in response to 20 mV test pulse were plotted as normalized current amplitude versus prepulse voltage and used to measure voltage-dependence of steady-state slow inactivation. A: slow inactivation of fast TTX-S Na+ current was determined in control neurons (open circles, n = 9) and neurons preincubated with 5 mM papa-NONOate (closed circles, n = 9) for 15 min at 37°C in culture medium by voltage clamping at room temperature. B: slow inactivation of slow TTX-R Na+ current was similarly determined in control neurons (open circles, n = 9) and neurons preincubated with 5 mM papa-NONOate (closed circles, n = 9).

Figure 9B shows the voltage dependence of slow Na⁺ channel slow inactivation before (, n = 9) and after 15-min exposure to 5 mM papa-NONOate (, n = 9). Slow Na⁺ channel current density before and after exposure of 5 mM papa-NONOate were 1.24 ± 0.31 and 0.26 ± 0.04 nA/pF (P < 0.05). The V_1/2 and the slope for control neurons and 5 mM papa-NONOate-exposed neurons were 80.98 ± 6.1 mV and 17.9 ± 3.5 mV/e-fold change and 78.54 ± 3.0 mV and 20.00 ± 2.7 mV/e-fold change, respectively. These results suggest that NO does not inhibit slow Na⁺ channels by facilitating the transition to slow inactivation. Unlike fast Na⁺ channels, the V_1/2 of slow inactivation is shifted to the hyperpolarized direction by approximately 40 mV compared with the V_1/2 of fast inactivation (500-ms prepulse), 39.9 mV, of the slow Na⁺ channels (slow inactivation, Table 2).

Inhibition of Na⁺ currents in DRG neurons held at resting potential (70 mV)

Experiments on the inhibition of Na⁺ currents were carried out by holding membrane potential at 130 mV to remove fast and slow inactivation of fast and slow Na⁺ channels. However, in vivo, DRG neurons do not see hyperpolarized membrane potentials for more than a few milliseconds. Because NO donors do not shift the voltage dependence of steady-state inactivation of fast (Fig. 1F) or slow Na⁺ channels (Fig. 2F), a similar extent of Na⁺ channel inhibition is expected at 130 and 70 mV. The steady-state voltage dependence of fast and slow Na⁺ channels indicate that under control conditions the availability of the fast Na⁺ channels is reduced to ~50% at 70 mV compared with 130 mV, whereas 100% of the slow Na⁺ channels are still available to open at 70 mV. Although the degree of papa-NONOate-mediated inhibition of fast and slow Na⁺ currents may be similar at 130 and 70 mV, the availability of fast Na⁺ channels may be smaller than for slow Na⁺ channels, which may have an effect on the excitability. Therefore we investigated NO-mediated inhibition of Na⁺ channels at 70 mV. NO-mediated inhibition of Na⁺ channels was determined by adding papa-NONOate to the culture medium rather than adding papa-NONOate to bath solution to minimize the effects of time-dependent intracellular dialysis and/or the associated shift in steady-state voltage-dependent inactivation. Under these conditions, resting membrane potential of the DRG neuron is probably close to 70 mV. Current densities of fast and slow Na⁺ currents of control neurons at 70 mV were 0.28 ± 0.08 nA/pF (n = 10) and 0.68 ± 0.24 nA/pF (n = 10), respectively. Current density of fast Na⁺ currents of 5 mM papa-NONOate-exposed DRG neurons was 0.05 ± 0.04 nA/pF (4 of 7 neurons). Fast Na⁺ currents were not present in 3 of 7 neurons tested. Current density of slow Na⁺ currents of 5 mM papa-NONOate-exposed DRG neurons was 0.15 ± 0.07 nA/pF (n = 10). Exposure to 5 mM papa-NONOate reduced both fast and slow Na⁺ current availability by 80% when the holding potential was set at 70 mV, similar to the inhibition observed at 130 mV holding potential with 5 mM papa-NONOate.

The persistent Na⁺ current is small and difficult to isolate with a holding potential of 70 mV, and therefore we were not able to determine the effect of NO on persistent Na⁺ currents under these conditions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We have demonstrated that the NO donor papa-NONOate inhibits three types of Na⁺ currents seen in C-type DRG neurons, i.e., fast TTX-sensitive, slow TTX-resistant, and persistent TTX-resistant Na⁺ currents. The NO scavenger hemoglobin abolished the effects of papa-NONOate, indicating that NO and/or NO-related species inhibit these Na⁺ currents. NO donor inhibition of all three types of Na⁺ currents was reversed by washout. Incubation of neurons with 8-bromo cGMP and cG-PKI had no effect on papa-NONOate-mediated Na⁺ current block, indicating that the block is independent of cGMP. Alkylation of free thiols with NEM prevented the actions of papa-NONOate, suggesting that NO, or a related reactive nitrogen species, modifies sulfhydryl groups on Na⁺ channels or a closely associated protein. Papa-NONOate-mediated block of Na⁺ currents is not due to a hyperpolarized shift in voltage-dependent inactivation. The absence of NO-mediated enhancement of slow inactivation in fast and slow Na⁺ channels suggest that NO does not inhibit fast and slow Na⁺ channels by facilitating the transition to a slow inactivated state. These results indicate that inhibition of Na⁺ currents is not due to modulation of fast or slow sodium channel inactivation. Together these results suggest that NO and/or NO-related products modify sulfhydryl groups of the Na⁺ channel and inhibit Na⁺ currents by blocking their conductance.

NO at an appropriate concentration serves as a key signaling molecule in physiological processes as diverse as host-defense, neuronal communication, and vascular regulation. However, NO overproduction has been implicated in inflammatory and neuro-degenerative disorders. The sodium channel blocking effects of NO described here may be relevant to the axonal conduction block observed in inflammatory disorders of the nervous system including experimental allergic encephalomyelitis (EAE), experimental autoimmune neuritis (EAN), and multiple sclerosis (MS). Macrophages isolated from the CNS of animals with EAE produce elevated levels of NO (Ruuls et al. 1996). NO levels in the CNS of animals with EAE are reported to be 30 times greater than in control animals (Hooper et al. 1995). Cerebrospinal fluid levels of NO metabolites are elevated in MS patients (Johnson et al. 1995). Effects of NO might be expected to be more marked in inflammatory diseases in which neurons may be exposed chronically to raised NO levels.

The time course for development of Na⁺ channel block and the dose dependence observed in this study is similar to results observed for Na⁺ current block of baroreceptor neurons using papa-NONOate as NO donor (Li et al. 1998). The time course of Na⁺ current inhibition (~20 min) closely matches the NO production by papa-NONOate (Gbadegesin et al. 1999). Na⁺ current block was seen within minutes on exposure of the DRG neurons to papa-NONOate and may reflect the time required for the release of NO from papa-NONOate. In vivo, NO release occurs in seconds (Liu e