ToxSci Advance Access originally published online on March 3, 2008
Toxicological Sciences 2008 103(2):346-353; doi:10.1093/toxsci/kfn045
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Na+/H+ Exchanger-1 Inhibitors Reduce Neuronal Excitability and Alter Na+ Channel Inactivation Properties in Rat Primary Sensory Neurons
Department of Investigative Toxicology, Drug Safety Research & Development, Pfizer Global R & D, Groton, Connecticut 06340
1 To whom correspondence should be addressed at Investigative Toxicology, Drug Safety Research & Development Groton, MS 8274-1328, PGRD, Eastern Point Road, Groton, CT 06340. Fax: (860) 715-3577. E-mail: christopher.j.somps{at}pfizer.com.
Received December 21, 2007; accepted February 22, 2008
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ABSTRACT |
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Inhibitors of the Na+/H+ exchanger isoform 1 (NHE-1) have been associated with peripheral neuropathy in rats and dogs. Recent studies suggest that NHE-1 plays an important role in mediating neuronal excitability. To investigate potential NHE-1-mediated mechanisms contributing to neuronal toxicity, we studied the effects of NHE-1 inhibitors on nerve and dorsal root ganglion (DRG) neurons isolated from the adult rat. Compound action potentials (CAPs) were recorded from electrically stimulated sections of isolated sciatic nerve/DRG/root preparations. Whole-cell patch-clamp technique was used to record fast and slow voltage-dependent Na+ currents from dissociated DRG neurons (29–41 µm). Exposures to 1 and 10µM of a selective NHE-1 inhibitor reduced the amplitude of the CAP recorded from the dorsal root by 33% and 58%, respectively (p < 0.05). The compound had no effect on CAPs recorded from the ventral root. Perfusion of dissociated DRG neurons with NHE-1 inhibitors at 10 and 100µM shifted voltage-dependent inactivation curves of fast Na+ current by as much as 11 mV (p < 0.001) in the hyperpolarizing direction. No shift was observed in slow Na+ currents. No statistically significant drug effects were observed on voltage-dependent activation or recovery from inactivation of either fast or slow Na+ currents. These results suggest that NHE-1 inhibitors may reduce peripheral neuronal excitability by shifting fast Na+ channels into the inactivated state under physiological conditions. Such effects may underlie peripheral neuropathies reported in rats and dogs with NHE-1 inhibitors.
Key Words: patch clamp; dorsal root ganglion; dorsal root; NHE-1 inhibitor; sodium current; Zoniporide.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUMMARYREFERENCES |
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Pharmacological inhibition of the Na+/H+ exchanger isoform 1, referred to as NHE-1, has been extensively demonstrated to protect the heart from ischemia-reperfusion injury (Karmazyn, 2001
; Moffat and Karmazyn, 1993) and is a promising therapeutic approach (Karmazyn, 2001; Knight et al., 2001). However, NHE-1 inhibitors have recently been observed to be associated with changes in peripheral nerve function and structure in vivo, including decreased patellar reflex in dogs, slowing of caudal nerve conduction velocity in rats and, with longer duration exposure, focal axonal degeneration in peripheral nerves, dorsal roots and dorsal root ganglion (DRG) of both rat and dog (Pettersen et al., in press).
NHE-1 is ubiquitously expressed in the plasma membranes of most mammalian cells (Fliegel and Wang, 1997; Mahnensmith and Aronson, 1985). NHE-1 inhibitors block the electroneutral exchange of intracellular protons and extracellular sodium ions. This is believed to underlie their therapeutic activity in cardiac tissue by reducing the accumulation of intracellular Na+ and Ca2+ during cardiac ischemia-reperfusion injury (Fliegel, 2005). However, NHE-1 inhibitors also produce a decrease in intracellular pH in a variety of tissues, including neurons (Schneider et al., 2004). It is known that changes in pH can modulate neuronal excitability (Gu et al., 2001). For example, decrease in intracellular pH produced by acute application of proprionic acid or by inhibition of the NHE in hippocampal slices of guinea pig results in decreased 0-Mg2+–induced spontaneous and epileptiform activity (Bonnet et al., 2000a, b). In contrast, knockout of the NHE-1 exchanger in mice produces an increase in seizure phenotype (Gu et al., 2001), possibly due to a compensatory increase in Na+ channel expression in the hippocampus and cortex (Xia et al., 2003). These later observations suggest a functional linkage between NHE-1, neuronal excitability and Na+ channels.
To determine if changes in the peripheral nervous system function and structure observed in vivo with NHE-1 inhibitors CP-597,396 (Pettersen et al., in press) and CP-628,319 (unpublished observations) are associated with altered neuronal excitability and/or Na+ channel properties, we investigated the acute effects of these compounds and a commercially available NHE-1 inhibitor, dimethylamiloride (DMA), on rat peripheral nerve excitability and DRG neuron sodium channel properties using electrophysiological techniques. We demonstrate here that NHE-1 inhibitors decrease peripheral neural excitability and alter the inactivating properties of fast sodium channels in DRG neurons.
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MATERIALS AND METHODS |
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Materials.
NHE-1 inhibitors CP-628,319 (Fig. 1A) and CP-597,396 (Zoniporide, Fig. 1B), were made in house by Pfizer, Inc., meeting stringent purity and quality criteria of candidate pharmaceuticals. CP-597,396 inhibits human NHE-1 with IC50's in the 10–100nM range (Tracey et al., 2003
) and has > 150-fold selectivity over NHE-2 and NHE-3 isoforms (Marala et al., 2002). Other than moderate binding to µ- and 
-opioid receptors, CP-597,396 had little affinity for a panel of additional receptors (Tracey et al., 2003). CP-628,319 is structurally similar to CP-597,396 with very similar potency and selectivity (unpublished). All other chemicals, including DMA (Fig. 1C) were purchased from Sigma Chemical Co. (St Louis, MO). Test compounds were dissolved in the external recording solution used for patch-clamp experiments to form 100-fold stock solutions. The stock solutions were stored at 4°C and diluted in the perfusion or external recording solution to the desired concentrations immediately before use.
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DRG-dorsal root-ventral root preparation.
Lumbar 4 and 5 DRG were dissected, with sciatic nerves and spinal dorsal and ventral roots attached, from adult male Sprague–Dawley rats (350–437 g; Charles River Lab, Wilmington, MA) anaesthetized with ketamine/xylazine (60/10 mg/Kg) and exsanguinated. During dissections nervous tissue was frequently irrigated with an ice-chilled, modified Krebs solution. This Krebs solution contained (in mM): 124 NaCl; 26 NaHCO3; 3 KCl; 1.3 NaH2PO4; 2 MgCl2; 10 dextrose, was bubbled with 95% O2 and 5% CO2, had a pH 7.4 and osmolality of 290–300 mmol/kg. Isolated tissue was removed to ice-cold Krebs solution and then allowed to recover to room temperature over the next 1–2 h. Ganglion and root preparations were then transferred to a home-made recording chamber and superfused at 2–3 ml/min with a perfusion solution which consisted of Krebs solution plus 2mM CaCl2. Compound action potentials (CAPs) were elicited by stimulation with glass suction electrodes closely fitted to the diameter of the dorsal roots or nerves (Liu et al., 2000
). CAPs were recorded with a tightly fitting suction electrode and amplified with an Axoclamp-2B amplifier (Molecular Devices, Sunnyvale, CA). Single brief constant current stimulation pulses (200 µs) of increasing intensity were applied to nerve or dorsal roots using pCLAMP 8.2 via a linear stimulus isolator (World Precision Instruments, Inc. Sarasota, FL), whereas the CAPs were recorded from sciatic nerve, dorsal roots or ventral roots using pCLAMP 8.2 (Molecular Devices). Stimulus intensity was increased until the response amplitude stopped increasing, reflecting maximal fiber recruitment. The largest CAP was chosen and its amplitude was measured from the pre-CAP baseline to the peak. The recordings were made at room temperature (21 ± 1°C). All experimental procedures regarding animal use in this study are conformed to the NIH Guide for the Care and Use of Laboratory Animals and the guidelines of the Pfizer Institutional Animal Care and Use Committee. Efforts were made to minimize animal suffering and to reduce the number of animals used.
DRG neuron dissociation and culture.
DRGs were isolated from adult rats and dissociated enzymatically as described previously (Liu et al., 2002). Briefly, after deep anesthesia with ketamine/xylazine (60/10 mg/kg; ip), rats were exsanguinated and the vertebral column was exposed by dorsal laminectomy. The lumbar ganglia (L4, L5) were excised, treated with collagenase A (1 mg/ml) for 25 min, and collagenase D (1 mg/ml) with trypsin (0.4 mg/ml) for 25 min, dissociated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and plated on glass cover slips (Warner Instrument Corp., Hamden, CT) (Honmou et al., 1994). The dissociated neurons were then maintained in a 5% CO2–95% O2 incubator at 37°C for 2–5 h, allowing cells to adhere (Oyelese et al., 1995). Recordings were made within 5 h of dissociation.
Whole-cell patch-clamp recording and data analysis.
The whole-cell patch clamp method (Hamill et al., 1981) was used to record voltage-dependent Na+ current from the cultured DRG neurons. Glass cover slips with DRG neurons attached were transferred to the bottom of a recording chamber fixed to an inverted microscope (TE300, Nikon, Japan). Membrane currents of DRG neurons were recorded using a computer controlled patch-clamp amplifier (Multiclamp 700A, Molecular Devices), and experiments were controlled by computer through a Digidata 1322A D/A interface. Microelectrodes were fabricated from borosilicate glass (World Precision Instruments, Inc., Sarasota, FL) using a P-97 micropipette puller (Sutter Instrument, San Rafael, CA), and fire-polished with the use of a MF-830 microforge (Narishige, Tokyo, Japan). Patch pipette resistances were between 0.5 and 1.2 M
. The external recording solution contained (in mM): 20 NaCl, 110 tetramethylammonium Cl, 3 KCl, 1 MgCl2, 1 CaCl2, 10 N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), pH 7.4; osmolality 305–315 mmol/kg (adjusted with glucose) The pipette solution contained (in mM): 135 CsF, 10 NaCl, 1 CaCl2, 11 Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid, 2 Mg-ATP, 10 HEPES, pH 7.3; osmolality 300–310 mmol/kg. Cells chosen for recording were between 29 and 41 µm in diameter. In this study, the tetramethylammonium-Cl in the bathing solution and the CsF in pipette solution were used to reduce Na+ currents and block competing K+ currents, respectively. Under these conditions, inward currents are referred to as voltage-gated sodium channel currents, INa (Leffler et al., 2002). All patch-clamp recordings were made at room temperature (21 ± 1°C).
After formation of gigaohm seal (> 4 G) and compensation of pipette capacitance and junction potential with amplifier circuitry, the whole-cell configuration was established. Whole-cell capacitance and series resistance was compensated at > 90%. The cells were held at –100 mV and various stimulation protocols were applied as described below. Data were sampled at 10K samples/second and stored on the computer hard disc. All recordings were initiated within 15–20 min of cells being placed in the recording chamber. Drug treatment solutions were perfused directly to the recording chamber.
The absolute currents evoked by series of voltage steps were converted to conductance by the equation gNa = INa/(Vm – ENa), where Vm is the command potential and ENa is the Na+ equilibrium potential (
+17.5 mV for [Na+]o = 20mM and [Na+]i = 10mM). Conductance values were normalized and fitted to a Boltzmann equation of the form g/gmax = {1 + exp[-(Vm – Va
)/Ka]} – 1, where g is the conductance, gmax is the maximal conductance, Vm is the command potential, Va1/2 is the voltage at which half-maximal activation is achieved and Ka is the slope factor of the activation curve.
A prepulse inactivation protocol (Cummins and Waxman, 1997; Renganathan et al., 2000; Roy and Narahashi, 1992) was applied to distinguish fast and slow Na+ currents. Single neuron inactivation curves were also fitted with a Boltzmann equation of the form (I)/(Imax) = {1+ exp[V – Vi1/2)/ki]} – 1, where I is current, Imax is the maximal current, V is the prepulse potential, Vi1/2 is the voltage for half-maximal inactivation, and ki is the slope factor of the inactivation curve. Values of Vi1/2 and ki for each neuron were obtained by curve fitting.
Data were analyzed using pCLAMP 8 (Molecular Devices), Origin (Microcal Software, Inc. Northampton, MA) and Excel (Microsoft, Seattle, WA) software and are presented as means ± SD or SE; n represents the number of cells examined. Statistical evaluations were based on Student's t-test, using a criterion of p < 0.05 for significance.
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RESULTS |
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Effect of CP-628,319 on CAPs
To determine if NHE-1 inhibitors acutely affect the excitability of peripheral nervous system tissue, we tested the effect of CP-628,319 on CAPs recorded from electrically stimulated sections of isolated rat sciatic nerve/DRG/root preparations. Combinations of stimulating and recording sites both proximal and distal to the DRG were used as depicted in Figure 2A. CAP amplitudes were measured as described in "Methods." Latencies between stimulus artifacts and the start of the mean response were measured and normalized to the distance between stimulation and recording sites to determine CAP conduction velocities for each combination of stimulating and recording sites. The conduction velocities in our preparations were between 10.3 and 36.6 m/s, which fall in the range of myelinated A-type fibers (Harper and Lawson, 1985b
). There were no statistical differences in the mean conduction velocities determined for the different stimulating and recording site combinations (p > 0.05, Table 1). CP-628,319 (at 1 and 10µM) did not affect the nerve conduction velocities in sciatic nerve/dorsal root or sciatic nerve/ventral root combinations (p > 0.05, Table 1).
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Exposure to 1 or 10µM CP-628,319 reduced the amplitude of the CAPs recorded from the dorsal root following sciatic nerve stimulation by 33% and 58%, respectively (p < 0.05), compared with pretreatment values (Figs. 2B and 2D). This effect also showed partial wash out over a 30-min period. Notably, CP-628,319 had no statistically significant effect on CAP amplitudes recorded from the ventral root (all p > 0.3, compared with pretreatment values, Figs. 2C and 2D) at 1 or 10µM, nor was any direct effect observed when stimulating and recording from sections of dorsal root or sciatic nerve (Fig. 2D). Thus, CP-628,319 reduced the response of sensory fibers "passing through" the DRG. It had no effect on motor fibers of the ventral root and no effect on sensory fibers proximal or distal to the DRG. Because CP-628,319 reduced the excitability of the sensory fibers in the region of the DRG, and Na+ channels play a significant role in determining membrane excitability (Devor, 2006
), we chose to examine the effect of CP-628,319, as well as additional NHE-1 inhibitors, CP-597,396 and DMA, on Na+ channel gating characteristics in neurons isolated from the DRG.
Effects of NHE-1 Inhibitors on Na+ Channels in DRG Neurons
Fast and slow INa.
Following dissociation, DRG neurons (Fig. 3A) were patch-clamped in the whole-cell configuration and transmembrane Na+ currents measured during depolarizing voltage steps from a holding potential of –100 mV. Sodium channel currents can be divided into fast and slow currents. The fast currents show rapid activation and inactivation kinetics and dominate the whole-cell currents in larger DRG neurons, whereas the slow currents are slower and dominate whole-cell currents in smaller DRG neurons (Cummins and Waxman, 1997; Everill et al., 2001; Honmou et al., 1994). Examples of currents acquired from larger and smaller DRG neurons are shown in Figure 3B. In the following experiments medium-to-large cells (30–41 µm) were patch-clamped for the study of fast current kinetics, which were confirmed by visual inspection of current vs. time traces. Small-to-medium neurons (29–36 µm) were patch-clamped for the study of slow current kinetics, which were confirmed by visual inspection of current vs. time traces. Cells with mixed kinetics were not studied.
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Effect of NHE-1 inhibitors on fast INa steady-state inactivation.
The effect of NHE-1 inhibitors on the voltage dependence of steady-state inactivation of fast INa was examined using a double-pulse stimulus protocol similar to that described previously (Cummins and Waxman, 1997
; Renganathan et al., 2000; Roy and Narahashi, 1992), and illustrated in Figure 3C. Briefly, medium-to-large DRG neurons were clamped to a series of prepulse potentials (–120 to –10 mV) for 50 ms to allow channel inactivation to develop prior to the application of a 20 ms test pulse to –10 mV. Peak currents recorded in response to the test pulse were normalized to the maximal peak current and then fitted by the Boltzmann equation to generate inactivation curves. The inactivation curves of fast INa before and after 10 and 100µM CP-628,319, CP-597,396, and DMA were compared as shown in Figures 4A-C. All three NHE-1 inhibitors shifted Na+ channel inactivation curves of fast Na+ currents to the left at these concentrations (Fig. 4C). Significant differences in Vi1/2 (voltage for half-maximal inactivation) were found between controls (–65.7 ± 4.5 mV, n = 8) and 10µM (–69.7 ± 4.5 mV, n = 8) or 100µM (–76.4 ± 3.9 mV, n = 3) CP-628,319 treatments. Likewise, significant differences in Vi1/2 were also observed between control (–69.1 ± 4.0 mV, n = 7) and 10µM (–72.5 ± 3.1 mV, n = 7) or 100µM (–74.2 ± 3.8 mV, n = 7) CP-597,396, and differences in Vi1/2 were observed between control (–66.7 ± 3.8 mV) and 10µM (–68.9 ± 3.2 mV) or 100µM (–70.5 ± 3.8 mV) DMA. Notably, the Kis (slope factors) of these fast INa inactivation curves were not affected by any compounds at concentrations tested (p > 0.05, Figs. 4A and 4B).
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Effect of CP-628,319 on other properties of fast INa.
To determine if NHE-1 inhibitors affect other properties of fast INa, we used CP-628,319 to study the effects on steady-state activation and recovery from inactivation. For activation properties of fast INa, there was no statistically significant difference for values of Va1/2 and Ka obtained before and after application of 10 and 100 µM CP-628,319 (n = 3–8, p > 0.05). We also compared overall fast INa current amplitude, measured as the peak amplitude from a standard holding potential (–100 mV) to the potential at which maximum Na+ current was achieved (–35 to –20 mV), for untreated cells and cells treated with 10 and 100µM CP-628,319 (data not shown). No significant difference was observed in peak inward currents (both p > 0.05).
The time course of recovery of the fast INa from inactivation was investigated using paired-pulse protocols described previously (Elliott and Elliott, 1993). A conditioning pulse (30 ms) from –100 to –20 mV was first employed to inactivate INa completely and then, after a variable length step to –100 mV for 2–2048 ms, a test pulse to –20 mV was applied. The amplitude of INa gradually returned to control values from about 50% of control values (data not shown). No significant difference was observed between the recovery curve obtained with control and that with 10 or 100µM CP-628,319 (p > 0.05, n = 6).
Effect of CP-628,319 on steady-state inactivation of slow INa.
To determine if the effects of NHE-1 inhibitors on steady-state inactivation were unique to fast INa we studied the effects of CP-628,319 on steady-state inactivation of slow INa. In this case, small-to-medium DRG neurons were clamped to a series of prepulse potentials (–50 to –10 mV) for 50 ms to allow channel inactivation to develop prior to the application of a 20-ms test pulse to –10 mV. The slow INa obtained following prepulse depolarization to levels above –50 mV were recorded and fitted. The inactivation curves before and after 10 and 100 µM CP-628,319 were compared as shown in Figure 5. CP-628,319 did not significantly shift the inactivation curve to the left. The differences in Vi1/2 between control (–33.7 ± 4.1 mV, n = 6), 10µM (–34.8 ± 3.9 mV, n = 5), and 100µM (–37.2 ± 1.9 mV, n = 2) are not statistically significant (both p > 0.05).
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DISCUSSION |
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In the present study, we show for the first time that NHE-1 inhibitors can reduce peripheral nerve excitability. Specifically, we showed that CP-628,319 decreased the amplitude of CAPs recorded from isolated dorsal roots, but not ventral roots, following stimulation of the sciatic nerve. In addition, we showed that NHE-1 inhibitors increased the amount of fast Na+ channel inactivation in medium-to-large sized DRG neurons. We propose that NHE-1 inhibitors reduce the amplitude of the CAP by removing the contribution of larger diameter sensory fibers whose fast Na+ channels have been shifted into a nonconducting, inactivated configuration. These acute effects of NHE-1 inhibitors may be related to the functional and structural changes in peripheral nervous system observed at similar free drug concentrations in rats and dogs following chronic exposure (1–4 weeks) to CP-597,396 (Pettersen et al., in press
).
Decreased Dorsal Root CAP
The amplitudes of CAPs recorded from acutely isolated dorsal roots following stimulation of sciatic nerves were decreased in the presence of NHE-1 inhibitor CP-628,319 at concentrations between 1 and 10µM. Notably, CAP amplitudes were not affected when recorded from the ventral roots following sciatic nerve stimulation. This observation suggests that CP-628,319 affects sensory, but not motor, pathway excitability. Conduction velocity measurements in these preparations were always well above 3 m/s indicating conductance by larger diameter, myelinated A-type fibers, and not A- or C- type fibers when recording at room temperature (Harper and Lawson, 1985a; Miyoshi and Goto, 1973). The lack of an effect on CAP when stimulating and recording on the same side of the DRG, that is, on either nerve or dorsal root tissue sections, points to an effect localized to regions near the neuronal soma within the DRG.
In the DRG neurons, axonal processes of the nerve do not pass directly through the neuronal cell body, but rather are offset and connect to the soma via a short axonal segment producing a "T"-shaped junction and a pseudounipolar geometry. In the region of the T-junction, fiber diameter decreases and nodes of Ranvier are located close to each other (Spencer et al., 1973). It has been suggested that this geometry is designed to assure that action potentials propagate past the soma and into dorsal root projections to the spinal cord (Amir and Devor, 2003). Voltage-gated Na+ channels are known to cluster at high densities within the axon hillock/initial segment region of neurons (Waxman, 2000; Wollner and Catterall, 1986) and within the nodes of Ranvier on myelinated axons (Ritchie and Rogart, 1977) where they are involved in action potential initiation and propagation, respectively. We suspect that our observation of selective effects of CP-628,319 on CAPs conducted through the DRG is somehow related to a unique combination of T-junction geometry and effects on local membrane excitability. Because Na+ channels play a significant role in determining membrane excitability (Devor, 2006), and because we were not able to study Na+ channels directly within the region of the T-junction, we chose to examine the effect of NHE-1 inhibitors on Na+ channel gating characteristics in neurons isolated from the DRG. The soma has long been used as a model of sensory fiber and terminal processes not readily accessible by traditional investigative methods (Richardson and Vasko, 2002).
Increased Steady-State Inactivation of Fast Na+ Current in DRG Neurons
NHE-1 inhibitors, CP-628,319, CP-597,396, and DMA, each produced a clear leftward shift in the steady-state inactivation curves in medium-to-large DRG neurons at concentrations between 10 and 100µM. Values for Vi1/2 (voltage for half-maximal inactivation) were significantly shifted to more negative potentials by between 4 and 11 mV. Slope factors were not changed. Thus, for a typical DRG neuron with a resting potential of –63 mV (Liu et al., 2000), a leftward shift in the steady-state Vi1/2 of 11 mV would decrease the fraction of Na+ channels available for opening from 43% to 16% for the inactivation slopes we measured. However, simulations of the effects of complete block of Na channel conductance in DRG neurons suggest little effect on action potential propagation in associated axonal fibers (Amir and Devor, 2003). If these simulations are correct then one might hypothesize that the decrease in available fast Na+ channels we see in DRG neurons in the presence of NHE-1 inhibitors also occurs in regions of the T-junction, where loss of available Na channels would likely result in conduction block in some fibers and reductions in CAP amplitudes as we have observed in isolated nerve/DRG/root preparations.
This effect of NHE-1 inhibitors on Na+ channel gating appears similar to that described for local anesthetics like lidocaine which block action potential propagation by shifting the equilibrium of Na+ channels toward the inactivated configuration and thereby reducing macroscopic Na+ currents (Butterworth and Strichartz, 1990; Castaneda-Castellanos et al., 2002; Vedantham and Cannon, 1999). However, in the case of NHE-1 inhibitors this is likely to be an indirect effect because there is no evidence that Zoniporide interacts directly with Na+ channels at concentrations up to 10µM (Tracey et al., 2003).
The modulation of sodium channel gating may be mediated via intracellular pH changes produced by inhibiting NHE-1 mediated extrusion of intracellular protons. NHE-1 is a ubiquitously expressed exchanger present in the plasma membranes of most mammalian cells (Fliegel and Wang, 1997; Mahnensmith and Aronson, 1985). In the central nervous system, NHE-1 has been described in many regions of CNS (Ma and Haddad, 1997), including hippocampus (Gu et al., 2001; Xia et al., 2003). NHE-1 has also been described in the peripheral nervous system, including inner and outer hair cells and spiral ganglion neurons (Bond et al., 1998), and NHE-1 transcript is found in both human and rat DRG (Gene Expression Atlas, Novartis, San Diego, CA, http://expression.gnf.org, Su et al., 2002). NHE-1 inhibitors produce a decrease in intracellular pH in a variety of tissues, including neurons (Schneider et al., 2004), and changes in pH can modulate neuronal excitability in the CNS (Bonnet et al., 2000a, b; Gu et al., 2001). In squid axon, internal alkalinization results in a decrease of the steady-state inactivation of sodium channels (Brodwick and Eaton, 1978).
Although these studies suggest that the alterations in Na+ channel inactivation properties we report here may be the result of decreased intracellular pH, we did not measure pH in DRG neurons in our studies. Additionally, despite the fact that we observed acute effects on rat sensory nervous system at NHE-1 inhibitor concentrations similar to those reported to cause peripheral nerve effects in vivo (Pettersen et al., in press), the compounds we tested here inhibit the electroneutral exchange of Na+ and H+ in vitro with IC50's in the 10–100nM range (Masereel et al., 2003; Tracey et al., 2003). Thus, the NHE-1 inhibitor concentrations we used were well above those that would be expected to block H+ extrusion via NHE-1 and it is possible that the effects we observed also reflect other reported roles of NHE-1, including roles in cytoskeletal organization, cell volume regulation and cell migration (Stock and Schwab, 2006).
Notably, NHE-1 inhibitors have recently been associated with changes in peripheral nerve function and structure in vivo, including decreased patellar reflex in dogs, slowing of caudal nerve conduction velocity in rats and, with longer duration exposure, focal axonal degeneration in peripheral nerves, dorsal roots and DRG of both rat and dog (Pettersen et al., in press). Our results suggest that these effects may reflect NHE-1–induced changes in the inactivation properties of fast Na+ channels which in turn block action potential propagation in large or medium sized sensory neurons within the DRG. How this conduction block is related to axonal degeneration is not clear, but may be due to chronic or repeated silencing of select neurons which then degenerate. Loss of larger diameter fibers from nerve has been reported following chronic muscle disuse produced by tenectomy (Tomanek and Tipson, 1967).
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SUMMARY |
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We have shown for the first time that NHE-1 inhibitors can reduce the excitability of the peripheral nervous system. We present evidence that NHE-1 inhibitors reduce the amplitude of the nerve CAP conducted past the DRG and increase the number of fast Na+ channels in the inactivated, nonconducting configuration in medium-to-large diameter DRG neurons. We propose that these two observations are mechanistically related. By stabilizing more fast Na+ channels in an inactivated state, NHE-1 inhibitors reduce the fraction of channels available for opening, producing an acute reduction in the amplitude of the CAP; an effect restricted to larger A-type fibers within the DRG. These effects of NHE-1 inhibitors may underlie the functional and, with longer duration dosing, structural changes in peripheral nervous system reported in rats and dogs dosed with NHE-1 inhibitor Zoniporide (Pettersen et al., in press
). Thus, inhibiting NHE-1 for the treatment of cardiac ischemia-reperfusion injury may carry risks of peripheral nervous system changes that limit dose and duration of therapy.
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