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Signaling Potentiates Voltage-Dependent Sodium Currents in Trigeminal Nociceptive Neurons
1Departments of Anesthesiology, 2Neurobiology and Center of Neuroengineering, and 3Neurology, and 4Center for Translational Neuroscience, Duke University Medical Center, Durham, North Carolina
Submitted 16 May 2005; accepted in final form 19 November 2005
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ABSTRACT |
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(IL-1) mediates inflammation and hyperalgesia, although the underlying mechanisms remain elusive. To better understand such molecular and cellular mechanisms, we investigated how IL-1 modulates the total voltage-dependent sodium currents (INa) and its tetrodotoxin-resistant (TTX-R) component in capsaicin-sensitive trigeminal nociceptive neurons, both after a brief (5-min) and after a chronic exposure (24-h) of 20 ng/ml IL-1. A brief exposure led to a 28% specific (receptor-mediated) reduction of INa in these neurons, which were found to contain type I IL-1 receptors (IL-1RI+) on both their soma and nerve endings. In marked contrast, after a 24-h exposure, the total sodium current was specifically increased by 67%, without significantly affecting the TTX-R component. This potentiation of INa was suppressed in the presence of selective inhibitors of protein kinase C and G-protein–coupled signaling pathways, thereby suggesting that INa can be modulated through multiple pathways. In summary, the potentiation of INa through chronic IL-1 signaling in nociceptive sensory neurons may be a critical component of inflammatory-associated hyperalgesia. |
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INTRODUCTION |
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cytokine. IL-1 is synthesized and released by a variety of cells including immune cells, glia, and neurons (Braddock and Quinn 2004
). Its release from these cells is frequently associated with inflammation and subsequently hyperalgesia. IL-1 binds to specific cell surface receptors, designated type I and type II IL-1 receptors (IL-1RI and IL-1RII, respectively) (Dinarello 2003). IL-1RI transduces the biological effects of IL-1, whereas IL-1RII serves as a decoy receptor that restricts the effect of the IL-1 on its target cells (Liege et al. 2000). IL-1ra is a naturally occurring IL-1RI receptor antagonist that has been shown to inhibit many biological responses to IL-1, including inflammatory hyperalgesia (Dinarello 2003; Smeets et al. 2003). Although IL-1 plays a central role in the generation of hyperalgesia (Bianchi et al. 1998; Wolf et al. 2003), the mechanisms underlying its action on nociceptors are relatively unexplored.
One reason for investigating its effect on nociceptors is that peripheral injections of IL-1 may induce hyperalgesia (Bal and Kusum 1998; Bianchi et al. 1998; Obreja et al. 2002b), and therefore excite or sensitize nociceptive neurons (Cuneyt Ozaktay et al. 2002; Fukuoka et al. 1994; Obreja et al. 2002b). Many if not most of IL-1's effects on sensory neurons were recorded a few minutes after it was applied (Tadano et al. 1999). In one study, soon after it was applied IL-1 induced an increase in the activity of C and A-
fibers (Cuneyt Ozaktay et al. 2002). In a subset of cultured dorsal root ganglion (DRG) neurons, a 90-s incubation of IL-1 was found to increase the sensitivity to noxious heat (Obreja et al. 2002a). In general, the response to acute injections of IL-1 is transient in that it decreases over the course of about 1 h (Ji et al. 2002; Tadano et al. 1999). The effects of longer-term incubations of IL-1 have not been as extensively studied. Chronic exposure can safely be assumed to be very relevant because it occurs under chronic inflammatory conditions. Many chronic pain states share a prominent inflammatory component such as arthritis, sciatica, dental pulpitis, or chronic infections of the eye, the skin, or bones (Brisby et al. 2002; Laughlin et al. 2000; Smeets et al. 2003). This notion is buttressed by the finding that, on injury, IL-1 mRNA expression peaks within 6–10 h and may remain elevated for several days (Acarin et al. 2000; Buttini et al. 2005; Minami et al. 1992; Vezzani et al. 1999).
Here we have explored the effects of short (5-min) and longer (24-h) incubations of IL-1 on voltage-gated sodium channels (VGSCs) from nociceptive trigeminal ganglion (TG) neurons.
We chose to investigate the effects of IL-1 on VGSCs because of their well-established role in nociceptor excitability as it pertains to nociception, pain, and inflammation (Decosterd et al. 2002; Gold 1999; Porreca et al. 1999; Waxman 1999). VGSCs are commonly characterized by their sensitivity to the neurotoxin, tetrodotoxin (TTX). VGSC subtypes that are inhibited by TTX (TTX-S) are usually rapidly activating and inactivating and are activated at relative hyperpolarizing voltages. VGSC subtypes that are resistant to TTX (TTX-R) are found in nociceptors and are additionally recognized by their slow inactivation kinetics and the relatively depolarizing voltages at which they are activated and deactivated (Roy and Narahashi 1992; Schild and Kunze 1997). In this study, we have found that a short-term exposure of IL-1 slightly decreases the total sodium current, whereas it increases significantly in longer-term exposures.
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METHODS |
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TG neurons from adult Sprague–Dawley rats were excised and cultured as described previously (Liu et al. 2004). Briefly, trigeminal ganglia were dissected aseptically and collected in modified Hank's balanced salt solution (mHBSS). After being washed in mHBSS, the ganglia were diced into small pieces and incubated in mHBSS for 30–50 min at 37°C in 0.1% collagenase (Type X1-S). Individual cells were dissociated by triturating the tissue through a fire-polished glass pipette, followed by a 10-min incubation at 37°C in 10 µg/ml DNase I (Type 1V) in F-12 medium (Life Technologies, Gaithersburg, MD). After they were centrifuged three times with F-12, the cells were cultured in F-12 supplemented with 10% fetal bovine serum. The cells were plated on poly-D-lysine–coated glass coverslips (diameter 15 mm) and cultured 24 h at 37°C in a water-saturated atmosphere with 5% CO2. The cell diameters (micrometer scale) were measured with a calibrated eyepiece under phase-contrast illumination. Neurons having projected soma diameters ranging between 18 and 31 µm were used. All experiments were carried out at room temperature (22–24°C).
Care of animals conformed to standards established by the National Institutes of Health. All animal protocols were approved by the Duke University Institutional Animal Care and Use Committee.
Gene expression studies
RT-PCR.
Gene expression of IL-1 receptors in TG neurons was verified by reverse transcription–polymerase chain reaction (RT-PCR) and immunohistochemistry. Total RNA was prepared from the TGs of six individual adult Sprague–Dawley rats. TG ganglions were removed aseptically and minced, and total RNA was extracted with Trizol Reagent (Life Technology, Gaithersburg, MD) according to standard procedures. To eliminate any residual DNA, the total RNA samples were treated with DNase I (Life Technology), and first-strand complementary DNA (cDNA) was synthesized with a commercially available kit (Roche Diagnostics, Indianapolis, IN). PCR was performed as described previously (Obreja et al. 2002a) using a commercially available kit and primers (PCR Core Kit, Boehringer Mannheim; primers from Life Technology). Amplicons were detected with ethidium bromide–stained agarose gels. Housekeeping gene GAPDH cDNA was amplified as a control. Water, instead of cDNA and cDNA synthesis without RT, served as a negative control. cDNA obtained from the spleen served as a positive control for amplification of IL-1RII because the spleen contains T and B cells that express this receptor (McMahan et al. 1991).
IMMUNOHISTOCHEMISTRY. Deeply anesthetized animals were transcardially perfused with ice-cold phosphate-buffered saline (PBS); trigeminal ganglia were dissected aseptically and embedded in OCT (optimal cutting temperature compound), rapidly frozen, and sectioned at 12-µm thickness with a cryostat (Leica, Wetzlar, Germany). Frozen sections were air-dried, fixated in ice-cold acetone for 1 min, and air-dried again; unspecific binding sites were blocked with 5% normal goat serum in PBS containing 0.1% Tween 20 (PBS-T). Primary antibody against IL-1RI was incubated for 24–48 h at 4°C. Primary antibody (Abcam, Cambridge, MA) specifically recognizes the extracellular portion of human IL-1RI, which is highly conserved between rodents and humans (67% identity, 80% homology). Rabbit IgG at the same concentration and rabbit preimmune serum were used as negative controls. Detection of the primary antibody was accomplished by immunofluorescence microscopy. For this, the primary antibody was detected with an anti-rabbit biotin–coupled antibody followed by streptavidin conjugated to the fluorescent dye Alexa 488 (reagents from Molecular Probes, Eugene, OR). The tissue was embedded in water-soluble mounting media and micrographs were taken with an Olympus BX60 upright microscope, at 10x, 20 x, and 40 x primary magnification. The microscope was equipped with a Coolpix CCD camera. ISEE software (Raleigh, NC) was used for image processing and measurements of the diameters of the somata. For this, ten nonconsecutive sections were evaluated.
Patch-clamp recordings
For whole cell voltage-clamp experiments we used N-51A borosilicate glass pipettes (Drummond Scientific, Broomall, PA) with resistances between 1 and 2 M
. The pipette solution was (in mM): CsCl, 140; NaCl, 10; CaCl2, 1.0; MgCl2, 2.0; EGTA, 5; HEPES, 10; Tris-ATP, 5 (pH 7.3). Recordings of the total sodium current (INa) were obtained using an Axopatch-200B patch clamp amplifier (Axon Instruments, Foster City, CA) and the output was digitized with a Digidata 1322A converter (Axon Instruments). The peak current–voltage (Ip–V) relationship was determined using 5-mV step depolarization increments from the holding potential of –80 to +40 mV. The time between two pulses was 2 s. To obtain the Ip–V relation of the sodium current activation, peak currents were analyzed using pCLAMP 8 and were plotted against the applied voltage. The voltage dependency of inactivation [h(
)] was determined by measuring the peak of the remaining maximal sodium current after the delivery of –100- to 40-mV prepulse voltage steps of 40-ms duration with 4-s interval time (Liu et al. 2001). The holding potential was –80 mV. Data obtained from neurons in which uncompensated series resistance resulted in voltage-clamp errors >5 mV were not used in further analysis. For all recordings the sampling rate was 10 kHz. The capacitance and series resistance (<90%) was compensated. At the end of each experiment 10 µM capsaicin (holding potential = –80 mV) was applied to determine whether the neuron is sensitive to capsaicin.
Chamber/solution delivery
The volume of the recording chamber containing the neurons was 370 µl and was continuously perfused by an external solution that contained (in mM): NaCl, 30; choline-Cl, 40; TEA-Cl, 70; KCl, 5; MgCl2, 1; CaCl2, 2; D-glucose, 10; HEPES, 10; CdCl2, 1, 4-AP, 3 (pH 7.4). With pipette solution, the ENa was calculated to be 27.9 mV. To obtain the distribution of TTX-S and TTX-R currents in capsaicin-sensitive neurons INa was first obtained, then 0.2 µM TTX was applied to obtain the TTX-R currents that were subsequently digitally subtracted from INa to obtain the TTX-S currents. This TTX concentration was chosen to be consistent with previous studies (Caffrey et al. 1992; Jeftinija 1994; Kim et al. 1999; Su et al. 1999), and also because it is essentially ineffective in blocking TTX-R currents with Nav1.8 and Nav1.9 subunits (Tate et al. 1998). In separate experiments TTX-R currents were obtained in the same manner, except that the neurons were bathed with 0.2 µM TTX to block TTX-S currents. TTX-R currents were not analyzed if they were 
10 pA. To observe the effects of IL-1 on the TTX-R currents it was added together with TTX. We chose this method to investigate the effects of IL-1 on TTX-R currents to reduce the time of the experiment and reduce rundown and other problems associated with longer-term recordings. At the end of these experiments, to determine whether the cell was capsaicin sensitive, 10 µM capsaicin was applied at a holding potential of –60 mV.
All lipid-soluble chemicals [such as capsaicin and 4
-phorbol 12,13-dibutyrate (PDBu)] were solubilized in 10 mM dimethylsulfoxide (DMSO) and diluted 1,000 x when injected into the chamber. No effect was found on INa for 0.1% DMSO. For the experiments involving the short-term delivery of IL-1 and/or IL-1ra, the reagents were simply dissolved in the extracellular buffer. All dissected neurons were cultured for 24 h. The difference between the short- and long-term exposures consisted only in the exposure time of IL-1 and IL-1ra. When recordings commenced, fresh agonists or antagonists were presented. This same procedural regimen was followed in the investigation of cytokines in neurons and lung epithelial cells (Kamosinska et al. 1997; Nicol et al. 1997).
Statistics and curve fitting
Data were analyzed using pClamp (Axon Instruments, Foster City, CA) and SigmaPlot (SPSS, Chicago IL) software. For 5-min exposures of IL-1 the effects were compared with the paired t-test. For those experiments in which the chemicals (agonists, antagonists, or modulators) were incubated 24 h, the effects of the treatments were compared with the unpaired t-test. In an attempt to reduce day-to-day variability, IL-1 incubation versus control was performed on small numbers of neurons on the same day; all reagents were aliquots from a larger batch so that we could increase the sample size by using identical stimulation conditions. Despite all attempts to reduce the variability, as seen in Tables 1–3, there was considerable variability in the current densities among the controls. For this reason the effects of the addition of IL-1 and other compounds were compared with controls done at the same time and treated in the same way. Another point regarding the variability is that, relative to TTX-S currents, the inactivation times of the maximum current TTX-R currents are relatively long, and the activation parameters as indicated by V0.5 are relatively depolarized (Gold 1999; Roy and Narahashi 1992; Schild and Kunze 1997).
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)]–voltage were fit to I/INa max = [1 + exp(V0.5 – Vm)/k]–1 or G/Gmax = 1/[1 + exp(V0.5 – Vm)/k], where V0.5 is the membrane potential (Vm) at which 50% of activation or (inactivation) was observed and k is the slope of the function. Chemicals
IL-1 (17 kD) and IL-1ra (17.5 kD) were obtained from R&D Systems (Minneapolis, MN). Calphostin C, D-erythro-sphingosine, and PDBu were purchased from Calbiochem (San Diego, CA). Other chemicals or reagents were purchased from Sigma Chemical (St. Louis, MO). Cell culture materials were purchased from Life Technologies (Rockville, MD).
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RESULTS |
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Although IL-1RI mRNA has been shown to be present in rat DRG neurons, including small soma diameter neurons (Copray et al. 2001; Obreja et al. 2002b), neither the message nor the expressed protein has been shown to be present in TG neurons. Figure 1A shows the results of an RT-PCR indicating that cells in the trigeminal ganglion harbor the message for IL-1RI, but not IL-1RII. The positive controls for IL-1RII expression obtained from rat spleen cDNA show that IL-1RII is not expressed in the rat trigeminal ganglion.
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). Thus the IL-1RI gene is expressed in the trigeminal ganglion and the respective receptor protein is expressed in the somata of neurons.
For pain perception, the presence of signaling molecules on nerve endings in the tissue is more relevant than the presence of these signaling molecules on the somata of nociceptive neurons. Therefore to determine whether the IL-1RI receptor protein is expressed on peripheral trigeminal nerve endings, we stained the skin of the rat snout. IL-1RI expression was found on peripheral sensory axons innervating the papillary layer of the snout epithelium (Fig. 1, D and E). It is also evident later in the basal cell, as would be expected, because it has been identified in mammalian keratinocytes (Grewe et al. 1996). Very thin nerve endings, perpendicular to the epithelial cell layer, could also be observed in the cornea of the eye (results not shown). With respect to the diameter of the somata of IL-1RI–expressing trigeminal neurons, 75.9% stained positive for IL-1RI and most of these neurons are predominantly of smaller diameter [i.e., <30 µm (Fig. 1F)], which indicates the presence of IL-1RI on nociceptor neurons, although to a lesser extent they are also present on some larger-diameter nerve cells.
Effects of IL-1 on INa, the total voltage-gated sodium current
EXPOSURE OF 5 MIN.
Three concentrations of IL-1 were tested on capsaicin-sensitive neurons: 1, 5, and 20 ng/ml. We found that a 5-min incubation of this cytokine decreased the total sodium current (INa) 1.0 ± 8.7% (n = 6), 13.7 ± 9.5% (n = 6), and 28.4 ± 5.3% (n = 7) (see Fig. 2 for 20 ng/ml). In all subsequent experiments we settled on using 20 ng/ml IL-1 because at this concentration it sensitized heat-activated currents in rat DRG neurons (Obreja et al. 2002a) and in TG neurons it did not activate either an inward or outward current (Fig. 2), but evoked an appreciable decrease in INa (see following text). For example, as seen in Fig. 2, INa decreased 26% after a 5-min incubation with 20 ng/ml IL-1. On average, after a 3-min wash the currents recovered to 8.4 ± 5.7% (n = 7) of their control values.
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. It was found that IL-1 produced small hyperpolarizing shifts in the conductance–voltage and inactivation–voltage relations (see Fig. 2 and Table 1).
The above data suggest that the inhibitory effects of IL-1 could either be a consequence of the specific activation of IL-1RI receptors or occur by less specific and direct mechanisms (Diem et al. 2003). To test whether it is indeed receptor mediated, we explored whether the inhibitory effect of IL-1 would be suppressed in the presence of the IL-1RI antagonist, IL-1ra (100 ng/ml). This antagonist concentration was used because in subfornical organ neurons it reversed the depolarization produced by IL-1 (Desson and Ferguson 2003). We found that INa did not significantly differ from control in the presence of 100 ng/ml IL-1ra ± 20 ng/ml IL-1 (Fig. 3 and Table 1). That 100 ng/ml IL-1ra did not affect INa also eliminates the possibility that IL-1 produced its effect by a nonspecific (i.e., membrane-perturbing) mechanism (Lundbaek and Andersen 1994). In addition, under all these conditions, the voltage-dependent parameters that characterize INa activation and inactivation were unchanged (Table 1). Thus the short-term effect of IL-1 is mediated through specific signaling through IL-1RI.
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in the short-term applications, we inquired whether or how a longer-term incubation of this cytokine would affect INa. This was accomplished by separating the neurons into a control group and an IL-1 group that was incubated for 24 h with 20 ng/ml IL-1. Under this condition we found that, on average, the magnitude of INa increased by a striking 67% (see Fig. 4 and Table 1). However, IL-1 did not induce statistically significant changes in the conductance–or inactivation–voltage relations (Fig. 4).
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. Under these conditions we found that IL-1ra alone did not produce a change in INa, but in its presence the IL-1–induced potentiation was essentially eliminated (Fig. 5A and Table 1). In addition, IL-1ra by itself or together with IL-1 did not produce statistically significant changes in the conductance–or inactivation–voltage relationships (Fig. 5, B and C and Table 1).
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on TTX-R currents in TG neurons
We first determined what fraction of INa in capsaicin-sensitive TG neurons is TTX-S and TTX-R, and found that TTX inhibited INa by 33.3 ± 13% (means ± SE, n = 23; range 16–62%) (data not shown), a value in good agreement with similar studies in DRGs (Arbuckle and Docherty 1995). The implication of this finding is that the INa values shown in these figures contain both types of currents, albeit to various extents. The G–V relationships between these two current types differed in that the TTX-S currents were activated at more depolarizing voltages. On average, the Boltzmann parameters, V0.5 and slope (k), that characterize the G–V relationship for the TTX-S and TTX-R currents were –27.8 ± 0.93 mV and 5.1 ± 0.67 (n = 16), and –10.2 ± 2.3 mV and 5.9 ± 1.1 (n = 16), respectively. The nearly 17-mV shift in the G–V curve and the magnitude of voltage dependency parameter k are reasonably consistent with a previous study in TG neurons, where V0.5 = –19.2 mV (k = 5.6) for TTX-S and 0.1 mV (k = 4.1) for TTX-R neurons (Kim et al. 1999) and also in good agreement with studies on DRGs where V0.5 values were –26.3 and –10.6 mV for TTX-S and TTX-R currents, respectively (Roy and Narahashi 1992).
Because we found that a 24-h incubation of IL-1 increased the total sodium current (INa), we looked exclusively to see its response on TTX-R currents (data not shown). To our surprise, we found no statistical difference (P = 0.57, t-test) in the magnitude of these currents in the presence or absence of IL-1 [control: –113 ± 22 pA/pF, n = 30: 20 ng/ml IL-1: –77 ± 17 pA/pF, n = 31; P = 0.20 t-test], which suggests that the changes in INa have to be primarily attributed to changes in the TTX-S currents.
Role of modulators of INa and their relation to IL-1 signaling
In many cells it has been found that a short-term exposure of IL-1 induced responses that are modulated by Ca2+, PKC, PLC, and protein tyrosine kinases (Amin et al. 2003; Grewe et al. 1996; Obreja et al. 2002a; Yang et al. 2000). Although there have been many studies of the effects of these modulators on INa after an acute administration, there have been far fewer reports on their effect after a 24-h incubation. To better understand how IL-1 signaling functions in capsaicin-sensitive trigeminal neurons, we tested how IL-1 signaling could be altered by modulation of selected signaling pathways.
PKC activators and inhibitors on IL-1–stimulated sodium currents
The effect on INa on a 24-h incubation of the PKC activator 0.5 µM PDBu is seen in Fig. 6. This PDBu concentration was chosen because in nociceptive neurons it was found that short applications affect voltage-dependent sodium and potassium channels (Gold et al. 1998; Liu and Simon 2003). However, whereas short-term applications activate PKC, with longer-term applications it appears to be downregulated (Liao et al. 1996; Manseau et al. 1998). Once again the TG neurons were divided into three groups: a control in which nothing was added, a group containing only PDBu, and a group containing PDBu and IL-1. Relative to the control, INa was reduced about 56% in the presence of PDBu. However, relative to the response of PDBu, we found that the coapplication of 0.5 µM PDBu and IL-1 caused an increase in the current density (Fig. 6A and Table 2). Taken together, inhibition of PKC pathways inhibited INa, yet even under this condition IL-1 retained its stimulatory properties on the total sodium current. We also found that a 24-h incubation of PDBu with TG nociceptive neurons did not result in statistically significant changes in either the conductance–or inactivation–voltage curves (Table 2).
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(see Fig. 6B). Once again, as in the PDBu experiments, the neurons were separated into three groups. Relative to the control, Calphostin C did not produce a statistically significant change of INa, although coincubation of Calphostin C together with IL-1 clearly suppressed the IL-1–induced increase in INa. Furthermore, Calphostin C by itself or together with IL-1 did not produce significant shifts in either the conductance–voltage or inactivation–voltage relation (Table 2).
Because these two different inhibitors of PKC gave different results regarding the ability of IL1- to sensitize INa (it was not prevented by PDBu but was by Calphostin C) in an attempt to clarify this issue, we tested the effect of D-erythro-sphingosine, a third well-known physiological inhibitor of PKC. Following the report of its activation effect on TRPM3 channels (Grimm et al. 2005) we incubated the neurons with 20 µM D-erythro-sphingosine. We found that in two separate experiments incubating the neurons for 24 h in the presence of 20 µM D-erythro-sphingosine was lethal to most of the TG neurons. We rejected the idea of reducing the concentration to nonlethal doses because we would not know whether it would work as a PKC inhibitor at these concentrations. Rather we tested another PKC antagonist, BIM {[2-(1–3)-dimethylaminopropyl]-1H-indol-3-yl-3–(1H-indol-3-yl)maleimide} at 1 µM (Grimm et al. 2005). We found that relative to the control, by itself BIM produced a statistically significant decrease in INa and together with IL-1 not only prevented the IL-1–sensitizing effect, but substantially reduced INa (Fig. 6C) without affecting the voltage-dependent parameters (Table 2).
Effect of IL-1 on INa with the G-protein inhibitor GDP--s
We also investigated whether G-protein–signaling pathways may contribute to the facilitatory effect of IL-1 by using GDP--s (1 mM), a nonspecific G-protein antagonist. This concentration was chosen based on findings from studies involving its effect on VGSCs (Ma et al. 1994; Shirayama et al. 1993). The experimental design was the same as that for the PKC experiments. We found that by itself GDP--s produced a 40% increase of the magnitude of INa (Fig. 6D). However, in the presence of GDP--s the facilitatory effect of IL-1 was eliminated (Fig. 6D and Table 3). In addition, neither GDP--s by itself nor in the presence of IL-1 had any significant change on the conductance–voltage or deactivation–voltage relations (Table 3).
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DISCUSSION |
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, is a proinflammatory cytokine that mediates a multitude of cellular responses that occur during inflammation. In vivo application of IL-1 and chronic inflammation are unambiguously associated with hyperalgesia. Several mechanisms may be involved in IL-1–induced hyperalgesia. These include synthesis of nitric oxide and prostaglandin, release of substance P, CGRP, or cytokines (Bal and Kusum 1998; Dinarello 2003) and, ultimately, plasticity of nociceptive signaling pathways (Decosterd et al. 2002). VGSCs in nociceptive neurons are potential molecular targets of IL-1 signaling because they are known to contribute to hyperalgesia during nerve injury or inflammation. Complex mechanisms cannot be excluded. For example, IL-1 may indirectly modulate INa by increasing the concentration of PGE2 (Dayer et al. 2005), which has been found to sensitize TTX-R sodium channels (Gold et al. 2002). Herein we provide evidence suggestive of a mechanism of IL-1 signaling in hyperalgesia. It was found that when acutely applied, which may have limited physiological relevancy, IL-1 decreases INa and therefore by itself is unlikely to account for the sensitizing effects of IL-1. However, when it is applied for longer time periods, as may occur in chronic inflammation, it produces a significant increase of INa in nociceptive trigeminal neurons, which could be regarded as an underlying electrophysiological substrate of hyperalgesia. Localization of IL1-RI in trigeminal nerve cells and their processes
Using RT-PCR we found that of the two IL-1 receptors, only IL-1RI is expressed in trigeminal ganglia (Fig. 1A). With respect to localization of the IL-1RI protein, the immunofluorescence data presented in Fig. 1 show that the IL-1RI is expressed in small and medium-sized TG neurons, to a lesser degree in larger neurons, and yet is not present in Schwann or other cell types in the trigeminal ganglion. Thus in dissociated cells it is very likely that IL-1 signals cells, autonomously in small- and medium-diameter neurons and not indirectly by Schwann or satellite cells. The observed effects of IL-1RI signaling are cell-autonomous to capsaicin-sensitive trigeminal neurons, most of which can be assumed to be nociceptors (Caterina et al. 1997; Waddell and Lawson 1989). With respect to DRG sensory neurons, a previous study reported a similar expression pattern in the sense that only IL-1RI was expressed (Copray et al. 2001; Obreja et al. 2002a). Whereas these investigations have demonstrated gene expression of IL1-RI by in situ hybridization of DRGs, we have localized the receptor protein by immunohistochemical methods on somata of trigeminal neurons as well as on nerve endings in trigeminally innervated pain-sensitive epithelia such as the skin of the snout (Fig. 1).
Acute (5-min) application
In capsaicin-sensitive TG neurons we did not find that by itself IL-1 would activate a current. However, in such types of neurons a 5-min application of IL-1 caused a concentration-dependent reduction in the magnitude of INa currents and at 20 ng/ml induced small hyperpolarizing shifts in the conductance–and inactivation–voltage curves (Fig. 2 and Table 1). Although the underlying reasons for the decrease are not known (but see following text), one explanation is simply that the number of sodium channels decreased, as has been shown in type II alveolar cells to occur for an IL-1–induced reduction in epithelial sodium channels (ENaCs) (Roux et al. 2005). Whatever the mechanism, the inhibition of INa was prevented by blocking the IL-1RI receptor with IL-1ra (Fig. 3 and Table 1). It was important to demonstrate that the response is receptor mediated because in retinal ganglion neurons, IL-1 inhibited voltage-gated sodium and potassium channels in a receptor-independent manner (Diem et al. 2003). In contrast, in rat subfornical organ (SFO) neurons IL-1 increased neuronal excitability, activated a nonselective cation current, and inhibited delayed rectifier potassium currents in a receptor-dependent manner (Desson and Ferguson 2003). In cortical neurons IL-1 (5 ng/ml) inhibited voltage-gated calcium channels (about 45%), although it was not determined whether the response was receptor mediated (MacManus et al. 2000). From these reports it is evident that IL-1 can affect several types of voltage-gated ion channels, including voltage-dependent TRPV1 receptors (Piper et al. 1999). In this respect, it is important that incubating IL-1 with DRG neurons for 24 h did not affect capsaicin-induced (TRPV1) currents (Nicol et al. 1997), whereas TRPV1 currents were sensitized with an acute IL-1 application (Obreja et al. 2002a). This underscores the fact that the application of an agonist for a specific receptor may produce vastly different responses depending on whether it is applied acutely or chronically.
Although the exogenous administration of low concentrations of IL-1 usually produces hyperalgesia, at higher concentrations it has been shown to produce an analgesic response (Bianchi et al. 1998). For example, in carrageenan-induced paw edema after injecting IL-1 (in a rat's paw) the nociceptive threshold was increased (Ji et al. 2002). Under this condition, the inhibition of INa in nociceptors may contribute to this analgesic behavior.
Chronic (24-h) effects of IL-1 on INa and TTX-R channels
In contrast to the short-term incubation with IL-1, a longer incubation of IL-1 with nociceptor neurons would more faithfully represent conditions found in chronic inflammation. In this regard, we found that a 24-h incubation of IL-1 produced a striking increase in INa and that this increase could be prevented by coincubation with the IL-1RI antagonist IL-1ra (Figs. 3 and 4). We note that this receptor-mediated increase occurred in spite of some desensitization of the IL-1RI receptors. This indicates that, like the acute response, the chronic response to IL-1 was receptor mediated. We also found that neither the conductance–voltage nor the inactivation–voltage curves were, on average, significantly altered (Fig. 5 and Table 1), suggesting that IL-1 increased either the number of channels or their conductance. We have not distinguished between these possibilities, although either mechanism could be operative in IL-1–induced sensitization.
It is well established that capsaicin-sensitive neurons contain both TTX-S and TTX-R sodium channels (Stucky and Lewin 1999). Because many studies have shown that during inflammation TTX-R channels become sensitized (Akopian et al. 1999; Gold et al. 1998; Lai et al. 2002), we expected to see IL-1 affect these channels. However, to our surprise, IL-1 did not affect TTX-R channels, indicating that the sensitizing effects of IL- on INa are mediated through TTX-S channels. In this regard, there have been studies showing that TTX-S VGSCs (such as Nav1.3 or Nav1.7) are involved in pain and/or inflammation (Hains et al. 2003; Nassar et al. 2004). Indeed, the nociception-specific Nav1.7 knockout exhibits deficits in inflammatory pain behavior (Nassar et al. 2004).
Downstream modulators that may modulate INa under chronic conditions
Here we have investigated the effects of four different PKC-inhibitors: PDBu, Calphostin C,D-erythro-sphingosine, and BIM in capsaicin-sensitive nociceptive neurons. One of them, D-erythro-sphingosine, simply killed the cells when they were exposed to it for 24 h. Acute exposure of the PKC activator, PDBu, in nociceptors was shown to increase the sensitivity of voltage-gated sodium channels (e.g., Gold et al. 1998). However, when left to incubate with cells for longer (say, 24-h) exposures, PKC pathways become downregulated (Liao et al. 1996; Wang et al. 1991). It was not known how a longer-term exposure of PDBu would affect INa by itself or in the presence of IL-1. We found that relative to control neurons treated in the same manner without PDBu, INa was considerably reduced in the presence of PDBu. As seen in Fig. 6, a similar response was seen with BIM. However, INa was unaffected by the more specific PKC antagonist, Calphostin C. One possibility is that these three antagonists were applied at different effective concentrations and thus exhibit different effects on PKC and other downstream pathways. That pathways other than PKC could be involved can be rationalized by the responses of the PKC antagonists in the presence of IL-1, where relative to their response in the presence of the antagonist they all exhibited different responses. That is, under these conditions the IL-1 response did not change, increased, or decreased in the presence of Calphostin C, PDBu, and BIM, respectively (see Fig. 6). It follows from these results that of three PKC antagonists tested the only one with any potential therapeutic value to prevent IL-1–induced hyperalgesia is Calphostin C. This is because it prevented the IL-1–induced sensitization and the remaining two antagonists, by inhibiting voltage-dependent sodium currents, would have deleterious effects on neuron excitability.
Inhibition of G-proteins on the IL-1 sensitization of INa
Because the IL-1RI receptor is a GPCR, we investigated whether the inhibition of G-proteins would contribute to the effect of IL-1. Although direct modulation by G-proteins has been proposed for many ion channels, it has been firmly established for only two families: voltage-activated Ca2+ channels and G-protein–activated inwardly rectifying K+ channels (Discal 2001). In contrast, there were only a few reports that found that VGSCs are modulated by G-proteins (Ma et al. 1994). When VGSCs, both in natively expressing hippocampal neurons or heterologously expressed in Chinese hamster ovary cells, were incubated with a G-protein stimulator for short periods, INa increased and the voltage-dependent parameters shifted in a hyperpolarizing direction. In contrast, the G-protein inhibitor, GDP--s, produced a decrease in INa and an 8- to 10-mV depolarizing shift in the voltage-dependent parameters (Ma et al. 1994).
In our experiments with GDP--s we found that after a 24-h incubation, INa increased 40% without producing shifts in the conductance–and deactivation–voltage curves (Fig. 6 and Table 2). One explanation of these data is simply that the number of VGSCs increased. However, relative to the currents evoked with only GDP--s, when IL-1 was coincubated with GDP--s an increase in INa was not observed. This suggests that a yet-to-be-determined G-protein–mediated pathway inhibits the IL-1–induced sensitization (Fig. 6D and Table 3).
In summary, the effect of IL-1 on voltage-dependent sodium channels in capsaicin-sensitive nociceptive neurons is critically dependent on the incubation time. During acute incubation with IL-1, a condition that is not likely to reflect the pathophysiology of inflammation, the alterations in voltage-dependent sodium channels are not likely to contribute to the hyperalgesic effects of IL-1. However, under more chronic conditions, INa, primarily through TTX-S channels, is strikingly facilitated and consequently may, at least partially, be responsible for the hyperalgesic effects of the inflammatory cytokine IL-1.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: Lieju Liu, 327 Bryan Research Building, Research Drive, Durham, NC 27710 (E-mail: Lieju{at}neuro.duke.edu)
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