The chemokine CXCL1/growth related oncogene increases sodium currents and neuronal excitability in small diameter sensory neurons
© Wang et al; licensee BioMed Central Ltd. 2008
Received: 10 September 2008
Accepted: 24 September 2008
Published: 24 September 2008
Altered Na+ channel expression, enhanced excitability, and spontaneous activity occur in nerve-injury and inflammatory models of pathological pain, through poorly understood mechanisms. The cytokine GRO/KC (growth related oncogene; CXCL1) shows strong, rapid upregulation in dorsal root ganglion in both nerve injury and inflammatory models. Neurons and glia express its receptor (CXCR2). CXCL1 has well-known effects on immune cells, but little is known about its direct effects on neurons.
We report that GRO/KC incubation (1.5 nM, overnight) caused marked upregulation of Na+ currents in acutely isolated small diameter rat (adult) sensory neurons in vitro. In both IB4-positive and IB4-negative sensory neurons, TTX-resistant and TTX-sensitive currents increased 2- to 4 fold, without altered voltage dependence or kinetic changes. These effects required long exposures, and were completely blocked by co-incubation with protein synthesis inhibitor cycloheximide. Amplification of cDNA from the neuronal cultures showed that 3 Na channel isoforms were predominant both before and after GRO/KC treatment (Nav 1.1, 1.7, and 1.8). TTX-sensitive isoforms 1.1 and 1.7 significantly increased 2 – 3 fold after GRO/KC incubation, while 1.8 showed a trend towards increased expression. Current clamp experiments showed that GRO/KC caused a marked increase in excitability, including resting potential depolarization, decreased rheobase, and lower action potential threshold. Neurons acquired a striking ability to fire repetitively; IB4-positive cells also showed marked broadening of action potentials. Immunohistochemical labelling confirmed that the CXCR2 receptor was present in most neurons both in dissociated cells and in DRG sections, as previously shown for neurons in the CNS.
Many studies on the role of chemokines in pain conditions have focused on their rapid and indirect effects on neurons, via release of inflammatory mediators from immune and glial cells. Our study suggests that GRO/KC may also have important pro-nociceptive effects via its direct actions on sensory neurons, and may induce long-term changes that involve protein synthesis.
Altered sodium channel expression in sensory neurons contributes to enhanced excitability and abnormal spontaneous activity after nerve injury or inflammation . However, little is known about mechanisms of this sodium channel regulation. Recent studies suggest important roles for cytokines and chemokines in the nervous system, in both neuropathic and inflammatory chronic pain. They may regulate pain via direct effects on neurons or glia, or via local release of modulators (e.g., prostaglandins; bradykinin) from immune cells [2–6].
We recently described a rat pain model in which the sensory neurons of the L4 and/or L5 dorsal root ganglia (DRG) are inflamed by deposition of zymosan, which stimulates a robust localized inflammatory response without overt nerve injury. This caused abnormal spontaneous activity, along with markedly increased sodium currents in small diameter, primarily nociceptive DRG neurons, an overall increase in excitability , and a very striking early increase (15-fold after one day) in the chemokine GRO/KC (growth-related oncogene/keratine-derived chemokine; CXCL1) . We observed similar large increases of GRO/KC in the spinal nerve ligation model .
GRO/KC is best known for its role in neutrophil chemotaxis and degranulation early during inflammation. In this regard its effects are similar to those of other CXC family cytokines with an ELF motif, including interleukin-8 (IL-8; CXCL8) in humans . GRO/KC may also have direct roles in the nervous system, including roles in pathological pain. Both GRO/KC and its primary receptor, CXCR2 (IL-8Rb) are expressed in neurons and other cells in the central nervous system, under normal and pathological conditions [11–17]. Less is known about GRO/KC in the peripheral nervous system. However, GRO/KC stimulates calcium influx , and release of the pain-related peptide calcitonin gene-related peptide (CGRP)  from cultured neonatal DRG neurons. Levels of GRO/KC in inflamed muscle tissue are well correlated with nociceptive behavior . Overall, these studies in peripheral nervous system suggest a pro-nociceptive role for GRO/KC (however, see ).
In view of the striking increases in GRO/KC observed in DRG in several pain models, the evidence that this chemokine may play important roles in pathological pain, and the relative dearth of information about its mechanisms of action in pain processing pathways (especially in the periphery), we initiated this study to examine effects of GRO/KC on the properties of small diameter DRG neurons. In light of our previous study showing marked increases in Na+ currents and excitability following local inflammation of the DRG , we focused on possible regulation of Na+ currents by GRO/KC in small diameter rat DRG neurons in acute primary culture. Here we report that overnight incubation with GRO/KC, but not acute application, leads to increased excitability, 2- to 4 fold increases in TTX-resistant and TTX-sensitive Na+ currents without altered voltage dependence or kinetic changes, and increases in mRNA abundance of Na channel isoforms that are already present in control cells. The increase in Na+ currents required protein synthesis. The results suggest that GRO/KC may have important pro-nociceptive effects through direct effects on neurons.
Young female Sprague-Dawley rats (body weight 100–150 g) were housed one or two per cage under a controlled diurnal cycle of 12 h light and 12 h dark with free access to water and food. The ambient environment was maintained at constant temperature (22 ± 0.5°C) and relative humidity (60–70%). All the surgical procedures and the experimental protocol were approved by the institutional animal care and use committee of the University of Cincinnati (Cincinnati, OH, USA).
Acute culture of sensory neurons
Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The bilateral L4 and L5 DRGs were isolated and the sheath was carefully removed in ice-cold normal Ringer solution. The connective tissue was digested by exposure to Ca2+-free solution containing 1.0% collagenase II (Fisher Scientific) for 30 min at 37°C followed by washout in normal Ringer solution for another 10 min. DRGs were then dissociated by trituration with fire-polished Pasteur pipettes. DRG cells were plated onto poly-D-lysine coated glass coverslips in Medium199 (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated FBS and 1000 U/ml each of penicillin and streptomycin. GRO/KC (rat recombinant, from Cell Sciences, Canton, MA, USA) and/or cycloheximide (Sigma) were added to the medium immediately after plating, as indicated, and the DRG cells were incubated at 37°C (5% CO2 balance air) before recording. Recordings were made within 30 hours of plating, and cells were selected for recording based on diameter (< 25 μm) and the absence of processes. Isolectin B4 (IB4) conjugated to fluorescein isothiocyante (FITC) (1 μg/μl; Sigma) was added to the culture medium for 30–60 min prior to start of recording sessions to identify non-peptidergic IB4-postive and peptidergic IB4-negative neurons [22, 23]. This procedure has been previously shown not to affect the currents measured . Neurons showing a robust fluorescence signal were classified as IB4-positive and those displaying no signal at all, IB4-negative. In general, the fluorescence was not examined until after completion of current recordings from each cell; i.e., IB4 binding was not used as a criterion in selecting which cells to record. In each individual experiment, we alternated between recording from control and experimental coverslips throughout the recording period. Every attempt was made to compare control and experimental cells taken from the same cultures, though in some cases it was not possible to obtain data from all four types of cells (experimental and control for both IB4-positive and IB4-negative) in a given experiment, particularly when IB4 status was not determined until the end of the recording. Additional controls for inter-animal variability are described in the Results section. Once it had been well established that the proportion of IB4-positive cells did not change with GRO/KC exposure, IB4 binding was sometimes examined prior to recording in order to ensure that approximately equal numbers of each cell type were recorded in each experiment.
After over-night culture (16–30 hours), coverslips were transferred to a recording chamber. Whole cell voltage-clamp recordings of small DRG neurons (diameter 15–25 μm) were conducted at room temperature with an AxoPatch-200B amplifier (Molecular Devices Corp, Union City, CA, USA). Patch pipettes (2.5–4.0 MΩ) were fabricated from borosilicate glass. Data were acquired on a Pentium IV computer with the Clampex 8 program (Molecular Devices Corp). The cell capacitance artifact was cancelled by the nulling circuit of the recording amplifier. Voltage errors were minimized by using ≥ 80% series resistance compensation. The current was filtered at 5 kHz and sampled at 50 kHz. The recording chamber was continuously perfused at room temperature with oxygenated bath solution at a flow rate of 1–2 ml/min.
For the current-clamp study of excitability, we measured the threshold current (rheobase), action potential (AP) threshold, resting membrane potential, AP rising rate, AP falling rate, afterhyperpolarization, and input resistance. Any cells without AP were excluded from the study. Only neurons that had stable membrane potential more negative than -45 mV after obtaining the whole cell configuration were used for further study.
For the voltage-clamp study, total Na+ current was divided into tetrodotoxin-resistant (TTX-R) and TTX-sensitive (TTX-S) currents, using the method previously described . Briefly, in most experiments, the TTX-R currents were isolated from the TTX-S currents by using a holding potential of -50 mV. The peak TTX-R current was measured at each potential. Both TTX-R and TTX-S current were evoked by a series of depolarizations from a holding potential of -80 mV. The amplitude of the TTX-S current in each cell was measured during depolarization to -10 mV from -80 mV, based on the distinct kinetic properties of the two currents. At this voltage, TTX-S current was measured as the difference between the early peak current and the current 5 msec after the beginning of the depolarization, a time window in which the TTX-S current essentially completely inactivates but TTX-R current changes little. In experiments in which TTX-S current evoked from -80 mV was measured at additional voltages, a time window of 11 msec (at -20 mV) or 27 msec (at -30 mV) was used, as determined in our previous studies using TTX to verify the separation of the currents.
Solutions for recording action potentials
The normal bath solution for recording AP contained (in mM) 130 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.4 with NaOH, and the osmolarity adjusted to ~300–310 mOsm with sucrose. The pipette solution contained (in mM) 140 KCl, 1 CaCl2, 2 MgCl2, 11 EGTA, 10 HEPES, 10 NaCl and 2 Mg ATP. The pH was adjusted to 7.3 with KOH, and osmolarity to ~290–300 mOsm with sucrose.
Solutions for recording sodium currents
Sodium currents were measured under a reduced sodium gradient to reduce space clamp problems. The Na+ bath solution contained (in mM) 30 NaOH, 110 TMA-Cl, 5 TEA-Cl, 2 MgCl2, 1 CaCl2, 0.1 CdCl2, 10 HEPES, and 10 glucose. The pH was adjusted to 7.4 with HCl, and the osmolarity was adjusted to ~300–310 mOsm with sucrose. The pipette solution contained (in mM) 140 CsCl, 5 NaOH, 2 MgCl2, 1 CaCl2, 11 EGTA, 10 HEPES, 2 Mg ATP, and 1 Li GTP. The pH was adjusted to 7.2 with CsOH, and the osmolarity to ~290 – 300 mOsm with sucrose. Voltages were not corrected for liquid junction potentials, which were calculated to be < 10 mV in all cases.
Immunohistochemical staining of GRO/KC receptor CXCR2 in sectioned DRG tissue
Rats were anesthetized and fixed by perfusing 200–300 ml of Zamboni's fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH = 7.4) through the left ventricle of the heart. The bilateral L4/L5 DRGs were removed and post-fixed in the perfusion fixative for 2 hours at room temperature. The ganglia were horizontally sectioned with a cryostat at a thickness of 8 μm. Tissue sections were incubated in polyclonal rabbit anti-rat antibody to CXCR2 (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:200 overnight at 4°C, followed by reaction with secondary antibody Alexa Fluor 594 goat anti-rabbit IgG (H+L) (1:1000, Invitrogen, Carlsbad, CA) for 1 hour.
Immunohistochemical staining of GRO/KC receptor CXCR2 in dissociated DRG cells
DRG cells from L4/L5 spinal levels were removed from rats, and neurons were isolated and cultured overnight. The cultured cells were fixed with 4% paraformaldeyhde for 30 min at room temperature, washed in ice cold acetone for 1 min, and permeablized with 1% Triton for 10 min. The cells were then incubated with antibody to CXCR2 (1:100, Santa Cruz Biotechnology) and neuronal marker anti-NeuN (1:100, Milipore, Billerica, MA) for 2 hours followed by incubation in secondary antibody conjugated to Alexa Fluor 594(1:1000, Milipore, Billerica, MA) for 1 hour. After drying, the cells were mounted on coverslips with Vector Hard Set mounting medium (Vector Laboratories Inc., Burlingame, CA, USA). In some experiments, IB4 conjugated to FITC (1 μg/μl), instead of anti-NeuN antibody, was added to the culture medium for 30 min before switching to the primary antibody, to identify IB4-positive and IB4 negative neurons. The immunoreactive products were visualized using a confocal microscope and Slidebook 4.1 imaging acquisition software (Intelligent Imaging Innovations, Denver, CO, USA).
Western blot analysis of CXCR2 in protein extracted from DRG
Western blot analysis was employed to confirm that the CXCR2 antibody used for immunohistochemistry recognized protein in the DRG of the size expected for CXCR2. Bilateral L4/L5 DRGs were isolated and homogenized in RIPA lysis buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, and 10 mM EDTA) (Millipore, Billerica, MA) supplemented just before running the assay with a protease and phosphatase inhibitor cocktail (100 μl of aprotinin, leupeptin, and pepstatin; 500 μl of PMSF, Na3VO4, and NaF) (Pierce, Rockford, IL, USA) followed by centrifugation at 4 °C for 40 min. Samples (15 μg of total protein per lane) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) running in Tris-HEPES-SDS buffer (Pierce, Rockford, IL) followed by electrophoretic transfer to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) in tris-glycine-SDS transfer buffer. Nonspecific binding sites were blocked with 3% nonfat dry milk (Bio-Rad) in Tween (0.1%)-phosphate-buffered saline (PBST) at room temperature for 1 hour. The CXCR2 antibody (1:300, Santa Cruz Biotechnology) was applied to the blot followed by incubation with immunopure peroxidase conjugated goat anti-rabbit IgG (H+L) (1:20,000, Pierce, Rockford, IL). The blot was developed by exposure to blue-light-sensitive Hyperfilm ECL (GE Healthcare Bio-Sciences Corp) using the enhanced Chemiluninescent (ECL) Western Blotting Detection System (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).
Quantitative analysis of sodium channel subtype expression
Primers used for qPCR experiments:
Rat gene symbol
Rat gene ID
Rat mRNA reference
Primers Forward, reverse
Predicted size (bp)
Position in mRNA
Incubation with GRO/KC enhances Na+ current density without affecting other parameters
Small diameter DRG neurons were acutely cultured and incubated overnight with 1.5 nM rat (recombinant) GRO/KC or vehicle. The Na+ currents were measured in small diameter neurons with whole cell patch clamp techniques. After incubation in GRO/KC, voltage-activated Na+ currents had overall properties (except for amplitude) similar to those we have previously described in cells cultured from control and locally inflamed DRG . The Na+ currents in both control and GRO/KC treated cells activated at -30 to -20 mV (Figure 1). Persistent Na+ currents attributed in previous studies to the Nav 1.9 isoform, which activate at potentials near -60 mV under recording conditions similar to ours [28, 29], were observed in neither control nor GRO/KC treated cells. We observed small sustained inward currents -40 mV in less than 10% of cells, and never observed inward currents at potentials of -50 or -60 mV (Figure 1; compare to [28, 29]). Na+ currents from GRO/KC treated cells (like those previously observed in control cells and inflamed cells) had a faster component that was blocked by 50 nM tetrodotoxin (TTX; n = 6 GRO/KC treated cells; data not shown) or by using a holding potential of -50 mV (Figure 1 and ). TTX-resistant currents, which have slower kinetics, could be isolated by using a holding potential of -50 mV which inactivated TTX-sensitive currents.
The higher (2 – 3 fold) TTX-S current density observed in IB4-negative compared to IB4-positive cells was significant in both control cells (p = 0.03, Mann-Whitney test) and GRO/KC treated cells (p = 0.02, Mann-Whitney test). There was a tendency for the peak TTX-R current (at 0 mV) to be higher in IB4-negative cells which however did not reach significance in either control cells (28% higher, p = 0.16, Mann-Whitney test) or in GRO/KC treated cells (32% higher, p = 0.19, t-test).
Enhancement of Na+ currents by GRO/KC is blocked by protein synthesis inhibitor
The Na channel expression profile indicates upregulation of previously expressed channel types
Distinct effects of GRO/KC on the excitability of IB4-positive and IB4-negative neurons
Membrane properties of isolated control and GRO/KC treated neurons in whole cell current clamp experiments
Cell and AP Properties
Number of Cells
20.0 ± 0.8
21.2 ± 0.7
22.8 ± 0.5
20.2 ± 0.9*
Cell Capacitance, pF
17.6 ± 1.3
20.0 ± 1.2
19.0 ± 1.3
16.8 ± 1.7*
Resting Potential, mV
-63.0 ± 1.6
-56.0 ± 1.4**
-64.6 ± 1.4
-55.4 ± 1.9 **
Input Resistance, MΩ
1244.0 ± 144.3
986.1 ± 99.4
1062.0 ± 64.6
1427.4 ± 92.3**
AP Rheobase, pA
60.4 ± 8.4
21.1 ± 2.1**
76.2 ± 14.2
51.1 ± 11.2*
AP Threshold, mV
-19.7 ± 0.9
-28.6 ± 1.4**
-16.6 ± 0.6
-25.4 ± 1.3**
AP Rising Rate, mV/ms
106.8 ± 9.6
112.5 ± 10.7
103.4 ± 8.8
102.4 ± 12.0
AP Falling Rate, mV/ms
49.7 ± 4.8
61.9 ± 7.3
43.4 ± 3.8
39.9 ± 5.0**
AP Amplitude, mV
64.8 ± 1.7
74.8 ± 1.8**
63.1 ± 1.9
71.1 ± 1.5 **
AP Duration, ms
6.6 ± 0.4
7.3 ± 0.5
8.3 ± 0.5
14.0 ± 1.0**
AHP Amplitude, mV
3.5 ± 0.5
5.9 ± 0.6 **
3.0 ± 0.8
4.5 ± 0.7
AHP Duration, ms
10.8 ± 2.4
14.7 ± 0.8*
7.7 ± 1.7
9.9 ± 1.4
CXCR2 is expressed in both IB4-positive and IB4-negative neurons
In DRG sections, CXCR2 was expressed in most neurons, whether small, medium, or large diameter (Figure 8C, D). An antibody directed to the C terminus of CXCR2 (supplier: Santa Cruz) displayed intense staining of plasma membrane and, in some cells, the nuclear membrane, with no immunoreactivity in the nucleoli. Although there was no apparent difference in CXCR2 staining of the plasma membrane between most small and large neurons, a subset of small DRG neurons displayed very high density staining, similar to observations made in the dissociated cells. Sections or dissociated cells incubated with primary antibody preabsorbed with 30 fold excess of antigen did not show any immunoreactivity (Figure 8E). In order to further validate the antibody used for the CXCR2 staining, we conducted western blot analysis of protein extracted from whole DRGs. Using the same antibody from Santa Cruz at 1:300 dilution, the western blot showed a strong doublet of 58–66 kDa and a lighter staining band of 35 kDa. An example is shown in Figure 8F; similar results were obtained in 3 replicate experiments.
In this study we found that overnight incubation with GRO/KC (CXCL1), a chemokine that is rapidly upregulated in the DRG in several different models of chronic pain, caused increased excitability and marked upregulation of Na+ current amplitudes in acutely isolated small diameter DRG neurons in short-term culture. This upregulation required protein synthesis. Many studies on the role of chemokines in pain conditions, including GRO/KC (e.g.,), have focused on their indirect effects on neurons, via stimulation of release of inflammatory mediators from immune and glial cells that then act on sympathetic and sensory neurons. Our study suggests that GRO/KC might also have important pro-nociceptive effects via its direct actions on sensory neurons. The majority of small diameter DRG neurons are nociceptors. The striking increase in excitability after GRO/KC incubation, including the ability to fire continuously during long depolarizations, would be expected to profoundly increase the sensitivity to painful stimuli in vivo – both by increasing the sensitivity of nociceptors to stimuli, and by increasing the release of excitatory neurotransmitters in the dorsal horn. It will be important to conduct experiments to interfere with GRO/KC production or action in the DRG in vivo, to confirm the pro-nociceptive roles for this chemokine suggested by this in vitro study.
Our results have some interesting similarities to those reported by Liu et al. , who found that chronic (24 h) exposure of cultured small diameter capsaicin-sensitive trigeminal neurons to another pro-inflammatory cytokine, interleukin 1-β (IL-1β), led to a 67% increase in TTX-S (but not the TTX-R) Na+ current, without changes in voltage dependence. As in our study, acute applications did not increase Na+ current (and in fact caused slight decreases in the Na+ current), and the cytokine receptor was shown to be expressed on the neurons.
GRO/KC actions are thought to be mediated by the CXCR2 receptor. As discussed in the Introduction, the presence of this receptor on neurons (including neonatal DRG neurons in vitro) has been previously observed with a variety of methods. The effects reported here are most likely to be due to a direct effect of GRO/KC on the neurons, because: (1) effects were obtained in low density, acute cultures, in which non-neuronal cells were very sparse and intercellular mediators would be diluted by the culture medium; and (2) using an antibody to the CXCR2 receptor, we demonstrated that the receptor was present on the membranes of virtually all of the small diameter neurons in our cultures. Western blot analysis of DRG proteins, using the same antibody, revealed a doublet band at 58–66 kDa and a lighter band of 35 kDa. For comparison, Xiong et al.  reported a band of approximately 58 kDa in Western blots of CXCR2 receptor in rat hippocampus. The human receptor, which has an almost identical predicted molecular weight of 40.7 kDa based on amino acid sequence (vs. 40.5 kDa for the rat receptor), has been reported to have an apparent molecular weight of 56 kDa when isolated from neutrophils, due to the presence of 2 N-linked 9 kDa carbohydrate moieties . Interestingly, this same group reported additional forms of the human receptor, of 38 and 40 kDa that were shown to differ in their carbohydrate groups.
The inability of GRO/KC to increase Na+ currents after acute application, or in the presence of cycloheximide, provides evidence that protein synthesis is required for the effect on Na+ currents. The simplest explanation would be that additional Na+ channels are synthesized after GRO/KC incubation, as this would be consistent with the observation that only the magnitudes of the currents are altered, not their activation range or decay time constants. Our measurements of Na channel subtype expression were consistent with this view. However, we cannot entirely rule out other possibilities such as synthesis of proteins that modulate channels already present. The CXCR2 receptor is a G-protein-coupled receptor that has been shown to activate a number of different signalling pathways in neurons, including intracellular Ca2+, inositol tris-phosphate, MAP kinases, and CREB (reviewed in ). In turn all of these pathways can lead to enhanced transcription and protein synthesis through a number of signal transduction pathways. In addition, some G-protein coupled receptors, including the closely related CXCR4 receptor, have been shown to be present in the nucleus as well as on the plasma membrane, where they are thought to activate nuclear effects through additional pathways (for review, see ). This is particularly interesting in light of our observation of nuclear staining for CXCR2 in some cells in addition to plasma membrane staining in DRG neurons. While artifactual nuclear staining can be a problem with immunohistochemical methods, this seems somewhat less likely given the observation that not all nuclei showed staining in our specimens.
We initially identified GRO/KC as a cytokine that might play important roles in pathological pain because it showed marked early increases in a pain model in which localized inflammation of the DRG is induced by zymosan . Using the same model, we found that localized inflammation led to marked increases in TTX-S and TTX-R currents in small diameter DRG neurons, as well as increased excitability, as determined by patch clamp studies in acutely isolated neurons . That study and the present study have certain common findings: both localized inflammation and GRO/KC incubation give striking increases in the magnitude of TTX-S current in both IB4-positive and IB4-negative cells, without obvious changes in inactivation time constant or voltage dependence. Both lead to increased excitability, though these effects are larger for GRO/KC treatment. One difference is that localized inflammation led to increased TTX-R current only in IB4-positive cells, while GRO/KC incubation also increased this current in IB4-negative cells. The comparison of these two studies suggests that GRO/KC elevations in the localized inflammation model provide one plausible explanation for some, but not all of the changes in ion currents and excitability observed in this model. We observed effects on excitability and Na+ currents at a relatively low GRO/KC concentration of 1.5 nM, which is well within the physiological range for most peptide receptor actions. In one study, GRO/KC-stimulated (acute) Ca2+ elevation in isolated DRG neurons could be observed at GRO/KC concentrations of 6.4 nM but was not significant at 1.2 nM . In cells expressing cloned CXCR2 receptors, the binding constant for GRO/KC was 0.2 nM . Thus it seems plausible that the effects reported here could occur in vivo.
The TTX-R currents described here, as in our previous study, had characteristics attributed to the Nav1.8 isoform. Our recording conditions would have minimized contributions from the persistent current (thought to be mediated by the Nav1.9 isoform) due to slow inactivation at the holding potentials used, and/or because the current may "washout" during whole cell patch clamp [28, 38]. Other studies also suggest that the persistent current would be quite small under our recording conditions (adenosine triphosphate in the pipette, Cl- as the anion) . In our qPCR experiments we observed that the Nav1.9 isoform was very low abundance under our culture conditions, though it had levels similar to Nav 1.8 in cDNA derived from whole DRG. Hence the lack of Nav 1.9 currents in our experiments may be related to the acute culture process; some studies of the persistent current used shorter culture times than we used in this study. The TTX-S current in DRG neurons is mediated by a number of different isoforms, which are not readily distinguished electrophysiologically. Some of the excitability changes we observed after GRO/KC incubation, such as resting membrane depolarization and increased input resistance, are not readily explained by the increased Na+ currents observed in the voltage clamp experiments. Hence it is highly likely that additional ion currents can be modulated by GRO/KC. This was also true of localized inflammation, which upregulates voltage activated K+ currents as well as Na+ currents .
In summary, the pro-inflammatory cytokine GRO/KC (CXCL1), which markedly increases in DRG during the early phase of several models of pathological pain, may exert important pro-nociceptive effects in part through its direct actions on nociceptive neurons.
This work was supported in part by NIH grants NS55860 and NS45594 (J.Z.), the University of Cincinnati Millennium Fund, and the University of Cincinnati Neuroscience Program/Summer Undergraduate Research Program in Neuroscience (DMW).
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