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 .