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The chemokine CXCL1/growth related oncogene increases sodium currents and neuronal excitability in small diameter sensory neurons.

Wang JG, Strong JA, Xie W, Yang RH, Coyle DE, Wick DM, Dorsey ED, Zhang JM - Mol Pain (2008)

Bottom Line: These effects required long exposures, and were completely blocked by co-incubation with protein synthesis inhibitor cycloheximide.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0531, USA. jungang.wang@uchsc.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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Effect of the protein synthesis inhibitor cycloheximide (CHX) on enhancement of Na+ currents by GRO/KC incubation. Cycloheximide (3.5 μM) or vehicle (DMSO) was added to cell cultures just before addition of 1.5 nM GRO/KC or vehicle, and TTX-S and TTX-R Na currents were measured 16 to 30 hours later. *, significantly different from all other groups; #, significantly different from control (one-way ANOVA followed by Tukey's multiple comparison test). Data are from 1 set of experiments (3 cultures) comparing 22 control and 29 CHX treated cells, and a second set of experiments (2 cultures) comparing 14 GRO/KC treated cells with 22 GRO/KC + CHX treated cells. Additional data, from Figure 1, are included in the control and GRO/KC data groups. Analysis omitting this additional data gave similar results, except that the TTX-R current in IB4-positive cells showed no significant differences between groups.
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Figure 5: Effect of the protein synthesis inhibitor cycloheximide (CHX) on enhancement of Na+ currents by GRO/KC incubation. Cycloheximide (3.5 μM) or vehicle (DMSO) was added to cell cultures just before addition of 1.5 nM GRO/KC or vehicle, and TTX-S and TTX-R Na currents were measured 16 to 30 hours later. *, significantly different from all other groups; #, significantly different from control (one-way ANOVA followed by Tukey's multiple comparison test). Data are from 1 set of experiments (3 cultures) comparing 22 control and 29 CHX treated cells, and a second set of experiments (2 cultures) comparing 14 GRO/KC treated cells with 22 GRO/KC + CHX treated cells. Additional data, from Figure 1, are included in the control and GRO/KC data groups. Analysis omitting this additional data gave similar results, except that the TTX-R current in IB4-positive cells showed no significant differences between groups.

Mentions: We were unable to observe increases in Na+ currents following acute (5 minute) application of 1.5 nM GRO/KC (n = 5 IB4-positive and 7 IB4-negative cells). The finding that GRO/KC enhanced the magnitude of Na+ currents without marked shifts in voltage dependence activation or kinetics (Figure 2, 3), and was observed only after a period of incubation, suggested that the primary effect was an increased number of Na+ channels, and hence that protein synthesis might be required. To test this idea, we conducted experiments using the protein synthesis inhibitor cycloheximide. The results are shown in Figure 5. In general, cycloheximide prevented the GRO/KC-induced increases in Na+ current density without significantly affecting baseline current densities. In both IB4-positive and IB4-negative cells, only the GRO/KC without CHX group differed significantly, from all three other groups.


The chemokine CXCL1/growth related oncogene increases sodium currents and neuronal excitability in small diameter sensory neurons.

Wang JG, Strong JA, Xie W, Yang RH, Coyle DE, Wick DM, Dorsey ED, Zhang JM - Mol Pain (2008)

Effect of the protein synthesis inhibitor cycloheximide (CHX) on enhancement of Na+ currents by GRO/KC incubation. Cycloheximide (3.5 μM) or vehicle (DMSO) was added to cell cultures just before addition of 1.5 nM GRO/KC or vehicle, and TTX-S and TTX-R Na currents were measured 16 to 30 hours later. *, significantly different from all other groups; #, significantly different from control (one-way ANOVA followed by Tukey's multiple comparison test). Data are from 1 set of experiments (3 cultures) comparing 22 control and 29 CHX treated cells, and a second set of experiments (2 cultures) comparing 14 GRO/KC treated cells with 22 GRO/KC + CHX treated cells. Additional data, from Figure 1, are included in the control and GRO/KC data groups. Analysis omitting this additional data gave similar results, except that the TTX-R current in IB4-positive cells showed no significant differences between groups.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562993&req=5

Figure 5: Effect of the protein synthesis inhibitor cycloheximide (CHX) on enhancement of Na+ currents by GRO/KC incubation. Cycloheximide (3.5 μM) or vehicle (DMSO) was added to cell cultures just before addition of 1.5 nM GRO/KC or vehicle, and TTX-S and TTX-R Na currents were measured 16 to 30 hours later. *, significantly different from all other groups; #, significantly different from control (one-way ANOVA followed by Tukey's multiple comparison test). Data are from 1 set of experiments (3 cultures) comparing 22 control and 29 CHX treated cells, and a second set of experiments (2 cultures) comparing 14 GRO/KC treated cells with 22 GRO/KC + CHX treated cells. Additional data, from Figure 1, are included in the control and GRO/KC data groups. Analysis omitting this additional data gave similar results, except that the TTX-R current in IB4-positive cells showed no significant differences between groups.
Mentions: We were unable to observe increases in Na+ currents following acute (5 minute) application of 1.5 nM GRO/KC (n = 5 IB4-positive and 7 IB4-negative cells). The finding that GRO/KC enhanced the magnitude of Na+ currents without marked shifts in voltage dependence activation or kinetics (Figure 2, 3), and was observed only after a period of incubation, suggested that the primary effect was an increased number of Na+ channels, and hence that protein synthesis might be required. To test this idea, we conducted experiments using the protein synthesis inhibitor cycloheximide. The results are shown in Figure 5. In general, cycloheximide prevented the GRO/KC-induced increases in Na+ current density without significantly affecting baseline current densities. In both IB4-positive and IB4-negative cells, only the GRO/KC without CHX group differed significantly, from all three other groups.

Bottom Line: These effects required long exposures, and were completely blocked by co-incubation with protein synthesis inhibitor cycloheximide.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anesthesiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0531, USA. jungang.wang@uchsc.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus