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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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GRO/KC incubated cells have Na+ currents that are qualitatively similar to those seen in control cells. A. Examples of current traces elicited from holding potential of -80 mV (darker, smaller traces) or -50 mV. As in control cells, the currents elicited from -80 mV have a faster component, which is TTX-sensitive (see text). B. Persistent Na+ currents activating at -60 to -50 mV were not observed. Average leak-subtracted steady state current is plotted against the step potential. Holding potential was -80 mV.
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Figure 1: GRO/KC incubated cells have Na+ currents that are qualitatively similar to those seen in control cells. A. Examples of current traces elicited from holding potential of -80 mV (darker, smaller traces) or -50 mV. As in control cells, the currents elicited from -80 mV have a faster component, which is TTX-sensitive (see text). B. Persistent Na+ currents activating at -60 to -50 mV were not observed. Average leak-subtracted steady state current is plotted against the step potential. Holding potential was -80 mV.

Mentions: Data were analyzed using Clampfit 9 (Molecular Devices Corp), Graphpad Prism (GraphPad Software, Inc., San Diego, CA, USA), and Origin 7 (Origin Lab Corp., Northampton, MA, USA). Currents were normalized by cell capacitance. The ohmic portion of the observed peak Na+ currents was measured from current steps to -100 through -60 mV and subtracted. Data are expressed as mean ± standard error of the mean (SEM). Differences in proportion of cells binding IB4 were examined with Fisher's exact test. Statistical significance of differences between average values in experimental and control neurons were analyzed by Student's t-test or, for data that were not normally distributed, the Mann-Whitney rank sum test, using SigmaStat Software (Systat Software, Inc., San Jose, CA, USA). In the case of multiple comparisons over a voltage range for activation data, the data were analyzed by two-way repeated measures ANOVA (RM ANOVA), with pairwise multiple comparison (Holm-Sidak method) to determine at which voltages the differences between experimental and control cells were significant if an overall effect of GRO/KC treatment was observed. ANOVA (or Kruskal-Wallis test on ranks, for data not showing normal distribution) was used to analyze experiments with more than two experimental groups. Activation data was fitted to the Boltzmann equation (Figure 1) was done using Graphpad Prism software. Significance of fold-changes in relative Na channel expression in qPCR experiments was done using a ratio t-test. Significance was ascribed for p < 0.05.


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)

GRO/KC incubated cells have Na+ currents that are qualitatively similar to those seen in control cells. A. Examples of current traces elicited from holding potential of -80 mV (darker, smaller traces) or -50 mV. As in control cells, the currents elicited from -80 mV have a faster component, which is TTX-sensitive (see text). B. Persistent Na+ currents activating at -60 to -50 mV were not observed. Average leak-subtracted steady state current is plotted against the step potential. Holding potential was -80 mV.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: GRO/KC incubated cells have Na+ currents that are qualitatively similar to those seen in control cells. A. Examples of current traces elicited from holding potential of -80 mV (darker, smaller traces) or -50 mV. As in control cells, the currents elicited from -80 mV have a faster component, which is TTX-sensitive (see text). B. Persistent Na+ currents activating at -60 to -50 mV were not observed. Average leak-subtracted steady state current is plotted against the step potential. Holding potential was -80 mV.
Mentions: Data were analyzed using Clampfit 9 (Molecular Devices Corp), Graphpad Prism (GraphPad Software, Inc., San Diego, CA, USA), and Origin 7 (Origin Lab Corp., Northampton, MA, USA). Currents were normalized by cell capacitance. The ohmic portion of the observed peak Na+ currents was measured from current steps to -100 through -60 mV and subtracted. Data are expressed as mean ± standard error of the mean (SEM). Differences in proportion of cells binding IB4 were examined with Fisher's exact test. Statistical significance of differences between average values in experimental and control neurons were analyzed by Student's t-test or, for data that were not normally distributed, the Mann-Whitney rank sum test, using SigmaStat Software (Systat Software, Inc., San Jose, CA, USA). In the case of multiple comparisons over a voltage range for activation data, the data were analyzed by two-way repeated measures ANOVA (RM ANOVA), with pairwise multiple comparison (Holm-Sidak method) to determine at which voltages the differences between experimental and control cells were significant if an overall effect of GRO/KC treatment was observed. ANOVA (or Kruskal-Wallis test on ranks, for data not showing normal distribution) was used to analyze experiments with more than two experimental groups. Activation data was fitted to the Boltzmann equation (Figure 1) was done using Graphpad Prism software. Significance of fold-changes in relative Na channel expression in qPCR experiments was done using a ratio t-test. Significance was ascribed for p < 0.05.

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