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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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Overnight incubation with GRO/KC (1.5 nM) increases Na+ current density. A. TTX-R current density significantly increased in IB4-negative cells after GRO/KC incubation. P < 0.0001 for overall effect. Individual voltages at which the difference between control and GRO/KC treated cells was significant are indicated by the * symbol (two-way RM ANOVA with Holm-Sidak method for Pairwise Multiple Comparisons). B. A similar but smaller enhancement of TTX-R current was observed in IB4-positive cells (overall p value = 0.0016). TTX-S current density measured at -30 to -10 mV as described in methods, also significantly increased after GRO/KC incubation in both IB4-negative (C; and IB4-positive cells (D). Data are from 9 cultures; N = 53 control cells and 54 GRO/KC treated cells.
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Figure 2: Overnight incubation with GRO/KC (1.5 nM) increases Na+ current density. A. TTX-R current density significantly increased in IB4-negative cells after GRO/KC incubation. P < 0.0001 for overall effect. Individual voltages at which the difference between control and GRO/KC treated cells was significant are indicated by the * symbol (two-way RM ANOVA with Holm-Sidak method for Pairwise Multiple Comparisons). B. A similar but smaller enhancement of TTX-R current was observed in IB4-positive cells (overall p value = 0.0016). TTX-S current density measured at -30 to -10 mV as described in methods, also significantly increased after GRO/KC incubation in both IB4-negative (C; and IB4-positive cells (D). Data are from 9 cultures; N = 53 control cells and 54 GRO/KC treated cells.

Mentions: As shown in Figure 2, GRO/KC incubation (16 to 30 hours) significantly increased the density both TTX-R and TTX-S currents. These effects were seen in both IB4-positive and IB4-negative neurons. These results are from 53 control cells (26 were IB4-positive) and 54 GRO/KC treated cells (28 were IB4-positive). In order to ensure that this primary finding was not confounded by inter-animal variability, we also analyzed the subset of data in which currents measured in control cells could be compared with that measured in GRO/KC treated cells from the same batch (animal), in side-by-side experiments. Statistical analysis similar to that shown in Figure 1 was then conducted using the batch averages (n = 4) instead of the individual values for each cell. Despite the much smaller N, the increase in TTX-R current was still statistically significant in both IB4-positive (overall p = 0.019) and IB4-negative (p = 0.002) cells. The GRO/KC induced increases in TTX-S currents showed the same trends as shown in Figure 2, though these did not reach significance with the smaller N value when batch averages were analyzed (p = 0.1 for IB4-negative cells and p = 0.2 for IB4-positive cells). The proportion of IB4-positive cells was not significantly altered by GRO/KC treatment (p = 0.8, Fisher's exact test).


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)

Overnight incubation with GRO/KC (1.5 nM) increases Na+ current density. A. TTX-R current density significantly increased in IB4-negative cells after GRO/KC incubation. P < 0.0001 for overall effect. Individual voltages at which the difference between control and GRO/KC treated cells was significant are indicated by the * symbol (two-way RM ANOVA with Holm-Sidak method for Pairwise Multiple Comparisons). B. A similar but smaller enhancement of TTX-R current was observed in IB4-positive cells (overall p value = 0.0016). TTX-S current density measured at -30 to -10 mV as described in methods, also significantly increased after GRO/KC incubation in both IB4-negative (C; and IB4-positive cells (D). Data are from 9 cultures; N = 53 control cells and 54 GRO/KC treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Overnight incubation with GRO/KC (1.5 nM) increases Na+ current density. A. TTX-R current density significantly increased in IB4-negative cells after GRO/KC incubation. P < 0.0001 for overall effect. Individual voltages at which the difference between control and GRO/KC treated cells was significant are indicated by the * symbol (two-way RM ANOVA with Holm-Sidak method for Pairwise Multiple Comparisons). B. A similar but smaller enhancement of TTX-R current was observed in IB4-positive cells (overall p value = 0.0016). TTX-S current density measured at -30 to -10 mV as described in methods, also significantly increased after GRO/KC incubation in both IB4-negative (C; and IB4-positive cells (D). Data are from 9 cultures; N = 53 control cells and 54 GRO/KC treated cells.
Mentions: As shown in Figure 2, GRO/KC incubation (16 to 30 hours) significantly increased the density both TTX-R and TTX-S currents. These effects were seen in both IB4-positive and IB4-negative neurons. These results are from 53 control cells (26 were IB4-positive) and 54 GRO/KC treated cells (28 were IB4-positive). In order to ensure that this primary finding was not confounded by inter-animal variability, we also analyzed the subset of data in which currents measured in control cells could be compared with that measured in GRO/KC treated cells from the same batch (animal), in side-by-side experiments. Statistical analysis similar to that shown in Figure 1 was then conducted using the batch averages (n = 4) instead of the individual values for each cell. Despite the much smaller N, the increase in TTX-R current was still statistically significant in both IB4-positive (overall p = 0.019) and IB4-negative (p = 0.002) cells. The GRO/KC induced increases in TTX-S currents showed the same trends as shown in Figure 2, though these did not reach significance with the smaller N value when batch averages were analyzed (p = 0.1 for IB4-negative cells and p = 0.2 for IB4-positive cells). The proportion of IB4-positive cells was not significantly altered by GRO/KC treatment (p = 0.8, Fisher's exact test).

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