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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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Expression of Na channels Nav1.1 through Nav 1.9 in cultured DRG cells before and after GRO/KC incubation. cDNA template was reverse-transcribed from RNA that had been isolated from control cells and cells from the sister cultures treated overnight with GRO/KC (1.5 nM). Expression of each gene was normalized to that of the housekeeping gene HPRT in the same batch of cDNA, determined during the same PCR amplification. A, profile of Na channel expression relative to HPRT. B, fold-change in expression of the 3 highly expressed Na channels after GRO/KC treatment. *, significantly different from control (= 1.0, dotted line), ratio t-test. Values shown are averages from 4 separate cultures.
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Figure 6: Expression of Na channels Nav1.1 through Nav 1.9 in cultured DRG cells before and after GRO/KC incubation. cDNA template was reverse-transcribed from RNA that had been isolated from control cells and cells from the sister cultures treated overnight with GRO/KC (1.5 nM). Expression of each gene was normalized to that of the housekeeping gene HPRT in the same batch of cDNA, determined during the same PCR amplification. A, profile of Na channel expression relative to HPRT. B, fold-change in expression of the 3 highly expressed Na channels after GRO/KC treatment. *, significantly different from control (= 1.0, dotted line), ratio t-test. Values shown are averages from 4 separate cultures.

Mentions: Nine different genes for voltage-gated Na channels have been described, with Nav1.1 – Nav1.7 mediating TTX-sensitive current and Nav 1.8 and 1.9 mediating TTX-resistant currents [30]. A simple explanation for the observed effects of GRO/KC on Na channel currents would be enhanced expression of channels with properties similar to those seen in control cells. As an initial test of this possibility, we used quantitative PCR methods to quantify relative changes in the amounts of Na channel mRNA present in DRG neurons after overnight incubation in 1.5 nM GRO/KC. Culture conditions were kept as close as possible to those used for the electrophysiological experiments. Expression data were normalized to that of the housekeeping gene HPRT. Of the nine Na channels, 3 showed much higher expression relative to HPRT than the others: Nav 1.1, 1.7, and 1.8 (Figure 6A). Nav 1.4 (a form found primarily in skeletal muscle), and 1.5 (a form found primarily in cardiac muscle), were undetectable or showed product only during the final few cycles. Nav 1.9 (which is thought to mediate persistent Na currents in nociceptors) was also very low abundance, with relative expression on the order of 10-6 times that of Nav 1.8. In contrast, in mRNA isolated from a whole DRG, we observed Nav1.9 mRNA with an abundance similar to that of Nav 1.8. This profile of expressed channels was grossly similar both before and after GRO/KC treatment; no previously low abundance channels were markedly upregulated by GRO/KC. The fold- increases in Nav 1.1, 1.7, and 1.8 were between 2 and 3 fold, roughly similar to the observed increase in current densities (Figure 6B); this measurement is less sensitive to the efficiency correction. These fold-increases were significant except in the case of Nav 1.8, which showed a nonsignificant trend to similarly increase (p = 0.08).


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)

Expression of Na channels Nav1.1 through Nav 1.9 in cultured DRG cells before and after GRO/KC incubation. cDNA template was reverse-transcribed from RNA that had been isolated from control cells and cells from the sister cultures treated overnight with GRO/KC (1.5 nM). Expression of each gene was normalized to that of the housekeeping gene HPRT in the same batch of cDNA, determined during the same PCR amplification. A, profile of Na channel expression relative to HPRT. B, fold-change in expression of the 3 highly expressed Na channels after GRO/KC treatment. *, significantly different from control (= 1.0, dotted line), ratio t-test. Values shown are averages from 4 separate cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Expression of Na channels Nav1.1 through Nav 1.9 in cultured DRG cells before and after GRO/KC incubation. cDNA template was reverse-transcribed from RNA that had been isolated from control cells and cells from the sister cultures treated overnight with GRO/KC (1.5 nM). Expression of each gene was normalized to that of the housekeeping gene HPRT in the same batch of cDNA, determined during the same PCR amplification. A, profile of Na channel expression relative to HPRT. B, fold-change in expression of the 3 highly expressed Na channels after GRO/KC treatment. *, significantly different from control (= 1.0, dotted line), ratio t-test. Values shown are averages from 4 separate cultures.
Mentions: Nine different genes for voltage-gated Na channels have been described, with Nav1.1 – Nav1.7 mediating TTX-sensitive current and Nav 1.8 and 1.9 mediating TTX-resistant currents [30]. A simple explanation for the observed effects of GRO/KC on Na channel currents would be enhanced expression of channels with properties similar to those seen in control cells. As an initial test of this possibility, we used quantitative PCR methods to quantify relative changes in the amounts of Na channel mRNA present in DRG neurons after overnight incubation in 1.5 nM GRO/KC. Culture conditions were kept as close as possible to those used for the electrophysiological experiments. Expression data were normalized to that of the housekeeping gene HPRT. Of the nine Na channels, 3 showed much higher expression relative to HPRT than the others: Nav 1.1, 1.7, and 1.8 (Figure 6A). Nav 1.4 (a form found primarily in skeletal muscle), and 1.5 (a form found primarily in cardiac muscle), were undetectable or showed product only during the final few cycles. Nav 1.9 (which is thought to mediate persistent Na currents in nociceptors) was also very low abundance, with relative expression on the order of 10-6 times that of Nav 1.8. In contrast, in mRNA isolated from a whole DRG, we observed Nav1.9 mRNA with an abundance similar to that of Nav 1.8. This profile of expressed channels was grossly similar both before and after GRO/KC treatment; no previously low abundance channels were markedly upregulated by GRO/KC. The fold- increases in Nav 1.1, 1.7, and 1.8 were between 2 and 3 fold, roughly similar to the observed increase in current densities (Figure 6B); this measurement is less sensitive to the efficiency correction. These fold-increases were significant except in the case of Nav 1.8, which showed a nonsignificant trend to similarly increase (p = 0.08).

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