Anesth Analg. 2014 Feb;118(2):318-24.

Up-regulation of NaV1.7 sodium channels expression by tumor necrosis factor-α in cultured bovine adrenal chromaffin cells and rat dorsal root ganglion neurons.


Tamura R1, Nemoto T, Maruta T, Onizuka S, Yanagita T, Wada A, Murakami M, Tsuneyoshi I.
  • 1From the Departments of *Anesthesiology and Intensive Care and †Pharmacology, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan; ‡Department of Anesthesiology, Division of Basic Research, Washington University School of Medicine, St. Louis, Missouri; §Department of Sports Health and Welfare, Faculty of Social Welfare, Kyusyu University of Health and Welfare, Miyazaki, Japan.



BACKGROUND: Tumor necrosis factor-α (TNF-α) is not only a key regulator of inflammatory response but also an important pain modulator. TNF-α enhances both tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant Na channel currents in dorsal root ganglion (DRG) neurons. However, it remains unknown whether TNF-α affects the function and expression of the TTX-S NaV1.7 Na channel, which plays crucial roles in pain generation.

METHODS: We used cultured bovine adrenal chromaffin cells expressing the NaV1.7 Na channel isoform and compared them with cultured rat DRG neurons. The expression of TNF receptor 1 and 2 (TNFR1 and TNFR2) in adrenal chromaffin cells was studied by Semiquantitative reverse transcription-polymerase chain reaction. The effects of TNF-α on the expression of NaV1.7 were examined with reverse transcription-polymerase chain reaction and Western blot analysis. Results were expressed as mean ± SEM.

RESULTS: TNFR1 and TNFR2 were expressed in adrenal chromaffin cells, as well as reported in DRG neurons. TNF-α up-regulated NaV1.7 mRNA by 132% ± 9% (N = 5, P = 0.004) in adrenal chromaffin cells, as well as 117% ± 2% (N = 5, P < 0.0001) in DRG neurons. Western blot analysis showed that TNF-α increased NaV1.7 protein up to 166% ± 24% (N = 5, corrected P < 0.0001) in adrenal chromaffin cells, concentration- and time-dependently.

CONCLUSIONS: TNF-α up-regulated NaV1.7 mRNA in both adrenal chromaffin cells and DRG neurons. In addition, TNF-α up-regulated the protein expression of the TTX-S NaV1.7 channel in adrenal chromaffin cells. Our findings may contribute to understanding the peripheral nociceptive mechanism of TNF-α.

PMID: 24445633



Voltage-gated sodium channels (VGSCs) initiate and propagate action potentials in neural circuits. VGSCs consist of the principal α-subunit, with or without auxiliary b-subunit. In mammals, 9 α-subunit isoforms (NaV1.1–NaV1.9) arise from 9 different genes (SCN1A–SCN5A and SCN8A–SCN11A). In dorsal root ganglion (DRG) neurons, which are afferent nerves in the peripheral nervous system and relay sensory information from peripheral sensory receptors into the central nervous system (spinal cord and brain), VGSCs also play important roles in pain generation. NaV1.3, NaV1.6, NaV1.7, NaV1.8, and NaV1.9 are expressed in DRG neurons. The dysregulation of expression or activity of these VGSCs on DRG neurons contribute to neuropathic, inflammatory, or diabetic neuropathy pain. For example, the changes in the physiological properties of NaV1.7 cause painful and painless channelopathies. Based on the animal studies, Nav1.7 was very involved in eliciting intolerable pain in inflammation (1, 2) and diabetic neuropathy (3); Although global Nav1.7 null mutant mice died shortly after birth, acute inflammatory pain responses evoked by chemical irritants (carrageenan, Freund’s adjuvant, or formalin) were reduced or abolished in knockout mice specifically lacking nociceptor Nav1.7 (1). In rat DRG neurons, herpes vector-mediated knockdown of Nav1.7 prevented the development of hyperalgesia in response to inflammation evoked by Freund’s adjuvant (2). Streptozotocin (STZ)-induced diabetic rats developed painful neuropathy such as allodynia and hyperalgesia to thermal stimulus between 4–8 weeks after onset of diabetes mellitus. In DRG neurons of these diabetic rats, the expression of Nav1.3 and Nav1.7 were significantly increased, compared to the decrease of the expression of Nav1.6 and Nav1.8 (3). However, all these findings were demonstrated in animal experimental models. So, it had been still unclear whether painful channelopathies actually existed in human. Surprisingly, in 2004 and 2006, gain-of-function/loss-of-function mutations of SCN9A encoding NaV1.7 were discovered for the first time in human (4). One gain-of-function mutation of NaV1.7 was responsible for “inherited erythermalgia”, an intermittent burning pain syndrome in the distal extremities. Another gain-of-function mutation of NaV1.7 accounted for “paroxysmal extreme pain disorder” characterized by paroxysmal visceral pain. “Congenital insensitivity to pain” was caused by loss-of-function mutation of NaV1.7; patients had never felt pain with correct perception of other sensations such as warm and cold. These discoveries are too interesting and important because they are the first human diseases, providing clear evidence that the mutations of the ion channels are conducive to painful and painless channelopathies. And furthermore, currently, it has been known that another gain-of-function mutation of NaV1.7 caused “small fiber neuropathy”, and the mutations of other VGSC isoforms such as NaV1.8, and NaV1.9 are also associated to human painful and painless channelopathies (5).

Tumor necrosis factor alpha (TNF-α) is one of proinflammatory cytokines, which promote systematic inflammation. Several studies reported that TNF-α enhanced Na+ channel currents in cultured DRG neurons. Our hypothesis was that TNF-α might be produced by the injection of chemical irritants and cause up-regulation of NaV1.7 on DRG neurons, leading to inflammatory pain. Our study demonstrated that in cultured bovine adrenal chromaffin cells and cultured rat DRG neurons, both of which predominantly expressed NaV1.7, the up-regulation of protein and mRNA of NaV1.7 was caused by TNF-α treatment. Our findings were further supported by the study by Huang Y et al., who demonstrated that NaV1.7, TNF-α, and phosphorylated-nucleus factor-kappa B (p-NF-kB) were up-regulated in DRG neurons of STZ-treated diabetic rats with mechanical allodynia and thermal hyperalgesia (6). In these diabetic rats, the inhibition of the synthesis of TNF-α by thalidomaide or NF-kB inhibitor dithiocarbamate strongly attenuated pain behaviors and up-regulation of NaV1.7, TNF-α, and p-NF-kB in DRG neurons, which means that in diabetes, TNF-α caused the up-regulation of NaV1.7 through NF-kB pathway on DRG neurons. In addition, these two studies strongly suggest that inflammatory and diabetic neuropathy pain are developed by parallel pathology (Figure 1). VGSCs including NaV1.7 are also expressed on pancreatic b cell and involved in glucose-stimulated insulin secretion (7). Small fiber neuropathy is acquired secondary to diabetes, whereas gain-of-function mutation of NaV1.7 was reported in 30% of small fiber neuropathy with unknown etiology (5). Some patients with painful neuropathy caused by gain-of-function mutation of NaV1.7 also have diabetes (7). These facts convince us to accept the hypothesis by Hoeijmakers JG et al., who suggested the possibility that mutation of NaV1.7 may increase susceptibility for development of diabetes by increasing the vulnerability of pancreatic b cells to injury and DRG neurons to damage (7).

Importance of our study: We first demonstrated that proinflammatory cytokine TNF-α up-regulated NaV1.7 in cultured DRG neurons.


TM fig1

Figure 1. Parallel pathology of inflammatory and diabetic neuropathy pain.

TNF-a induces inflammatory pain via the up-regulation of NaV1.7. Diabetic neuropathy pain is induced by the up-regulation of NaV1.7 via TNF-a/NF-kB pathway.



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