Improving NKCC1 Function Increases the Excitability of DRG Neurons Exacerbating Pain Induced After TRPV1 Activation of Primary Sensory Neurons

Abstract

Background Our aim was to investigate the effects of the protein expression and the function of sodium, potassium, and chloride co-transporter (NKCC1) in the dorsal root ganglion (DRG) after activation of transient receptor potential vanilloid 1 receptor (TRPV1) in capsaicin-induced acute inflammatory pain and the possible mechanism of action.Methods Male Sprague–Dawley rats were randomly divided into control, capsaicin, and inhibitor groups. The expression and distribution of TRPV1 and NKCC1 in rat DRG were observed by immunofluorescence. Thermal radiation and acetone test were used to detect the pain threshold of heat and cold noxious stimulation in each group. The expressions of NKCC1 mRNA, NKCC1 protein, and p-NKCC1 in the DRG were detected by PCR and western blotting (WB). Patch clamp and chloride fluorescent probe were used to observe the changes of GABA activation current and intracellular chloride concentration. After intrathecal injection of protein kinase C (PKC) inhibitor (GF109203X) or MEK/extracellular signal-regulated kinase (ERK) inhibitor (U0126), the behavioral changes and the expression of NKCC1 and p-ERK protein in L4–6 DRG were observed.Result: TRPV1 and NKCC1 were co-expressed in the DRG. Compared with the control group, the immunofluorescence intensity of NKCC1 and p-NKCC1 in the capsaicin group was significantly higher, and the expression of NKCC1 in the nuclear membrane was significantly higher than that in the control group. The expression of NKCC1 mRNA and protein of NKCC1 and p-NKCC1 in the capsaicin group were higher than those in the control group. After capsaicin injection, GF109203X inhibited the protein expression of NKCC1 and p-ERK, while U0126 inhibited the protein expression of NKCC1. In the capsaicin group, paw withdrawal thermal latency (WTL) was decreased, while cold withdrawal latency (CWL) was prolonged. Bumetanide, GF109203X, or U0126 could reverse the effect. GABA activation current significantly increased in the DRG cells of the capsaicin group, which could be reversed by bumetanide. The concentration of chloride in the DRG cells of the capsaicin group increased, but decreased after bumetanide, GF109203X, and U0126 were administered.Conclusion Activation of TRPV1 by exogenous agonists can increase the expression and function of NKCC1 protein in DRG, which is mediated by activation of PKC/p-ERK signaling pathway. These results suggest that DRG NKCC1 may participate in the inflammatory pain induced by TRPV1.

Inhibition of NKCC1 in spinal dorsal horn and dorsal root ganglion results in alleviation of neuropathic pain in rats with spinal cord contusion

“…A recent report shows that activation of TRPV1 by exogenous agonists can increase the expression and function of the NKCC1 protein in DRGs, which is mediated by the activation of the PKC/pERK signaling pathway. 43 This needs to be confirmed in future studies. Although more experiments are needed, our data have provided evidence to demonstrate that changes in the peripheral nervous system also play a major role in promoting pain sensitization after SCI.…”

“…[40][41][42] Intrathecal delivery of the NKCC1 antagonist BU decreased the concentration of chloride in the DRGs and inhibited the hyperalgesia caused by capsaicin. 43 An in vitro study found that inflammatory mediators increase the NKCC1 channel expression of DRGs within three hours. 44 Taken together, these findings indicate that the NKCC1 of DRGs might be an important player in pain.…”

Pre-Synaptic GABAA in NaV1.8+ Primary Afferents Is Required for the Development of Punctate but Not Dynamic Mechanical Allodynia following CFA Inflammation

“…Mechanistically, the basis of the reduction in ADS in C-fibres after inflammation has not been determined. However, this could be related to an elevation of intracellular chloride that occurs in DRG neurons [ 56 ] upon increased phosphorylation of NKCC1 following inflammation [ 27 ]. Our previous work has shown that ADS is increased by both the conditional deletion of GABA A -R in NaV1.8 + neurons and the NKCC1 blockade while, akin to CFA, GABA A -R agonists reduce ADS [ 21 ].…”

The role of KCC2 and NKCC1 in spinal cord injury: From physiology to pathology

“…To achieve this, key pathways are blocked/activated by pharmacological means, such as the P2X4-BDNF-KCC2 pathway ( Ferrini et al, 2013 ; Romeo-Guitart and Casas, 2020 ), BDNF-trkb ( Miletic and Miletic, 2008 ; Rivera et al, 2002 ; Rivera et al, 2004 ; Huang et al, 2017 ; Wenner, 2014 ; Sánchez-Brualla et al, 2018 ) or direct regulation of KCC2 activity, such as N and C termin ( Ferrini et al, 2013 ) N-terminal loop Chi et al, 2021, phosphorylation of serine 940 ( Leonzino et al, 2016 ) will likely be potential targets for clinical treatment of spinal cord injury and its complications. In addition, through exercise training, modulation of the 5-HTergic system ( Bos et al, 2013 ; Sánchez-Brualla et al, 2018 ; Ryu et al, 2021 ), the GABAergic system, and TRPV1 ( Deng et al, 2021 ) to indirectly affect NKCC1 and KCC2 expression levels are also feasible intervention strategies. The targeted regulation of different types of neurons may become a future treatment strategy for SCI and its complications ( Chen et al, 2018 ).…”

“…Transient receptor potential vanilloid 1 (TRPV1) is an important protein in the perception of pain ( Takayama et al, 2015 ) and its expression in the dorsal horn of the spinal cord is an essential link in the emergence of neuropathic pain after SCI ( Wu et al, 2013 ; Lee et al, 2021 ). The regulation of NKCC1 expression mediated by TRPV1 through the activation of the PKC/phosphorylated extracellular signal-regulated kinase (pERK) signaling pathway may be the mechanism underlying its involvement in SCI-related neuropathic pain ( Deng et al, 2021 ).…”