Acid-Sensing Ion Channel Contributes To Central Sensitization And Inflammatory Pain Hypersensitivity

Inflammation and trauma lead to enhanced pain sensitivity (hyperalgesia), which is in part due to altered plasticity in the spinal cord. The molecular mechanism underlying this nociceptive plasticity is not fully understood. An important component in inflammation-induced hypersensitivity to thermal and mechanical stimuli (inflammatory pain hypersensitivity) is the amplification of pain-related information resulting from modulation and expression of specific ion channels in the spinal dorsal horn (SDH) and hyperexcitability of spinal dorsal horn neuron.The acid-sensing ion channels (ASICs) belong to the epithelial Na⁺ channel/ degenerin superfamily. Four genes (ASIC1, ASIC2, ASIC3, ASIC4) encoding seven different ASIC subunits have been cloned to date: ASIC1a, 1b, 1b2, 2a, 3b, 3, 4. The major structural features of various ASICs include two transmembrane domains (TM1 and TM2), a large extracellular loop, and the C and N termini facing the intracellular space. The recent crystallography study showed trimer assembly of ASIC1a. Besides ASIC3 and ASIC1b are specifically expressed in the peripheral nervous system (PNS), other subunits of ASICs exist both in the peripheral and central nervous system (CNS). The ASICs in peripheral nerve system have been suggested the transmission of sensation, such as pain, tasting and mechanical reception. However, it is little known about the functions of ASICs in CNS, especially under pathological conditions. In the previous study, we found that ASICs are expressed in spinal cord and acid-activated currents have been detected in the cultured spinal neurons. However, the molecular identity and the functional roles of ASICs in dorsal horn neurons remain poorly understood.In the present study, we first identified that Ca²⁺-permeable, homomeric ASIC1a channels as the predominant ASICs in rat SDH neurons. Furthermore, we elucidate the contribution of ASIC1a in the SDH to inflammatory pain hypersensitivity. The main results were summarized as followings: 1. Ca²⁺-permeable ASIC1a Channels Are the Major ASIC in SDH Neurons Using electrophysiological recording. we found that PcTX1, a specific antagonist of homomeric ASIC1a channel, mostly inhibited acid-induced currents (at pH6.0) in acutely dissociated spinal dorsal horn neurons. Further experiments were performed to examine the effect of suppressing ASIC1a expression in cultured SDH neurons by RNA interference. We reduced ASIC1a expression in cultured SDH neurons via transfection of specific ASIC1a siRNA. Whole-cell recordings in neurons 4 days after transfection showed that ASIC1a silencing resulted in a marked reduction of the acid-induced currents (at pH 6.0). Taken together, these results indicate that ASIC1a subunit is the major functional subunit underlying ASIC currents in SDH neurons. Moreover, we demonstrated that homomeric ASIC1a channel render acid-induced Ca²⁺ elevation in the SDH through Ca²⁺ imaging experiment.2. Spinal ASIC1a Channel Contributes to Inflammatory Pain Hypersensitivity To address the role of Ca²⁺-permeable ASIC1a channels of SDH neurons in nociception, we tested the effect of local blockade of homomeric ASIC1a channels with PcTX1 on thermal and mechanical hypersensitivity induced by subcutaneous injection of CFA into one of the rat hindpaws. The paw withdrawal latency (PWL) and the paw withdrawal threshold (PWT) in response to heat or mechanical stimulation were significantly decreased 1 day after CFA injection and the effect persisted for at least 3 days in CFA-treated rats, as compared with that in control rats injected with saline. Intrathecal injection of PcTX1 significantly attenuated both thermal and mechanical hypersensitivity induced by the CFA treatment. Moreover, Intrathecal injection of amiloride, the non-specific ASIC antagonist, also reversed CFA-induced thermal hypersensitivity with a similar time course. To investigate whether ASIC1a subunit in the SDH plays an essential role in inflammatory pain hypersensitivity, we further performed antisense knock-down experiments. A specific antisense oligonucleotide suppressed the protein expression of ASIC1a. In parallel experiments, we found that intrathecal administration of ASIC1a AS significantly reduced the thermal and mechanical hypersensitivity in the CFA-inflamed rats, as indicated by higher values of PWL and PWT. In contrast to that observed in inflamed rats, blockade of ASIC1a channel or suppressing ASIC1a expression did not affect either PWL or PWT in naive rats without CFA injection.Further support of spinal ASIC1a function in nociception came from the formalin test. Subcutaneous injection of formalin normally produces two distinct phases of spontaneous nociceptive behavioral responses: an early/acute phase (0-5 min post-injection, phase I) attributable to the direct irritant effect of the chemical, and a late/tonic phase (15-60 min, phase II) that reflects a combination of ongoing sensory input and central sensitization. Intrathecal injection of PcTX1 only reduced the second phase of formalin-induced pain behaviour. Furthermore, we injected PcTX1 subcutaneously into the hindpaw treated with CFA. Neither the normal baseline PWL nor the CFA-induced reduction in PWL was affected. Thus ASIC1a channels appear to play a major role in central but not peripheral nociception.3. Upregulation of ASIC1a Channel Contributes to Central Sensitization—Effects on Excitability and PlasticityHow can ASIC1a channel in the SDH contribute to the nociceptive transmission? Immunohistochemistry experiments indicated an increased neuronal ASIC1a staining in superficial layers of SDH ipsilateral to the inflammatory side, as compared with that of contralateral SDH or the SDH of sham-operated rats without CFA injection. However, ASIC1a is less expressed in the central terminal of DRG neurons or the glia in the SDH. Thus, we concluded that peripheral inflammation selectively increased the expression of ASIC1a in SDH neurons.To address the relevance of ASIC1a in the development of inflammatory pain hypersensitivity, we next investigated the mechanism by which ASIC1a channels participate in nociceptive processing in SDH neurons. In vivo single unit recordings were made from "wide dynamic range" (WDR) SDH neurons. We found that the "wind-up" of single unit activity evoked by prolonged repetitive C-fiber stimulation was mostly attenuated by infusion with PcTX1. In addition, PcTX1 infusion of naive rats had no effect on WDR neuronal activity in response to non-noxious (brushing) and noxious (pressing and pinching) mechanical stimuli applied to peripheral receptive fields. In contrast, in rats with CFA-induced inflammation, PcTX1 significantly attenuated the hyperactivity of WDR neurons in response to noxious mechanical stimuli, whereas the brush-evoked hyperactivity of WDR neurons was not significantly affected.4. Mechanism of Upregulation of ASIC1a Channel in the SDH Following Peripheral InflammationAfter inflammation, the central terminals of DRG neurons release neurotransmitter (Glu) and neuromodulators substance P (SP) and BDNF, which alter the synaptic transmission in the SDH. We found that chronic treatment of cultured SDH neurons with BDNF caused upregluation of ASIC1a in a concentration-dependent manner. Furthermore, chronic intrathecal injection of K252a, a TrkB receptor inhibitor, suppressed ASIC1a expression and pain behavior following peripheral inflammation. Moreover, intrathecal injection of PcTX1 did not further affect pain behavior of K252a-treated rats. The results indicate that BDNF may mediate the upregulation of ASIC1a. In addition, pre-treatment of BDNF enhanced ASIC1a-mediated currents and PI3K, but not PKA or CaMKII pathway mediated the enhancement.Taken together, our study demonstrated that ASIC1a channels in SDH neurons are essential for inflammation-induced pain hypersensitivity and spinal neuron sensitization. The result indicates a novel mechanism for the development and maintenance of inflammatory pain and offers an alternative therapeutic target for the drug research.