Channels In Dorsal Root Ganglion Research Articles

Involvement of HDAC2-mediated kcnq2/kcnq3 genes transcription repression activated by EREG/EGFR-ERK-Runx1 signaling in bone cancer pain

Bone cancer pain (BCP) represents a prevalent symptom among cancer patients with bone metastases, yet its underlying mechanisms remain elusive. This study investigated the transcriptional regulation mechanism of Kv7(KCNQ)/M potassium channels in DRG neurons and its involvement in the development of BCP in rats. We show that HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes, which encode Kv7(KCNQ)/M potassium channels in dorsal root ganglion (DRG), contributes to the sensitization of DRG neurons and the pathogenesis of BCP in rats. Also, HDAC2 requires the formation of a corepressor complex with MeCP2 and Sin3A to execute transcriptional regulation of kcnq2/kcnq3 genes. Moreover, EREG is identified as an upstream signal molecule for HDAC2-mediated kcnq2/kcnq3 genes transcription repression. Activation of EREG/EGFR-ERK-Runx1 signaling, followed by the induction of HDAC2-mediated transcriptional repression of kcnq2/kcnq3 genes in DRG neurons, leads to neuronal hyperexcitability and pain hypersensitivity in tumor-bearing rats. Consequently, the activation of EREG/EGFR-ERK-Runx1 signaling, along with the subsequent transcriptional repression of kcnq2/kcnq3 genes by HDAC2 in DRG neurons, underlies the sensitization of DRG neurons and the pathogenesis of BCP in rats. These findings uncover a potentially targetable mechanism contributing to bone metastasis-associated pain in cancer patients.

NGF contributes to activities of acid-sensing ion channels in dorsal root ganglion neurons of male rats with experimental peripheral artery disease.

A feature of peripheral artery diseases (PAD) includes limb ischemia/reperfusion (I/R) and ischemia. Both I/R and ischemia amplify muscle afferent nerve-activated reflex sympathetic nervous and blood pressure responses (termed as exercise pressor reflex). Nevertheless, the underlying mechanisms responsible for the exaggerated autonomic responses in PAD are undetermined. Previous studies suggest that acid-sensing ion channels (ASICs) in muscle dorsal root ganglion (DRG) play a leading role in regulating the exercise pressor reflex in PAD. Thus, we determined if signaling pathways of nerve growth factor (NGF) contribute to the activities of ASICs in muscle DRG neurons of PAD. In particular, we examined ASIC1a and ASIC3 currents in isolectin B4 -negative muscle DRG neurons, a distinct subpopulation depending on NGF for survival. Hindlimb I/R and ischemia were obtained in male rats. In results, femoral artery occlusion increased the levels of NGF and NGF-stimulated TrkA receptor in DRGs, whereas they led to upregulation of ASIC3 but not ASIC1a. In addition, application of NGF onto DRG neurons increased the density of ASIC3 currents and the effect of NGF was significantly attenuated by TrkA antagonist GW441756. Moreover, the enhancing effect of NGF on the density of ASIC3-like currents was decreased by the respective inhibition of intracellular signaling pathways, namely JNK and NF-κB, by antagonists SP600125 and PDTC. Our results suggest contribution of NGF to the activities of ASIC3 currents via JNK and NF-κB signaling pathways in association with the exercise pressor reflex in experimental PAD.

Piezo2 Channel Upregulation is Involved in Mechanical Allodynia in CYP-Induced Cystitis Rats

Mechanical sensing Piezo2 channel in primary sensory neurons has been shown contribute to mechanical allodynia in somatic chronic pain conditions. Interstitial cystitis (IC)-associated pain is often triggered by bladder filling, a presentation that mimics the mechanical allodynia. In the present study, we aimed to examine the involvement of sensory Piezo2 channel in IC-associated mechanical allodynia using a commonly employed cyclophosphamide (CYP)-induced IC model rat. Piezo2 channels in dorsal root ganglia (DRGs) was knocked down by intrathecal injections of Piezo2 anti-sense oligodeoxynucleotides (ODNs) in CYP-induced cystitis rats, and mechanical stimulation–evoked referred bladder pain was measured in the lower abdomen overlying the bladder using von Frey filaments. Piezo2 expression at the mRNA, protein, and functional levels in DRG neurons innervating the bladder was detected by RNA-fluorescence in situ hybridization, western blotting, immunofluorescence, and Ca2+ imaging, respectively. We found that Piezo2 channels were expressed on most (> 90%) of the bladder primary afferents, including afferents that express CGRP, TRPV1 and stained with isolectin B4. CYP-induced cystitis was associated with Piezo2 upregulation in bladder afferent neurons at the mRNA, protein, and functional levels. Knockdown of Piezo2 expression in DRG neurons significantly suppressed mechanical stimulation–evoked referred bladder pain as well as bladder hyperactivity in CYP rats compared to CYP rats treated with mismatched ODNs. Our results suggest upregulation of Piezo2 channels is involved in the development of bladder mechanical allodynia and bladder hyperactivity in CYP-induced cystitis. Targeting Piezo2 might be an attractive therapeutic approach for IC-related bladder pain.

Multi-target modulation of ion channels underlying the analgesic effects of α-mangostin in dorsal root ganglion neurons

Backgroundα-Mangostin is a xanthone isolated from the pericarps of mangosteen fruit with, and has analgesic properties. Although the effects suggest an interaction of α-mangostin with ion channels in the nociceptive neurons, electrophysiological investigation of the underlying mechanism has not been performed. HypothesisWe hypothesized that α-Mangostin exerts its analgesic effects by modulating the activity of various ion channels in dorsal root ganglion (DRG) neurons. MethodsWe performed a whole-cell patch clamp study using mouse DRG neurons, HEK293T cells overexpressing targeted ion channels, and ND7/23 cells. Molecular docking (MD) and in silico absorption, distribution, metabolism, and excretion (ADME) analyses were conducted to obtain further insights into the binding sites and pharmacokinetics, respectively. ResultsApplication of α-mangostin (1–3 µM) hyperpolarized the resting membrane potential (RMP) of small-sized DRG neurons by increasing background K+ conductance and thereby inhibited action potential generation. At micromolar levels, α-mangostin activates TREK-1, TREK-2, or TRAAK, members of the two-pore domain K+ channel (K2P) family known to be involved in RMP formation in DRG neurons. Furthermore, capsaicin-induced TRPV1 currents were potently inhibited by α-mangostin (0.43 ± 0.27 µM), and partly suppressed tetrodotoxin-sensitive voltage-gated Na+ channel (NaV) currents. MD simulation revealed that multiple oxygen atoms in α-mangostin may form stable hydrogen bonds with TREKs, TRAAK, TRPV1, and NaV channels. In silico ADME tests suggested that α-mangostin may satisfy the drug-likeness properties without penetrating the blood–brain barrier. ConclusionThe analgesic properties of α-mangostin might be mediated by the multi-target modulation of ion channels, including TREK/TRAAK activation, TRPV1 inhibition, and reduction of the tetrodotoxin-sensitive NaV current. The findings suggest that the phytochemical can be a multi-ion channel-targeting drug and an alternative drug for effective pain management.

Covalently Binding Adenosine A3 Receptor Agonist ICBM Irreversibly Reduces Voltage-Gated Ca2+ Currents in Dorsal Root Ganglion Neurons

Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed.

A novel high throughput combined voltage-clamp/current-clamp analysis of single primary neurons.

The patch-clamp technique is the gold-standard methodology for biophysical investigations of channels and receptors studied by voltage-clamp, and for analysis of excitability of cells, such as neurons studied via current-clamp. However, the throughput of manual patch-clamp is slow, and high-throughput robotic patch-clamp, while helpful for assessments, such as drug screening, has been primarily used to study channels and receptors expressed in heterologous expression systems. In this study, we introduce a novel approach to automated high throughput patch-clamping that substantially enhances high throughput analysis of excitable cells at the channel and cellular level. As a proof-of-concept, we apply this approach to investigate the detailed biophysical properties of voltage-gated sodium (Nav) channels in dorsal root ganglion (DRG) neurons, which are among the most diverse and complex neuronal cells. Our approach enables high throughput, unbiased, fast, simultaneous, and head-to-head electrophysiological recordings from a wide range of primary neurons. Furthermore, our approach eliminates the need for culturing of cells on coverslips and provides the ability to perform both voltage- and current-clamp recordings on the same neuron. This approach can be used for many applications, including both physiological and pharmacological analyses of primary DRG neurons.

High-throughput combined voltage-clamp/current-clamp analysis of freshly isolated neurons

The patch-clamp technique is the gold-standard methodology for analysis of excitable cells. However, throughput of manual patch-clamp is slow, and high-throughput robotic patch-clamp, while helpful for applications like drug screening, has been primarily used to study channels and receptors expressed in heterologous systems. We introduce an approach for automated high-throughput patch-clamping that enhances analysis of excitable cells at the channel and cellular levels. This involves dissociating and isolating neurons from intact tissues and patch-clamping using a robotic instrument, followed by using an open-source Python script for analysis and filtration. As a proof of concept, we apply this approach to investigate the biophysical properties of voltage-gated sodium (Nav) channels in dorsal root ganglion (DRG) neurons, which are among the most diverse and complex neuronal cells. Our approach enables voltage- and current-clamp recordings in the same cell, allowing unbiased, fast, simultaneous, and head-to-head electrophysiological recordings from a wide range of freshly isolated neurons without requiring culturing on coverslips.

Involvement of TRPM7 Channel on the Induction of Diabetic Neuropathic Pain in Mice: Protective Role of Selenium and Curcumin.

Excessive levels of the mitochondrial reactive oxygen radical (mitSOX) and Ca2+ influx were found to cause neuropathic pain in patients with diabetes mellitus (DM). Naltriben (NLT) and mitSOX activate the transient receptor (TRP) melastatin 7 (TRPM7) channel, but antioxidants and carvacrol inhibit it. Selenium (Se) and curcumin (CRC) have been thoroughly studied for their modulator effects on streptozotocin (STZ)-induced neuropathic pain, apoptosis, and oxidative stress through the blockage of TRP channels in dorsal root ganglion (DRG) neurons. It has not yet been fully understood how Se and CRC protect against STZ-induced neuropathic pain by modulating TRPM7. Here, we assessed how Se and CRC affected the Ca2+ influx, mitSOX-mediated oxidative damage, and apoptosis in the DRGs of mice through modifying TRPM7 activity. Seven groups (control, Se, CRC, STZ, STZ + Se, STZ + CRC, and STZ + Se + CRC) were induced from the 56 male mice. We observed that the STZ-induced stimulation of TRPM7 increased mechanical neuropathic pain (von Frey), thermal neuropathic pain (hot plate), cytosolic Ca2+, TRPM7 current density, TRPM7 expression, lipid peroxidation, mitSOX, cytosolic ROS, apoptosis, caspase-3, caspase-8, and caspase-9 concentrations, whereas Se and CRC therapies diminished the alterations. The STZ-mediated decreases of DRG viability, brain glutathione, glutathione peroxidase, vitamin A, and vitamin E concentrations were also upregulated in the treatment groups by the therapies. These findings collectively imply that an imbalance of neuropathic pain, oxidative neurotoxicity, and apoptosis in the mice is caused by the STZ-mediated activation of TRPM7. However, the downregulation of TRPM7 activity caused by the injections of Se and CRC reduced the neurotoxicity and apoptosis.

Properties and Differential Expression of H+ Receptors in Dorsal Root Ganglia: Is a Labeled-Line Coding for Acid Nociception Possible?

Pain by chemical irritants is one of the less well-described aspects of nociception. The acidic substance is the paradigm of the chemical noxious compound. An acidic insult on cutaneous, subcutaneous and muscle tissue results in pain sensation. Acid (or H+) has at least two main receptor channels in dorsal root ganglia (DRG) nociceptors: the heat receptor transient receptor potential vanilloid 1 (TRPV1) and the acid-sensing ionic channels (ASICs). TRPV1 is a low-sensitivity H+ receptor, whereas ASIC channels display a higher H+ sensitivity of at least one order of magnitude. In this review, we first describe the functional and structural characteristics of these and other H+-receptor candidates and the biophysics of their responses to low pH. Additionally, we compile reports of the expression of these H+-receptors (and other possible complementary proteins) within the DRG and compare these data with mRNA expression profiles from single-cell sequencing datasets for ASIC3, ASIC1, transient receptor potential Ankiryn subtype 1 (TRPA1) and TRPV1. We show that few nociceptor subpopulations (discriminated by unbiased classifications) combine acid-sensitive channels. This comparative review is presented in light of the accumulating evidence for labeled-line coding for most noxious sensory stimuli.

Oxaliplatin Depolarizes the IB4- Dorsal Root Ganglion Neurons to Drive the Development of Neuropathic Pain Through TRPM8 in Mice.

Use of chemotherapy drug oxaliplatin is associated with painful peripheral neuropathy that is exacerbated by cold. Remodeling of ion channels including TRP channels in dorsal root ganglion (DRG) neurons contribute to the sensory hypersensitivity following oxaliplatin treatment in animal models. However, it has not been studied if TRP channels and membrane depolarization of DRG neurons serve as the initial ionic/membrane drives (such as within an hour) that contribute to the development of oxaliplatin-induced neuropathic pain. In the current study, we studied in mice (1) in vitro acute effects of oxaliplatin on the membrane excitability of IB4+ and IB4– subpopulations of DRG neurons using a perforated patch clamping, (2) the preventative effects of a membrane-hyperpolarizing drug retigabine on oxaliplatin-induced sensory hypersensitivity, and (3) the preventative effects of TRP channel antagonists on the oxaliplatin-induced membrane hyperexcitability and sensory hypersensitivity. We found (1) IB4+ and IB4– subpopulations of small DRG neurons displayed previously undiscovered, substantially different membrane excitability, (2) oxaliplatin selectively depolarized IB4– DRG neurons, (3) pretreatment of retigabine largely prevented oxaliplatin-induced sensory hypersensitivity, (4) antagonists of TRPA1 and TRPM8 channels prevented oxaliplatin-induced membrane depolarization, and (5) the antagonist of TRPM8 largely prevented oxaliplatin-induced sensory hypersensitivity. These results suggest that oxaliplatin depolarizes IB4– neurons through TRPM8 channels to drive the development of neuropathic pain and targeting the initial drives of TRPM8 and/or membrane depolarization may prevent oxaliplatin-induce neuropathic pain.

Peripheral kappa opioid receptor activation drives cold hypersensitivity in mice

Noxious cold sensation is commonly associated with peripheral neuropathies, however, there has been limited progress in understanding the mechanism of cold pain. Here we identify a role for kappa opioid receptors (KOR) in driving cold hypersensitivity. First, we show that systemic activation of KOR by U50,488 (U50), increases the latency to jump and the number of jumps on a cold plate at 3°C. KOR antagonist, NorBNI, attenuates U50-induced cold hypersensitivity. However, the central administration of NorBNI does not block U50-induced cold hypersensitivity suggesting that peripheral KORs may modulate this effect. To directly test this, we use the peripherally-restricted KOR agonist, ff(nle)r-NH2 and also show cold hypersensitivity. To begin to understand how peripheral KORs drive cold hypersensitivity we investigated whether KORs interact with transient receptor potential ankyrin 1(TRPA1) channels, known to facilitate the perception of noxious cold, in dorsal root ganglion (DRG). Using fluorescent in situ hybridization, we show that KOR mRNA colocalizes with the transcripts for the cold-activated TRPA1 channels in DRG. We also show a potentiation in intracellular calcium release in DRG during the simultaneous application of the TRPA1 agonist, mustard oil (MO), and a KOR agonist, U50, when compared to MO alone. Together our data suggest that peripheral KORs may induce cold hypersensitivity through modulation of TRPA1 channels.

SNI and CFA induce similar changes in TRPV1 and P2X3 expressions in the acute phase but not in the chronic phase of pain.

Peripheral inflammation and nerve injury usually accompany each other. However, whether inflammatory and neuropathic pain share similar mechanisms at all stages is unknown. TRPV1 and P2X3 are two major ion channels in dorsal root ganglia (DRGs) and are involved in chronic pain. Here, their function and expression in DRGs at different phases of the two types of pain were investigated. Both the paw withdrawal threshold (PWT) and paw withdrawal latency were decreased in rats injected with complete Freud's adjuvant (CFA). However, only the PWT was decreased in rats with spared nerve injury (SNI). CFA increased the magnitude of the TRPV1-mediated Ca2+ response but not the P2X3-mediated Ca2+ response 14 days after injection. Consistent with this result, the P2X3 expression level in CFA rats was increased only at 3 days after injection. SNI surgery increased the magnitudes of the TRPV1- and P2X3-mediated Ca2+ responses and upregulated both TRPV1 and P2X3 expression in lumbar DRGs. The distributions of TRPV1 and P2X3 in DRGs after modeling were observed, and TRPV1 was found to be highly expressed mainly in the L4-L5 DRGs in CFA rats and in the L5-L6 DRGs in SNI rats. P2X3 was highly expressed in the L4-L6 DRGs in CFA rats 3 days after injection but was only highly expressed in the L4 DRG 14 days after modeling. On the other hand, SNI promoted the P2X3 expression L4-L5 DRGs 3 days after surgery, but only L6 DRG 14 days after modeling. All the results indicate that P2X3 and TPRV1 are involved in inflammatory and neuropathic pain by different expression levels and distributions in the lumbar DRG in the chronic stage.

1-O-Acetylgeopyxin A, a derivative of a fungal metabolite, blocks tetrodotoxin-sensitive voltage-gated sodium, calcium channels and neuronal excitability which correlates with inhibition of neuropathic pain

Chronic pain can be the result of an underlying disease or condition, medical treatment, inflammation, or injury. The number of persons experiencing this type of pain is substantial, affecting upwards of 50 million adults in the United States. Pharmacotherapy of most of the severe chronic pain patients includes drugs such as gabapentinoids, re-uptake blockers and opioids. Unfortunately, gabapentinoids are not effective in up to two-thirds of this population and although opioids can be initially effective, their long-term use is associated with multiple side effects. Therefore, there is a great need to develop novel non-opioid alternative therapies to relieve chronic pain. For this purpose, we screened a small library of natural products and their derivatives in the search for pharmacological inhibitors of voltage-gated calcium and sodium channels, which are outstanding molecular targets due to their important roles in nociceptive pathways. We discovered that the acetylated derivative of the ent-kaurane diterpenoid, geopyxin A, 1-O-acetylgeopyxin A, blocks voltage-gated calcium and tetrodotoxin-sensitive voltage-gated sodium channels but not tetrodotoxin-resistant sodium channels in dorsal root ganglion (DRG) neurons. Consistent with inhibition of voltage-gated sodium and calcium channels, 1-O-acetylgeopyxin A reduced reduce action potential firing frequency and increased firing threshold (rheobase) in DRG neurons. Finally, we identified the potential of 1-O-acetylgeopyxin A to reverse mechanical allodynia in a preclinical rat model of HIV-induced sensory neuropathy. Dual targeting of both sodium and calcium channels may permit block of nociceptor excitability and of release of pro-nociceptive transmitters. Future studies will harness the core structure of geopyxins for the generation of antinociceptive drugs.

Sleep deprivation of rats increases postsurgical expression and activity of L-type calcium channel in the dorsal root ganglion and slows recovery from postsurgical pain

Perioperative sleep disturbance is a risk factor for persistent pain after surgery. Clinical studies have shown that patients with insufficient sleep before and after surgery experience more intense and long-lasting postoperative pain. We hypothesize that sleep deprivation alters L-type calcium channels in the dorsal root ganglia (DRG), thus delaying the recovery from post-surgical pain. To verify this hypothesis, and to identify new predictors and therapeutic targets for persistent postoperative pain, we first established a model of postsurgical pain with perioperative sleep deprivation (SD) by administering hind paw plantar incision to sleep deprivation rats. Then we conducted behavioral tests, including tests with von Frey filaments and a laser heat test, to verify sensory pain, measured the expression of L-type calcium channels using western blotting and immunofluorescence of dorsal root ganglia (an important neural target for peripheral nociception), and examined the activity of L-type calcium channels and neuron excitability using electrophysiological measurements. We validated the findings by performing intraperitoneal injections of calcium channel blockers and microinjections of dorsal root ganglion cells with adeno-associated virus. We found that short-term sleep deprivation before and after surgery increased expression and activity of L-type calcium channels in the lumbar dorsal root ganglia, and delayed recovery from postsurgical pain. Blocking these channels reduced impact of sleep deprivation. We conclude that the increased expression and activity of L-type calcium channels is associated with the sleep deprivation-mediated prolongation of postoperative pain. L-type calcium channels are thus a potential target for management of postoperative pain.

Reversal of Peripheral Neuropathic Pain by the Small-Molecule Natural Product Physalin F via Block of CaV2.3 (R-Type) and CaV2.2 (N-Type) Voltage-Gated Calcium Channels

No universally efficacious therapy exists for chronic pain, a disease affecting one-fifth of the global population. An overreliance on the prescription of opioids for chronic pain despite their poor ability to improve function has led to a national opioid crisis. In 2018, the NIH launched a Helping to End Addiction Long-term plan to spur discovery and validation of novel targets and mechanisms to develop alternative nonaddictive treatment options. Phytochemicals with medicinal properties have long been used for various treatments worldwide. The natural product physalin F, isolated from the Physalis acutifolia (family: Solanaceae) herb, demonstrated antinociceptive effects in models of inflammatory pain, consistent with earlier reports of its anti-inflammatory and immunomodulatory activities. However, the target of action of physalin F remained unknown. Here, using whole-cell and slice electrophysiology, competition binding assays, and experimental models of neuropathic pain, we uncovered a molecular target for physalin F's antinociceptive actions. We found that physalin F (i) blocks CaV2.3 (R-type) and CaV2.2 (N-type) voltage-gated calcium channels in dorsal root ganglion (DRG) neurons, (ii) does not affect CaV3 (T-type) voltage-gated calcium channels or voltage-gated sodium or potassium channels, (iii) does not bind G-protein coupled opioid receptors, (iv) inhibits the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in spinal cord slices, and (v) reverses tactile hypersensitivity in models of paclitaxel-induced peripheral neuropathy and spinal nerve ligation. Identifying CaV2.2 as a molecular target of physalin F may spur its use as a tool for mechanistic studies and position it as a structural template for future synthetic compounds.

Beneficial effect of mirtazapine on diabetes-induced hyperalgesia: involvement of TRPV1 and ASIC1 channels in the spinal cord and dorsal root ganglion

ABSTRACTObjectives: Neuropathic pain reduces the life qualities of patients with Diabetes mellitus. Clinical guidelines recommend relief in diabetic neuropathic pain through the use of some antidepressants, anticonvulsants, opioids as well as capsaicin cream or lidocaine patches. However, since the majority of patients do not or partially respond to current treatments, there is a growing necessity for new drugs increasing the pain relief in patients with diabetes. Therefore, based on the therapeutic potential of antidepressants on neuropathic pain, we investigated the promising antihyperalgesic effect of mirtazapine (MRT) in painful diabetic neuropathy.Methods: Experimental diabetes was induced in rats by single intraperitoneal injection of 55 mg/kg dose of streptozocin (STZ). After 4 weeks of injection of STZ, MRT was administrated for 14 days at 40 mg/kg dose. Randall–Selitto and Hargreaves tests were applied for paw-withdrawal threshold and paw-withdrawal latency measurement. TRPV1 and ASIC1 expressions measured by Western blot in dorsal root ganglion and spinal cord.Results: Administration of MRT significantly improved both of the decreased paw-withdrawal threshold and shortened the paw-withdrawal latency of diabetic rats, respectively. Besides, increased levels of TRPV1 and ASIC1 channels in dorsal root ganglion and spinal cord of diabetic rats, evaluated by Western blot method, were decreased following the MRT treatment.Discussion: These data show, for the first time, that MRT has beneficial effects against diabetes-induced hyperalgesia, and that suppressive effect of this drug on TRPV1 and ASIC1 levels, which are increased in diabetic rats, may be some of the pharmacological mechanisms underlying the exhibited antihyperalgesic effect of MRT.

Possible involvement of peripheral TRP channels in the hydrogen sulfide-induced hyperalgesia in diabetic rats

BackgroundPeripheral diabetic neuropathy can be painful and its symptoms include hyperalgesia, allodynia and spontaneous pain. Hydrogen sulfide (H2S) is involved in diabetes-induced hyperalgesia and allodynia. However, the molecular target through which H2S induces hyperalgesia in diabetic animals is unclear. The aim of this study was to determine the possible involvement of transient receptor potential (TRP) channels in H2S-induced hyperalgesia in diabetic rats.ResultsStreptozotocin (STZ) injection produced hyperglycemia in rats. Intraplantar injection of NaHS (an exogenous donor of H2S, 3–100 µg/paw) induced hyperalgesia, in a time-dependent manner, in formalin-treated diabetic rats. NaHS-induced hyperalgesia was partially prevented by local intraplantar injection of capsazepine (0.3–3 µg/paw), HC-030031 (100–316 µg/paw) and SKF-96365 (10–30 µg/paw) blockers, at 21 days post-STZ injection. At the doses used, these blockers did not modify formalin-induced nociception. Moreover, capsazepine (0.3–30 µg/paw), HC-030031 (100–1000 µg/paw) and SKF-96365 (10–100 µg/paw) reduced formalin-induced nociception in diabetic rats. Contralateral injection of the highest doses used did not modify formalin-induced flinching behavior. Hyperglycemia, at 21 days, also increased protein expression of cystathionine-β-synthase enzyme (CBS) and TRPC6, but not TRPA1 nor TRPV1, channels in dorsal root ganglia (DRG). Repeated injection of NaHS enhanced CBS and TRPC6 expression, but hydroxylamine (HA) prevented the STZ-induced increase of CBS protein. In addition, daily administration of SKF-96365 diminished TRPC6 protein expression, whereas NaHS partially prevented the decrease of SKF-96365-induced TRPC6 expression. Concordantly, daily intraplantar injection of NaHS enhanced, and HA prevented STZ-induced intraepidermal fiber loss, respectively. CBS was expressed in small- and medium-sized cells of DRG and co-localized with TRPV1, TRPA1 and TRPC6 in IB4-positive neurons.ConclusionsOur data suggest that H2S leads to hyperalgesia in diabetic rats through activation of TRPV1, TRPA1 and TRPC channels and, subsequent intraepidermal fibers loss. CBS enzyme inhibitors or TRP-channel blockers could be useful for treatment of painful diabetic neuropathy.

The paracetamol metabolite N-acetylp-benzoquinone imine reduces excitability in first- and second-order neurons of the pain pathway through actions on KV7 channels.

Paracetamol (acetaminophen, APAP) is one of the most frequently used analgesic agents worldwide. It is generally preferred over nonsteroidal anti-inflammatory drugs because it does not cause typical adverse effects resulting from the inhibition of cyclooxygenases, such as gastric ulcers. Nevertheless, inhibitory impact on these enzymes is claimed to contribute to paracetamols mechanisms of action which, therefore, remained controversial. Recently, the APAP metabolites N-arachidonoylaminophenol (AM404) and N-acetyl-p-benzoquinone imine (NAPQI) have been detected in the central nervous system after systemic APAP administration and were reported to mediate paracetamol effects. In contrast to nonsteroidal anti-inflammatory drugs that rather support seizure activity, paracetamol provides anticonvulsant actions, and this dampening of neuronal activity may also form the basis for analgesic effects. Here, we reveal that the APAP metabolite NAPQI, but neither the parent compound nor the metabolite AM404, reduces membrane excitability in rat dorsal root ganglion (DRG) and spinal dorsal horn (SDH) neurons. The observed reduction of spike frequencies is accompanied by hyperpolarization in both sets of neurons. In parallel, NAPQI, but neither APAP nor AM404, increases currents through KV7 channels in DRG and SDH neurons, and the impact on neuronal excitability is absent if KV7 channels are blocked. Furthermore, NAPQI can revert the inhibitory action of the inflammatory mediator bradykinin on KV7 channels but does not affect synaptic transmission between DRG and SDH neurons. These results show that the paracetamol metabolite NAPQI dampens excitability of first- and second-order neurons of the pain pathway through an action on KV7 channels.

Regulation of Nociceptive Glutamatergic Signaling by Presynaptic Kv3.4 Channels in the Rat Spinal Dorsal Horn.

Presynaptic voltage-gated K+ (Kv) channels in dorsal root ganglion (DRG) neurons are thought to regulate nociceptive synaptic transmission in the spinal dorsal horn. However, the Kv channel subtypes responsible for this critical role have not been identified. The Kv3.4 channel is particularly important because it is robustly expressed in DRG nociceptors, where it regulates action potential (AP) duration. Furthermore, Kv3.4 dysfunction is implicated in the pathophysiology of neuropathic pain in multiple pain models. We hypothesized that, through their ability to modulate AP repolarization, Kv3.4 channels in DRG nociceptors help to regulate nociceptive synaptic transmission. To test this hypothesis, we investigated Kv3.4 immunoreactivity (IR) in the rat cervical superficial dorsal horn (sDH) in both sexes and implemented an intact spinal cord preparation to investigate glutamatergic synaptic currents from second order neurons in the sDH under conditions that selectively inhibit the Kv3.4 current. We found presynaptic Kv3.4 IR in peptidergic and nonpeptidergic nociceptive fibers of the sDH. The Kv3.4 channel is hypersensitive to 4-aminopyridine and tetraethylammonium (TEA). Accordingly, 50 μm 4-aminopyridine and 500 μm TEA significantly prolong the AP, slow the maximum rate of repolarization in small-diameter DRG neurons, and potentiate monosynaptic excitatory postsynaptic currents (EPSCs) in dorsal horn laminae I and II through a presynaptic mechanism. In contrast, highly specific inhibitors of BK, Kv7, and Kv1 channels are less effective modulators of the AP and have little to no effect on EPSCs. The results strongly suggest that presynaptic Kv3.4 channels are major regulators of nociceptive synaptic transmission in the spinal cord.SIGNIFICANCE STATEMENT Intractable neuropathic pain can result from disease or traumatic injury and many studies have been conducted to determine the underlying pathophysiological changes. Voltage-gated ion channels, including the K+ channel Kv3.4, are dysregulated in multiple pain models. Kv3.4 channels are ubiquitously expressed in the dorsal root ganglion (DRG), where they are major regulators of DRG excitability. However, little is known about the ionic mechanisms that regulate nociceptive synaptic transmission at the level of the first synapse in the spinal cord, which is critical to pain transmission in both intact and pathological states. Here, we show that Kv3.4 channels have a significant impact on glutamatergic synaptic transmission in the dorsal horn, further illuminating its potential as a molecular pain therapeutic target.

Pharmacologic Characterization of AMG8379, a Potent and Selective Small Molecule Sulfonamide Antagonist of the Voltage-Gated Sodium Channel NaV1.7.

Potent and selective antagonists of the voltage-gated sodium channel NaV1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human NaV1.7 channels with an IC50 of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC50 of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other NaV family members, including NaV1.4 expressed in muscle and NaV1.5 expressed in the heart, as well as TTX-resistant NaV channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple NaV1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective NaV1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.