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Background: There is growing evidence supporting a role for TRPA1 receptors in the neurotransmission of peripheral mechanical stimulation. In order to enhance understanding of TRPA1 contributions to mechanotransmission, we examined the effects a selective TRPA1 receptor antagonist, A-967079, on spinal neuronal activity following peripheral mechanical stimulation in uninjured, CFA-inflamed, and osteoarthritc (OA) rats. Results: Systemic injection of A-967079 (30 μmol/kg, i.v.) decreased the responses of wide dynamic range (WDR), and nociceptive specific (NS) neurons following noxious pinch stimulation of the ipsilateral hind paw in uninjured and CFA-inflamed rats. Similarly, A-967079 reduced the responses of WDR neurons to high-intensity mechanical stimulation (300 g von Frey hair) of the knee joint in both OA and OA-sham rats. WDR neuronal responses to low-intensity mechanical stimulation (10 g von Frey hair) were also reduced by A-967079 administration to CFA-inflamed rats, but no effect was observed in uninjured rats. Additionally, the spontaneous activity of WDR neurons was decreased after A-967079 injection in CFA-inflamed rats but was unaltered in uninjured, OA, and OA-sham animals. Conclusions: Blockade of TRPA1 receptors disrupts transmission of high-intensity mechanical stimulation to the spinal cord in both uninjured and injured rats indicating that TRPA1 receptors have an important role in noxious mechanosensation in both normal and pathological conditions. TRPA1 receptors also contribute to the transmission of low-intensity mechanical stimulation, and to the modulation of spontaneous WDR firing, but only after an inflammatory injury.

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Nav1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC₅₀ = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Nav1.8 (IC₅₀ = 8 nM) and was >100-fold selective vs. human Nav1.2, Nav1.3, Nav1.5, and Nav1.7 (IC₅₀ values >=1 μM). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED₅₀ = 47 mg/kg, i.p.), sciatic nerve injury (ED₅₀ = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED₅₀ [almost equal to] 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freund's adjuvant injection (ED₅₀ = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Nav1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

P2X 3 and P2X 2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X 3 and P2X 2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X 3 and P2X 2/3 receptor-mediated calcium flux ( K i = 22–92 nM) and was highly selective (IC 50 >10 μM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X 3 and P2X 2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X 3 containing channels was stereospecific because the R -enantiomer (A-317344) of A-317491 was significantly less active at P2X 3 and P2X 2/3 receptors. A-317491 dose-dependently (ED 50 = 30 μmol/kg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED 50 = 10–15 μmol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R -enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED 50 >100 μmol/kg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X 3 and P2X 2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X 3 and P2X 2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

We have recently reported that systemic delivery of A‐317491, the first non‐nucleotide antagonist that has high affinity and selectivity for blocking P2X3 homomeric and P2X2/3 heteromeric channels, is antinociceptive in rat models of chronic inflammatory and neuropathic pain. In an effort to further evaluate the role of P2X3/P2X2/3 receptors in nociceptive transmission, A‐317491 was administered either intrathecally or into the hindpaw of a rat in several models of acute and chronic nociception. Intraplantar (ED50=300 nmol) and intrathecal (ED50=30 nmol) injections of A‐317491 produced dose‐related antinociception in the CFA model of chronic thermal hyperalgesia. Administration of A‐317491 by either route was much less effective to reduce thermal hyperalgesia in the carrageenan model of acute inflammatory hyperalgesia. Intrathecal, but not intraplantar, delivery of A‐317491 attenuated mechanical allodynia in both the chronic constriction injury and L5‐L6 nerve ligation models of neuropathy (ED50=10 nmol for both models). Intrathecal injections of A‐317491 did not impede locomotor performance. Both routes of injection were effective in reducing the number of nocifensive events triggered by the injection of formalin into a hindpaw. Nocifensive behaviors were significantly reduced in both the first and second phases of the formalin assay (intrathecal ED50=10 nmol, intraplantar ED50>300 nmol). Nocifensive behaviors induced by the P2X receptor agonist α,β‐meATP were also significantly reduced by intraplantar injection of A‐317491. These data indicate that both spinal and peripheral P2X3/P2X2/3 receptors have significant contributions to nociception in several animal models of nerve or tissue injury. Intrathecal administration of A‐317491 appears to be more effective than intraplantar administration to reduce tactile allodynia following peripheral nerve injury. British Journal of Pharmacology (2003) 140, 1381–1388. doi:10.1038/sj.bjp.0705574

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Na v 1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC 50 = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Na v 1.8 (IC 50 = 8 nM) and was >100-fold selective vs. human Na v 1.2, Na v 1.3, Na v 1.5, and Na v 1.7 (IC 50 values ≥1 μM). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED 50 = 47 mg/kg, i.p.), sciatic nerve injury (ED 50 = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED 50 ≈ 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freund's adjuvant injection (ED 50 = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Na v 1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against${\\rm Na}_{{\\rm v}}1.8$have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC₅₀ = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human${\\rm Na}_{{\\rm v}}1.8$(IC₅₀ = 8 nM) and was > 100-fold selective vs. human${\\rm Na}_{{\\rm v}}1.2$,${\\rm Na}_{{\\rm v}}1.3$,${\\rm Na}_{{\\rm v}}1.5$, and${\\rm Na}_{{\\rm v}}1.7$(IC₅₀ values ≥1 μM). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED₅₀ = 47 mg/kg, i.p.), sciatic nerve injury (ED₅₀ = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED₅₀ ≈ 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freund's adjuvant injection (ED₅₀ = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of${\\rm Na}_{{\\rm v}}1.8$sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Na sub(v)1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC sub(50) = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Na sub(v)1.8 (IC sub(50) = 8 nM) and was >100-fold selective vs. human Na sub(v)1.2, Na sub(v)1.3, Na sub(v)1.5, and Na sub(v)1.7 (IC sub(50) values greater than or equal to 1 mu M). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED sub(50) = 47 mg/kg, i.p.), sciatic nerve injury (ED sub(50) = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED sub(50) approximately 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freund's adjuvant injection (ED sub(50) = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Na sub(v)1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

P2X 3 and P2X 2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X 3 and P2X 2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X 3 and P2X 2/3 receptor-mediated calcium flux ( K i = 22–92 nM) and was highly selective (IC 50 >10 μM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X 3 and P2X 2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X 3 containing channels was stereospecific because the R -enantiomer (A-317344) of A-317491 was significantly less active at P2X 3 and P2X 2/3 receptors. A-317491 dose-dependently (ED 50 = 30 μmol/kg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED 50 = 10–15 μmol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R -enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED 50 >100 μmol/kg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X 3 and P2X 2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X 3 and P2X 2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

P2X3and P2X2/3receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3and P2X2/3receptor activation. A-317491 potently blocked recombinant human and rat P2X3and P2X2/3receptor-mediated calcium flux (Ki= 22-92 nM) and was highly selective$(IC_{50} > 10\\;\\mu M)$over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3and P2X2/3receptors in rat dorsal root ganglion neurons. Blockade of P2X3containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3and P2X2/3receptors. A-317491 dose-dependently (ED50= 30 μ mol/kg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50= 10-15 μ mol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective$(ED_{50} > 100\\;\\mu mol/kg\\;s.c.)$in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3and P2X2/3receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3and P2X2/3receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.