Opioid-Induced Hyperalgesia and Tolerance Are Driven by HCN Ion Channels.

Prolonged exposure to opioids causes an enhanced sensitivity to painful stimuli (opioid-induced hyperalgesia, OIH) and a need for increased opioid doses to maintain analgesia (opioid-induced tolerance, OIT), but the mechanisms underlying both processes remain obscure. We found that pharmacological block or genetic deletion of HCN2 ion channels in primary nociceptive neurons of male mice completely abolished OIH but had no effect on OIT. Conversely, pharmacological inhibition of central HCN channels alleviated OIT but had no effect on OIH. Expression of C-FOS, a marker of neuronal activity, was increased in second-order neurons of the dorsal spinal cord by induction of OIH, and the increase was prevented by peripheral block or genetic deletion of HCN2, but block of OIT by spinal block of HCN channels had no impact on C-FOS expression in dorsal horn neurons. Collectively, these observations show that OIH is driven by HCN2 ion channels in peripheral nociceptors, while OIT is driven by a member of the HCN family located in the CNS. Induction of OIH increased cAMP in nociceptive neurons, and a consequent shift in the activation curve of HCN2 caused an increase in nociceptor firing. The shift in HCN2 was caused by expression of a constitutively active µ-opioid receptor (MOR) and was reversed by MOR antagonists. We identified the opioid-induced MOR as a six-transmembrane splice variant, and we show that it increases cAMP by coupling constitutively to Gs HCN2 ion channels therefore drive OIH, and likely OIT, and may be a novel therapeutic target for the treatment of addiction.

STIM1 and ORAI1 form a novel cold transduction mechanism in sensory and sympathetic neurons.

Moderate coolness is sensed by TRPM8 ion channels in peripheral sensory nerves, but the mechanism by which noxious cold is detected remains elusive. Here, we show that somatosensory and sympathetic neurons express two distinct mechanisms to detect noxious cold. In the first, inhibition by cold of a background outward current causes membrane depolarization that activates an inward current through voltage-dependent calcium (CaV ) channels. A second cold-activated mechanism is independent of membrane voltage, is inhibited by blockers of ORAI ion channels and by downregulation of STIM1, and is recapitulated in HEK293 cells by co-expression of ORAI1 and STIM1. Using total internal reflection fluorescence microscopy we found that cold causes STIM1 to aggregate with and activate ORAI1 ion channels, in a mechanism similar to that underlying store-operated calcium entry (SOCE), but directly activated by cold and not by emptying of calcium stores. This novel mechanism may explain the phenomenon of cold-induced vasodilation (CIVD), in which extreme cold increases blood flow in order to preserve the integrity of peripheral tissues.

Artemisinin inhibits neutrophil and macrophage chemotaxis, cytokine production and NET release.

Immune cell chemotaxis to the sites of pathogen invasion is critical for fighting infection, but in life-threatening conditions such as sepsis and Covid-19, excess activation of the innate immune system is thought to cause a damaging invasion of immune cells into tissues and a consequent excessive release of cytokines, chemokines and neutrophil extracellular traps (NETs). In these circumstances, tempering excessive activation of the innate immune system may, paradoxically, promote recovery. Here we identify the antimalarial compound artemisinin as a potent and selective inhibitor of neutrophil and macrophage chemotaxis induced by a range of chemotactic agents. Artemisinin released calcium from intracellular stores in a similar way to thapsigargin, a known inhibitor of the Sarco/Endoplasmic Reticulum Calcium ATPase pump (SERCA), but unlike thapsigargin, artemisinin blocks only the SERCA3 isoform. Inhibition of SERCA3 by artemisinin was irreversible and was inhibited by iron chelation, suggesting iron-catalysed alkylation of a specific cysteine residue in SERCA3 as the mechanism by which artemisinin inhibits neutrophil motility. In murine infection models, artemisinin potently suppressed neutrophil invasion into both peritoneum and lung in vivo and inhibited the release of cytokines/chemokines and NETs. This work suggests that artemisinin may have value as a therapy in conditions such as sepsis and Covid-19 in which over-activation of the innate immune system causes tissue injury that can lead to death.

Imbalance of proresolving lipid mediators in persistent allodynia dissociated from signs of clinical arthritis.

ABSTRACT: Rheumatoid arthritis-associated pain is poorly managed, often persisting when joint inflammation is pharmacologically controlled. Comparably, in the mouse K/BxN serum-transfer model of inflammatory arthritis, hind paw nociceptive hypersensitivity occurs with ankle joint swelling (5 days after immunisation) persisting after swelling has resolved (25 days after immunisation). In this study, lipid mediator (LM) profiling of lumbar dorsal root ganglia (DRG), the site of sensory neuron cell bodies innervating the ankle joints, 5 days and 25 days after serum transfer demonstrated a shift in specialised proresolving LM profiles. Persistent nociception without joint swelling was associated with low concentrations of the specialised proresolving LM Maresin 1 (MaR1) and high macrophage numbers in DRG. MaR1 application to cultured DRG neurons inhibited both capsaicin-induced increase of intracellular calcium ions and release of calcitonin gene-related peptide in a dose-dependent manner. Furthermore, in peritoneal macrophages challenged with lipopolysaccharide, MaR1 reduced proinflammatory cytokine expression. Systemic MaR1 administration caused sustained reversal of nociceptive hypersensitivity and reduced inflammatory macrophage numbers in DRG. Unlike gabapentin, which was used as positive control, systemic MaR1 did not display acute antihyperalgesic action. Therefore, these data suggest that MaR1 effects observed after K/BxN serum transfer relate to modulation of macrophage recruitment, more likely than to direct actions on sensory neurons. Our study highlights that, in DRG, aberrant proresolution mechanisms play a key role in arthritis joint pain dissociated from joint swelling, opening novel approaches for rheumatoid arthritis pain treatment.

Hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channels drive pain in mouse models of diabetic neuropathy.

Diabetic patients frequently suffer from continuous pain that is poorly treated by currently available analgesics. We used mouse models of type 1 and type 2 diabetes to investigate a possible role for the hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) ion channels as drivers of diabetic pain. Blocking or genetically deleting HCN2 channels in small nociceptive neurons suppressed diabetes-associated mechanical allodynia and prevented neuronal activation of second-order neurons in the spinal cord in mice. In addition, we found that intracellular cyclic adenosine monophosphate (cAMP), a positive HCN2 modulator, is increased in somatosensory neurons in an animal model of painful diabetes. We propose that the increased intracellular cAMP drives diabetes-associated pain by facilitating HCN2 activation and consequently promoting repetitive firing in primary nociceptive nerve fibers. Our results suggest that HCN2 may be an analgesic target in the treatment of painful diabetic neuropathy.

Alleviating pain hypersensitivity through activation of type 4 metabotropic glutamate receptor.

Hyperactivity of the glutamatergic system is involved in the development of central sensitization in the pain neuraxis, associated with allodynia and hyperalgesia observed in patients with chronic pain. Herein we study the ability of type 4 metabotropic glutamate receptors (mGlu4) to regulate spinal glutamate signaling and alleviate chronic pain. We show that mGlu4 are located both on unmyelinated C-fibers and spinal neurons terminals in the inner lamina II of the spinal cord where they inhibit glutamatergic transmission through coupling to Cav2.2 channels. Genetic deletion of mGlu4 in mice alters sensitivity to strong noxious mechanical compression and accelerates the onset of the nociceptive behavior in the inflammatory phase of the formalin test. However, responses to punctate mechanical stimulation and nocifensive responses to thermal noxious stimuli are not modified. Accordingly, pharmacological activation of mGlu4 inhibits mechanical hypersensitivity in animal models of inflammatory or neuropathic pain while leaving acute mechanical perception unchanged in naive animals. Together, these results reveal that mGlu4 is a promising new target for the treatment of chronic pain.

A novel selective metabotropic glutamate receptor 4 agonist reveals new possibilities for developing subtype selective ligands with therapeutic potential.

Metabotropic glutamate (mGlu) receptors are promising targets to treat numerous brain disorders. So far, allosteric modulators are the only subtype selective ligands, but pure agonists still have strong therapeutic potential. Here, we aimed at investigating the possibility of developing subtype-selective agonists by extending the glutamate-like structure to hit a nonconsensus binding area. We report the properties of the first mGlu4-selective orthosteric agonist, derived from a virtual screening hit, LSP4-2022 using cell-based assays with recombinant mGlu receptors [EC(50): 0.11 ± 0.02, 11.6 ± 1.9, 29.2 ± 4.2 µM (n>19) in calcium assays on mGlu4, mGlu7, and mGlu8 receptors, respectively, with no activity at the group I and -II mGlu receptors at 100 µM]. LSP4-2022 inhibits neurotransmission in cerebellar slices from wild-type but not mGlu4 receptor-knockout mice. In vivo, it possesses antiparkinsonian properties after central or systemic administration in a haloperidol-induced catalepsy test, revealing its ability to cross the blood-brain barrier. Site-directed mutagenesis and molecular modeling was used to identify the LSP4-2022 binding site, revealing interaction with both the glutamate binding site and a variable pocket responsible for selectivity. These data reveal new approaches for developing selective, hydrophilic, and brain-penetrant mGlu receptor agonists, offering new possibilities to design original bioactive compounds with therapeutic potential.