ABSTRACT
BACKGROUND: The origin of αSMA-positive myofibroblasts, key players within organ fibrosis, is still not fully elucidated. Pericytes have been discussed as myofibroblast progenitors in several organs including the lung. METHODS: Using tamoxifen-inducible PDGFRß-tdTomato mice (PDGFRß-CreERT2; R26tdTomato) lineage of lung pericytes was traced. To induce lung fibrosis, a single orotracheal dose of bleomycin was given. Lung tissue was investigated by immunofluorescence analyses, hydroxyproline collagen assay and RT-qPCR. RESULTS: Lineage tracing combined with immunofluorescence for nitric oxide-sensitive guanylyl cyclase (NO-GC) as marker for PDGFRß-positive pericytes allows differentiating two types of αSMA-expressing myofibroblasts in murine pulmonary fibrosis: (1) interstitial myofibroblasts that localize in the alveolar wall, derive from PDGFRß+ pericytes, express NO-GC and produce collagen 1. (2) intra-alveolar myofibroblasts which do not derive from pericytes (but express PDGFRß de novo after injury), are negative for NO-GC, have a large multipolar shape and appear to spread over several alveoli within the injured areas. Moreover, NO-GC expression is reduced during fibrosis, i.e., after pericyte-to-myofibroblast transition. CONCLUSION: In summary, αSMA/PDGFRß-positive myofibroblasts should not be addressed as a homogeneous target cell type within pulmonary fibrosis.
Subject(s)
Pulmonary Fibrosis , Mice , Animals , Pulmonary Fibrosis/metabolism , Pericytes/metabolism , Myofibroblasts/metabolism , Guanylate Cyclase/metabolism , Fibrosis , Collagen/metabolismABSTRACT
BACKGROUND: Slick, a sodium-activated potassium channel, has been recently identified in somatosensory pathways, but its functional role is poorly understood. The authors of this study hypothesized that Slick is involved in processing sensations of pain and itch. METHODS: Immunostaining, in situ hybridization, Western blot, and real-time quantitative reverse transcription polymerase chain reaction were used to investigate the expression of Slick in dorsal root ganglia and the spinal cord. Mice lacking Slick globally (Slick-/-) or conditionally in neurons of the spinal dorsal horn (Lbx1-Slick-/-) were assessed in behavioral models. RESULTS: The authors found Slick to be enriched in nociceptive Aδ-fibers and in populations of interneurons in the spinal dorsal horn. Slick-/- mice, but not Lbx1-Slick-/- mice, showed enhanced responses to noxious heat in the hot plate and tail-immersion tests. Both Slick-/- and Lbx1-Slick-/- mice demonstrated prolonged paw licking after capsaicin injection (mean ± SD, 45.6 ± 30.1 s [95% CI, 19.8 to 71.4]; and 13.1 ± 16.1 s [95% CI, 1.8 to 28.0]; P = 0.006 [Slick-/- {n = 8} and wild-type {n = 7}, respectively]), which was paralleled by increased phosphorylation of the neuronal activity marker extracellular signal-regulated kinase in the spinal cord. In the spinal dorsal horn, Slick is colocalized with somatostatin receptor 2 (SSTR2), and intrathecal preadministration of the SSTR2 antagonist CYN-154806 prevented increased capsaicin-induced licking in Slick-/- and Lbx1-Slick-/- mice. Moreover, scratching after intrathecal delivery of the somatostatin analog octreotide was considerably reduced in Slick-/- and Lbx1-Slick-/- mice (Slick-/- [n = 8]: 6.1 ± 6.7 bouts [95% CI, 0.6 to 11.7]; wild-type [n =8]: 47.4 ± 51.1 bouts [95% CI, 4.8 to 90.2]; P = 0.039). CONCLUSIONS: Slick expressed in a subset of sensory neurons modulates heat-induced pain, while Slick expressed in spinal cord interneurons inhibits capsaicin-induced pain but facilitates somatostatin-induced itch.
Subject(s)
Capsaicin , Posterior Horn Cells , Animals , Capsaicin/adverse effects , Capsaicin/metabolism , Ganglia, Spinal/metabolism , Mice , Pain , Posterior Horn Cells/metabolism , Potassium Channels , Pruritus/chemically induced , Sensory Receptor Cells/metabolism , Sodium Channels , Somatostatin/adverse effects , Somatostatin/metabolism , Spinal Cord/metabolismABSTRACT
Previous studies suggest that adenosine A1 receptors (A1R) modulate the processing of pain. The aim of this study was to characterize the distribution of A1R in nociceptive tissues and to evaluate whether targeting A1R with the partial agonist capadenoson may reduce neuropathic pain in mice. The cellular distribution of A1R in dorsal root ganglia (DRG) and the spinal cord was analyzed using fluorescent in situ hybridization. In behavioral experiments, neuropathic pain was induced by spared nerve injury or intraperitoneal injection of paclitaxel, and tactile hypersensitivities were determined using a dynamic plantar aesthesiometer. Whole-cell patch-clamp recordings were performed to assess electrophysiological properties of dissociated DRG neurons. We found A1R to be expressed in populations of DRG neurons and dorsal horn neurons involved in the processing of pain. However, administration of capadenoson at established in vivo doses (0.03-1.0 mg/kg) did not alter mechanical hypersensitivity in the spared nerve injury and paclitaxel models of neuropathic pain, whereas the standard analgesic pregabalin significantly inhibited the pain behavior. Moreover, capadenoson failed to affect potassium currents in DRG neurons, in contrast to a full A1R agonist. Despite expression of A1R in nociceptive neurons, our data do not support the hypothesis that pharmacological intervention with partial A1R agonists might be a valuable approach for the treatment of neuropathic pain.
Subject(s)
Adenosine A1 Receptor Agonists/therapeutic use , Neuralgia/drug therapy , Neuralgia/metabolism , Receptor, Adenosine A1/biosynthesis , Adenosine A1 Receptor Agonists/pharmacology , Animals , Cells, Cultured , Female , Male , Mice , Mice, Inbred C57BL , Pain Measurement/drug effects , Pain Measurement/methods , Receptor, Adenosine A1/genetics , Treatment OutcomeABSTRACT
The sodium-activated potassium channel Slack (KNa1.1, Slo2.2, or Kcnt1) is highly expressed in populations of sensory neurons, where it mediates the sodium-activated potassium current (IKNa) and modulates neuronal activity. Previous studies suggest that Slack is involved in the processing of neuropathic pain. However, mechanisms underlying the regulation of Slack activity in this context are poorly understood. Using whole-cell patch-clamp recordings we found that Slack-mediated IKNa in sensory neurons of mice is reduced after peripheral nerve injury, thereby contributing to neuropathic pain hypersensitivity. Interestingly, Slack is closely associated with ATP-sensitive P2X3 receptors in a population of sensory neurons. In vitro experiments revealed that Slack-mediated IKNa may be bidirectionally modulated in response to P2X3 activation. Moreover, mice lacking Slack show altered nocifensive responses to P2X3 stimulation. Our study identifies P2X3/Slack signaling as a mechanism contributing to hypersensitivity after peripheral nerve injury and proposes a potential novel strategy for treatment of neuropathic pain.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Calcium/pharmacology , Nerve Tissue Proteins/metabolism , Neuralgia/metabolism , Potassium Channels, Sodium-Activated/metabolism , Receptors, Purinergic P2X3/metabolism , Sensory Receptor Cells/physiology , Adenosine Triphosphate/pharmacology , Animals , Behavior Rating Scale , Ganglia, Spinal/drug effects , Ganglia, Spinal/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/genetics , Patch-Clamp Techniques , Peripheral Nerves/pathology , Potassium Channels/metabolism , Potassium Channels/physiology , Potassium Channels, Sodium-Activated/genetics , Receptors, Purinergic P2X3/physiology , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
The alkaloid narciclasine has been characterized extensively as an anticancer compound. Accumulating evidence suggests that narciclasine has anti-inflammatory potential; however, the underlying mechanism remains poorly understood. We hypothesized that narciclasine affects the activation of endothelial cells (ECs), a hallmark of inflammatory processes, which is a prerequisite for leukocyte-EC interaction. Thus, we aimed to investigate narciclasine's action on this process in vivo and to analyze the underlying mechanisms in vitro. In a murine peritonitis model, narciclasine reduced leukocyte infiltration, proinflammatory cytokine expression, and inflammation-associated abdominal pain. Moreover, narciclasine decreased rolling and blocked adhesion and transmigration of leukocytes in vivo. In cultured ECs, narciclasine inhibited the expression of cell adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin and blocked crucial steps of the NF-κB activation cascade: NF-κB promotor activity, p65 nuclear translocation, inhibitor of κB α (IκBα) phosphorylation and degradation, and IκBα kinase ß and TGF-ß-activated kinase 1 phosphorylation. Interestingly, these effects were based on the narciclasine-triggered loss of TNF receptor 1 (TNFR1). Our study highlights narciclasine as an interesting anti-inflammatory compound that effectively inhibits the interaction of leukocytes with ECs by blocking endothelial activation processes. Most importantly, we showed that the observed inhibitory action of narciclasine on TNF-triggered signaling pathways is based on the loss of TNFR1.-Stark, A., Schwenk, R., Wack, G., Zuchtriegel, G., Hatemler, M. G., Bräutigam, J., Schmidtko, A., Reichel, C. A., Bischoff, I., Fürst, R. Narciclasine exerts anti-inflammatory actions by blocking leukocyte-endothelial cell interactions and down-regulation of the endothelial TNF receptor 1.
Subject(s)
Amaryllidaceae Alkaloids/pharmacology , Anti-Inflammatory Agents/pharmacology , Cell Adhesion , Human Umbilical Vein Endothelial Cells/drug effects , Phenanthridines/pharmacology , Receptors, Tumor Necrosis Factor, Type I/metabolism , Animals , Cell Movement , Cells, Cultured , Down-Regulation , E-Selectin/genetics , E-Selectin/metabolism , Female , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Jurkat Cells , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Receptors, Tumor Necrosis Factor, Type I/genetics , THP-1 Cells , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
Impaired NO-cGMP signaling has been linked to several neurological disorders. NO-sensitive guanylyl cyclase (NO-GC), of which two isoforms-NO-GC1 and NO-GC2-are known, represents a promising drug target to increase cGMP in the brain. Drug-like small molecules have been discovered that work synergistically with NO to stimulate NO-GC activity. However, the effects of NO-GC stimulators in the brain are not well understood. In the present study, we used Förster/fluorescence resonance energy transfer (FRET)-based real-time imaging of cGMP in acute brain slices and primary neurons of cGMP sensor mice to comparatively assess the activity of two structurally different NO-GC stimulators, IWP-051 and BAY 41-2272, in the cerebellum, striatum and hippocampus. BAY 41-2272 potentiated an elevation of cGMP induced by the NO donor DEA/NO in all tested brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP increases in the cerebellum and striatum, but not in the hippocampal CA1 area or primary hippocampal neurons. The brain-region-selective activity of IWP-051 suggested that it might act in a NO-GC isoform-selective manner. Results of mRNA in situ hybridization indicated that the cerebellum and striatum express NO-GC1 and NO-GC2, while the hippocampal CA1 area expresses mainly NO-GC2. IWP-051-potentiated DEA/NO-induced cGMP signals in the striatum of NO-GC2 knockout mice but was ineffective in the striatum of NO-GC1 knockout mice. These results indicate that IWP-051 preferentially stimulates NO-GC1 signaling in brain slices. Interestingly, no evidence for an isoform-specific effect of IWP-051 was observed when the cGMP-forming activity of whole brain homogenates was measured. This apparent discrepancy suggests that the method and conditions of cGMP measurement can influence results with NO-GC stimulators. Nevertheless, it is clear that NO-GC stimulators enhance cGMP signaling in the brain and should be further developed for the treatment of neurological diseases.
Subject(s)
Brain/metabolism , Cyclic GMP/analysis , Guanylate Cyclase/metabolism , Nitric Oxide/metabolism , Animals , Cells, Cultured , Cyclic GMP/metabolism , Fluorescence Resonance Energy Transfer/methods , Mice, Knockout , Neuroimaging/methods , Neurons , Purkinje CellsABSTRACT
Slack (Slo2.2) is a sodium-activated potassium channel that regulates neuronal firing activities and patterns. Previous studies identified Slack in sensory neurons, but its contribution to acute and chronic pain in vivo remains elusive. Here we generated global and sensory neuron-specific Slack mutant mice and analyzed their behavior in various animal models of pain. Global ablation of Slack led to increased hypersensitivity in models of neuropathic pain, whereas the behavior in models of inflammatory and acute nociceptive pain was normal. Neuropathic pain behaviors were also exaggerated after ablation of Slack selectively in sensory neurons. Notably, the Slack opener loxapine ameliorated persisting neuropathic pain behaviors. In conclusion, Slack selectively controls the sensory input in neuropathic pain states, suggesting that modulating its activity might represent a novel strategy for management of neuropathic pain.
Subject(s)
Hyperalgesia/metabolism , Nerve Tissue Proteins/metabolism , Neuralgia/metabolism , Potassium Channels/metabolism , Sensory Receptor Cells/metabolism , Animals , Hot Temperature , Hyperalgesia/genetics , Hyperalgesia/physiopathology , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Neuralgia/physiopathology , Pain Measurement , Pain Threshold/physiology , Physics , Potassium Channels/genetics , Potassium Channels, Sodium-ActivatedABSTRACT
A large body of evidence indicates that nitric oxide (NO) plays an important role in the processing of persistent inflammatory and neuropathic pain in the spinal cord. Several animal studies revealed that inhibition or knockout of NO synthesis ameliorates persistent pain. However, spinal delivery of NO donors caused dual pronociceptive and antinociceptive effects, pointing to multiple downstream signaling mechanisms of NO. This review summarizes the localization and function of NO-dependent signaling mechanisms in the spinal cord, taking account of the recent progress made in this field.
Subject(s)
Nitric Oxide/physiology , Pain/physiopathology , Spinal Cord/physiology , Animals , Cyclic GMP/physiology , Humans , Peroxynitrous Acid/biosynthesis , Signal TransductionABSTRACT
BACKGROUND: Phosphodiesterase 2A (PDE2A) is an evolutionarily conserved enzyme that catalyzes the degradation of the cyclic nucleotides, cyclic adenosine monophosphate, and/or cyclic guanosine monophosphate. Recent studies reported the expression of PDE2A in the dorsal horn of the spinal cord, pointing to a potential contribution to the processing of pain. However, the functions of PDE2A in spinal pain processing in vivo remained elusive. METHODS: Immunohistochemistry, laser microdissection, and quantitative real-time reverse transcription polymerase chain reaction experiments were performed to characterize the localization and regulation of PDE2A protein and messenger RNA in the mouse spinal cord. Effects of the selective PDE2A inhibitor, BAY 60-7550 (Cayman Chemical, Ann Arbor, MI), in animal models of inflammatory pain (n = 6 to 10), neuropathic pain (n = 5 to 6), and after intrathecal injection of cyclic nucleotides (n = 6 to 8) were examined. Also, cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in spinal cord tissues were measured by liquid chromatography tandem mass spectrometry. RESULTS: The authors here demonstrate that PDE2A is distinctly expressed in neurons of the superficial dorsal horn of the spinal cord, and that its spinal expression is upregulated in response to hind paw inflammation. Administration of the selective PDE2A inhibitor, BAY 60-7550, increased the nociceptive behavior of mice in animal models of inflammatory pain. Moreover, BAY 60-7550 increased the pain hypersensitivity induced by intrathecal delivery of cyclic adenosine monophosphate, but not of cyclic guanosine monophosphate, and it increased the cyclic adenosine monophosphate levels in spinal cord tissues. CONCLUSION: Our findings indicate that PDE2A contributes to the processing of inflammatory pain in the spinal cord.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 2/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 2/physiology , Inflammation/enzymology , Inflammation/physiopathology , Pain/enzymology , Pain/physiopathology , Spinal Cord/enzymology , Animals , Behavior, Animal/drug effects , Behavior, Animal/physiology , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 2/biosynthesis , Drug Hypersensitivity/physiopathology , Imidazoles/administration & dosage , Imidazoles/pharmacology , Immunohistochemistry , Inflammation/complications , Injections, Spinal , Male , Mice , Mice, Inbred C57BL , Microdissection , Neuralgia/enzymology , Neuralgia/physiopathology , Neuralgia/psychology , Pain/etiology , Pain Measurement , Phosphodiesterase Inhibitors/administration & dosage , Phosphodiesterase Inhibitors/pharmacology , Posterior Horn Cells/enzymology , Real-Time Polymerase Chain Reaction , Triazines/administration & dosage , Triazines/pharmacology , Up-Regulation/genetics , Up-Regulation/physiology , ZymosanABSTRACT
The plant alkaloid homoharringtonine (HHT) is a Food and Drug Administration (FDA)-approved drug for the treatment of hematologic malignancies. In addition to its well-established antitumor activity, accumulating evidence attributes anti-inflammatory effects to HHT, which have mainly been studied in leukocytes to date. However, a potential influence of HHT on inflammatory activation processes in endothelial cells, which are a key feature of inflammation and a prerequisite for the leukocyte-endothelial cell interaction and leukocyte extravasation, remains poorly understood. In this study, the anti-inflammatory potential of HHT and its derivative harringtonine (HT) on the TNF-induced leukocyte-endothelial cell interaction was assessed, and the underlying mechanistic basis of these effects was elucidated. HHT affected inflammation in vivo in a murine peritonitis model by reducing leukocyte infiltration and proinflammatory cytokine expression as well as ameliorating abdominal pain behavior. In vitro, HT and HHT impaired the leukocyte-endothelial cell interaction by decreasing the expression of the endothelial cell adhesion molecules intracellular adhesion molecule -1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was mediated by a bipartite mechanism. While HHT did not affect the prominent TNF-induced pro-inflammatory NF-ĸB signaling cascade, the compound downregulated the VCAM1 mRNA expression in an IRF-1-dependent manner and diminished active ICAM1 mRNA translation as determined by polysome profiling. This study highlights HHT as an anti-inflammatory compound that efficiently hampers the leukocyte-endothelial cell interaction by targeting endothelial activation processes.
Subject(s)
Down-Regulation , Homoharringtonine , Inflammation , Interferon Regulatory Factor-1 , RNA, Messenger , Vascular Cell Adhesion Molecule-1 , Animals , Down-Regulation/drug effects , Vascular Cell Adhesion Molecule-1/metabolism , Vascular Cell Adhesion Molecule-1/genetics , Inflammation/drug therapy , Inflammation/pathology , Inflammation/metabolism , RNA, Messenger/metabolism , RNA, Messenger/genetics , Humans , Interferon Regulatory Factor-1/metabolism , Interferon Regulatory Factor-1/genetics , Mice , Homoharringtonine/pharmacology , Male , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Anti-Inflammatory Agents/pharmacology , Intercellular Adhesion Molecule-1/metabolism , Intercellular Adhesion Molecule-1/genetics , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Mice, Inbred C57BL , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/genetics , Leukocytes/drug effects , Leukocytes/metabolismABSTRACT
AIMS: Na+-activated Slack potassium (K+) channels are increasingly recognized as regulators of neuronal activity, yet little is known about their role in the cardiovascular system. Slack activity increases when intracellular Na+ concentration ([Na+]i) reaches pathophysiological levels. Elevated [Na+]i is a major determinant of the ischemia and reperfusion (I/R)-induced myocardial injury, thus we hypothesized that Slack plays a role under these conditions. METHODS: and results: K+ currents in cardiomyocytes (CMs) obtained from wildtype (WT) but not from global Slack knockout (KO) mice were sensitive to electrical inactivation of voltage-sensitive Na+-channels. Live-cell imaging demonstrated that K+ fluxes across the sarcolemma rely on Slack, while the depolarized resting membrane potential in Slack-deficient CMs led to excessive cytosolic Ca2+ accumulation and finally to hypoxia/reoxygenation-induced cell death. Cardiac damage in an in vivo model of I/R was exacerbated in global and CM-specific conditional Slack mutants and largely insensitive to mechanical conditioning maneuvers. Finally, the protection conferred by mitochondrial ATP-dependent K+ channels required functional Slack in CMs. CONCLUSIONS: Collectively, our study provides evidence for Slack's crucial involvement in the ion homeostasis of no or low O2-stressed CMs. Thereby, Slack activity opposes the I/R-induced fatal Ca2+-uptake to CMs supporting the cardioprotective signaling widely attributed to mitoKATP function.
ABSTRACT
Various disorders are accompanied by histamine-independent itching, which is often resistant to the currently available therapies. Here, it is reported that the pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch-sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack-activating antipsychotic drug, loxapine, a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles is designed that enables to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibits negligible dopamine D2 and D3 receptor binding, unlike loxapine. Notably, compound 6 displays potent on-target antipruritic activity in multiple mouse models of acute histamine-independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.
Subject(s)
Potassium Channels , Pruritus , Animals , Mice , Antipruritics/therapeutic use , Histamine/metabolism , Loxapine/therapeutic use , Potassium Channels/metabolism , Pruritus/drug therapy , Pruritus/metabolismABSTRACT
Reactive oxygen species (ROS) contribute to sensitization of pain pathways during neuropathic pain, but little is known about the primary sources of ROS production and how ROS mediate pain sensitization. Here, we show that the NADPH oxidase isoform Nox4, a major ROS source in somatic cells, is expressed in a subset of nonpeptidergic nociceptors and myelinated dorsal root ganglia neurons. Mice lacking Nox4 demonstrated a substantially reduced late-phase neuropathic pain behavior after peripheral nerve injury. The loss of Nox4 markedly attenuated injury-induced ROS production and dysmyelination processes of peripheral nerves. Moreover, persisting neuropathic pain behavior was inhibited after tamoxifen-induced deletion of Nox4 in adult transgenic mice. Our results suggest that Nox4 essentially contributes to nociceptive processing in neuropathic pain states. Accordingly, inhibition of Nox4 may provide a novel therapeutic modality for the treatment of neuropathic pain.
Subject(s)
NADPH Oxidases/metabolism , Neuralgia/metabolism , Neurons/metabolism , Peripheral Nerve Injuries/metabolism , Sciatic Nerve/metabolism , Animals , Behavior, Animal/physiology , Cell Count , Ganglia, Spinal/metabolism , Hyperalgesia/metabolism , Mice , Mice, Transgenic , Microglia/metabolism , Motor Activity/physiology , NADPH Oxidase 4 , NADPH Oxidases/genetics , Nociceptors/metabolism , Pain Measurement , Reactive Oxygen Species/metabolismABSTRACT
Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidonic acid that act as endogenous signaling molecules in multiple biological systems. Here we have investigated the specific contribution of 5,6-EET to transient receptor potential (TRP) channel activation in nociceptor neurons and its consequence for nociceptive processing. We found that, during capsaicin-induced nociception, 5,6-EET levels increased in dorsal root ganglia (DRGs) and the dorsal spinal cord, and 5,6-EET is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux (100 nm) in cultured DRG neurons that was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices, 5,6-EET dose dependently enhanced the frequency, but not the amplitude, of spontaneous EPSCs (sEPSCs) in lamina II neurons that also responded to mustard oil (allyl isothiocyanate), indicating a presynaptic action. Furthermore, 5,6-EET-induced enhancement of sEPSC frequency was abolished in TRPA1-null mice, suggesting that 5,6-EET presynaptically facilitated spinal cord synaptic transmission by TRPA1. Finally, in vivo intrathecal injection of 5,6-EET caused mechanical allodynia in wild-type but not TRPA1-null mice. We conclude that 5,6-EET is synthesized on the acute activation of nociceptors and can produce mechanical hypersensitivity via TRPA1 at central afferent terminals in the spinal cord.
Subject(s)
8,11,14-Eicosatrienoic Acid/analogs & derivatives , Action Potentials/physiology , Afferent Pathways/physiopathology , Hyperalgesia/physiopathology , Sensory Receptor Cells/metabolism , 8,11,14-Eicosatrienoic Acid/metabolism , Animals , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
Inhibitor-κB kinase ε (IKKε) was only recently identified as an enzyme with high homology to the classical I-κB kinase subunits, IKKα and IKKß. Despite this similarity, it is mainly discussed as a repressor of viral infections by modulating type I IFNs. However, in vitro studies also showed that IKKε plays a role in the regulation of NF-κB activity, but the distinct mechanisms of IKKε-mediated NF-κB activation are not clear. Given the paramount role of NF-κB in inflammation, we investigated the regulation and function of IKKε in models of inflammatory hyperalgesia in mice. We found that IKKε was abundantly expressed in nociceptive neurons in the spinal cord and in dorsal root ganglia. IKKε mRNA and protein levels rapidly increased in spinal cord and dorsal root ganglia during hind paw inflammation evoked by injection of zymosan or formalin. IKKε knockout mice showed normal nociceptive responses to acute heat or mechanical stimulation. However, in inflammatory pain models, IKKε-deficient mice exhibited a significantly reduced nociceptive behavior in comparison with wild type mice, indicating that IKKε contributed to the development of inflammatory hyperalgesia. Antinociceptive effects were associated with reduced activation of NF-κB and attenuated NF-κB-dependent induction of cyclooxygenase-2, inducible NO synthase, and metalloproteinase-9. In contrast, IRF-3, which is an important IKKε target in viral infections, was not regulated after inflammatory nociceptive stimulation. Therefore, we concluded that IKKε modulates inflammatory nociceptive sensitivity by activation of NF-κB-dependent gene transcription and may be useful as a therapeutic target in the treatment of inflammatory pain.
Subject(s)
Hyperalgesia/enzymology , I-kappa B Kinase/metabolism , Neurons/metabolism , Animals , Blotting, Western , Cytokines/biosynthesis , Enzyme-Linked Immunosorbent Assay , Ganglia, Spinal/metabolism , Hyperalgesia/etiology , In Situ Hybridization , Inflammation/complications , Inflammation/enzymology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/immunology , NF-kappa B/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/immunology , Spinal Cord/metabolismABSTRACT
Increasing cGMP is a unique therapeutic principle, and drugs inhibiting cGMP-degrading enzymes or stimulating cGMP production are approved for the treatment of various diseases such as erectile dysfunction, coronary artery disease, pulmonary hypertension, chronic heart failure, irritable bowel syndrome, or achondroplasia. In addition, cGMP-increasing therapies are preclinically profiled or in clinical development for quite a broad set of additional indications, e.g., neurodegenerative diseases or different forms of dementias, bone formation disorders, underlining the pivotal role of cGMP signaling pathways. The fundamental understanding of the signaling mediated by nitric oxide-sensitive (soluble) guanylyl cyclase and membrane-associated receptor (particulate) guanylyl cyclase at the molecular and cellular levels, as well as in vivo, especially in disease models, is a key prerequisite to fully exploit treatment opportunities and potential risks that could be associated with an excessive increase in cGMP. Furthermore, human genetic data and the clinical effects of cGMP-increasing drugs allow back-translation into basic research to further learn about signaling and treatment opportunities. The biannual international cGMP conference, launched nearly 20 years ago, brings all these aspects together as an established and important forum for all topics from basic science to clinical research and pivotal clinical trials. This review summarizes the contributions to the "10th cGMP Conference on cGMP Generators, Effectors and Therapeutic Implications," which was held in Augsburg in 2022 but will also provide an overview of recent key achievements and activities in the field of cGMP research.
Subject(s)
Cyclic GMP , Guanylate Cyclase , Male , Humans , Guanylate Cyclase/metabolism , Soluble Guanylyl Cyclase/metabolism , Cyclic GMP/metabolism , Signal Transduction , Research , Nitric Oxide/metabolismABSTRACT
A large body of evidence indicates that nitric oxide (NO) and cGMP contribute to central sensitization of pain pathways during inflammatory pain. Here, we investigated the distribution of cyclic nucleotide-gated (CNG) channels in the spinal cord, and identified the CNG channel subunit CNGA3 as a putative cGMP target in nociceptive processing. In situ hybridization revealed that CNGA3 is localized to inhibitory neurons of the dorsal horn of the spinal cord, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. CNGA3 expression is upregulated in the superficial dorsal horn of the mouse spinal cord and in dorsal root ganglia following hindpaw inflammation evoked by zymosan. Mice lacking CNGA3 (CNGA3(-/-) mice) exhibited an increased nociceptive behavior in models of inflammatory pain, whereas their behavior in models of acute or neuropathic pain was normal. Moreover, CNGA3(-/-) mice developed an exaggerated pain hypersensitivity induced by intrathecal administration of cGMP analogs or NO donors. Our results provide evidence that CNGA3 contributes in an inhibitory manner to the central sensitization of pain pathways during inflammatory pain as a target of NO/cGMP signaling.
Subject(s)
Cyclic GMP/metabolism , Cyclic Nucleotide-Gated Cation Channels/metabolism , Inflammation/complications , Nitric Oxide/metabolism , Pain/etiology , Signal Transduction/physiology , Spinal Cord/metabolism , Analysis of Variance , Animals , Cyclic GMP/adverse effects , Cyclic GMP/analogs & derivatives , Cyclic GMP/pharmacology , Cyclic Nucleotide-Gated Cation Channels/deficiency , Cyclic Nucleotide-Gated Cation Channels/genetics , Disease Models, Animal , Enzyme Inhibitors/pharmacology , Ganglia, Spinal/metabolism , Gene Expression Regulation/drug effects , Glial Fibrillary Acidic Protein/metabolism , Inflammation/chemically induced , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microdissection , Naphthalenes/metabolism , Natriuretic Peptides/adverse effects , Pain/drug therapy , Pain/pathology , Pain Measurement/drug effects , Pain Measurement/methods , Pain Perception/drug effects , Peripheral Nervous System Diseases/drug therapy , Peripheral Nervous System Diseases/pathology , Physical Stimulation/adverse effects , RNA, Messenger/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Stathmin/metabolism , Statistics, Nonparametric , Thionucleotides/pharmacology , Triazenes/pharmacology , Vesicular Inhibitory Amino Acid Transport Proteins/metabolismABSTRACT
Inflammation or injury to the somatosensory nervous system may result in chronic pain conditions, which affect millions of people and often cause major health problems. Emerging lines of evidence indicate that reactive oxygen species (ROS), such as superoxide anion or hydrogen peroxide, are produced in the nociceptive system during chronic inflammatory and neuropathic pain and act as specific signaling molecules in pain processing. Among potential ROS sources in the somatosensory system are NADPH oxidases, a group of electron-transporting transmembrane enzymes whose sole function seems to be the generation of ROS. Interestingly, the expression and relevant function of the Nox family members Nox1, Nox2, and Nox4 in various cells of the nociceptive system have been demonstrated. Studies using knockout mice or specific knockdown of these isoforms indicate that Nox1, Nox2, and Nox4 specifically contribute to distinct signaling pathways in chronic inflammatory and/or neuropathic pain states. As selective Nox inhibitors are currently being developed and investigated in various physiological and pathophysiological settings, targeting Nox1, Nox2, and/or Nox4 could be a novel strategy for the treatment of chronic pain. Here, we summarize the distinct roles of Nox1, Nox2, and Nox4 in inflammatory and neuropathic processing and discuss the effectiveness of currently available Nox inhibitors in the treatment of chronic pain conditions.
ABSTRACT
Cyclic GMP (cGMP) is a second messenger that regulates numerous physiological and pathophysiological processes. In recent years, more and more studies have uncovered multiple roles of cGMP signalling pathways in the somatosensory system. Accumulating evidence suggests that cGMP regulates different cellular processes from embryonic development through to adulthood. During embryonic development, a cGMP-dependent signalling cascade in the trunk sensory system is essential for axon bifurcation, a specific form of branching of somatosensory axons. In adulthood, various cGMP signalling pathways in distinct cell populations of sensory neurons and dorsal horn neurons in the spinal cord play an important role in the processing of pain and itch. Some of the involved enzymes might serve as a target for future therapies. In this review, we summarise the knowledge regarding cGMP-dependent signalling pathways in dorsal root ganglia and the spinal cord during embryonic development and adulthood, and the potential of targeting these pathways. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.
Subject(s)
Cyclic GMP , Ganglia, Spinal , Axons/metabolism , Cyclic GMP/metabolism , Female , Ganglia, Spinal/metabolism , Humans , Pregnancy , Sensory Receptor Cells/metabolism , Spinal Cord/metabolismABSTRACT
The transient receptor potential (TRP) ankyrin type 1 (TRPA1) channel is highly expressed in a subset of sensory neurons where it acts as an essential detector of painful stimuli. However, the mechanisms that control the activity of sensory neurons upon TRPA1 activation remain poorly understood. Here, using in situ hybridization and immunostaining, we found TRPA1 to be extensively co-localized with the potassium channel Slack (KNa1.1, Slo2.2, or Kcnt1) in sensory neurons. Mice lacking Slack globally (Slack-/-) or conditionally in sensory neurons (SNS-Slack-/-) demonstrated increased pain behavior after intraplantar injection of the TRPA1 activator allyl isothiocyanate. By contrast, pain behavior induced by the TRP vanilloid 1 (TRPV1) activator capsaicin was normal in Slack-deficient mice. Patch-clamp recordings in sensory neurons and in a HEK cell line transfected with TRPA1 and Slack revealed that Slack-dependent potassium currents (IKS) are modulated in a TRPA1-dependent manner. Taken together, our findings highlight Slack as a modulator of TRPA1-mediated, but not TRPV1-mediated, activation of sensory neurons.