Mechanism of gabapentinoid potentiation of opioid effects on cyclic AMP signaling in neuropathic pain.

Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.

Major Differences in Transcriptional Alterations in Dorsal Root Ganglia Between Spinal Cord Injury and Peripheral Neuropathic Pain Models.

Chronic, often intractable, pain is caused by neuropathic conditions such as traumatic peripheral nerve injury (PNI) and spinal cord injury (SCI). These conditions are associated with alterations in gene and protein expression correlated with functional changes in somatosensory neurons having cell bodies in dorsal root ganglia (DRGs). Most studies of DRG transcriptional alterations have utilized PNI models where axotomy-induced changes important for neural regeneration may overshadow changes that drive neuropathic pain. Both PNI and SCI produce DRG neuron hyperexcitability linked to pain, but contusive SCI produces little peripheral axotomy or peripheral nerve inflammation. Thus, comparison of transcriptional signatures of DRGs across PNI and SCI models may highlight pain-associated transcriptional alterations in sensory ganglia that do not depend on peripheral axotomy or associated effects such as peripheral Wallerian degeneration. Data from our rat thoracic SCI experiments were combined with meta-analysis of published whole-DRG RNA-seq datasets from prominent rat PNI models. Striking differences were found between transcriptional responses to PNI and SCI, especially in regeneration-associated genes (RAGs) and long noncoding RNAs (lncRNAs). Many transcriptomic changes after SCI also were found after corresponding sham surgery, indicating they were caused by injury to surrounding tissue, including bone and muscle, rather than to the spinal cord itself. Another unexpected finding was of few transcriptomic similarities between rat neuropathic pain models and the only reported transcriptional analysis of human DRGs linked to neuropathic pain. These findings show that DRGs exhibit complex transcriptional responses to central and peripheral neural injury and associated tissue damage. Although only a few genes in DRG cells exhibited similar changes in expression across all the painful conditions examined here, these genes may represent a core set whose transcription in various DRG cell types is sensitive to significant bodily injury, and which may play a fundamental role in promoting neuropathic pain.

POPDC1 scaffolds a complex of adenylyl cyclase 9 and the potassium channel TREK-1 in heart.

The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain-containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. We show that TREK-1 binds the AC9:POPDC1 complex and copurifies in a POPDC1-dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP-independent, TREK-1 association with AC9 and POPDC1 is reduced upon stimulation of the ß-adrenergic receptor (ßAR). AC9 activity is required for ßAR reduction of TREK-1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK-1 currents. Finally, deletion of the gene-encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress-induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK-1 to regulate heart rate control.

Macrophage Migration Inhibitory Factor (MIF) Makes Complex Contributions to Pain-Related Hyperactivity of Nociceptors after Spinal Cord Injury.

Neuropathic pain is a major, inadequately treated challenge for people with spinal cord injury (SCI). While SCI pain mechanisms are often assumed to be in the central nervous system, rodent studies have revealed mechanistic contributions from primary nociceptors. These neurons become chronically hyperexcitable after SCI, generating ongoing electrical activity (OA) that promotes ongoing pain. A major question is whether extrinsic chemical signals help to drive OA after SCI. People living with SCI exhibit acute and chronic elevation of circulating levels of macrophage migration inhibitory factor (MIF), a cytokine implicated in preclinical pain models. Probable nociceptors isolated from male rats and exposed to a MIF concentration reported in human plasma (1 ng/ml) showed hyperactivity similar to that induced by SCI, although, surprisingly, a ten-fold higher concentration failed to increase excitability. Conditioned behavioral aversion to a chamber associated with peripheral MIF injection suggested that MIF stimulates affective pain. A MIF inhibitor, Iso-1, reversed SCI-induced hyperexcitability. Unlike after SCI, acute MIF-induced hyperexcitability was only partially abrogated by inhibiting ERK signaling. Unexpectedly, MIF concentrations that induced hyperactivity in nociceptors from Naïve animals, after SCI induced a long-lasting conversion from a highly excitable nonaccommodating type to a rapidly accommodating, hypoexcitable type, possibly as a homeostatic response to prolonged depolarization. Treatment with conditioned medium from cultures of dorsal root ganglion (DRG) cells obtained after SCI was sufficient to induce MIF-dependent hyperactivity in neurons from Naïve rats. Thus, changes in systemic and DRG levels of MIF may help to maintain SCI-induced nociceptor hyperactivity that persistently promotes pain.Significance Statement:Chronic neuropathic pain is a major challenge for people with spinal cord injury (SCI). Pain can drastically impair quality of life, and produces substantial economic and social burdens. Available treatments, including opioids, remain inadequate. This study shows that the cytokine macrophage migration inhibitory factor (MIF) can induce pain-like behavior and plays an important role in driving persistent ongoing electrical activity in injury-detecting sensory neurons (nociceptors) in a rat SCI model. The results indicate that SCI produces an increase in MIF release within sensory ganglia. Low MIF levels potently excite nociceptors, but higher levels trigger a long-lasting hypoexcitable state. These findings suggest that therapeutic targeting of MIF in neuropathic pain states may reduce pain and sensory dysfunction by curbing nociceptor hyperactivity.

Oncostatin M induces hyperalgesic priming and amplifies signaling of cAMP to ERK by RapGEF2 and PKA.

Hyperalgesic priming is characterized by enhanced nociceptor sensitization by pronociceptive mediators, prototypically PGE2 . Priming has gained interest as a mechanism underlying the transition to chronic pain. Which stimuli induce priming and what cellular mechanisms are employed remains incompletely understood. In adult male rats, we present the cytokine Oncostatin M (OSM), a member of the IL-6 family, as an inducer of priming by a novel mechanism. We used a high content microscopy based approach to quantify the activation of endogenous PKA-II and ERK of thousands sensory neurons in culture. Incubation with OSM increased and prolonged ERK activation by agents that increase cAMP production such as PGE2 , forskolin, and cAMP analogs. These changes were specific to IB4/CaMKIIα positive neurons, required protein translation, and increased cAMP-to-ERK signaling. In both, control and OSM-treated neurons, cAMP/ERK signaling involved RapGEF2 and PKA but not Epac. Similar enhancement of cAMP-to-ERK signaling could be induced by GDNF, which acts mostly on IB4/CaMKIIα-positive neurons, but not by NGF, which acts mostly on IB4/CaMKIIα-negative neurons. In vitro, OSM pretreatment rendered baseline TTX-R currents ERK-dependent and switched forskolin-increased currents from partial to full ERK-dependence in small/medium sized neurons. In summary, priming induced by OSM uses a novel mechanism to enhance and prolong coupling of cAMP/PKA to ERK1/2 signaling without changing the overall pathway structure.

Depolarization-Dependent C-Raf Signaling Promotes Hyperexcitability and Reduces Opioid Sensitivity of Isolated Nociceptors after Spinal Cord Injury.

Chronic pain caused by spinal cord injury (SCI) is notoriously resistant to treatment, particularly by opioids. After SCI, DRG neurons show hyperactivity and chronic depolarization of resting membrane potential (RMP) that is maintained by cAMP signaling through PKA and EPAC. Importantly, SCI also reduces the negative regulation by Gαi of adenylyl cyclase and its production of cAMP, independent of alterations in G protein-coupled receptors and/or G proteins. Opioid reduction of pain depends on coupling of opioid receptors to Gαi/o family members. Combining high-content imaging and cluster analysis, we show that in male rats SCI decreases opioid responsiveness in vitro within a specific subset of small-diameter nociceptors that bind isolectin B4. This SCI effect is mimicked in nociceptors from naive animals by a modest 5 min depolarization of RMP (15 mm K+; -45 mV), reducing inhibition of cAMP signaling by µ-opioid receptor agonists DAMGO and morphine. Disinhibition and activation of C-Raf by depolarization-dependent phosphorylation are central to these effects. Expression of an activated C-Raf reduces sensitivity of adenylyl cyclase to opioids in nonexcitable HEK293 cells, whereas inhibition of C-Raf or treatment with the hyperpolarizing drug retigabine restores opioid responsiveness and blocks spontaneous activity of nociceptors after SCI. Inhibition of ERK downstream of C-Raf also blocks SCI-induced hyperexcitability and depolarization, without direct effects on opioid responsiveness. Thus, depolarization-dependent C-Raf and downstream ERK activity maintain a depolarized RMP and nociceptor hyperactivity after SCI, providing a self-reinforcing mechanism to persistently promote nociceptor hyperexcitability and limit the therapeutic effectiveness of opioids.SIGNIFICANCE STATEMENT Chronic pain induced by spinal cord injury (SCI) is often permanent and debilitating, and usually refractory to treatment with analgesics, including opioids. SCI-induced pain in a rat model has been shown to depend on persistent hyperactivity in primary nociceptors (injury-detecting sensory neurons), associated with a decrease in the sensitivity of adenylyl cyclase production of cAMP to inhibitory Gαi proteins in DRGs. This study shows that SCI and one consequence of SCI (chronic depolarization of resting membrane potential) decrease sensitivity to opioid-mediated inhibition of cAMP and promote hyperactivity of nociceptors by enhancing C-Raf activity. ERK activation downstream of C-Raf is necessary for maintaining ongoing depolarization and hyperactivity, demonstrating an unexpected positive feedback loop to persistently promote pain.

Monocytes/Macrophages control resolution of transient inflammatory pain.

UNLABELLED: Insights into mechanisms governing resolution of inflammatory pain are of great importance for many chronic pain-associated diseases. Here we investigate the role of macrophages/monocytes and the anti-inflammatory cytokine interleukin-10 (IL-10) in the resolution of transient inflammatory pain. Depletion of mice from peripheral monocytes/macrophages delayed resolution of intraplantar IL-1ß- and carrageenan-induced inflammatory hyperalgesia from 1 to 3 days to >1 week. Intrathecal administration of a neutralizing IL-10 antibody also markedly delayed resolution of IL-1ß- and carrageenan-induced inflammatory hyperalgesia. Recently, we showed that IL-1ß- and carrageenan-induced hyperalgesia is significantly prolonged in LysM-GRK2(+/-) mice, which have reduced levels of G-protein-coupled receptor kinase 2 (GRK2) in LysM(+) myeloid cells. Here we show that adoptive transfer of wild-type, but not of GRK2(+/-), bone marrow-derived monocytes normalizes the resolution of IL-1ß-induced hyperalgesia in LysM-GRK2(+/-) mice. Adoptive transfer of IL-10(-/-) bone marrow-derived monocytes failed to normalize the duration of IL-1ß-induced hyperalgesia in LysM-GRK2(+/-) mice. Mechanistically, we show that GRK2(+/-) macrophages produce less IL-10 in vitro. In addition, intrathecal IL-10 administration attenuated IL-1ß-induced hyperalgesia in LysM-GRK2(+/-) mice, whereas it had no effect in wild-type mice. Our data uncover a key role for monocytes/macrophages in promoting resolution of inflammatory hyperalgesia via a mechanism dependent on IL-10 signaling in dorsal root ganglia. PERSPECTIVE: We show that IL-10-producing monocytes/macrophages promote resolution of transient inflammatory hyperalgesia. Additionally, we show that reduced monocyte/macrophage GRK2 impairs resolution of hyperalgesia and reduces IL-10 production. We propose that low GRK2 expression and/or impaired IL-10 production by monocytes/macrophages represent peripheral biomarkers for the risk of developing chronic pain after inflammation.

Low nociceptor GRK2 prolongs prostaglandin E2 hyperalgesia via biased cAMP signaling to Epac/Rap1, protein kinase Cepsilon, and MEK/ERK.

Hyperexcitability of peripheral nociceptive pathways is often associated with inflammation and is an important mechanism underlying inflammatory pain. Here we describe a completely novel mechanism via which nociceptor G-protein-coupled receptor kinase 2 (GRK2) contributes to regulation of inflammatory hyperalgesia. We show that nociceptor GRK2 is downregulated during inflammation. In addition, we show for the first time that prostaglandin E2 (PGE2)-induced hyperalgesia is prolonged from <6 h in wild-type (WT) mice to 3 d in mice with low GRK2 in Nav1.8+ nociceptors (SNS-GRK2+/- mice). This prolongation of PGE2 hyperalgesia in SNS-GRK2+/- mice does not depend on changes in the sensitivity of the prostaglandin receptors because prolonged hyperalgesia also developed in response to 8-Br-cAMP. PGE2 or cAMP-induced hyperalgesia in WT mice is PKA dependent. However, PKA activity is not required for hyperalgesia in SNS-GRK2+/- mice. SNS-GRK2+/- mice developed prolonged hyperalgesia in response to the Exchange proteins directly activated by cAMP (Epac) activator 8-pCPT-2'-O-Me-cAMP (8-pCPT). Coimmunoprecipitation experiments showed that GRK2 binds to Epac1. In vitro, GRK2 deficiency increased 8-pCPT-induced activation of the downstream effector of Epac, Rap1, and extracellular signal-regulated kinase (ERK). In vivo, inhibition of MEK1 or PKCε prevented prolonged PGE2, 8-Br-cAMP, and 8-pCPT hyperalgesia in SNS-GRK2+/- mice. In conclusion, we discovered GRK2 as a novel Epac1-interacting protein. A reduction in the cellular level of GRK2 enhances activation of the Epac-Rap1 pathway. In vivo, low nociceptor GRK2 leads to prolonged inflammatory hyperalgesia via biased cAMP signaling from PKA to Epac-Rap1, ERK/PKCε pathways.

[Lead inhibits proton gated currents (ASIC) in dorsal root ganglion neurons]. / El plomo inhibe la corriente activada por protones (ASIC) en las neuronas de los ganglios dorsales.

BACKGROUND: Acid sensing ion channels (ASIC) are ionic channels activated by transient pH reductions in the ext raceilularenvi ronment. Although the activation mechanism is not fully elucidated, it is clear that the channel is activated by proton binding to its extraceilular domain, a process that is modulated by calcium and zinc. OBJECTIVE: The fact that divalent cations are able to modify ASIC operation, lead us to consider if lead, anotherdivalent cation and widely distributed neurotoxicant, is also capable to affect ASIC function. METHODS: For this purpose, we recordedASiC currents in rat dorsal root ganglion neurons using the whole cell patch-clamp technique. RESULTS: The results indicated that lead inhibits ASIC currents in a concentration -dependent fashion. CONCLUSIONS: These results contribute to the understanding of the activation mechanism of ASIC and to explain some of the toxic mechanisms of lead in the organism.

El plomo inhibe la corriente activada por protones (ASIC) en las neuronas de los ganglios dorsales / Lead inhibits proton gated currents (ASIC) in dorsal root ganglion neurons

Antecedentes. Los canales iónicos ASIC (del inglés Acid Sensing Ion Channel) son canales iónicos activados por reducciones transitorias en el pH extracelular. Pese a no conocerse con exactitud su mecanismo, la activación ocurre por medio de la unión de protones al dominio extracelular del canal y es modulada por iones calcio y zinc. Objetivo. El hecho de que los cationes divalentes modifiquen el funcionamiento del canal nos llevó a preguntar si el plomo, otro catión divalente, sería capaz de alterar el funcionamiento de los ASIC. Métodos y resultados. Mediante el uso de la técnica de fijación de voltaje en configuración de célula completa en las neuronas de los ganglios de la raíz dorsal de la rata, encontramos que el plomo inhibe la corriente ASIC en forma dependiente de la concentración. Conclusiones. Estos resultados contribuyen a definir los mecanismos de activación de los canales ASIC y a explicar algunos de los mecanismos tóxicos del plomo en el organismo.

BACKGROUND: Acid sensing ion channels (ASIC) are ionic channels activated by transient pH reductions in the ext raceilularenvi ronment. Although the activation mechanism is not fully elucidated, it is clear that the channel is activated by proton binding to its extraceilular domain, a process that is modulated by calcium and zinc. OBJECTIVE: The fact that divalent cations are able to modify ASIC operation, lead us to consider if lead, anotherdivalent cation and widely distributed neurotoxicant, is also capable to affect ASIC function. METHODS: For this purpose, we recordedASiC currents in rat dorsal root ganglion neurons using the whole cell patch-clamp technique. RESULTS: The results indicated that lead inhibits ASIC currents in a concentration -dependent fashion. CONCLUSIONS: These results contribute to the understanding of the activation mechanism of ASIC and to explain some of the toxic mechanisms of lead in the organism.