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

Abstract

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 Gai 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 Gai/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 1 ; 245 mV), reducing inhibition of cAMP signaling by l-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.

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

“…Spinal cord injury procedure Rat SCI surgeries (SCI group) were performed as previously described (Bedi et al, 2010;Bavencoffe et al, 2016;Berkey et al, 2020;Garza Carbajal et al, 2020). Animals were anesthetized with isoflurane (induction 4%-5%; maintenance 1%-2%, Covetrus) before a laminectomy at the vertebral T10 level was performed, followed by a dorsal contusive spinal impact (150 kilodyne, 1 s dwell time) using an Infinite Horizon Spinal Cord Impactor (Precision Systems and Instrumentation).…”

“…FIBSI was written using the Anaconda (version 2019.7.0.0, Anaconda) distribution of Python (version 3.5.2) with dependencies on the NumPy and matplotlib.pyplot libraries (Cassidy et al, 2020). The FIBSI program incorporates the previously published algorithm used to analyze DSFs in our prior publications (Odem et al, 2018;Berkey et al, 2020;Garza Carbajal et al, 2020;Lopez et al, 2021;North et al, 2022). FIBSI uses the Ramer-Douglas-Peucker algorithm to detect DSFs, which were obtained from 30 s recordings sampled at 10 kHz with PatchMaster version 2x90.1 (HEKA Elektronik) and filtered with a 10 kHz Bessel filter.…”

“…Persistent activity of the cAMP pathway (PKA and EPAC) and activation by C-Raf of ERK are necessary for nociceptor hyperactivity after SCI (Bavencoffe et al, 2016;Berkey et al, 2020;Garza Carbajal et al, 2020). Hyperactivity after SCI may involve intrinsic alterations that include increased sensitivity to extrinsic excitatory signals (Walters, 2012(Walters, , 2019, as was shown by hypersensitivity to the TRPV1 agonist, capsaicin, after SCI (Wu et al, 2013).…”

Chai-Hu-San-Shen Capsule Ameliorates Ventricular Arrhythmia Through Inhibition of the CaMKII/FKBP12.6/RyR2/Ca2+ Signaling Pathway in Rats with Myocardial Ischemia

“…However, isolation of primary cardiomyocytes is very difficult, and H9C2 cells are a generally accepted model for evaluation of cardiomyocyte biochemistry. Since H-89 has been demonstrated to effectively inhibit PKA phosphorylation [60][61][62][63], the expression levels of PKA and p-PKA were not evaluated in this study to confirm that PKA activity was inhibited. is study showed that the occurrence of arrhythmia after ischemia was closely related to phosphorylation of RyR2 (Ser2808) and RyR2 (Ser2814) by CaMKII, which promoted dissociation of the FKBP12.6-RyR2 complex, and increased intracellular Ca 2+ content.…”

RNA Profiling of Neuropathic Pain-Associated Human DRGs Reveal Sex-differences in Neuro-immune Interactions Promoting Pain

“…Ectopic activity can originate from axons, in particular early after injury, but it can also emerge from the DRG soma (Amir et al, 2005). This likely happens due to changes in expression of ion channels in the DRG neuronal soma (Devor, 2006), or due to signaling events that cause the neuron to generate ectopic action potentials and instability in the resting membrane potential (Devor and Yarom, 2002; Odem et al, 2018; Garza Carbajal et al, 2020; Laumet et al, 2020; Lopez et al, 2021). Our previous work clearly showed that human DRG neuron somata from neuropathic pain patients could display ectopic activity, even after surgical removal and culturing (North et al, 2019).…”