Sustained morphine‐mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal treatment with Raf‐1‐selective siRNA

Abstract

BACKGROUND AND PURPOSELong-term morphine treatment enhances pain neurotransmitter [such as calcitonin gene-related peptide (CGRP)] levels in the spinal cord. It has been suggested previously that increased spinal CGRP may contribute to sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance. Previous in vitro studies from our group indicated that Raf-1 kinase-mediated adenylyl cyclase superactivation played a crucial role in sustained morphine-mediated augmentation of basal and evoked CGRP release from cultured primary sensory neurons. The present study was aimed to evaluate the physiological significance of this molecular mechanism in vivo, in rats. EXPERIMENTAL APPROACHRats were intrathecally (i.th) injected with a Raf-1-selective small interfering RNA (siRNA) mixture for 3 days and were subsequently infused with saline or morphine, s.c. for 7 days. Thermal and mechanical sensory thresholds of the animals were assessed by daily behavioural tests. After final behavioural testing (day 6), spinal cords were isolated from each animal group and spinal CGRP and Raf-1 protein levels were measured using ELISA and immunohistochemistry. KEY RESULTSSelective knockdown of spinal Raf-1 protein levels by i.th Raf-1-selective siRNA pretreatment significantly attenuated sustained morphine-mediated up-regulation of CGRP immunoreactivity in the spinal cord of rats and prevented the development of thermal hyperalgesia, mechanical allodynia and antinociceptive tolerance. CONCLUSIONS AND IMPLICATIONSRaf-1 played a significant role in sustained morphine-mediated paradoxical pain sensitization and antinociceptive tolerance in vivo. These findings suggest novel pharmacological approaches to improve the long-term utility of opioids in the treatment of chronic pain. AbbreviationsAC, adenylyl cyclase; CGRP, calcitonin gene-related peptide; DRG, dorsal root ganglion; dsRNA, double-stranded RNA; ELISA, enzyme-linked immunosorbent assay; i.th, intrathecal; PKA, protein kinase A; Raf-1, c-Raf or Raf-1 kinase; siRNA, small interfering RNA

Opioid-induced hypernociception is associated with hyperexcitability and altered tetrodotoxin-resistant Na+ channel function of dorsal root ganglia

“…The requirement of lower pulse strength to induce AP suggests that in vivo even a mild noxious stimulus that normally does not excite the sensory neurons may be enough to excite them, leading to hypernociception following chronic morphine. Supporting this interpretation, recent studies demonstrate development of tactile allodynia on the sixth day following sustained morphine delivery in rats (55). Apart from lower rheobase and multiple APs, chronic morphine did not affect any other characteristics of the APs.…”

GluN2B N-methyl-D-aspartate receptor and excitatory amino acid transporter 3 are upregulated in primary sensory neurons after 7 days of morphine administration in rats

“…The pump delivered the solution at a constant rate of 10 μl/h and lasted for 1 week. Continuous morphine administration for 7 days was previously shown to reliably induce OIH [60; 75]. Before implantation, the pumps were weighed.…”

Molecular Pharmacology ofδ-Opioid Receptors

“…One of such mechanisms has been well characterized in CHO cells, in which sustained opioid activation leads to AC5/AC6 superactivation via AC phosphorylation by rapidly accelerated fibrosarcoma (Raf)-1 , whose activation is in turn induced by Rous sarcoma oncogene cellular homolog (Src), PKC, and/or calmodulin (Varga, 2003) via Gbg-and PLCb-dependent pathways (Rubenzik et al, 2001). As a consequence of Raf-1-dependent AC superactivation, cultured sensory neurons display increased PKA activity and enhanced release of CGRP (Yue et al, 2008), which may contribute to the development of analgesic tolerance (Tumati et al, 2010). Other PKA-dependent mechanisms contributing to opioid tolerance as well as pain sensitization include AMPA receptor phosphorylation and targeting of AMPA receptors to the membrane (Asiedu et al, 2011;Zhuo, 2012) as well as PKA-dependent phosphorylation of NMDA receptors (Qiu et al, 2013), which lead to increased neuronal excitability.…”