Sustained morphine treatment augments basal CGRP release from cultured primary sensory neurons in a Raf-1 dependent manner

Abstract

Recent studies suggest that sustained morphine-mediated paradoxical pain may play an important role in the development of analgesic tolerance. The intracellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) release are not fully clarified. Cyclic AMP (cAMP)-dependent protein kinase (PKA) plays an important role in the modulation of presynaptic neurotransmitter release. Moreover, we have shown earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). Therefore, in the present study we examined the role of Raf-1 in sustained morphine-mediated regulation of cAMP formation and basal CGRP release in vitro, in cultured neonatal rat dorsal root ganglion (DRG) neurons. We found that sustained morphine treatment significantly augments intracellular cAMP production as well as basal CGRP release from cultured neonatal rat DRG neurons. The selective PKA inhibitor, H-89, attenuates the sustained morphine-mediated augmentation of basal CGRP release, indicating that the cAMP/PKA pathway plays an important role in regulation of CGRP release from sensory neurons. Since our present data also demonstrated that selective Raf-1 inhibitor, GW 5074, attenuated both the cAMP overshoot and the augmentation of CGRP release mediated by sustained morphine in neonatal rat DRG neurons, we suggest that Raf-1-mediated sensitization of the intracellular cAMP formation may play an important role in sustained morphine-mediated augmentation of spinal pain neurotransmitter release.

Sustained Morphine Treatment Augments Capsaicin-Evoked Calcitonin Gene-Related Peptide Release from Primary Sensory Neurons in a Protein Kinase A- and Raf-1-Dependent Manner

“…7). Accordingly, we hypothesize that, similar to our previous findings in recombinant cell lines (Rubenzik et al, 2001;Varga et al, 2003a,b;Yue et al, 2006Yue et al, , 2008, sustained morphine-mediated activation of Raf-1 may lead to phosphorylation and sensitization of adenylyl cyclase isoenzyme(s) in neonatal rat DRG neurons (AC superactivation). The resulting increase in basal and/or stimulated cellular cAMP formation (cAMP overshoot) increases cAMP-dependent protein kinase (PKA) activity that in turn leads to phosphorylation and sensitization of the heat-sensing TRPV1 vanilloid receptors (Fig.…”

“…The concentration of morphine was adopted from our previous studies (Rubenzik et al, 2001;Varga et al, 2003a;Yue et al, 2006). To test the involvement of opioid receptors, DRG cells were also treated with the nonselective opioid receptor antagonist naltrexone (10 M) in the presence (Mor ϩ NTX group) or absence (NTX group) of morphine for 24 h. The concentrations of morphine and naltrexone were selected according to our previous study (Yue et al, 2008). Control cells were incubated in Neurobasal A/LG/PS/B27 medium in the absence or presence (GW or H-89 or PKI groups) of the kinase inhibitors.…”

“…CGRP release was measured by modifications to the method described by Yue et al (2008). Capsaicin (100 mM) stock solution was prepared in ethanol and diluted to the final required concentrations in Neurobasal A/LG/PG medium.…”

“…We found that sustained opioid agonist (SNC 80 or morphine) treatment leads to phosphorylation and sensitization of cellular adenylyl cyclase(s) (AC superactivation) in these cells in a Raf-1-dependent manner (Varga et al, 2003b;Yue et al, 2006). Recently, we showed that sustained morphine treatment also leads to AC superactivation in cultured neonatal rat primary sensory neurons in a Raf-1-dependent manner (Yue et al, 2008).…”

“…Thus, sustained incubation of NG108-15 neuroblastoma ϫ glioma hybrid cells (Sharma et al, 1977) or recombinant human -opioid/Chinese hamster ovary (CHO) cells (Yue et al, 2006) with morphine or recombinant human ␦-opioid/CHO cells with SNC 80 or deltorphin (Rubenzik et al, 2001) was shown to lead to cAMP overshoot after removal of the opioid ligand. In addition, our recent investigations demonstrate that sustained morphine treatment also causes cAMP overshoot in cultured primary sensory DRG neurons (Yue et al, 2008).…”