Phosphoproteomics and Bioinformatics Analyses of Spinal Cord Proteins in Rats with Morphine Tolerance

Abstract

IntroductionMorphine is the most effective pain-relieving drug, but it can cause unwanted side effects. Direct neuraxial administration of morphine to spinal cord not only can provide effective, reliable pain relief but also can prevent the development of supraspinal side effects. However, repeated neuraxial administration of morphine may still lead to morphine tolerance.MethodsTo better understand the mechanism that causes morphine tolerance, we induced tolerance in rats at the spinal cord level by giving them twice-daily injections of morphine (20 µg/10 µL) for 4 days. We confirmed tolerance by measuring paw withdrawal latencies and maximal possible analgesic effect of morphine on day 5. We then carried out phosphoproteomic analysis to investigate the global phosphorylation of spinal proteins associated with morphine tolerance. Finally, pull-down assays were used to identify phosphorylated types and sites of 14-3-3 proteins, and bioinformatics was applied to predict biological networks impacted by the morphine-regulated proteins.ResultsOur proteomics data showed that repeated morphine treatment altered phosphorylation of 10 proteins in the spinal cord. Pull-down assays identified 2 serine/threonine phosphorylated sites in 14-3-3 proteins. Bioinformatics further revealed that morphine impacted on cytoskeletal reorganization, neuroplasticity, protein folding and modulation, signal transduction and biomolecular metabolism.ConclusionsRepeated morphine administration may affect multiple biological networks by altering protein phosphorylation. These data may provide insight into the mechanism that underlies the development of morphine tolerance.

The Proteomics of Intrathecal Analgesic agents for Chronic Pain

“…GFAP is down regulated [33] and hypophosphorylated [31] in rats receiving intrathecal morphine. Nervous system plasticity depends on phosphorylation of key cytoskeletal proteins, and the hypophosphorylation of GFAP associated with morphine may play a role in opioid tolerance.…”

“…Some members of this family are up-regulated in neuropathic pain models [ 16 ]. They are also hyperphosphorylated following intrathecal morphine therapy [ 31 ].…”

“…Intrathecal morphine therapy also alters the expression of these enzymes. Gamma-enolase, and fructose-bisphophate aldolase C are both hyper- phosphorylated in rats receiving intrathecal morphine [ 31 ].…”

“…HSPs are up-regulated in the nervous system in response to stress or injury, and HSP 70 protects against oxidative damage [ 57 ]. In morphine tolerant rats, after intrathecal morphine therapy, HSP 70 is down regulated [ 30 ] and hyperphosphorylated [ 31 ]. Morphine therapy may negate the protective effects of HSP 70, and allow the apoptosis of dysfunctional neurons in neuropathic pain states.…”

“…Liaw et al studied the effect of intrathecal morphine on protein phosphorylation [ 31 ]. The authors hypothesised that specific phosphorylated proteins were involved in the development of morphine tolerance.…”