title: Evidence that intrathecal morphine-3-glucuronide may cause pain enhancement via toll-like receptor 4/MD-2 and interleukin-1beta.
abstract: Morphine-3-glucoronide (M3G) is a major morphine metabolite detected in cerebrospinal fluid of humans receiving systemic morphine. M3G has little-to-no affinity for opioid receptors and induces pain by unknown mechanisms. The pain-enhancing effects of M3G have been proposed to significantly and progressively oppose morphine analgesia as metabolism ensues. We have recently documented that morphine activates toll-like receptor 4 (TLR4), beyond its classical actions on mu-opioid receptors. This suggests that M3G may similarly activate TLR4. This activation could provide a novel mechanism for M3G-mediated pain enhancement, as (a) TLR4 is predominantly expressed by microglia in spinal cord and (b) TLR4 activation releases pain-enhancing substances, including interleukin-1 (IL-1). We present in vitro evidence that M3G activates TLR4, an effect blocked by TLR4 inhibitors, and that M3G activates microglia to produce IL-1. In vivo, intrathecal M3G (0.75 microg) induced potent allodynia and hyperalgesia, blocked or reversed by interleukin-1 receptor antagonist, minocycline (microglial inhibitor), and (+)-and (-)-naloxone. This latter study extends our prior demonstrations that TLR4 signaling is inhibited by naloxone nonstereoselectively. These results with (+)-and (-)-naloxone also demonstrate that the effects cannot be accounted for by actions at classical, stereoselective opioid receptors. Hyperalgesia (allodynia was not tested) and in vitro M3G-induced TLR4 signaling were both blocked by 17-DMAG, an inhibitor of heat shock protein 90 (HSP90) that can contribute to TLR4 signaling. Providing further evidence of proinflammatory activation, M3G upregulated TLR4 and CD11b (microglial/macrophage activation marker) mRNAs in dorsal spinal cord as well as IL-1 protein in the lumbosacral cerebrospinal fluid. Finally, in silico and in vivo data support that the glucuronic acid moiety is capable of inducing TLR4/MD-2 activation and enhanced pain. These data provide the first evidence for a TLR4 and IL-1 mediated component to M3G-induced effects, likely of at least microglial origin.
authors.affiliation: Department of Psychology and Neuroscience, University of Colorado at Boulder, Boulder, CO, USA. susannah.lewis@colorado.edu
authors.forename: S S
authors.initials: SS
authors.lastname: Lewis
authors.forename: M R
authors.initials: MR
authors.lastname: Hutchinson
authors.forename: N
authors.initials: N
authors.lastname: Rezvani
authors.forename: L C
authors.initials: LC
authors.lastname: Loram
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: S F
authors.initials: SF
authors.lastname: Maier
authors.forename: K C
authors.initials: KC
authors.lastname: Rice
authors.forename: L R
authors.initials: LR
authors.lastname: Watkins
mesh_terms: D000818:Animals; D039481:CD11b Antigen / Q000378:metabolism; D000697:Central Nervous System Stimulants / Q000008:administration & dosage / Q000009:adverse effects*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D006930:Hyperalgesia / Q000139:chemically induced / Q000188:drug therapy / Q000378:metabolism; D007278:Injections, Spinal; D053583:Interleukin-1beta / Q000134:cerebrospinal fluid / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000378:metabolism*; D008297:Male; D017628:Microglia / Q000187:drug effects / Q000378:metabolism; D009022:Morphine Derivatives / Q000008:administration & dosage / Q000009:adverse effects*; D010146:Pain / Q000139:chemically induced* / Q000188:drug therapy / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D039481:CD11b Antigen; D000697:Central Nervous System Stimulants; D018841:HSP90 Heat-Shock Proteins; D053583:Interleukin-1beta; D051218:Lymphocyte Antigen 96; D009022:Morphine Derivatives; D012333:RNA, Messenger; D017472:Receptors, Interleukin-1; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; C018108:morphine-3-glucuronide