Neuroexcitatory effects of morphine-3-glucuronide are dependent on Toll-like receptor 4 signaling.
BACKGROUND
Multiple adverse events are associated with the use of morphine for the treatment of chronic non-cancer pain, including opioid-induced hyperalgesia (OIH). Mechanisms of OIH are independent of opioid tolerance and may involve the morphine metabolite morphine-3-glucuronide (M3G). M3G exhibits limited affinity for opioid receptors and no analgesic effect. Previous reports suggest that M3G can act via the Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) heterodimer in the central nervous system to elicit pain.


METHODS
Immunoblot and immunocytochemistry methods were used to characterize the protein expression of TLR4 present in lumbar dorsal root ganglion (DRG). Using in vitro intracellular calcium and current clamp techniques, we determined whether TLR4 activation as elicited by the prototypical agonists of TLR4, lipopolysaccharide (LPS) and M3G, contributed to changes in intracellular calcium and increased excitation. Rodents were also injected with M3G to determine the degree to which M3G-induced tactile hyperalgesia could be diminished using either a small molecule inhibitor of the MD-2/TLR4 complex in rats or TLR4 knockout mice. Whole cell voltage-clamp recordings were made from small- and medium-diameter DRG neurons (25 μm < DRG diameter <45 μm) for both control and M3G-treated neurons to determine the potential influence on voltage-gated sodium channels (NaVs).


RESULTS
We observed that TLR4 immunoreactivity was present in peptidergic and non-peptidergic sensory neurons in the DRG. Non-neuronal cells in the DRG lacked evidence of TLR4 expression. Approximately 15% of assayed small- and medium-diameter sensory neurons exhibited a change in intracellular calcium following LPS administration. Both nociceptive and non-nociceptive neurons were observed to respond, and approximately 40% of these cells were capsaicin-insensitive. Increased excitability observed in sensory neurons following LPS or M3G could be eliminated using Compound 15, a small molecule inhibitor of the TLR4/MD-2 complex. Likewise, systemic injection of M3G induced rapid tactile, but not thermal, nociceptive behavioral changes in the rat, which were prevented by pre-treating animals with Compound 15. Unlike TLR4 wild-type mice, TLR4 knockout mice did not exhibit M3G-induced hyperalgesia. As abnormal pain sensitivity is often associated with NaVs, we predicted that M3G acting via the MD-2/TLR4 complex may affect the density and gating of NaVs in sensory neurons. We show that M3G increases tetrodotoxin-sensitive and tetrodotoxin-resistant (NaV1.9) current densities.


CONCLUSIONS
These outcomes provide evidence that M3G may play a role in OIH via the TLR4/MD-2 heterodimer complex and biophysical properties of tetrodotoxin-sensitive and tetrodotoxin-resistant NaV currents.
Department of Anesthesia, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Michael R
MR
Due
Andrew D
AD
Piekarz
Natalie
N
Wilson
Polina
P
Feldman
Matthew S
MS
Ripsch
Sherry
S
Chavez
Hang
H
Yin
Rajesh
R
Khanna
Fletcher A
FA
White
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D000697:Central Nervous System Stimulants / Q000494:pharmacology*; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects; D006930:Hyperalgesia / Q000503:physiopathology; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics; D037102:Lectins / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051218:Lymphocyte Antigen 96 / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009022:Morphine Derivatives / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics; D010147:Pain Measurement; D017288:Pain Threshold / Q000187:drug effects; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010812:Physical Stimulation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011984:Sensory Receptor Cells / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D026941:Sodium Channel Blockers / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014110:Touch / Q000187:drug effects
D000697:Central Nervous System Stimulants; D037102:Lectins; D008070:Lipopolysaccharides; C411728:Ly96 protein, mouse; D051218:Lymphocyte Antigen 96; D009022:Morphine Derivatives; D026941:Sodium Channel Blockers; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D013779:Tetrodotoxin; D010751:Phosphopyruvate Hydratase; D015740:Calcitonin Gene-Related Peptide; C018108:morphine-3-glucuronide; D002118:Calcium