Pharmacological characterization of LPS and opioid interactions at the toll-like receptor 4.
OBJECTIVE
Previous work in our laboratory showed opioid agents inhibit cytokine expression in astrocytes. Recently, Watkins and colleagues hypothesized that opioid agonists activate toll-like receptor 4 (TLR4) signalling, which leads to neuroinflammation. To test this hypothesis, we characterized LPS and opioid effects on TLR4 signalling in reporter cells.


METHODS
NF-κB reporter cells expressing high levels of TLR4 were used to compare LPS and opioid effects on NF-κB activation, a pathway activated by TLR4 stimulation.


RESULTS
LPS increased TLR4 signalling in a concentration-dependent manner and was antagonized by LPS antagonist (LPS-RS, from Rhodobacter sphaeroides). A concentration ratio analysis showed that LPS-RS was a competitive antagonist. The opioid agonists, morphine and fentanyl, produced minor activation of TLR4 signalling when given alone. When tested following LPS stimulation, opioid agonists inhibited NF-κB activation but this inhibition was not blocked by the general opioid antagonist, naloxone, nor by the selective μ opioid receptor antagonist, β-FNA. Indeed, both naloxone and β-FNA also inhibited NF-κB activation in reporter cells. Further examination of fentanyl and β-FNA effects revealed that both opioid agents inhibited LPS signalling in a non-competitive fashion.


CONCLUSIONS
These results show that LPS-RS is a competitive antagonist at the TLR4 complex, and that both opioid agonists and antagonists inhibit LPS signalling in a non-competitive fashion through a non-GPCR, opioid site(s) in the TLR4 signalling pathway. If confirmed, existing opioid agents or other drug molecules more selective at this novel site may provide a new therapeutic approach to the treatment of neuroinflammation.
Department of Pharmacology and Physiology, Oklahoma State University-Center for Health Sciences, Tulsa, OK 74107, USA. cw.stevens@okstate.edu
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D000701:Analgesics, Opioid / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D001667:Binding, Competitive; D004336:Drug Antagonism; D048168:Escherichia coli K12 / Q000378:metabolism; D017930:Genes, Reporter / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000235:genetics / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D009292:Narcotic Antagonists / Q000737:chemistry / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D011994:Recombinant Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D000701:Analgesics, Opioid; C114999:K-12 lipopolysaccharide; C495377:LY96 protein, human; D008024:Ligands; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D009292:Narcotic Antagonists; D009419:Nerve Tissue Proteins; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4