title: Activation of adult rat CNS endothelial cells by opioid-induced toll-like receptor 4 (TLR4) signaling induces proinflammatory, biochemical, morphological, and behavioral sequelae.
abstract: CNS immune signaling contributes to deleterious opioid effects including hyperalgesia, tolerance, reward, and dependence/withdrawal. Such effects are mediated by opioid signaling at toll-like receptor 4 (TLR4), presumptively of glial origin. Whether CNS endothelial cells express TLR4 is controversial. If so, they would be well positioned for activation by blood-borne opioids, contributing to opioid-induced pro-inflammatory responses. These studies examined adult primary rat CNS endothelial cell responses to (-)-morphine or its mu opioid receptor (MOR)-inactive metabolite morphine-3-glucuronide (M3G), both known TLR4 agonists. We demonstrate that adult rat CNS endothelial cells express functional TLR4. M3G activated nuclear factor kappaB (NF-κB), increased tumor necrosis factor-α (TNFα) and cyclooxygenase-2 (COX2) mRNAs, and released prostaglandin E2 (PGE2) from these cells. (-)-Morphine-induced upregulation of TNFα mRNA and PGE2 release were unmasked by pre-treatment with nalmefene, a MOR antagonist without TLR4 activity (unlike CTAP, shown to have both MOR- and TLR4-activity), suggestive of an interplay between MOR and TLR4 co-activation by (-)-morphine. In support, MOR-dependent Protein Kinase A (PKA) opposed TLR4 signaling, as PKA inhibition (H-89) also unmasked (-)-morphine-induced TNFα and COX2 mRNA upregulation. Intrathecal injection of CNS endothelial cells, stimulated in vitro with M3G, produced TLR4-dependent tactile allodynia. Further, cortical suffusion with M3G in vivo induced TLR4-dependent vasodilation. Finally, endothelial cell TLR4 activation by lipopolysaccharide and/or M3G was blocked by the glial inhibitors AV1013 and propentofylline, demonstrating endothelial cells as a new target of such drugs. These data indicate that (-)-morphine and M3G can activate CNS endothelial cells via TLR4, inducing proinflammatory, biochemical, morphological, and behavioral sequelae. CNS endothelial cells may have previously unanticipated roles in opioid-induced effects, in phenomena blocked by presumptive glial inhibitors, as well as TLR4-mediated phenomena more broadly.
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA. Electronic address: peter.grace@colorado.edu.
authors.forename: P M
authors.initials: PM
authors.lastname: Grace
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Ramos
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Rodgers
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA; Department of Chemistry and Biochemistry, and the BioFrontiers Institute, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.affiliation: Discipline of Physiology, School of Medical Sciences, University of Adelaide, South Australia, Australia.
authors.forename: M R
authors.initials: MR
authors.lastname: Hutchinson
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: M T
authors.initials: MT
authors.lastname: Lewis
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: K N
authors.initials: KN
authors.lastname: Morgan
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Kroll
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: F R
authors.initials: FR
authors.lastname: Taylor
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Strand
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: D
authors.initials: D
authors.lastname: Berkelhammer
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: M G
authors.initials: MG
authors.lastname: Huey
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: L I
authors.initials: LI
authors.lastname: Greene
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Cochran
authors.affiliation: Department of Chemistry and Biochemistry, and the BioFrontiers Institute, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: H
authors.initials: H
authors.lastname: Yin
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: D S
authors.initials: DS
authors.lastname: Barth
authors.affiliation: MediciNova, Inc., San Diego, CA, USA.
authors.forename: K W
authors.initials: KW
authors.lastname: Johnson
authors.affiliation: Chemical Biology Research Branch, National Institute on Drug Abuse and National Institute on Alcohol Abuse and Alcoholism, Rockville, MD, USA.
authors.forename: K C
authors.initials: KC
authors.lastname: Rice
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: S F
authors.initials: SF
authors.lastname: Maier
authors.affiliation: Department of Psychology and the Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.
authors.forename: L R
authors.initials: LR
authors.lastname: Watkins
mesh_terms: D000818:Animals; D002490:Central Nervous System / Q000187:drug effects* / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology; D006930:Hyperalgesia / Q000188:drug therapy / Q000503:physiopathology; D008297:Male; D009020:Morphine / Q000494:pharmacology*; D009022:Morphine Derivatives / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism; D009294:Narcotics / Q000494:pharmacology*; D009457:Neuroglia / Q000187:drug effects / Q000502:physiology; D015213:Neuroimmunomodulation / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D017450:Receptors, Opioid, mu / Q000037:antagonists & inhibitors / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology
chemical_list: D009022:Morphine Derivatives; D016328:NF-kappa B; D009294:Narcotics; D012333:RNA, Messenger; D017450:Receptors, Opioid, mu; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D009020:Morphine; D051546:Cyclooxygenase 2; C489691:Ptgs2 protein, rat; D017868:Cyclic AMP-Dependent Protein Kinases; D015232:Dinoprostone; C018108:morphine-3-glucuronide
keywords: CTAP; M3G; TLR4; endothelial cell; morphine; nalmefene