Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBrain Behav Immun. Author manuscript; available in PMC 2011 January 1. preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.

Recently, we demonstrated that ceramide kinase, and its product, ceramide 1-phosphate (Cer-1-P), were mediators of arachidonic acid released in cells in response to interleukin-1␤ and calcium ionophore (Pettus, B. J., Bielawska, A., Spiegel, S., Roddy, P., Hannun, Y. A., and Chalfant, C. E. (2003) J. Biol. Chem. 278, 38206 -38213). In this study, we demonstrate that down-regulation of cytosolic phospholipase A 2 (cPLA 2 ) using RNA interference technology abolished the ability of Cer-1-P to induce arachidonic acid release in A549 cells, demonstrating that cPLA 2 is the key phospholipase A 2 downstream of Cer-1-P. Treatment of A549 cells with Cer-1-P (2.5 M) induced the translocation of full-length cPLA 2 from the cytosol to the Golgi apparatus/perinuclear regions, which are known sites of translocation in response to agonists. Cer-1-P also induced the translocation of the CaLB/C2 domain of cPLA 2 in the same manner, suggesting that this domain is responsive to Cer-1-P either directly or indirectly. In vitro studies were then conducted to distinguish these two possibilities. In vitro binding studies disclosed that Cer-1-P interacts directly with full-length cPLA 2 and with the CaLB domain in a calcium-and lipid-specific manner with a K Ca of 1.54 M. Furthermore, Cer-1-P induced a calcium-dependent increase in cPLA 2 enzymatic activity as well as lowering the EC 50 of calcium for the enzyme from 191 to 31 nM. This study identifies Cer-1-P as an anionic lipid that translocates and directly activates cPLA 2 , demonstrating a role for this bioactive lipid in the mediation of inflammatory responses.

Increased intracellular

In migrating eukaryotic cells, phosphatidylinositol 3-kinase (PI3K), filamentous actin (F-actin), and monomeric Rho GTPases are key components of a complex positive-feedback system that maintains and amplifies a phosphatidylinositol-3,4,5-trisphosphate signal at the leading edge of the cell. This lipid signal is required for cell polarization and movement. In leukocytes and Dictyostelium, activation or inhibition of any one of these components leads to the activation or inhibition, respectively, of the others via undefined feedback interactions. The role of Ca 2؉ signals in migrating leukocytes is controversial, and there has been no indication that Ca 2؉ participates in positive feedback. Here, we demonstrate that an extracellular Ca 2؉ influx is required for positive feedback at the leading edge of spontaneously polarized macrophages. Inhibition of extracellular Ca 2؉ influx leads to loss of leading-edge PI3K activity, disassembly of F-actin, cessation of ruffling, and decay of chemoattractant signals. Conversely, increasing cytosolic Ca 2؉ enhances membrane ruffling, PI3K activity, and F-actin accumulation.Overall, these findings demonstrate that an extracellular Ca 2؉ influx is an essential component, together with PI3K and F-actin, of the positive-feedback cycle that maintains leading-edge structure and ruffling activity and that supports the chemoattractant response. Strikingly, the Ca 2؉ -sensitive enzyme protein kinase C␣ (PKC␣) is enriched at the leading edge, and its enrichment is sensitive to blockade of Ca 2؉ influx, to inhibition of PI3K activity, and to F-actin depolymerization. These findings support the working hypothesis that a local, leading-edge Ca 2؉ signal recruits PKC␣ as a central player in the positive-feedback loop.actin ͉ calcium ͉ chemotaxis ͉ phosphoinositide 3-kinase ͉ phosphatidylinositol-3,4,5-trisphosphate