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1.
J Biol Chem ; 292(15): 6123-6134, 2017 04 14.
Article in English | MEDLINE | ID: mdl-28242764

ABSTRACT

Sensitization of the heat-activated ion channel transient receptor potential vanilloid 1 (TRPV1) through lipids is a fundamental mechanism during inflammation-induced peripheral sensitization. Leukotriene B4 is a proinflammatory lipid mediator whose role in peripheral nociceptive sensitization is not well understood to date. Two major G-protein-coupled receptors for leukotriene B4 have been identified: the high-affinity receptor BLT1 and the low-affinity receptor BLT2. Transcriptional screening for the expression G-protein-coupled receptors in murine dorsal root ganglia showed that both receptors were among the highest expressed in dorsal root ganglia. Calcium imaging revealed a sensitization of TRPV1-mediated calcium increases in a relative narrow concentration range for leukotriene B4 (100-200 nm). Selective antagonists and neurons from knock-out mice demonstrated a BLT1-dependent sensitization of TRPV1-mediated calcium increases. Accordingly, leukotriene B4-induced thermal hyperalgesia was mediated through BLT1 and TRPV1 as shown using the respective knock-out mice. Importantly, higher leukotriene B4 concentrations (>0.5 µm) and BLT2 agonists abolished sensitization of the TRPV1-mediated calcium increases. Also, BLT2 activation inhibited protein kinase C- and protein kinase A-mediated sensitization processes through the phosphatase calcineurin. Consequently, a selective BLT2-receptor agonist increased thermal and mechanical withdrawal thresholds during zymosan-induced inflammation. In accordance with these data, immunohistochemical analysis showed that both leukotriene B4 receptors were expressed in peripheral sensory neurons. Thus, the data show that the two leukotriene B4 receptors have opposing roles in the sensitization of peripheral sensory neurons forming a self-restricting system.


Subject(s)
Calcium Signaling/physiology , Ganglia, Spinal/metabolism , Leukotriene B4/metabolism , Receptors, Leukotriene B4/metabolism , Sensory Receptor Cells/metabolism , Animals , Calcineurin/genetics , Calcineurin/metabolism , Calcium Signaling/drug effects , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Hyperalgesia/chemically induced , Hyperalgesia/genetics , Hyperalgesia/metabolism , Leukotriene B4/pharmacology , Mice , Mice, Knockout , Protein Kinase C/genetics , Protein Kinase C/metabolism , Receptors, Leukotriene B4/genetics , TRPV Cation Channels/genetics , TRPV Cation Channels/metabolism
2.
Proc Natl Acad Sci U S A ; 113(44): 12544-12549, 2016 11 01.
Article in English | MEDLINE | ID: mdl-27791151

ABSTRACT

Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a severe dose- and therapy-limiting side effect of widely used cytostatics that is particularly difficult to treat. Here, we report increased expression of the cytochrome-P450-epoxygenase CYP2J6 and increased concentrations of its linoleic acid metabolite 9,10-EpOME (9,10-epoxy-12Z-octadecenoic acid) in dorsal root ganglia (DRGs) of paclitaxel-treated mice as a model of CIPNP. The lipid sensitizes TRPV1 ion channels in primary sensory neurons and causes increased frequency of spontaneous excitatory postsynaptic currents in spinal cord nociceptive neurons, increased CGRP release from sciatic nerves and DRGs, and a reduction in mechanical and thermal pain hypersensitivity. In a drug repurposing screen targeting CYP2J2, the human ortholog of murine CYP2J6, we identified telmisartan, a widely used angiotensin II receptor antagonist, as a potent inhibitor. In a translational approach, administration of telmisartan reduces EpOME concentrations in DRGs and in plasma and reverses mechanical hypersensitivity in paclitaxel-treated mice. We therefore suggest inhibition of CYP2J isoforms with telmisartan as a treatment option for paclitaxel-induced neuropathic pain.


Subject(s)
Benzimidazoles/pharmacology , Benzoates/pharmacology , Cytochrome P-450 Enzyme System/genetics , Neuralgia/prevention & control , Paclitaxel/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/toxicity , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/metabolism , Female , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Gene Expression Regulation, Enzymologic/drug effects , HEK293 Cells , Humans , Linoleic Acids/blood , Linoleic Acids/metabolism , Male , Mice, Inbred C57BL , Molecular Targeted Therapy/methods , Neuralgia/chemically induced , Paclitaxel/toxicity , Pain Threshold/drug effects , Telmisartan
3.
J Lipid Res ; 46(3): 603-14, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15627655

ABSTRACT

The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.


Subject(s)
Adipocytes/metabolism , Glycerides/chemistry , Oxadiazoles/chemistry , Sterol Esterase/analysis , Sterol Esterase/metabolism , Adipocytes/chemistry , Adipocytes/enzymology , Animals , Cell Extracts/chemistry , Glycerides/chemical synthesis , Humans , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Liposomes/chemistry , Molecular Structure , Oxadiazoles/chemical synthesis , Rats , Recombinant Proteins/analysis , Recombinant Proteins/metabolism , Sensitivity and Specificity , Sonication , Spectrophotometry/methods , Substrate Specificity/physiology , Time Factors
4.
Biochimie ; 85(12): 1245-56, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14739077

ABSTRACT

For facilitation of the experimental analysis of the mechanism and regulation of mobilization of fatty acids from adipose triacylglycerol (TAG) stores, which also represents important targets for pharmacological intervention with the pathogenesis of diabetes and obesity, we developed a convenient and reliable non-radioactive cell-based assay. Isolated rat adipocytes are incubated with the fluorescent fatty acid derivative, 12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoic acid (NBD-FA), in the presence of insulin. The resulting NBD-FA-labeled TAG is efficiently cleaved by hormone-sensitive lipase (HSL) in vitro. After removal of insulin and excess of free NBD-FA, lipolysis is initiated by addition of isoproterenol and/or adenosine deaminase. The amount of NBD-FA generated in total or released into the incubation medium in the presence of modulatory hormones or compounds is then monitored by thin layer chromatography and fluorescence imaging. Release of NBD-FA, glycerol and [3H]oleic acid from TAG follows similar kinetics and concentration dependence in response to various lipolytic and anti-lipolytic stimuli as well as inhibitors of HSL. Release of NBD-FA from adipocytes correlates well to translocation of HSL from the cytosol to TAG droplets. In addition, we found that a cell-free system consisting of NBD-FA-labeled TAG droplets with endogenous associated HSL closely reflects the lipolytic state of the adipocytes used for its preparation. In conclusion, release of NBD-FA from TAG in vivo and in vitro can be used as accurate index for (regulation of) lipolysis in primary and cultured adipocytes.


Subject(s)
4-Chloro-7-nitrobenzofurazan/analogs & derivatives , 4-Chloro-7-nitrobenzofurazan/metabolism , Adipocytes/metabolism , Fatty Acids/metabolism , Fluorescent Dyes/analysis , Lauric Acids/metabolism , Lipolysis/physiology , 4-Chloro-7-nitrobenzofurazan/analysis , 4-Chloro-7-nitrobenzofurazan/chemistry , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/enzymology , Animals , Cells, Cultured , Fatty Acids/analysis , Fluorescent Dyes/metabolism , Glycerol/metabolism , Isoproterenol/pharmacology , Lauric Acids/analysis , Lauric Acids/chemistry , Lipolysis/drug effects , Oleic Acid/metabolism , Rats , Rats, Wistar , Sterol Esterase/metabolism
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