title: Proteomic analysis of PKCγ-related proteins in the spinal cord of morphine-tolerant rats.
abstract: BACKGROUND
  Morphine tolerance is a common drawback of chronic morphine exposure, hindering use of this drug. Studies have shown that PKCã may play a key role in the development of morphine tolerance, although the mechanisms are not fully known.
  
  
  RESULTS
  In a rat model of morphine tolerance, PKCã knockdown in the spinal cord was successfully carried out using RNA interference (RNAi) with lentiviral vector-mediated short hairpin RNA of PKCã (LV-shPKCã). Spinal cords (L4-L5) were obtained surgically from morphine-tolerant (MT) rats with and without PKCã knockdown, for comparative proteomic analysis. Total proteins from the spinal cords (L4-L5) were extracted and separated using two-dimensional gel electrophoresis (2DGE); 2D gel images were analyzed with PDQuest software. Seven differential gel-spots were observed with increased spot volume, and 18 spots observed with decreased spot volume. Among these, 13 differentially expressed proteins (DEPs) were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), comparing between MT rats with and without PKCã knockdown. The DEPs identified have roles in the cytoskeleton, as neurotrophic factors, in oxidative stress, in ion metabolism, in cell signaling, and as chaperones. Three DEPs (GFAP, FSCN and GDNF) were validated with Western blot analysis, confirming the DEP data. Furthermore, using immunohistochemical analysis, we reveal for the first time that FSCN is involved in the development of morphine tolerance.
  
  
  CONCLUSIONS
  These data cast light on the proteins associated with the PKCã activity during morphine tolerance, and hence may contribute to clarification of the mechanisms by which PKCã influences MT.
authors.affiliation: Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha, China.
authors.forename: Zongbin
authors.initials: Z
authors.lastname: Song
authors.forename: Qulian
authors.initials: Q
authors.lastname: Guo
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Maoyu
authors.initials: M
authors.lastname: Li
authors.forename: Chang
authors.initials: C
authors.lastname: Liu
authors.forename: Wangyuan
authors.initials: W
authors.lastname: Zou
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004361:Drug Tolerance; D015180:Electrophoresis, Gel, Two-Dimensional; D055785:Gene Knockdown Techniques; D008297:Male; D008969:Molecular Sequence Data; D009020:Morphine / Q000494:pharmacology*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013116:Spinal Cord / Q000378:metabolism*
chemical_list: D009020:Morphine; C087857:protein kinase C gamma; D011493:Protein Kinase C