title: Isoflurane inhibits cyclic adenosine monophosphate response element-binding protein phosphorylation and calmodulin translocation to the nucleus of SH-SY5Y cells.
abstract: BACKGROUND
  Calmodulin (CaM) activation by Ca(2+), its translocation to the nucleus, and stimulation of phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) (P-CREB) are necessary for new gene expression and have been linked to long-term potentiation, a process important in memory formation. Because isoflurane affects memory, we tested whether isoflurane interfered with the translocation of CaM to the neuronal cell nucleus and attenuated the formation P-CREB.
  
  
  METHODS
  SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western blotting, enzyme-linked immunosorbant assay, and immunocytochemistry. The translocation of CaM from the cytosol to the nucleus was also examined after depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Before and during KCl depolarization, cells were exposed to isoflurane, isoflurane plus Bay K 8644, nitrendipine, and omega-conotoxin GVIa, respectively.
  
  
  RESULTS
  P-CREB increased after KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 s. The increase in P-CREB formation was inhibited by nitrendipine, but not omega-conotoxin, and by isoflurane in a concentration-dependent fashion. Pretreatment with the L-type Ca(2+) channel agonist, Bay K 8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred after KCl depolarization. CaM translocation was inhibited by nitrendipine and attenuated by isoflurane. Bay K 8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus.
  
  
  CONCLUSIONS
  Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca(2+)entry through L-type Ca(2+) channels.
authors.affiliation: Department of Anesthesiology, NYU School of Medicine, 550 1st Ave., New York, NY 10016, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: Jhon-Jairo
authors.initials: JJ
authors.lastname: Sutachan
authors.forename: Jose
authors.initials: J
authors.lastname: Montoya-Gacharna
authors.forename: Chong-Feng
authors.initials: CF
authors.lastname: Xu
authors.forename: Fang
authors.initials: F
authors.lastname: Xu
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Neubert
authors.forename: Esperanza
authors.initials: E
authors.lastname: Recio-Pinto
authors.forename: Thomas J J
authors.initials: TJ
authors.lastname: Blanck
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D006801:Humans; D007530:Isoflurane / Q000494:pharmacology*; D008564:Membrane Potentials; D009447:Neuroblastoma / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D009568:Nitrendipine / Q000494:pharmacology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D013997:Time Factors; D020866:omega-Conotoxin GVIA / Q000494:pharmacology
chemical_list: D018685:Anesthetics, Inhalation; C115163:CREB1 protein, human; D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D002147:Calmodulin; D017362:Cyclic AMP Response Element-Binding Protein; D011189:Potassium Chloride; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D020866:omega-Conotoxin GVIA; D009568:Nitrendipine; D007530:Isoflurane; D002118:Calcium