title: Activation of PI3-K/Akt pathway for thermal preconditioning to protect cultured cerebellar granule neurons against low potassium-induced apoptosis.
abstract: OBJECTIVE
  This study was designed to investigate whether the activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is required for thermal preconditioning to protect rat cerebellar granule neurons (CGN) against apoptosis induced by low potassium, and to explore the possibility of a link between the upregulated heat shock protein (HSP)70 expression and Akt activation in the acquisition of neuroprotection induced by thermal preconditioning.
  
  
  METHODS
  CGN cultured for 8 d in vitro were switched to 5K medium for 24 h after thermal preconditioning (TP; 43.5 degree for 90 min, then 37 degree for 1 h). To study the role of the PI3-K/Akt pathway, a PI3-K inhibitor, LY294002 (20 micromol/L) was added into the cultures 1 h before TP. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay and fluorescein diacetate staining were used to determine cell viability. Hoechst 33258 staining and agar gel electrophoresis were used to test the morphological and biological characters of CGN. Western blot analysis was employed to detect the levels of phospho-Akt, phospho-glycogen synthase kinase 3beta (GSK3beta) Akt, GSK3beta, and HSP70.
  
  
  RESULTS
  TP protected CGN against apoptosis induced by low potassium. LY294002 inhibited the neuroprotective effect on CGN induced by TP. TP induced a robust activation of Akt and the inactivation of GSK3beta via PI3-K. Furthermore, the activation of the PI3-K/Akt pathway by TP persisted for 24 h in the 5K cultures. LY294002 (20 micromol/L) failed to inhibit the upregulated HSP70 expression induced by TP.
  
  
  CONCLUSIONS
  The activation of the PI3-K/Akt pathway is required for TP to protect CGN against apoptosis induced by low potassium, but the neuroprotective effect by Akt activation is not mediated through the downstream induction of HSP70 expression.
authors.affiliation: Department of Pharmacology, Zhongshan Medical School, Sun Yat-sen University, Guangzhou 510080, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Cao
authors.forename: De-xiong
authors.initials: DX
authors.lastname: Cao
authors.forename: Xing-wen
authors.initials: XW
authors.lastname: Su
authors.forename: Li-jun
authors.initials: LJ
authors.lastname: Chen
authors.forename: Ai-lin
authors.initials: AL
authors.lastname: Liu
authors.forename: Wei-jian
authors.initials: WJ
authors.lastname: Jian
authors.forename: Yu-ping
authors.initials: YP
authors.lastname: Wu
authors.forename: Peng-xin
authors.initials: PX
authors.lastname: Qiu
authors.forename: Guang-mei
authors.initials: GM
authors.lastname: Yan
mesh_terms: D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D002531:Cerebellum / Q000473:pathology*; D002867:Chromones / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis; D006358:Hot Temperature; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010766:Phosphorylation; D011188:Potassium; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D002867:Chromones; D018840:HSP70 Heat-Shock Proteins; D009025:Morpholines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D011188:Potassium