title: A unique modulator of endoplasmic reticulum stress-signalling pathways: the novel pharmacological properties of amiloride in glial cells.
abstract: OBJECTIVE
  Stress on the endoplasmic reticulum (ER) can trigger rescuer responses such as the unfolded protein response (UPR). However, pharmacological modulators of these ER-regulated stress responses are not well understood. In the present study, we found that amiloride, a potassium-sparing diuretic, has unique properties relating to such stress.
  
  
  METHODS
  We treated mouse primary cultured glial cells with amiloride, in the absence and presence of the ER stress-inducing reagents tunicamycin (Tm) or dithiothreitol, and measured UPR and ER stress-induced cell death. IRE1alpha phosphorylation, eIF2alpha phosphorylation, X-box binding protein 1 (XBP1) splicing, glucose regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression by reverse transcription-polymerase chain reaction and Western blotting were used to assess UPR and lactate dehydrogenase activity was determined to measure ER stress-induced cell death.
  
  
  RESULTS
  Amiloride completely inhibited ER stress-induced activation of IRE1alpha, an ER-localized stress sensor protein, splicing of XBP1, and subsequent expression of GRP78 at the mRNA and protein levels. ER stress induces the phosphorylation of eIF2alpha, leading to the expression of CHOP or an attenuation of translation in cells. Surprisingly, treatment with amiloride alone markedly promoted the phosphorylation but actually inhibited ER stress-induced CHOP expression. Finally, we found that amiloride (200 microM) synergistically enhanced ER stress-induced cell death, which was mediated through caspases. On the other hand, a low dose of amiloride (20 microM) significantly prevented Tm-induced cell death.
  
  
  CONCLUSIONS
  These results suggest that amiloride can modulate UPR. They also suggest amiloride to be an important pharmacological agent and provide basic information for understanding and preventing ER stress-related diseases.
authors.affiliation: Department of Pharmacotherapy, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi, Minami-ku, Hiroshima, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Hosoi
authors.forename: Ayaka
authors.initials: A
authors.lastname: Kume
authors.forename: Kayo
authors.initials: K
authors.lastname: Otani
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Oba
authors.forename: Koichiro
authors.initials: K
authors.lastname: Ozawa
mesh_terms: D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D016923:Cell Death; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000502:physiology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D004722:Endoribonucleases / Q000502:physiology; D006360:Heat-Shock Proteins / Q000502:physiology; D051379:Mice; D009457:Neuroglia / Q000187:drug effects* / Q000502:physiology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D000071701:Regulatory Factor X Transcription Factors; D015398:Signal Transduction; D051742:Transcription Factor CHOP / Q000096:biosynthesis / Q000502:physiology; D014157:Transcription Factors / Q000096:biosynthesis / Q000502:physiology; D000071717:X-Box Binding Protein 1
chemical_list: D004268:DNA-Binding Proteins; C497250:Ddit3 protein, mouse; C103652:Elf2 protein, mouse; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605544:Xbp1 protein, mouse; D051742:Transcription Factor CHOP; D000584:Amiloride; C510185:Ern1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases