title: Amiloride derivatives induce apoptosis by depleting ER Ca(2+) stores in vascular endothelial cells.
abstract: OBJECTIVE
  Amiloride derivatives are blockers of the Na(+)/H(+) exchanger (NHE) and at micromolar concentrations have protective effects on cardiac and brain ischaemia/reperfusion injury but at higher concentrations also induce apoptosis. Here, we aimed to elucidate the mechanism related to this cytotoxic action.
  
  
  METHODS
  We quantified the expression of genes associated with endoplasmic reticulum (ER) stress and measured changes in luminal ER Ca(2+) concentration ([Ca(2+)](ER)) with a 'cameleon' indicator, D1ER.
  
  
  RESULTS
  Amiloride derivatives induced apoptosis in vascular endothelial cells, an effect that increased at alkaline extracellular pH. The potency order for cytotoxicity was 5-(N,N-hexamethylene)-amiloride (HMA) > 5-(N-methyl-N-isobutyl) amiloride > 5-(N-ethyl-N-isopropyl) amiloride (EIPA) >> amiloride. HMA dose-dependently increased the transcription of the ER stress genes GADD153 and GADD34 and rapidly depleted [Ca(2+)](ER), mimicking the effects of the sarco/endoplasmic reticulum ATPase (SERCA) inhibitor thapsigargin. The NHE1-specific inhibitor HOE 694 inhibited NHE activity by 87% but did not alter [Ca(2+)](ER). The decrease in [Ca(2+)](ER) evoked by amiloride derivatives was also observed in HeLa cells and was mirrored by an increase in cytosolic Ca(2+) concentration.
  
  
  CONCLUSIONS
  Amiloride derivatives disrupt ER and cytosolic Ca(2+) homeostasis by a mechanism unrelated to NHE inhibition, most likely by interfering with the activity of SERCA. We propose that ER Ca(2+) depletion and subsequent ER stress provide a rationale framework for the apoptotic effects of amiloride derivatives.
authors.affiliation: Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.
authors.forename: K S
authors.initials: KS
authors.lastname: Park
authors.forename: D
authors.initials: D
authors.lastname: Poburko
authors.forename: C B
authors.initials: CB
authors.lastname: Wollheim
authors.forename: N
authors.initials: N
authors.lastname: Demaurex
mesh_terms: D000584:Amiloride / Q000031:analogs & derivatives / Q000633:toxicity*; D000943:Antigens, Differentiation / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics; D004232:Diuretics / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D054645:Protein Phosphatase 1; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D051742:Transcription Factor CHOP / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D000943:Antigens, Differentiation; D018797:Cell Cycle Proteins; C497249:DDIT3 protein, human; D004232:Diuretics; D004791:Enzyme Inhibitors; D006146:Guanidines; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C074107:3-methylsulfonyl-4-piperidinobenzoyl guanidine; C057360:5-(N,N-hexamethylene)amiloride; D051742:Transcription Factor CHOP; D019284:Thapsigargin; D000584:Amiloride; C087519:PPP1R15A protein, human; D054645:Protein Phosphatase 1; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium; C039614:ethylisopropylamiloride