title: Endoplasmic reticulum Ca2+ depletion induces endothelial cell apoptosis independently of caspase-12.
abstract: OBJECTIVE
  Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca(2+) content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca(2+) is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca(2+) signalling and caspase-12 cleavage in apoptosis of endothelial cells.
  
  
  METHODS
  Cytosolic Ca(2+) concentration ([Ca(2+)](i)) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques.
  
  
  RESULTS
  Thapsigargin (5 microM), an inhibitor of the ER Ca(2+)-ATPase, depleted ER Ca (2+) content, increased [Ca(2+)](i), cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca(2+)](i) but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca(2+) was chelated with BAPTA/AM (100 microM), bradykinin caused ER Ca(2+) depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 microM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 microM), inhibited caspase-12 cleavage but not apoptosis.
  
  
  CONCLUSIONS
  Thus, increases in intracellular Ca(2+) concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca(2+) depletion appears to induce apoptosis independently of caspase-12.
authors.affiliation: Department of Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan.
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Nakano
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Watanabe
authors.forename: Mariko
authors.initials: M
authors.lastname: Ozeki
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Asai
authors.forename: Hideki
authors.initials: H
authors.lastname: Katoh
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Satoh
authors.forename: Hideharu
authors.initials: H
authors.lastname: Hayashi
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D001011:Aorta; D017209:Apoptosis; D015153:Blotting, Western / Q000379:methods; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002154:Calpain / Q000037:antagonists & inhibitors; D053454:Caspase 12; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D004151:Dipeptides / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology*; D004730:Endothelium, Vascular / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D013552:Swine; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D002614:Chelating Agents; D004151:Dipeptides; D004791:Enzyme Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004533:Egtazic Acid; D019284:Thapsigargin; D002154:Calpain; D053454:Caspase 12; D020169:Caspases; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D001920:Bradykinin; D002118:Calcium; C058076:calpain inhibitor III