title: A subpopulation of mitochondria prevents cytosolic calcium overload in endothelial cells after cold ischemia/reperfusion.
abstract: BACKGROUND
  Calcium represents a key mediator of cold ischemia/reperfusion (CIR) injury presumably by affecting mitochondrial function. In this study, we investigated cellular and mitochondrial changes of calcium homeostasis in sublethally damaged human endothelial cells.
  
  
  METHODS
  Changes in cellular and mitochondrial calcium concentrations were studied after cold ischemia in University of Wisconsin solution for 12 hr and reperfusion in ringer solution. Cytosolic-free calcium concentration ([Ca2+]c) and mitochondrial-free calcium content ([Ca2+]m) were analyzed by fura-2 and rhod-2 fluorescence, respectively. Pretreatment of cells with ruthenium red (RR) or a H+-ionophore was used to inhibit mitochondrial calcium uptake. Mitochondrial membrane potential (DeltaPsim) was measured by 5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide and 3,3'-dihexyloxacarbocyanine iodide fluorescence.
  
  
  RESULTS
  Twelve-hr cold ischemia did not induce apoptosis in endothelial cells. In such sublethally damaged cells, [Ca2+]c rose from approximately 20 nmol/L after cold ischemia to approximately 120 nmol/L during reperfusion. Pretreatment with RR leads to an approximately 5-fold rise in [Ca2+]c. Image analysis revealed a significant increase of [Ca2+]m in a subpopulation of mitochondria during reperfusion. This was not the case in RR-pretreated cells. DeltaPsim decreased significantly during cold ischemia and was sustained during reperfusion. The loss of DeltaPsim can be related to a reduced portion of mitochondria exhibiting high DeltaPsim.
  
  
  CONCLUSIONS
  Our results suggest that cytosolic calcium influx during CIR is buffered by a selective portion of mitochondria in human umbilical vein endothelial cells. These mitochondria protect cells against cytosolic calcium overload and probably against subsequent cell injury.
authors.affiliation: D. Swarovski Research Laboratory, Department of Transplant and General Surgery, University Hospital Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Amberger
authors.forename: H
authors.initials: H
authors.lastname: Weiss
authors.forename: T
authors.initials: T
authors.lastname: Haller
authors.forename: G
authors.initials: G
authors.lastname: Köck
authors.forename: M
authors.initials: M
authors.lastname: Hermann
authors.forename: M
authors.initials: M
authors.lastname: Widschwendter
authors.forename: R
authors.initials: R
authors.lastname: Margreiter
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003080:Cold Temperature; D003600:Cytosol / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D006706:Homeostasis; D006801:Humans; D007511:Ischemia / Q000378:metabolism* / Q000503:physiopathology; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology*; D009994:Osmolar Concentration; D015427:Reperfusion Injury / Q000378:metabolism* / Q000503:physiopathology; D014471:Umbilical Veins
chemical_list: D002118:Calcium