Mitochondrial defects by intracellular calcium overload versus endothelial cold ischemia/reperfusion injury

Abstract Questions as to the critical stress factor and primary targets of cold ischemia/reperfusion (CIR) injury were addressed by comparing mitochondrial defects caused by (1) CIR injury and (2) intracellular Ca²⁺ overload. CIR was simulated in transformed human umbilical vein endothelial cell cultures (tEC) by 8 h cold anoxia in University of Wisconsin solution and reoxygenation at 37°C. Intracellular Ca²⁺ concentrations were changed by permeabilization of suspended cells with digitonin in culture medium (RPMI, 0.4 mM Ca²⁺). Binding of free Ca²⁺ by ethylene glycol‐bis(β‐aminoethylether)‐N,N,N',N'‐tetraacetic acid in RPMI or mitochondrial incubation medium served as controls. Extracellular Ca²⁺ protected the cell membrane against permeabilization. Mitochondrial functions were determined before and after permeabilization of the cell membrane. After CIR, mitochondrial respiratory capacity declined, but oxygen consumption remained coupled to adenosine triphosphate (ATP) production. In contrast, Ca²⁺ overload caused uncoupling of mitochondrial respiration. High intracellular Ca²⁺ overload, therefore, does not reproduce cold ischemia/reperfusion injury in endothelial cells.