Little evidence for a major role of Ca2+ in cold-induced injury of liver cells.

We have previously shown that cold-induced injury to hepatocytes and liver endothelial cells occurs predominantly via an iron-dependent pathway. However, other groups have reported evidences suggesting that Ca(2+) ions could be involved in the process of cold-induced injury of liver cells. We here assessed the relative importance and potential interaction of both pathways in cultured primary hepatocytes and cultured liver endothelial cells. The sequence cold incubation/rewarming of hepatocytes and endothelial cells led to an increase in the cytosolic calcium concentration during the early rewarming phase, but the increased cytosolic calcium concentration did not correlate with cell injury. A partial protection from cold-induced cell injury was achieved by the intracellular calcium chelators Quin-2 and BAPTA. However, additional experiments showed that the ability of these chelators to bind iron was probably responsible for a major part of this protection. Incubation in calcium-free media led to an increased cell injury and a physiological calcium concentration (2.5mM) was protective. In addition, targeting suggested downstream pathways of calcium-dependent cold-induced injury, i.e. by the addition of Ruthenium Red, an inhibitor of mitochondrial Ca(2+) uniporter, or by inhibiting Bax translocation to the mitochondria, did not provide protection from cold-induced injury in both cell types. Taken together, our data suggest that calcium increases but does not play a major role in cold-induced cell injury to hepatocytes and liver endothelial cells.