Transplant graft dysfunction is a significant problem despite tremendous advances in organ procurement and preservation in recent years. It has been postulated that massive calcium influx into the cytoplasm after ischemia-reperfusion in both kidney and liver allografts is a major mediator of cell dysfunction and death, which adversely effects graft survival. Maintenance of low intracellular calcium is vital for the proper functioning of cells after reperfusion, and this depends on the maintenance of efficient systems to transport calcium out of the cytosol or into the endoplasmic reticulum (ER). These systems include intracellular calcium release channels (ICRC) on the ER. The proposed research will test the hypothesis that the function of the ICRC is altered during the cold preservation-induced ischemia of both the kidney and liver. To test this hypothesis, three studies are proposed with the following aims: 1. To characterize the effects of cold preservation-induced ischemia on the function of the two subsets of ICRC: inositol 1,4,5-triphosphate (IP3)- and ryanodine-receptor (RyR)-ICRC, 2. To determine if the ischemia-induced changes in ICRC function are associated with the direct effect of kidney and liver ischemia on the ICRC or the indirect effects secondary to the metabolic disturbances in the cells, and 3. To determine how hypothermic preservation influences the intracellular calcium dynamics in intact porcine hepatocytes and renal tubular epithelial cells. Three experiments are proposed. In the first experiment, kidneys and livers procurred from 9-12 weeks old cross-bred farm pigs under isoflurane anesthesia will be perfused with University of Wisconsin (UW) solution and stored on ice for specified periods of time (0, 4, 8, 12, 18 hrs). ER fractions will then be prepared from these organs by gradient ultra-