J S Kim1, J H Southard. 1. Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison 53792, USA.
Abstract
BACKGROUND: Preservation injury is multifactorial and its mechanism is still incompletely defined. Calcium may play an important role in preservation injury. METHODS: The effects of hypothermia on cytosolic free calcium concentration ([Ca2+]I) and total cellular calcium content in isolated rat hepatocytes were investigated by using fura-2 fluorescence and atomic absorption spectroscopy. Fura-2 loaded cells were placed into a prechilled (7 degrees C) cuvette equipped with a stirrer or preserved in the University of Wisconsin (UW) solution for up to 48 hr. In some experiments, cells were pretreated with inhibitors of Ca2+ release from mitochondria (m-iodobenzylguanidine [MIBG]) and from endoplasmic reticulum (ryanodine [RYA]) for 20 min at 37 degrees C. Mitochondrial functions after preservation were evaluated by measuring ATP and respiratory rates. RESULTS: Cooling to 7 degrees C caused a rapid increase in [Ca2+]I that was substantially blocked by MIBG and RYA pretreatment. The elevated calcium gradually leaked out of the cells into the Ca2+-free medium. In long-term storage of the cells in the UW solution, there was a marked decrease in both cytosolic free calcium and total cellular calcium. Pretreatment of the livers with MIBG before cold preservation in the UW solution resulted in a stimulation of ATP regeneration in tissue slices. MIBG pretreatment also improved mitochondrial respiratory functions after cold preservation. CONCLUSIONS: Thus, the loss of mitochondrial function after liver preservation in the UW solution may be related to the effects of hypothermia on calcium metabolism. Approaches to help maintain calcium homeostasis during storage may improve organ preservation.
BACKGROUND: Preservation injury is multifactorial and its mechanism is still incompletely defined. Calcium may play an important role in preservation injury. METHODS: The effects of hypothermia on cytosolic free calcium concentration ([Ca2+]I) and total cellular calcium content in isolated rat hepatocytes were investigated by using fura-2 fluorescence and atomic absorption spectroscopy. Fura-2 loaded cells were placed into a prechilled (7 degrees C) cuvette equipped with a stirrer or preserved in the University of Wisconsin (UW) solution for up to 48 hr. In some experiments, cells were pretreated with inhibitors of Ca2+ release from mitochondria (m-iodobenzylguanidine [MIBG]) and from endoplasmic reticulum (ryanodine [RYA]) for 20 min at 37 degrees C. Mitochondrial functions after preservation were evaluated by measuring ATP and respiratory rates. RESULTS: Cooling to 7 degrees C caused a rapid increase in [Ca2+]I that was substantially blocked by MIBG and RYA pretreatment. The elevated calcium gradually leaked out of the cells into the Ca2+-free medium. In long-term storage of the cells in the UW solution, there was a marked decrease in both cytosolic free calcium and total cellular calcium. Pretreatment of the livers with MIBG before cold preservation in the UW solution resulted in a stimulation of ATP regeneration in tissue slices. MIBG pretreatment also improved mitochondrial respiratory functions after cold preservation. CONCLUSIONS: Thus, the loss of mitochondrial function after liver preservation in the UW solution may be related to the effects of hypothermia on calcium metabolism. Approaches to help maintain calcium homeostasis during storage may improve organ preservation.
Authors: Christopher D Anderson; Janene Pierce; Ian B Nicoud; Andrey E Belous; Christopher M Jones; Ravi S Chari Journal: J Surg Res Date: 2007-06-14 Impact factor: 2.192
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