Biphasic mechanism for hypothermic induced loss of protein synthesis in hepatocytes

Background. A complication in liver transplantation is increased clotting t imes due to inhibition of protein synthesis resulting from prolonged hypoth ermic preservation. Protein synthesis is also blocked in cold preserved hep atocytes, In this study, the mechanism of inhibition of protein synthesis i n cold preserved hepatocytes was investigated. Methods. Hepatocytes prepared from rat liver were cold preserved in Univers ity of Wisconsin solution for 4, 24, and 48 hr, Protein synthesis was measu red as incorporation of radiolabeled leucine into acid precipitable protein s. Hepatocytes were treated with antioxidants dithiothreitol, trolox or def eroxamine, nitric oxide synthase inhibitor (N-G-monomethyl-L-arginine monoa cetate), steroids (dexamethasone or methylprednisolone), methods to keep ad enosine triphosphate high (aerobic storage), and cytoskeletal disrupting ag ents (cytochalasin D or colchicine). Results. There was a 26% decrease in protein synthesis after only 4 hr of c old storage and a further 25% decrease at 24 hr, Antioxidants, elevated ade nosine triphosphate, and NG-monomethyl-L-arginine monoacetate did not affec t the rate of loss of protein synthesis. Protein synthesis was not due to i nhibition of amino acid transport or lack of amino acids in the storage med ium. Steroid pretreatment of hepatocytes had no effect on the loss of prote in synthesis occurring in the first 4 hr of storage but did suppress the lo ss occurring during the next 44 hr of storage. Cytoskeletal disrupting agen ts, added to freshly isolated cells, inhibited protein synthesis. Conclusion. The mechanism of loss of protein synthesis in cold preserved li ver cells is not mediated by: (1) oxygen free radical generation or improve d by antioxidant therapy, (2) nitric oxide generation in hepatocytes, (3) a n adenosine triphosphate-sensitive destruction of cell viability, and (4) d ecreased permeability of amino acids or loss of amino acids from the cells. Loss of protein synthesis due to hypothermic storage appears biphasic, The first phase, occurring within 4 hr of storage, may be the result of the ef fects of hypothermia on the cell cytoskeletal system and may be untreatable . The second phase, which occurs during the next 24 to 48 hr is sensitive t o steroid pretreatment. This phase may be amenable to improved preservation methodology. Improved preservation of the liver may require the use of ste roids to conserve protein synthetic capabilities.